Citation
Clonal propagation of endangered native plants rhododendron chapmanii gray, taxus floridana nutt., and torreya taxifolia arn.

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Title:
Clonal propagation of endangered native plants rhododendron chapmanii gray, taxus floridana nutt., and torreya taxifolia arn.
Creator:
Barnes, Lee Roy, 1954-
Copyright Date:
1985
Language:
English

Subjects

Subjects / Keywords:
Embryos ( jstor )
Plantlets ( jstor )
In vitro fertilization ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Lee Roy Barnes. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
13317791 ( OCLC )
ACU6768 ( LTUF )
0020892879 ( ALEPH )

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Full Text










CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS
RHODODENDRON CHAPMANII GRAY, TAXUS FLORIDANA NUTT.,
AND TORREYA TAXIFOLIA ARN.









by


LEE ROY BARNES, JR.

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1985
















ACKNOWLEDGEMENTS


I would like to acknowledge the support and advice of my graduate

committee, Drs. Charles Johnson, Thomas Sheehan, Mike Young, Indra Vasil, as well as, Norman Schenck. I also thank the Department of Ornamental Horticulture for financial support, Mr. Jim Stevenson and Dr. Greg Brock with the Florida Department of Natural Resources for their friendly cooperation, and the Park Manager and staff of Torreya State Park for their assistance.

I would also like to thank the following for their patient and

enthusiastic help, Ms. Nancy Philman, Ms. Shirley Anderson, Ms. Jackie Host, Mr. T..D. Townsend, Mr. Jan Weinbrecht, Ms. Barbara Prichard, Ms. Debbie Gaw and Ms. Robin Reddick. Also I thank Ms. Anne Whealy, Ms. Sara Rosenbaum, Mr. Chris Fooshee, Mr. Ed Duke and Ms. Beth Logan for human support.

Special thanks are offered to Dr. Rita Hummel and Dr. Jasper Joiner for their humor, laughter, and personal concern.

Finally, I would like to acknowledge the timeless support and love of my parents and sister.


ii

















TABLE OF CONTENTS


PAGE

ACKNOWLEDGEMENTS............................................. ii

LIST OF TABLES AND FIGURES........................................ vi

KEY TO ABBREVIATIONS........................................ viii

ABSTRACT.................................................... ix

1.0 INTRODUCTION.............................................. 1

2.0 LITERATURE REVIEW......................................... 3

2.1 Endangered and Threatened Native Plants................... 3

2.2 Advantages of Micropropagation Techniques................. 4

2.3 Species Description and Distribution...................... 5

2.3.1 Rhododendron chapmanii Gray............................. 5
2.3.2 Taxus floridana Nutt. .................................. 5
2.3.3 Torreya taxifolia Arn. ................................. 6

2.4 Woody Plant Tissue Culture................................ 6

2.4.1 Organogenesis frouii Callus and Suspension Cultures ...... 7 2.4.2 Organogenesis from Plant Organs and Organ Sections...... 8 2.4.3 Stimulation of Axillary Meristens....................... 15
2.4.4 Taxus Embryo Culture.................................... 18
2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets...... 19 3.0 MATERIALS AND METHODS..................................... 21

3.1 Rhododendron chapmanii Gray............................... 21

3.1.1 Establishment of Stock Plants........................... 21
3.1.2 Disinfestation....................................... 22
3.1.3 Media Selection..................................... 22
3.1.4 Selection of Growth Regulator Levels.................... 23
3.1.5 Stage II Subcultures.................................... 23
3.1.6 Rooting and Establishment............................... 24
3.1.7 Colonization with Ericoid Mycorrhizae................... 24


iii











3.2 Taxus floridana Nutt. ................................. 26

3.2.1 Conventional Propagation by Cuttings.................... 26
3.2.2 Culture of Quiescent Shoot-tips......................... 26
3.2.3 Culture of Expanding Vegetative Buds.................... 27
3.2.4 Microsporangia Culture............................... 28
3.2.5 Embryo Culture...................................... 28

3.3 Torreya taxifolia Arn. ................................. 29

3.3.1 Conventional Propagation by Cuttings.................... 29
3.3.2 Culture of Quiescent Shoot-tips......................... 30
3.3.3 Culture of Expanding Vegetative Buds.................... 30
3.3.4 Culture of Micro- and Megasporangia..................... 30
3.3.5 Embryo Culture...................................... 30
3.3.6 Effect of Cytokinin Sprays on Stock Plants.............. 31

4.0 RESULTS.............................................. 33

4.1 Rhododendron chapmanii Gray............................ 33

4.1.1 Disinfestation.. ....................................... 33
4.1.2 Selection of Growth Regulators Levels................... 33
4.1.3 Stage II Subcultures................................. 36
4.1.4 Rooting and Establishment............................... 36
4.1.5 Colonization with Ericoid Mycorrhizae................... 39

4.2 Taxus floridana Nutt.................................. 43

4.2.1 Conventional Propagation by Cuttings.................... 43
4.2.2 Micropropagation of Taxus Shoot-tips.................... 43
4.2.3 Culture of Expanding-Vegetative Buds.................... 43
4.2.4 Culture of Microsporangia............................ 46
4.2.5 Embryo Culture.......................................... 46

4.3 Torreya taxifolia Arn................................... 46

4.3.1 Conventional Propagation by Cuttings.................... 46
4.3.2 Micropropagation of Shoot-tips........................ 48
4.3.3 Culture of Expanding Vegetative Buds.................... 48
4.3.4 Culture of Microsporangia and Megasporangia............. 48
4.3.5 Embryo Culture ...................................... 48
4.3.6 Cytokinin Sprayed Seedlings ............................ 51

5.0 DISCUSSION........................................... 53

5.1 Rhododendron chapmanii................................. 53

5.2 Taxus floridana..................................... 57

5.3 Torreya taxifolia................................... 60


CONCLUSIONS........................................


64


6.0












LIST OF REFERENCES.......................... .............. 66

BIOGRAPHICAL SKETCH....................................... 74


V


















LIST OF TABLES AND FIGURES


Table Page

4.1.1 Percent contamination of in vitro cultured Rhododendron
chapmanii shoot-tips grown in shade house and growth
chambers................................................ 34

4.1.2 Growth response of in vitro cultured Rhododendron
chapmanii shoot-tips cultured on Woody Plant Medium
with 5 levels of the cytokinin, 2-i-P................... 35

4.1.3 Growth response of in vitro cultured Rhododendron
chapmanii shoot-tips grown in 3x3 factorial combination
of auxin (IAA) and cytokinin (2-i-P).................... 37

4.1.4 Survival and rooting response of microcuttings of
Rhododendron chapmanii following treatment with auxins
from 2 sources.......................................... 40

4.1.5 Summary of micropropagation system developed for
Rhododendron chapmanii.................................. 41

4.1.6 Response of Rhododendron chapmanii plantlets produced
in vitro to 2 establishment media and inoculation with
or Without ericoid mycorrhizae.......................... 42

4.2.1 Rooting response of cuttings from 2 aged groups of
Taxus floridana to 3 levels of the auxin, IBA........... 44

4.2.2 In vitro growth response of Taxus floridana shoot-tips
on 4 nutrient media..................................... 45

4.3.1 Rooting response of cuttings of Torreya taxifolia
treated with 3 levels of the auxin, IBA................. 47

4.3.2 Growth response of cultured shoot-tips of Torreya
taxifolia grown in vitro on 4 nutrient media............ 49

4.3.3 Average number of axillary shoots of Torreya taxifolia
developing in vitro on 3 nutrient media and 5
concentrations of the cytokinin, BA...................... 50

Figure

4.1 Multiplication rates of in vitro cultured Rhododendron
chapmanii during initial and additional SII subcultures.. 38 vi


















KEY TO ABBREVIATIONS


GROWTH REGULATORS ABA Abscisic acid BA Benzyl adenine GA3 Gibberellic acid IAA Indole acetic acid IBA Indole butanoic acid 2-i-P 2-isopentyl adenine NAA Naphthalene acetic acid


NUTRIENT MEDIA H Heller's LS Linsmaier and Skoog 1/2LS 1/2 Strength Macronutrients
Linsmaier and Skoog MS Murashige and Skoog WPM Woody Plant Medium 1/2WPM 1/2 Strength Macronutrients
Woody Plant Medium


vii

















Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS RHODODENDRON
CHAPMANII GRAY, TAXUS FLORIDANA NUTT. AND TORREYA TAXIFOLIA ARN.

By

Lee Roy Barnes, Jr.
August 1985

Chairman: Dr. Charles R. Johnson Major Department: Horticultural Science

Clonal propagation utilizing conventional and micropropagation techniques was investigated for three endemic and endangered Florida plant species, Rhododendron chapmanii Gray, Taxus floridana Nutt. and Torreya taxifolia Arn. This is critical for their preservation since these species exist in limited numbers but have high ornamental value which has threatened their survival due to illegal collection.

Micropropagation of R. chapmanii was investigated using shoot-tip

cultures. An average of 7.6 fold increase in lateral shoots was achieved for three additional Stage II multiplication cycles on low salts Woody Plant Medium (WPM) with 2-i-P (10 mg/l) and adenine sulfate (80 mg/1). Microcuttings rooted in 4-6 weeks when direct-stuck into autoclaved 1:1:1 (v:v:v) Canadian peat:vermiculite:perlite medium in covered flats. A 5 second quick-dip in 1000 mg/l IBA (20% EtOH + 80% H20) increased survival and rooting of microcuttings. Inoculation of plantlets with the mycorrhizal fungus, Pezizella ericae increased survival and growth when plantlets were established in a less than optimal soil-less medium (1:1 v:v Fired nontmorillonite clay:Canadian peat) but not in MetroMix-500.
viii











Torreya and Taxus cuttings could be easily propagated when treated with 2,000-4,000 mg/1 IBA. However, cuttings taken from lateral branches demonstrated strict plagiotropic growth. Best rooting and growth of Torreya cuttings (20%) occurred following treatment with 4000 mg/i IBA. Taxus cuttings rooted best (67%) following treatment with 2000 mg/i IBA.

Shoot-tip cultures (3-5 cm long) of Taxus and Torreya developed only 2-3 lateral shoots, apparently from preformed buds. Cultured "embryonic

shoots" from expanding vegetative buds failed to produce multiple shoots. Excised embryos did not germinate following liquid culture to leach inhibitors. Micro- and megasporangia did not grow or differentiate in culture.

The most promising technique for Torreya involved application of cytokinins (4 weekly sprays with 100-200 mg/i BA or 2-i-P) to seedlings before culture. Newly stimulated axillary shoots developed additional shoots and multiple "bud-masses" when cultured on WPM with BA (1 mg/1) and NAA (0.01 mg/1). These were slow to develop but could be subcultured with additional growth of multiple shoots. No rooted plantlets were obtained.


ix

















1.0 INTRODUCTION


Preservation of indigenous endangered plant species is a critical issue requiring timely investigation of propagation techniques to increase the numbers of these rare species before they become extinct. Three endangered plant species of immediate concern are Rhododendron chapmanii Gray, Taxus floridana Nutt. and Torreya taxifolia Arn. All are endemic to restricted areas of northern Florida. Presently only 3000 Rhododendron chapmanii (Chapman's rhododendron) plants are known in situ and these are endangered due to habitat destruction or alteration, and over-collection for use in ornamental plantings. Taxus floridana (Florida yew) is endangered due to limited numbers and distribution but is highly desirable for landscape use and is subsequently subject to over-collection. Torreya taxifolia (Florida torreya) is endangered due not only to very limited numbers but also due to being decimated by a fungal disease which has destroyed most seed bearing trees. Today only 1200 apparently diseased seedlings and weak stump sprouts remain.

Presently, all three species are protected by state and federal

laws. However, in spite of legal protection, the remaining specimens of each species is declining. Numerous R. chapmanii have been illegally dug from the wild. Torreya taxifolia does not appear to be reproducing in the wild and is all but extinct, except for a few apparently healthy plants located in botanical gardens elsewhere in the United States.

The purpose of this investigation was to determine the

feasibility of clonal propagation techniques for the mass production of
1






2



these native ornamental species. Micropropagation techniques could potentially increase the number of these species with little impact on the present populations, as well as offer the possibility of eliminating the systemic pathogen(s) associated with Torreya die-back. If large numbers of these highly desirable plants could be produced, then they would be available for stabilizing natural populations, landscape plantings, and germ plasm preservation via meristem culture and cryopreservation.
















2.0 LITERATURE REVIEW


2.1 Endangered and Threatened Native Plants

One group of native plants of particular interest are those which are rare, threatened or endangered in their present environments. Endangered and threatened plants are often legally classified at both federal and state levels. "Endangered" plants are defined as "species in imminent danger of extinction or extirpation and whose survival is unlikely if the causal factors presently at work continue operating" (Ward, 1978. p. xiii). "Threatened" plants are defined as "species which are believed likely to move into the above endangered catagory in the near future if the causal factors now at work continue operating" (Ward, 1978. p. xiii). A "rare" species classification includes those species which are not presently endangered or threatened, but are potentially at risk.

Congress first attempted to protect endangered plant species by

enacting the Endangered Species Act of 1973 (House Document 95-41, 1975). Ayensu and de Filipps (1978) revised this initial listing and estimated that approximately 10% of the native vascular flora in the United States (approximately 20,000 spp.) or 2,000 species, subspecies and varieties are presently endangered or threatened.

Ayensu and DeFilipps (1978) concluded there are numerous reasons for protecting these species including the advantages of maintaining a broad gene pool and genetic diversity within and among species,


3






4


especially with important food, fiber and timber species. Many of these

threatened plants show great potential for ornamental use.

Within the state of Florida, there are an estimated 3,500 vascular plant species which are native or naturalized (Ward, 1978). Of these, 124 species are presently listed at the state level as being endangered or threatened. Ward (1978) concludes that this number represents only

3.5% of the total plant species but further estimates that the true number of endangered, threatened or rare species may vary from the listed number by a factor of 2 or as large as 7.

In 1978, the State of Florida (s. 581.185 Florida Statutes)

revised its listing of "endangered" plants to include over 30 species. They listed as "threatened" species all bromeliads, all cacti (except Opuntia), all orchids, all native rhododendrons, most ferns, most native Ilex spp., all Zamia spp., all Zephranthes spp. and 48 other species.


2.2 Advantages of Micropropagation Techniques

While many of the endangered and threatened plants are best

protected by procurement and management of sensitive habitats, many of these species can be propagated and introduced into the horticultural trade with minimal impact on the remaining populations or their genetic diversity. Seedage and conventional vegetative propagation by cuttings can be used to increase the number of plants. However, these techniques are often of limited value due to the lack of propagation and cultural information for these rare species. Also the expense of collecting seed

or vegetative propagules would be prohibitive due to limited numbers and travel expense.

Micropropagation of endangered species offers many advantages

over both seed and conventional vegetative propagation methods, such as






5


(1) high initial multiplication rates from limited plant material in a small area; (2) clonal propagation of selected phenotypes via stimulation of existing axillary meristems; (3) potential to force

rejuvenation of selected mature plants resulting in increased rooting ability of shoots with juvenile rooting capacity (Rhodes, 1968; Lyrene, 1981); (4) ability to propagate infertile or low fertility species (Wochok, 1981); (5) high degree of environmental control to minimize variability with limited plant material and (6) possibility of obtaining disease-free material via meristem culture (Abbott, 1977; Boxus and Druart, 1980; Styer and Chin, 1984).

Wochok (1981) suggested that tissue culture can be very useful in preserving threatened and endangered species and developed micropropagation techniques for 4 native species (Atriplex canescens, Mahonia aquifolium 'compacta', Mahonia repens, and Populus tremuloides).


2.3 Species Description and Distribution

2.3.1. Rhododendron chapmanii Gray

Rhododendron chapmanii Gray (Chapman's rhododendron) is a small

evergreen shrub, 1-2 meters in height, which produces a showy display of

terminal clusters of clear pink to rose-pink flowers in late spring (April-May). This species is apparently very drought, heat, and pest resistant (Simon, 1983).

Conventional propagation by seeds and cuttings has been successful (Salter, 1982 per.com.; Gensel, 1983 per. com.), albeit with a low rate of multiplication.







6


2.3.2 Taxus floridana Nutt.

Taxus floridana Nutt. (Florida yew; Savin) is an evergreen,

needle-bearing shrub or small tree 3-8 m (rarely 10 m) in height. It is highly adapted to heavy shade and tolerates severe pruning.

Conventional clonal propagation by cuttings is possible; however, cuttings from lateral branches grow only plagiotrophically and few orthotropic shoots are available. Seed propagation is limited by a double dormancy within the seed which usually requires two seasons to germinate. Seed set is limited and heavy fruiting has been observed only once in the last ten years in Torreya State Park (Womble, 1982, per. com.). While over 200 seeds were collected in 1982, only 25 were collected in 1983 following an extensive, tree to tree search.


2.3.3 Torreya taxifolia Arn.

Torreya taxifolia Arn. (Florida torreya; Stinking Cedar; Polecat Wood; Gopherwood; Savin; Tumion) is a medium sized, pungent smelling (when bruised or crushed) evergreen tree which grows to 15 'n.

Conventional propagation by cuttings is possible; however, lateral cuttings have been found to continue growing only plagiotrophically. Seed propagation has been successful but is limited by extreme scarcity of seed and a poorly understood double dormancy which usually prevents germination until the second year.


2.4 Woody Plant Tissue Culture

Tissue culture techniques have been applied to three general areas of interest, namely clonal propagation of (1) woody ornamentals (Murashige, 1974; Abbott, 1977; Debergh and Maene, 1981; McComb, 1978; Lazarte, 1981; 3riggs and McCullock, 1983), (2) fruit trees (McComb,







7


1978; Zimmerman, 1978; Lazarte, 1981; Loreti and Mozini, 1982), and

(3) superior genotypes of selected forest species (Durzan and Campbell, 1974; Abbott, 1977; Brown and Sommer, 1977; Cheng, 1978; Bonga, 1980; Durzan, 1980).

Propagation of woody species has been investigated by three different approaches: (1) organogenesis from callus or suspension cultures, (2) organogenesis directly from plant organ or organ sections and (3) axillary shoot stimulation. Attempts have been made to propagate from both selected mature tissues and from the more competent or plastic juvenile tissues. Although it is more desirable to obtain plants from elite mature genotypes, these tissues have been difficult to root. Rejuvenation treatments and the use of juvenile zones within the plants have been successful in some cases (Lyrene, 1981). By far the greatest amount of success has occurred with the use of explants from juvenile tissues, especially embryos and young seedlings. Although morphogenically flexible and generally easy to root, these juvenile sources are genetically variable and untested as to their mature phenotype.


2.4.1 Organogenesis from Callus and Suspension Cultures

Organogenesis from callus or suspension cultures established from elite trees would offer the greatest advantages. This would allow large scale and cost efficient production of superior trees. This would be especially important if somatic embryogenesis could be obtained and microencapsulation developed so that clonal "seeds" could be produced. However, at least two factors prevent the wide scale use of these techniques. First, organogenesis from callus and suspension cultures has not been successful for most important forest species (Wilkins et al.






8


1985). Secondly, the genetic stability of adventitious organogenesis is often questionable (Wilkins et al., 1985; Larkin and Scowcroft, 1981).

Successful regeneration of plantlets via somatic embryogenesis from suspension and callus cultures has been reported for Santalum album (Lakshmi Sita et al., 1980), and Liquidambar styraciflua (Sommer and Brown, 1980). Regeneration of shoots and roots from callus has been reported for triploid Populus tremuloides (Winton, 1970), Citrus grandis and C. sinensis (Chaturvedi and Mitra, 1974), Ulmus americana (Durzan and Lopushanski, 1975), Ephedra gerardiana (Ramawat and Arya, 1976), Larix decidua (Bonga, 1981), Malus domestica (Liu et al., 1983), Pinus eldarica (Herrara and Phillips, 1984), Eugenia jambos and E. malaccenis (Litz, 1984).

In summary, organogenesis from true cell suspensions and callus

cultures has only been successful for a limited number of woody species (especially with timber, tropical fruit, and arborescent monocotyledonous species), usually at low frequencies and often with the inability to root shoots from callus derived from mature trees (Wilkins et al., 1985).


2.4.2 Organogenesis from Plant Organs and Organ Sections

Direct organogenesis from plant organs and organ sections has been widely accomplished among woody species. In most cases, this has occurred from very juvenile explants, especially embryos or young seedlings. Rejuvenation from mature tissues has been successful from inflorescenes or micro- and megasporangia, tissues temporally associated with meiosis (Bonga, 1980).

Organogenesis has occurred in widely dispersed plant families.

LaRue (1948) obtained low frequency shoot regeneration from the megagametophyte of Zamia floridana. He later reported (1954) regeneration of







9


shoots and roots from megagametophytes of both Zamia floridana and Cycas revoluta. Norstog (1965) cultured megagainetophytes of Zamia integrifolia

and was able to obtain diploid and haploid roots and leaves in 35% of his cultures. He was also able to obtain diploid and haploid embryoids from cultures of excised embryos and haploid megagametophytes, respectively. Plants were successfully established in soil.

Konar and Oberoi (1965) obtained embryoids on cotyledons of Biota orientalis Endl. (now Platycladus orientalis). Further development of these embryoids did not occur until transfer to a different medium. These "embryoids" may have been inappropriately named since they did

not appear to have a root apex although normal appearing cotyledons and shoot apex developed into a young shoot.

Norstog and Rhamstine (1967) induced proliferation of callus from haploid and diploid tissues of Zamia integrifolia and haploid tissues of Cycas species.

Hu and Sussex (1971) were able to stimulate embryogenesis from cultured cotyledons of Ilex aquifolium. They noted that embryoids developed only on cotyledons where the shoot-tip failed to develop, suggesting an antagonism between shoot growth and initiation, possibly due to apical dominance of the active shoot-tip.

Isikawa (1974) reported the formation of adventitious buds and roots on the hypocotyls of Cryptomeria japonica, primarily in illuminated cultures. This organogenesis required low levels of the growth regulators ABA, NAA and BA. He concluded that callus formation may be antagonistic to shoot formation of conifers.

Sommer et al. (1975) reported high frequency adventitious bud formation on cultured cotyledons of Pinus palustris when grown on






10


modified Gresshoff and Doy medium. If maintained on media with high auxin/cytokinin ratios, excess callus began to form after 5-6 weeks. They also mentioned success in obtaining adventitious buds on cotyledons of P. elliotii, P. taeda, P. virginiana, P. rigida, P. strobus and Pseudotsuga menziesii but did not mention if plantlets were obtained from these species.

Cheng (1975) obtained adventitious bud formation on cotyledons and cotyledon slices of Pseudotsuga menziesii during primary culture, and was able to maintain the capacity to produce buds after 3 and 4 subcultures. This system required 3 separate steps: (1) culture initiation with equal amounts of auxins and cytokinins, (2) organ initiation with high cytokinin levels, and (3) further growth of adventitious buds on 1/2 strength modified MS medium and no growth regulators for further growth of adventitious buds. Plantlets were obtained by treating shoot cuttings with IBA (amounts not given). The necessity for separate steps suggests that conditions necessary for initiation of one step were inhibitory to further development.

Cheng (1976) reported adventitious buds from cotyledons of Tsuga heterophylla using techniques similar to those employed for Pseudotsuga menziesii. In addition, she was able to root shoots directly in soil mix thus saving one culture step.

Winton and Verhagen (1976) induced adventitious shoots directly front cotyledons of Pseudotsuga meniesii and also from subcultured callus, obtaining up to 100-200 clonal shoots from each seed. They also obtained callus on needles from 3 month old seedlings and from needles from a 25 year old tree. However, they were only able to stimulate adventitious shoots from the 3 month old needle callus.







11


Bonga (1977) initiated a new approach to obtain adventitious shoots from 15-20 year old Abies balsamea. "Embryonic shoots," composed of dormant buds with scale leaves excised, were soaked either in sterile water for 24 hours, or in aqueous solutions of growth regulators. Soaked buds formed additional axillary shoots when transferred to a growth medium while non-soaked buds only expanded as a single shoot.

Reilly and Washer (1977) cultured embryos of Pinus radiata and

obtained adventitious buds directly from cotyledons and hypocotyls and from meristematic callus which proliferated from these organs. This callus could be subcultured on cytokinin-free medium every 4 weeks. When shoot primordia were separated from these meristematic masses, they developed into well formed shoots. By serial subculture of callus, they were able to obtain over 200 shoots from 1 embryo in 6 months. These shoots could be rooted on medium with auxin (1-25 mg/1l IBA).

Brown and Sommer (1977) reported that 9 Pinus species developed buds on cotyledons at a frequency of 26-100% depending on the species and medium. They were unable to root these elongating shoots except very rarely (less than 1% rooted).

Mott et al. (1977) summarized their work with Pinus taeda whereby they obtained more buds on excised cotyledons vs intact embryos. They found significant differences in organogenic response between seed families but these differences could generally be reduced by adjusting the growth regulator combinations.

Coleman and Thorpe (1977) cultured cotyledons from seeds and

shoot-tips from 4 to 10 year old trees of Thuja plicata. Using lateral shoot-tips from 4 to 10 year old trees, they obtained adventitious shoots which grew only orthotrophically. The addition of high levels of






12


GA3 (0.01 mg/1) to the medium induced the development of male strobili from the vegetative apex. Fifty percent of the adventitious buds from juvenile tissues rooted when transferred to a medium with 1/2 strength MS and 1.6 mg/1l BA, while only 11% of those from the mature shoots rooted. They concluded that the successful formation of plantlets

requires 3 distinct sequential treatments (1) meristematic induction,

(2) bud elongation and (3) rooting and establishment.

Arnold and Eriksson (1978) collected vegetative buds of Picea abies from 5 year old trees, 75 year old hedged trees (2 m tall) and from trees in 4 age classes: 5-10, 10-15, 15-20, and 20-50 year old trees. No differences were found in the organogenic ability of these explants with an average of 30% of cultures producing buds. They made no attempt to test the rooting ability of these buds.

Aitken et al. (1981) compared explant sources for regeneration of Pinus radiata buds. Excised whole embryos, and intact and excised cotyledons from 1 week old seedlings were cultured, resulting in an average of 9 rootable shoots from the embryos, 18 from intact cotyledons and 180 from the excised cotyledons from 1 week old seedlings. Induction of nodular, smooth-surfaced meristematic callus appeared necessary for large-scale propagation. After 24 weeks they had obtained over 1300 shoots from the excised cotyledons from 1 seed thus greatly increasing the efficiency of this technique.

Bonga (1981) published a second paper on organogenesis from 15-20 year old mature trees including Abies balsamea. Morphogenesis occurred rarely from female cones of Pinus mugo and Picea abies. Vegetative shoots from A. balsamae, P. glauca and Pseudotsuga menziesii produced buds at the base of young expanding needles. His methods included







13


collecting buds before bud break, excising underwater and soaking for 15 minutes in 1 g/l malonic acid solution or for 24 hours under water.

Mott and Amerson (1981) updated their work with Pinus taeda,

summarizing the need for sequential transfer of explants. They stressed the point that 1 medium and combination of growth regulators initiated a response but further development was inhibited until they were transferred to a different medium. They stressed the need for judicious observation of the timing and duration of growth regulator exposure and termed the short term exposure to certain growth regulator combinations as "pulsing."

Patel and Berlyn (1982) questioned the genetic stability of

multiple buds regenerated from embryo explants of Pinus coulteri even when no cytokinins were used in the medium. Cells from callus and regenerated buds showed a progressive increase in DNA levels over time. After 6 weeks in culture, 80% of cells in regenerated buds had DNA levels between 4C and 12C as detennined with a microspectrophotometer. They did not determine whether this resulted in mutated whole plants.

David et al. (1982) reported adventitious budding on Pinus pinaster cotyledons and from short-shoots and elongating needles collected from a 10 year old tree which had been sprayed weekly with BA. Meristems of cultured short-shoots produced buds from 2 locations, from the apical meristem of the short-shoot and from mitotically active cells at the base of the expanding needles. When they altered the NH4/K+ ratio of the medium to 1, they obtained increased adventitious bud formation. They emphasized the importance of having mitotically active and morphogenically nondetermined cells.








14


Jansson and Bornman (1983) reported improved morphogenesis of spring collected "embryonic shoots" (bud with budscales removed) of Picea abies by inclusion of the "crown," a layer of 4 to 7 extremely thickened collenchyma cells located at the base of the shoot. This anatomical feature is common to Abies, Cedrus, Larix, Pseudotsuga, Picea, Sequoia, Taxus, Torreya and Tsuga. Following excision and culture of the embryonic shoots with attached crowns, an average of 10 buds per explant was produced while crown-minus explants produced only 3 buds per explant. They reiterated comments by Chalupa and Durzan (1973) that the crown may act as a filter or translocation barrier to nutrients and hormonal stimuli. They also suggest that the crown may be a barrier to basipetal transport.

In summary, organogenesis from juvenile tissues, especially

cotyledons and hypocotyls, is common among many conifers. Mitotically active cells of expanding needles also appear morphogenically flexible. However, the number of buds which can be produced by this method is limited (few to dozens) or rarely, hundreds from a single explant. By subculture of meristematic masses derived from these organs, up to 1300 plantlets have been obtained from a single seed of Pinus radiata (Aitken et al., 1981). The ability to obtain plantlets usually requires several different media and growth regulator regimes with careful attention paid to the duration of each exposure. Shoot initiation, shoot elongation,

root initiation and root elongation require different conditions to maximize the propagation rates. For many species, rooting has occurred at low frequencies but evidence suggests that more careful attention to duration of auxin exposure and light levels may increase rooting.








15


2.4.3 Stimulation of Axillary Meristems

The stimulation of axillary meristems has been most successful with ornamental and fruit tree species. Styer and Chin (1983) list over 145 genera which have been cultured from excised meristems and shoot-tips. 3riggs and McCulloch (1984) list 63 genera and 106 species and cultivars which are being propagated commercially or in research laboratories in the United States and Western Canada.

Haines and de Fossard (1977) reported stimulation of axillary buds from orthotrophic stem sections of Araucaria cunninghamia. Shoots which developed from bud traces of ortotrophic branches grew upright while

shoots derived from plagiotrophic branches grew only laterally.

Vieitez and Vieitez (1980a) propagated Castanea sativa by culture fron lateral buds from 3 to 4 month old seedlings and from axillary buds of cultured embryos. Keys and Cech (1981,1982) propagated Castanea dentata by stimulating axillary neristens from mature embryos and from 4 month old seedlings grown in a greenhouse. Chevre et al. (1983) micropropagated C. sativa and obtained increased axillary

multiplication and elongation by doubling the calcium and magnesium concentrations in MS medium and by lowering the pH to 4.0.

Rancillac et al. (1982) developed methods to induce axillary shoot development from 1 month old seedlings of Pinus pinaster. Rooting of these shoots was most successful when shoots were cultured for 17 days on media with 2 mg/l NAA followed by transfer to an auxin-free media.

Wang and Hu (1983) cultured shoot-tips of 5 year old Sassafras

randaiense and obtained "multiple-bud-inass" proliferation on MS plus 60 mg/l kinetin within 2 months of incubation. The rooting percentage








16


remained below 20%, but a 95% survival rate was achieved following

transfer to vermiculite in flats.

While explants from mature trees are generally difficult to root, Hearne (1982) reported the advantages of using mature tissues. He suggested that for many species where mature tissues could be air layered, these mature tissues are desirable since they retain their mature features and often root easily in culture. He cited the advantages of earlier flowering, and that plantlets commonly have reduced canopy size which reduced the need for pruning and eased harvesting. He cited precocious flowering roses within 49 days following deflasking, and fruiting grapes and papaya within 12 weeks from deflasking. His observations suggest that axillary shoot explants from mature plants retain their mature qualities yet root as if they had been "phenotypically" rejuvenated while in culture.

Amos and McCown (1981) reported their attempts to micropropagate conifers by shoot-tip and axillary meristem culture. They obtained shoot multiplication from species of Juniperus, Sequoia, Taxus, Thuja, and Tsuga when cultured with very low levels of BA. Picea and Pinus did not show rapid shoot development. They concluded that as a group, conifers are more sensitive to intermediate levels of cytokinins than woody dicot species. They were able to root harvested shoots, but did not comment on the further growth of these cultures (i.e. whether orthotrophic or plagiotropic shoots developed).

Micropropagation of rhododendrons has been a great commercial success. Anderson (1975) first reported micropropagation of Rhododendron using shoot-tip cultures on a modified, approximately 1/4 strength MS with a much reduced K content, obtaining an average of 6.2








17


new shoots per culture within 8 weeks. He noted that BA proved phytotoxic while 2-i-P was not, and that the addition of 80 mg/l adenine sulfate was necessary for superior shoot development. Anderson (1978) also reported successful direct rooting of micropropagated rhododendrons from Stage II culture thus saving a very labor intensive step. Several rooting media were successful as long as they were well drained and porous, for example 1:1 (v:v) perlite:peat.

Strode (1979) reported on the commercial micropropagation of

rhododendrons using a revised Anderson (1975) medium amended with 5 mg/l 2-i-P and 1 mg/l IAA. After 4 subcultures at 6 week intervals, he obtained an average of 22.9 plants which could be attained when 4 shoot-tips were cultured in a single container. Harvested shoots rooted 90% within 5 weeks.

Meyer (1982) reported the successful use of tissues from flower buds and inflorescences for rhododendron cultivars. Flower buds were chosen as they had been used successfully with several herbaceous species and resulted in lesser initial contamination. Meyer excised buds from dormant plants between October and April and obtained "granular masses" of tissues on Anderson's medium with 1-4 mg/1l IAA and

5-15 mg/l 2-i-P.

McCown and LLoyd (1983) surveyed the response of 7 genotypes of

rhododendron to micropropagation. They used Woody Plant Medium (WPM- a modified Gresshoff and Doy medium), which was originally developed for micropropagation of Kalmia and other woody species (McCown and Lloyd, 1981). They observed that 2-i-P stimulated the greatest shoot multiplication, while BA was phytotoxic to all but elipidote and lepidote rhododendrons. Optimal cytokinin levels varied little with the







18


genotype, with 1-3.5 mg/l 2-i-P generally optimal. They observed that adventitious shoot development occurred occasionally when 2-i-P greater than 2.75 mg/i was used. Average multiplication rates were 7 to 21 depending on cultivar. It was estimated that with an average of 40 shoots per 6 week subculture period, over 75,000 shoots could be generated per square meter of culture space per year. Plantlets from callus cultures were also obtained; however, many aberrant types developed. With axillary shoot stimulation, plants remained true-to-type even after 6 years of culture, except for occasional albino/green chimeras which were readily observed and easily rouged. It was noted that the resulting rooted plantlets grew vigorously with a high degree of basal branching, similar to the growth habit of seedling rhododendrons. They stated their uncertainty as to whether this branching was induced by growth regulators or whether the seedling morphology was being mimicked.


2.4.4 Taxus Embryo Culture

Taxus species have been cultured in vitro by several researchers. Tulecke (1959) cites LaRue's work with cultured pollen from Taxus brevifolia whereby he obtained an unusual "pincushion callus" with cells resembling pollen tubes sticking out at right angles to the surface of the callus. LaRue maintained long term cultures of this slow growing callus but never obtained regeneration of shoots.

Zenkteler and Guzowska (1970) cultured mature female gametophytes of Taxus baccata on White's medium and obtained proliferation of callus from 2-5% of the explants. Variation in chromosome number from n to 8n was found in callus from the 14th and 15th 8-10 week subcultures. Regeneration of shoots or roots was not observed. Cultured embryos did







19


not develop even when placed next to growing gametophytic tissues.

LePage-Degivry (1968,), LePage-Degivry (1970), 1973abc) and

LePage-Degivry and Garello (1973) initiated a series of experiments in which she cultured excised embryos from Taxus baccata. She (LePage, 1968) found that by culturing mature excised embryos in liquid Heller's

medium with sucrose she was able to leach out inhibitors to germination. Later, it was determined that the culture of excised embryos for 15-20 days in liquid media was necessary to remove inhibitors (LePage-Degivry, 1970). The inhibitor was identified as ABA by paper chromatography. After subculturing to solid medium with 1 mg/l GA3 or after a chilling period, the embryos readily germinated. When

embryos which had begun germination were treated with extracts of the conditioned liquid medium, growth ceased (LePage-Degivry, 1970). However, an additional GA3 treatment resulted in resumption of germination. Paper chromatography of the extract revealed that ABA was leached from the explant during liquid culture.

It was later determined that ABA was not leached out by distilled or mineral water alone, and that sucrose and Ca++ or K+ ions in the medium were required to remove inhibition (LePage-Degivry, 1973b). LePage-Degivry (1973c) reported renewal of inhibition of leached and germinating embryos with the inclusion of ABA in the solid medium. No adventitious organogenesis was reported.


2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets

Several micropropagated woody species have been inoculated with mycorrhizal fungi in vitro and following rooting and establishment in soil. Inoculation of micropropagated plantlets with mycorrhizae could potentially increase survival, growth, and tolerance to environmental







20


stresses. Morandi et al. (1979) observed that the colonization of micropropagated raspberries (Rubus idaeus) with vesicular arbuscular mycorrhizae (VAM) resulted in more vigorous and uniform sized plants, with a 53-1300 increase in shoot dry weight depending on the specific endophyte and cultivar used. Granger et al. (1983) noted that colonization of micropropagated apple plantlets (Malling clones) with VAM resulted in increased plant heights, total leaf areas, leaf dry weights and leaf content of Cu and P. This increase in growth occurred with the cultivar Malling 7; however, colonization did not increase growth of Malling Merton 111. Pons et al. (1983) noted colonization of micropropagated sweet cherry plantlets (Prunus avium L.) with VAM in vitro but did not obtain any observed growth enhancement. In preliminary experiments where cherry plantlets were inoculated at time of transplanting into soil media, a "marked improvement in growth" occurred.

Inoculation of rhododendron with ericoid mycorrhizae has been

successful in situ (Duddridge and Read, 1982) and in vitro (Pearson and Read, 1973; Moore-Parkhurst and Englander, 1981). Ericoid plants appear to be require the specific endophyte, Pezizella ericae (Read, 1982). Colonization of rhododendron with Pezizella resulted in enhanced nitrogen and phosphorus supply to the plant as well as increased resistance to heavy metal toxicity (Duddridge and Read, 1982; Read, 1983). Moore-Parkhurst and Englander (1981) developed a technique for in vitro colonization of rhododendron and Pezizella; however, this technique is impractical for commercial use. The most efficient time to inoculate plantlets would be at the time of transfer to soil.


















3.0 MATERIALS AND METHODS


3.1 Rhododendron chapmanii Gray

3.1.1 Establishment of Stock Plants

Stock plants were established froin seeds collected in late October, 1982, from cultivated plants located around the Doyle Conner Building, Division of Plant Industry, Gainesville, Florida. They were obtained as soon as the brown capsules began to dehisce, disinfested with 15t Clorox solution (0.79% by weight sodium hypochlorite) for 5 minutes, and rinsed with 3 changes of sterile deionized water. Seeds were spread out on sterile moistened filter papers in petri plates and incubated in light (16 hrs light/3 hrs dark at 25 C +/- 5 C with irradi-ance of 50-90 pMm-2 s ). Germination occurred within 2 weeks and when seedlings reached a height of 5 cm, they were transferred individually to 4-litar jr M-2 -1I
pots maintained in a growth chamber illuminated with 150 pMm-s cool white fluorescent lighting for 16 hours daily. Temperatures were maintained at 25 C +/- 1 C (day) and 20 C +/- 1 C (night). Plants were watered from below to avoid wetting the foliage.

Succulent shoot-tips, approximately 3-5 cm in length (5-10 nodes), were pruned by hand and then washed in soapy water, then rinsed in cool running tap water for 1 hour. Pruned stock plants subsequently developed

numerous additional axillary shoots, which developed into succulent shoot-tips of sufficient size to culture in 4-6 weeks.


21







22


3.1.2 Disinfestation

An experiment was designed to compare contamination rates of shoot-tip explants from 2 sources. Explants from plants grown both outside under 40% polypropylene (Saran) shade cloth and in a growth chamber were used.

Culture rooin conditions remained the same for all experiments. Cultures were illuminated with cool white fluorescent lights at 90 pMm2 s~I for 16 hours daily, and a temperature of 25 C + /- 5 C. Unless otherwise indicated, 20 ml of medium was aliquoted into 25x150 min Pyrex test tubes and capped with Kaputs. Media were autoclaved at 1.05 kg/cm2, at 110 C for 20 minutes and then allowed to cool on a slant.

Shoot-tips from both 60% shade (grown out of doors) and growth

chamber grown stock plants were disinfested for 15 minutes in 15% or 20% Clorox then rinsed in sterile distilled water 3 times. Shoot-tips were further trimined to remove the bleached petiole stubs, stein base and apex resulting in a 5 node stein section. Explants were inserted vertically

into agar to approximately half their total length. Culture medium consisted of basal Woody Plant Medium (WPM McCown and Lloyd; 1981) plus 2% sucrose, 0.8% Bacto aar and pH adjusted to 5.2 prior to autoclaving. Ten replications per treatment were used. Data on contamination were collected after 2 weeks under culture conditions described previously.


3.1.3 Media Selection

An experiment was inititated to determine the best medium for

micropropagation of Rhododendron chapmanii. Four media used for woody plants were selected : Woody Plant Medium (WPM-McCown and Lloyd, 1981), 1/2 strength nacronutrients of WPM (1/2WPM), Linsmaier and Skoog medium








23


(LS-Linsmaier and Skoog, 1964) and 1/2 strength macronutrients of LS (1/2LS). Culture vessels were 25x150 mm Pyrex test tubes containing 20 mis of medium. Each medium was amended with 1, 5, 10, 15, or 20 mg/1l 2-i-P, and (in mg/1) myo-inositol (100), NaH2PO4 (100), adenine sulfate

(80), sucrose (30,000) and Bacto-agar (8,000). The pH of the medium was adjusted to 5.2 before autoclaving. Ten replications per treatment were used with individual 5 node shoot-tips used as experimental units. Data were collected after 6 weeks.


3.1.4 Selection of Growth Regulators Levels

Two experiments were performed to determine effects of growth regulators on rapid multiplication. In the first experiment, the cytokinin, 2-i-P, was examined at 0, 5, 10, and 15 mg/i with ten replications per treatment. Individual 5 node shoot-tips were used as experimental units. In the second experiment, the interaction of cytokinins with auxins was investigated in a 3X3 factorial with 2-i-P at 5, 10, and 15 mg/1 and IAA at 0, 1, and 2 mg/i. Each treatment was replicated 10 times. Data were collected after 60 days in culture.


3.1.5 Stage II Subcultures

Following initial culture on multiplication medium (SII), explants were subcultured through 4 additional SII cycles on WPM with (in Mg/1) 2-i-P (10) and adenine sulfate (80). The initial SII culture utilized 25x150 mm test tubes, after which explants were transferred and cultured in small baby food jars (approximately 125 ml) containing 40 ml of

medium and covered with aluminum foil and wrapped with Parafilm. Thirty to 50 mm tall icrocuttings consisting of a single primary shoot with developing lateral shoots were subcultured. Uniform sized explants,








24


consisting of a single primary shoot with developing lateral shoots, were subcultured. These explants were placed 3 to a baby food jar with modified WPM medium. The explants were oriented horizontally and press d down slightly so that most nodes were in direct contact with agar. Ten or more replicates were made. Cultures were transferred onto fresh multiplication medium at 6 to 8 week intervals. Multiplication rates were evaluated over 5 subcultures on WPM. Data were collected at each

subculture time every 6-8 weeks.


3.1.6 Rooting and Establishment

Stage II microcuttings were given one of the following treatments:

(1) control, (2) 0.1% IBA in talc or (3) 0.1% IBA (in 10% ethonol) for 5 seconds. They then were stuck directly into 54x28x6.8 cm plastic flats containing 5 cm deep of autoclaved rooting medium, 1:1:1 (v:v:v) Canadian peat:vermiculite:perlite. Ninety-six explants were used for each treatment. Microcuttings were protected from drying by covering the flat with an inverted translucent plastic flat. They were misted daily by hand to increase humidity and were watered as needed with deionized water. Flats were illuminated under cool white fluorescent lights at approximately 90 pMm-2 s with a 16 hour photoperiod at 25 C +/- 2 C.


3.1.7 Colonization with Ericoid Mycorrhizae

An experiment was established to determine the growth response of

rooted plantlets to inoculation with mycorrhizae and establishment in two different container media. Well-rooted plantlets were inoculated with the ericoid mycorrhizal fungus, Pezizella ericae Read, and transplanted individually into 6 cm2 plastic "cell-pack" (4 cells/pack). Two soilless media were used: (1) Metro-Mix 500 (MM500) and (2) 2:1 (v:v) fired







25


montmorillinite clay: Canadian peat (FMC:CP). Fungal inoculum of P. ericae was prepared according to Marx and Kenny (1981). Pure cultures of P. ericae were obtained from the American Type Tissue Collection (culture no. 32985). The cultures were increased in 250 ml flasks containing 100 ml of modified M-elin-Norkrans fungal medium with a 1 centimeter layer of broken glass in the bottom of the container. Cultures were maintained at 25 C in the dark for 4 weeks, but were shaken weekly to fragment the hyphae. A mixture of 28:1 (v:v) vermiculite:finely screened Canadian peat was moistened with 750 ml of Melin-Norkrans medium and autoclaved. After cooling, 100 ml of prepared P. ericae inoculum was added and this mixture was cultured for 3 weeks in the dark at 25 C. This inoculum was

then leached by wrapping in several layers of cheese cloth and rinsing for 3 to 4 minutes under cool, running tap water to remove excess glucose and nutrients which might support growth of intrusive saprophytes. The veriniculite:peat inoculum was then air dried at 25 C for 3 days. Approximately 1 gm of inoculum was placed immediately below the transplanted plantlet's root zone. Non-inoculated plantlets had 1 gT of autoclaved vermiculite added below the roots as a control. Colonization and growth responses were evaluated after 16 weeks, using the technique described by Englander (1982). Roots were stained with Lactophenol-Trypan blue stain for 10 minutes at 90 C. Percent colonization was estimated by use of the modified gridline intersection method of Giovanettii and Mosse (1980). Each treatment was replicated 12 tines, with 4 plantlets per replication.







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3.2 Taxus floridana Nutt.

3.2.1 Conventional Propagation by Cuttings

An experiment was initiated to determine the optimal auxin

treatment for root formation on cuttings. Dormant, evergreen cuttings of Taxus floridana were collected in early February, 1982, from native populations in Torreya State Park and from plantings on the University of Florida campus. Ten to 15 cm long terminal and subterminal cuttings were collected from upright and lateral branches. The bases were trimmed and quick dipped for 10 seconds in 2000, 4000, or 8000 mg/l 13A (in 20% EtOH). Each treatment was replicated 10 times with individual cuttings as experimental units. Cuttings were stuck 5 cm deep in plastic flats containing 10 cm of a mixture of 1:1 (v:v) course sand:Canadian peat. They were misted intermittently (5 sec every 2 min) during daylight hours. Bottom heat (28 C) was supplied from electric cables placed 5 cm below the flats. Rooting was evaluated 120 days after sticking. Well rooted cuttings were transplanted into 1:1:1 (v:v:v) Canadian peat:perlite:sandy loam medium in 4-liter containers. After establishment, stock plants were placed in a growth chamber with the same cultural conditions mentioned for R. chapmanii (3.1.2).


3.2.2 Culture of Quiescent Shoot-tips

Four different plant organs were investigated--(1) quiescent

shoot-tips (3.2.2), (2) developing vegetative buds just before bud break (3.2.3), (3) swelling microsporangium (3.2.4)and (4) excised embryos (3.2.5).

Quiescent shoot-tips were collected in October, 1982, from

the most recently matured branches of plants located on University of Florida campus. Three to 4 cm long shoot-tips were washed in soapy







27


water, rinsed in running tap water for 1 hour and then disinfested with a 15-20% Clorox solution (with 5 drops Tween-20 per liter) for 15-20 minutes. Shoot-tips were rinsed in 3 separate changes of sterile deionized water. The bleached bases were trimmed and explants were inserted vertically to half their length into 20 ml of solidified WPM medium in 25x150 Pyrex test tubes.

An experiment was initiated to determine the best medium for

culture of Taxus floridana. The 4 nedia selected were the same used for Rhododendron chapmanii (Sect. 3.1.3). Data were collected after 6 weeks in culture. Twenty explants per medium were cultured with individual shoot-tips as experimental units. Basal media were amended with (in ng/1) myo-inositol (100), NaH2P04 (100), sucrose (30,000), Bacto-agar (3,000). The pH was adjusted before autoclaving to 5.2 with the addition of 1.0 or 0.1 N solution of NaOH or 1.0 or 0.1 N HCl prior to autoclaving. Data were collected on length of new growth, number of developing shoots and explant color.


3.2.3 Culture of Expanding Vegetative Buds

Swelling vegetative buds were collected weekly for 3 successive weeks preceding bud break in late February and into the second week of March, 1983. Buds were washed and disinfested as described previously (Sect. 3.2.3) and outer bud-scales were carefully excised under water as described by Bonga (1977,1981) and Jansson and Bornman (1983). Four preculture treatments were used. Trimmed buds were allowed to soak for 24 hours in sterile distilled water or 3 hours in either 22.5, 28.0, or 34.0 mg/1l BA solutions before culturing on WPM amended with myo-inositol (100), NaH2PO4 (100), sucrose (20,000), agar (8,000) and 5.2 pH. Ten







28


replications per treatment were used. Data were collected on overall growth and specific morphogenic changes.


3.2.4 Microsporagium Culture

Expanding microsporangia were collected and disinfested as described in Section 3.2.2. Microsporangia were trimned of outer scales and soaked in the 4 previously described solutions (3.2.3). Explants were established on basal WPM with agar (8,000). Each treatment involved 10 replications with individual microsporangium and data were collected after 6 weeks in culture.


3.2.5 Embryo Culture

Attempts were made to promote germination of excised embryos by leaching of inhibitors to seed germination. Seeds were surface disinfested using 20% Clorox for 20 minutes. The 1-2 mm long embryos were excised and further disinfested in 5% Clorox for 10 minutes. Embryos were rinsed 3 times with sterile deionized water, then placed into 20 ml of liquid Heller's medium with 2% sucrose. Medium pH was adjusted to 5.5 before autoclaving. The small embryos were most easily handled by catching them in a drop of liquid held in a 5 mm wide

bacterial transfer loop. Cultures were maintained in the dark without shaking (LePage, 1968). Due to limited seed availability, only 5 excised embryos were used per treatment with individual embryos as

experimental units.

Embryos were subjected to 3 different treatments. Following an initial 2 week culture period, they were transferred to either (1) Heller's medium supplemented with 1 mg/1l GA3 and solidified with 0.8% agar or (2) Heller's solidified -nedium and stored for 1 month in a







29


refrigerator at 4 C before returning to culture room conditions. Treatment (3) involved a second leaching cycle in liquid Heller's medium for 2 weeks before placement on solid Heller's medium with 1 mg/i GA3' GA3 was filtered sterilized and added to the cooling but still liquid medium. Data were collected from the cultures grown for 40 days in the dark at 25 C +/- 2 C.


3.3 Torreya taxifolia Arn.

3.3.1 Conventional Propagation by Cuttings

Evergreen cuttings were collected in early February, 1982, from

trees growing in Maclay Gardens, Tallahassee, and from plantings on the University of Florida campus. Ten to 15 cm long terminal and subterminal cuttings were collected from lateral branches. The cuttings were wounded twice along opposite sides by scraping the basal 2.5 cm of the cuttings to expose the lighter green cambium. Cuttings were treated in the same manner as described for Taxus (Sect. 3.2.1) involving treatment with 3 concentrations of IBA (2,000, 4,000, and 8,000 mg/1). Individual cuttings were used as experimental units, with 5 replications per treatment.

Cuttings were evaluated 120 days following sticking and well-rooted cuttings were transplanted into 1:1:1 (v:v:v) Canadian peat:perlite:sandy loam medium in 4-liter containers. Data collection included percent rooting, number and total length of roots.


3.3.2 Culture of Quiescent Shoot-tips

Five explant sources were investigated to determine the morphogenic capacity of various organs as reported in the literature--(l) quiescent shoot-tips (3.3.2), (2) expanding vegetative buds (3.3.3), (3)







30


expanding mega- and microsporangia (3.3.4), (4) excised embryos (3.3.5), and (5) seedling stock plants sprayed with cytokinins before culture (3.3.6).

In the first experiment (3.3.2), quiescent shoot-tips were

collected in October, 1982, from plantings located on the University of Florida campus. Three to 4 cm shoot-tips were washed and disinfested following the procedures previously described for quiescent shoot-tips of Taxus floridana (Sect. 3.2.2). They were cultured in each of four media (WPM, 1/2WPM, LS, and 1/2LS).


3.3.3 Culture of Expanding Vegetative Buds

Expanding vegetative buds were collected weekly over a 3 week

period preceeding budbreak in early spring (mid- to late March, 1984). Buds were trimmed of bud-scales and were handled following the procedures of Boulay (1977,1981) and Jansson and Bornman (1983) as described for Taxus in Sect. 3.2.4. Ten buds were used per treatment with individual buds as experimental units. Thus, 3 collection times were combined with 4 preculture soaks.


3.3.4 Culture of Micro- and Megasporangia

Expanding micro- and megasporangia were collected as they swelled following budbreak in the middle of March, 1984. These were washed and disinfested as with Taxus floridana microsporangia (3.2.4). Ten explants of each were treated with individual sporangia as experimental units.


3.3.5 Embryo Culture

An experiment was initiated to determine if germination inhibitors could be leached from excised embryos. They were excised following






31


the techniques of LePage (1968), LePage-Degivry (1970, 1973ab) and LePage-Degivry and Garello (1973) for Taxus baccata. Seeds, which had been warm stratified for 2 months followed by 2 months of cold stratification were surface sterilized in 20% Clorox followed by 3 sterile water rinses. Embryos, 2-5 mm long, were aseptically excised and further disinfested in 5% Clorox for 10 minutes, followed by 3 rinses in sterile deionized water. The small embryos could be most easily handled with a bacterial transfer loop. Excised embryos were cultured in 20 ml liquid Heller's medium as described for Taxus (Sect. 3.2.6). Excised embryos were exposed to the 3 treatment regimes described for Taxus floridana (3.2.5). Individual embryos were used as experimental units with 5 embryos per treatment.


3.3.6 Effect of Cytokinin Sprays on Stock Plants

Torreya seedling stock plants were sprayed weekly for 4 weeks with 2 concentrations of 2 cytokinins (BA and 2-i-P at 100 mg/1l and 200 mg/l plus 5 drops Tween-20 per liter). These treatments were suggested by Dr. M. A. El-Nil (1983, per. com.). Plants were sprayed until run off.

Only 5 one-year old seedlings were available for experimentation, limiting the treatments to only 1 plant per treatment. A single control plant was sprayed weekly with the same 10% EtOH carrier solution as the cytokinins. All developing basal and axillary shoots were removed and placed in culture. Explants consisted of shoot-tips and stem sections 1-2 cm in length. They were cultured on solidified WPM (0.8% agar) in 25x150 ml Pyrex test tubes with 4 concentrations of growth regulators in the medium: (1) WPM with 1 mg/l BA and 0.01 mg/l NAA, (2) WPM with 10 mg/l BA, (3) WPM with 1 mg/l 2-i-P, and (4) WPM with 10 mg/l 2-i-P.







32


-2 -1
Explants were cultured at 25 C (+/- 5 C), and illuminated 50 pMm s for 16 hours a day.
















4.0 RESULTS


4.1 Rhododendron chapmanii

4.1.1 Disinfestation

There was an average of 55% reduction in amount of contamination (30-40%) when explants were collected from stock plants grown in a growth chamber as compared to field grown explants (Table 4.1.1). There was no difference in contamination % between the two levels of Clorox used. Contamination of explants from shade house grown plants averaged 90% while those from growth chamber plants contaminated only 35%.

Field grown material remained very difficult to disinfest. In a later experiment (Section 4.1.2), leaves were removed from growth chamber grown shoot-tips prior to Clorox treatment. Contamination in this case was reduced to only 4% (data not given), when leaves were removed from explants prior to treatment with 15% clorox for 15 minutes. For all subsequent experiments in this section, leaf removal prior to Clorox treatment virtually eliminated contamination. Subcultures from visually contamination-free cultures remained clear and presumably without containination.


4.1.2 Selection of Growth Regulator Levels

There was a difference between basal medium without cytokinins vs all levels of 2-i-P tested, but no differences between the 2-i-P levels (Table 4.1.2). There was a significant reduction in the number of leaves formed when with 2-i-P levels greater than 10 :ng/l were used.


33






34


Table 4.1.1 Percent contamination of in vitro cultured Rhododendron chapmanii shoot-tips grown in shade house and growth chambers


Treatment Contamination
/0
Time Clorox Chamber Field
(min) ( %)


15 15 30 100

15 20 40 80





35


Table 4.1.2 Growth response of in vitro cultured Rhododendron chapmanii shoot-tips cultured on Woody Plant Medium with 5 levels of the cytokinin, 2-i-P



2-i-P concentration No. expanding Mean no.

(mg/1) axillary buds leaves



0 1.4a 8.4bZ

5 3.0b 13.7a

10 3.6b 16.6a 15 2.2b 7.3b 20 2.2b 7.6b


YMeans in a colomn followed by the same letter different at the 5% level by Duncan's Multiple


are not significantly Range Test.


ZMeans in a column followed by the same letter are not significantly different at the 10% level by Duncan's Multiple Range Test.






36


There were no differences between the total number of leaves and shoots produced for the 2-i-P and IAA combinations (Table 4.1.3). Differences between IAA means were significant at the 0.10 level, however, inclusion of IAA in the medium generally resulted in declining numbers of shoots and leaves.

Optimal shoot and leaf production occurred between 5-10 mg/i 2-i-P. With higher 2-i-P levels, axillary shoots remained short and elongated

much more slowly than plantlets cultured on 10 mg/i 2-i-P.


4.1.3 Stage II Subcultures

Following initial SII culture in test tubes, the medium in all

additional multiplication cycles contained adenine sulfate (80 mg/1) and explants were established in small baby food jars. Basal nodes and especially those embedded in the agar produced abundant axillary shoots. When explants were cultured upright in individual test tubes on WPM witn 10 mg/l 2-i-P and no adenine sulfate (Fig. 4.1) an average 5.8 shoots developed per explant in 6-8 weeks. For additional SII subcultures, increase in shoots was especially high when the explants were placed horizonally and pushed slightly into the medium (Fig. 4.1.5). This also

resulted in more uniform elongation of shoots.

Each additional multiplication cycle for the next three SII

subcultures in baby food jars averaged a 7.6 fold increase in number of shoots, however, this multiplication rate decreased to 5.0 during the fourth subculture (Fig. 4.1).


4.1.4 Rooting and establishment

Preliminary rooting attempts using an agar substrate were

unsuccessful. In a few cases where cultures were left undisturbed for 4






37


Table 4.1.3 Growth response of in vitro cultured Rhododendron chapmanii grown on a 3x3 factorial combination of auxin IAA) and cytokinin (2-i-P)



2-i-P IAA
(mg/1) (mg/1)

0 1 2


Total No. of Leaves


5 38.9aZ 35.2a 30.6a 10 37.2a 38.4a 39.4a 15 38.2a 34.6a 30.0a


Ave. No. Shoots Greater than 5 mm


5 3.2aZ 2.8a 2.8a 10 2.8a 2.6a 2.6a 15 3.6a 2.5a 1.6a


ZMeans in a column and row followed by the same letter are not significantly different at the 1% level using Duncan's Multiple Range Test.






38


9,.


Fold increase in no. of subculturable shoot-clumps


7

6-

5-

4-

31


/7.6 7.7 7. '


0
5


0
5.0


SII1 S112 S113 S114 SII5


Stage II Multiplication Cycles


Figure 4.1 Multiplication rates of in vitro cultured Rhododendron
chapmanii during initial and 4 additTonal SII subcultures.






39


to 6 months, roots appeared spontaneously above the agar surface. Also, the extremely fine "hair roots" characteristic of rhododendrons did not develop properly in poorly aerated substrates.

Direct sticking of microcuttings into 1:1:1 (v:v:v) Canadian

peat:venniculite:perlite resulted in well-rooted plantlets within 4 to 6 weeks. Treatment of microcuttings before sticking with 1000 mg/i IBA (dissolved in 10% EtOH) resulted in improved survival and rooting (Table

4.1.4).

A quick dip treatment (5 seconds) with 1000 mg/i IBA resulted in greatest survival and approximately 60% rooting (Table 4.1.4). Treatment with 1000 mg/l of IBA in talc resulted in poorer rooting and survival than controls. Survival improved slightly with more careful observation and daily misting with deionized water. Best survival to date has been 75-80% (data not given).

Table 4.1.5 summarizes the micropropagation staging system developed for R. chapmanii. With this rate of multiplication and rooting, it can be conservatively estimated that over 10,000 rooted plantlets could be produced from a single shoot-tip in one year (1 x 5.8 x 7.6 x 7.6 x 7.6 = 19350 x 60% rooting = 11610 plantlets in 9.5 months + 2 1/2 months additional growth).


4.1.5 Colonization with Ericoid Mycorrhizae

Inoculation of rooted plantlets with the ericoid mycorrhizae,

Pezizella ericae, resulted in colonization in two potting riedia, MM500 and FMC:CP. There were considerable differences (PF > 0.0001) between iedia for height of main stem, total stem lengths, number of leaves, and main stem caliper after 12 weeks of growth in the greenhouse (Table

4.1.5). Differences in number of stems was significant at the .05 level.


I






40


Table 4.1.4 Survival and rooting response of microcuttings of Rhododendron chapmanii following treatment with auxins from 2 sources



Treatment Survival and Rooting



none 26.0Z 1000 mg/l IBA 19.8 (in talc)

1000 mg/l IBA 59.4 (liquid-dip)


ZPercent survival and rooting were significantly different at the 1% level using analysis of catagorical data.






41


Table 4.1.5 Summary of Rhododendron chapmanii


micropropagation system developed for


STAGE EXPLANT DURATION RATE MEDIA GROWTH CULTURE
(weeks) REG. CONDITIONS


SI shoot-tips 6-8 5.8x WPM 10 mg/i 20 pMm-2s-1
3-5 cm 2-i-P
25 + 5 C

SII axillary 6-8 7.6x WPM 10 mg/i 20 pMm-2s-1
shoot-clumps 2-i-P
25 + 5 C

SIV axillary 4-6 80% 1:1:1 Quick- 90 pMm-21
shoots Rooting dip
(>15mm) Medium 25 + 5 C 1000 mg/i in high
IBA humidity
tent


XMedium WPM = Woody Plant


Medi um


YGrowth Regulators ZCulture conditions--light intensity in VMm-2s1
light duration 16 hrs light/ 8 hrs dark temperature in degrees C






42


Table 4.1.6 Response of Rhododendron chapmanii produced in vitro to 2 establishment media and with or without ericoid mycorrhizae


plantlets inoculation


MediaV Trt. WPt.X Ht. No.z Total No. Main %X
Surv. Main Stems Stem Lvs Stem Col.
Stem Length Caliper
(mm) (mm) (mm) M500 +M 91.7a 67.Oa 1.77a 96.la 23.6a 0.98a 61.4a M500 -M 95.8a 60.8a 2.14a 98.5a 26.7a 1.Oa 0 FMC:CP +M 80.9a 28.3b 1.40b 40.Ob 14.9b 0.35b 41.la FMC:CP -M 65.3a 23.Ob 1.18b 34.1b 12.8b 0.28b 0


VMedia


M500 = FMC:CP


MetroMix-500
= Fired montmorillinite clay:Canadian peat


W
Trt. = mycorrhizal treatment +M = with inoculation
-M = without inoculation

Plt. surv. % = Plantlet survival % XMeans in a column followed by the same letter are not significantly different at the 5% level by analysis of catagorical data. YMeans in a column followed by the same letter are not significantly different at the 1% level by Duncan's Multiple Range Test. ZMeans in a column followed by the same letter are not significantly different at the 5% level by Duncan's Multiple Range Test.






43


Plantlets grown in the less fertile FMC:CP potting mix were consistently smaller regardless of colonization.


4.2 Taxus floridana

4.2.1. Conventional Propagation by Cuttings

Conventional vegetative propagation resulted in rooting within 120 days under mist (Table 4.2.1). Cuttings in the "mixed clones" group were collected from smaller and younger trees 2-4 m tall. Cuttings fron the younger mixed clones rooted at better (best 671) and with greater number of roots. The best rooting percentage froen the older Champion tree was 40% but with fewer roots initiated and of shorter length than from the younger clones.


4.2.2 Micropropagation of Taxus Shoot-tips

The response of shoot-tips grown for 6 months on 4 different nedia supplemental with 0.5 mg/l IAA is shown in Table 4.2.2. There were no difference in shoot size, average number of shoots produced or length of new growth. WPM was chosen for additional experiments primarily due to

the better color quality and general overall appearance of the explants. Explants on WPM or 1/2WPM were light green, the normal color of new growth on whole plants.


4.2.3 Culture of Expanding Vegetative Shoot-tips

Culture of expanding vegetative shoot-tips collected 3 successive weeks before bud break resulted in either death of the explant, contamination of cultures or no growth. Shoot-tips were very small (2-3 mm), were not easily dissected and may have been damaged by disinfestation treatments or by removal of outer bud scales.







44




Table 4.2.1 Rooting response of Taxus floridana cuttings from 2 aged groups of plants to 3 levels of the auxin, IBA



IBA Rooting Mean root Mean root Total root (mg/1) no. length length
(cm) (cm)



Champ i on
Tree


2000 0 0 0 0 4000 40 4 1.0 4.0 8000 40 5 1.5 3.3 Mixed Clones


2000 67 4 1.0 4.0 4000 33 4 2.0 8.0 8000 25 14 3.0 42.0







45


Table 4.2.2 shoot-tips


In vitro growth response on 4 nutrient media


of T. floridana


Medium Size Mean no. Color Length
(mm) shoots new growth
(mm)


WPMIZ 70.a Y 1.2a LGX 17.5a

1/2WPM 42.Oa 2.4a LG 5.7a

LS 61.2a 2.8a BR 21.2a

1/2LS 63.Oa 1.4a LG/BR 30.Oa


ZWPM = Woody Plant Medium
1/2WPM = 1/2 strength macronutrients of Woody Plant Media LS = Linsmaier and Skoog Medium 1/2LS = 1/2 strength macronutrients of Linsmaier and Skoog Yp
Means in a column followed by the same letter are not significantly different at the 1% level by Duncan's Multiple Range Test.


XColor Key LG = Light Green


BR = Brown







46


4.2.4 Culture of Microsporangia

Culture of expanding microsporangia was attempted successive for 3 weeks preceding budbreak. Microsporangia were very small (2-3mm) and difficult to excise. Clean cultures could be consistently obtained, but no further development was observed. Explants treated with chemicals or

water soak did not develop further in culture and no differences were noted between treatments.


4.2.5 Embryo culture

Attempts to culture excised embryos were unsuccessful. None of the

3 treatments were successful in promoting germination. E.nbryos which showed no signs of growth after 1 month on solidified Heller's medium with GA3 or following removal from cold treatment were recultured into a second cycle of fresh liquid medium. After 3 weeks in liquid medium, these embryos were again transferred to solidified medium with GA3, but

no growth occurred.


4.3 Torreya taxifolia

4.3.1 Conventional Propagation by Cuttings

Rooting results are summarized in Table 4.3.1. The greatest percent rooting (56%) occurred with 8000 mg/l IBA. The number and average length of roots with this treatment were substantually less than those produced with 4000 mg/l IBA treatment. Cuttings treated with 4000 ing/l IBA rooted at a low percentage (20%) but produced nore numerous

roots with greater average and total length.







47


Table 4.3.1 Rooting response of cuttings of Torreya taxifolia treated with 3 levels of the auxin, IBA



IBA Treatment Rooting Mean no. Mean length
(mg/1) roots roots
(cm)


2000 14 3.5 1.0 4000 20 13.0 2.5 8000 56 4.5 1.5







48


4.3.2 Micropropagation of Shoot-tips

The response of shoot-tips collected from lateral branches of

nature trees (est. 15-20 years old) in culture is summarized in Table 4.3.2. The color and apparent health of explants cultured on WPM or 1/2WPM were superior to LS or 1/2LS, being darker green.

In a second experiment, shoot-tips collected in October and grown on 3 nutrient nedia each with 5 levels of BA was recorded after 2 months in culture (Table 4.3.3). Little or no growth occurred on either LS or 1/2LS. A few axillary shoots developed on WPM at low to medium levels of BA (0.5-1.0 mg/1) but declined with higher levels.


4.3.3 Culture of Expanding Vegetative Buds

All cultures of expanding buds were either contaminated or were killed by disinfestation procedures. Contamination continues to be a

serious problem from field grown plants but is much less of a probl em when stock plants are grown inside with lower light intensities and lower night temperatures.


4.3.4 Culture of Microsporangia and Megasporangia

Most of these cultures became contaminated or were killed by

disinfestation procedures. Microsporangia were sufficiently large enough to be handled and were plentiful in early spring.


4.3.5 Embryo culture

Embryo culture of T. taxifolia also proved unsuccessful. Although embryos could be easily disinfested with treatment with 10% Clorox following aseptic excision, they did not generally respond to the treatment used successfully for Taxus baccata embryos (LaPage, 1968). in 1 case among 10 attempts, the cotyledons parted and appeared to







49


Table 4.3.2 Growth response of shoot-tips of T. taxifolia on 4 nutrient media



Medium Mean size Mean no. Color Length
(mm) shoots new growth
(mm)



WPMZ 44.5ab 2.0a DGX 20.5a 1/2WPM 18.7b 2.la LG 9.4a LS 56.3a 1.6a YG 38.2a 1/2LS 42.8ab 2.Oa LG to YG 28.9a


ZWPM = Woody Plant Medium
1/2WPM = 1/2 strength macronutrients Woody Plant Medium LS = Linsmaier and Skoog Medium 1/2LS = 1/2 strength macronutrients Linsmaier and Skoog Medium

YMeans in a column followed by the same letter are not significantly different at the 5% level using Duncan's Multiple Range Test.
XColor Key DG = Dark Green, LG = Light Green, YG = Yellow Green







50


Table 4.3.3 Average number of axillary shoots of Torreya taxifolia developing in vitro on 3 nutrient media and 5 concentrations of the cytokinin, BA



BA No. axillary shoots

WPMZ LS 1/2LS


0 1.0a 1.0 1.0 0.5 1.3a 0 0 1.0 1.7a 0 0 5.0 1.0a 0 0 10.0 0.3a 0 0


ZWPM = Woody Plant Medium
LS = Linsmaier and Skoog Medium 1/2LS = 1/2 strength macronutrients Media um


of Linsmaier and Skoog


YMeans in a column followed by the same letter are not significantly different at the 5% level by analysis of
catagorical data.







5.


begin to germinate. When transferred to solid medium with 1.0 mg/l GA3' a shoot-tip appeared but degenerated shortly thereafter.


4.3.6 Cytokinin Sprayed Seedlings

Only five seedlings were available for investigation so each treatment was applied to a single seedling. Treatments were based on suggestions from Dr. M. A. El-Nil (1982, per. com.). Stock plants produced numerous basal shoots following treatment with cytokinin sprays. The control plant produced a few basal shoots which could be cultured. Excised basal shoots developed axillary bud masses after 30 days when cultured on media with cytokinins. Best results occurred when explants were collected from plants sprayed weekly with 1000 mg/l BA and then cultured on medium with 1 mg/1l BA and 0.01 mg/l NAA. Higher concentrations resulted in much smaller masses which did not enlarge much until transferred to media with lower or no cytokinin. Culture on media with 2-i-P resulted in little development until these were transferred to WPM medium with 1.0 mg/l BA and 0.01 mg/l NAA after which bud elongation occurred and occasional multiple shoots were observed.

Two morphologically different types of axillary bud development were observed. In the first case, normally shaped axillary shoots elongated with additional axillary shoot development from the leaf axils at the base of the cultured buds. The second type of development inaoived the development of compact "bud-masses" consisting of small, tightly appressed scale-like leaves with the ultimate emergence of multiple shoots after 3 months in culture. When buds from the same treatments were subcultured to the same or lower cytokinin medium, the buds on the lower cytokinin medium always began to show signs of growth







52



sooner. These buds were transferred to fresh medium but have since degenerated. No rooted plantlets were obtained.

















5.0 DISCUSSION


5.1 Rhododendron chapmanii

Conventional propagation using cuttings was atteinpted but was

unsuccessful due to failure of misting equipment. The experiment was not repeated because of insufficient cutting material and the development of a highly successful micropropagation system using shoot-tips. Microcuttings could be rooted and established with 60% or greater survival. Conventional propagation of evergreen Rhododendron species usually involves softwood cuttings (Hartman and Kester, 1981) or leafly hardwood cuttings collected in the fall which are treated with concentrated NaOH and IBA solution (Gray, 1974).

Micropropagation of R. chapmanii was highly successful using

shoot-tip explants and modifying the techniques of Anderson (1975) and the medium of McCown and Lloyd (1981). Technique improvements included decapitation of shoot apex and the horizontal placement of the explants in the medium so that most nodes were slightly embedded in it.

Horizontal placement of shoots into multiplication medium greatly increased the number and uniformity of shoots produced during each multiplication cycle (SII). The addition of 80-100 mg/l adenine sulfate improved the number and vigor of shoots produced. BA proved to be phytotoxic to R. chapmanii as has been previously reported for most Rhododendron species (Anderson, 1975; Lloyd and McCown, 1980; and McCown and Lloyd, 1983). Decapitated shoot-clump bases could be recultured as suggested by Strode (1979), however; this technique did


53







54


not result in as rapid an increase in shoots per cycle as when elongated shoots were laid horizontally on and pressed slightly into the medium.

The use of higher levels of 2-i-P (greater than 15-20 mg/1)

resulted in a greater number of shoots, however, these grew slower and took 2 to 3 months vs 6 to 8 weeks to reach a subculturable size. The reduced stem elongation could have been due to high cytokinin levels which inhibited shoot elongation. It is also probable that the amounts

of nutrients available to the numerous shoots were limiting and that culture on a greater volume of medium or more frequent transfers might hasten growth and multiplication. Ma and Wang (1977) suggested the use of short term, agitated liquid culture to increase survivability and multiplicationn.

Using the micropropagation system shown in Table 4.1.7, one can

calculate that over 11,000 plants could be obtained from 1 shoot-tip in 9.5 months. With 2.5 months additional growth in the greenhouse, these plantlets would be large enough to be transplanted to larger containers. Alternately, a square meter of lighted shelf space could produce 15,16,000 SII nicrocuttings in 6 to 8 weeks (225 bottles/M X 60-70 microcutting/bottle).

Although large numbers of shoots could be produced and routinely subcultured, a 60-80% survival rate on transfer to soil is still inefficient. All attempts to root R. chap;nanii in Stage III culture have failed even though several Pedia were investigated along with a range of auxin treatments. This differs from reports of 801 rooting in SIII by Ma and Wang (1977) and Strode et al. (1978). Rhododendron roots are extremely fine and thread-like and do not appear to grow easily into agar iledia.







55


Best rooting and survival of microcuttings of R. chapmanii

occurred when explants consisting of 3 to 5 shoots were direct-stuck in rooting medium with at least 30% Canadian peat moss. This agrees with the findings of Anderson (1978), Lloyd and McCown (1980) and Wong (1981). Direct rooting of microcuttings vs use of SIII cultures results in considerable savings in labor since at this stage explants must be handled individually only once for direct rooting vs twice when SIII cultures are used (Strode et al., 1979; Wong, 1981).

Microcuttings were rooted in flats with clear plastic covers or covered with inverted translucent flats to prevent drying. If covered flats were maintained under light levels greater that 100 pMm- 2sec~ excess heating occurred and plantlets declined and fungal contamination was greater. If microcuttings were initially maintained under lower light intensities (e.g. 25 Mm- 2s 1) for the first 2 weeks then maintained at 90 pMm -21, better survival and rooting occurred. Placement of microcuttings directly under intermittent mist resulted in poor rooting and survival and the development of chlorotic leaves due to either excessive leaching or possibly due to poor root-zone aeration. The use of fog systems or high humidity tents should be further

investigated.

In this investigation, the use of rooting compounds on

microcuttings resulted in better rooting and survival when a 1000 mg/l solution of IBA was applied as a 5 second quick-dip. Treatment with the same concentration of IBA (in talc) resulted in lower survival and rooting. Wong (1981) mentioned that the use of hormone powder increased proliferation of roots but not time to root. He also mentioned that the economic advantages of such treatments were yet to be determined.







56



Poor rooting and survival may be attributed to the small size and succulence of the microcuttings. Commercial producers of micropropagated rhododendrons (Briggs, per. com.) use lower cytokinin levels which result in reduced multiplication rates but these

microcuttings are much larger and hardier. Determination of optimal multiplication rates should not necessarily be based on optimal shoot numbers, but must include duration of cycle times, labor and space requirements, rooting survival rates and duration, as well as post-transplanting growth rates.

When shoot-clumps containing 3 to 5 shoots are used for rooting, better survival and rooting occurs, possibly due to reduced handling shock, greater carbohydrate (or minerals, etc.) reserves within each propagule, and due to the fact that if one shoot dies, another shoot is available for replacement. Once rooted and transplanted to containers, the plants from shoot-clumps form a multi-stem liner which is more desirable than a single-stem plant.

Root emergence in rooting medium became visible after 3 to 4 weeks, and plants were well rooted by 6 weeks. After root intiation, the explants began to produce new leaves which were slightly larger and less succulent. After these new leaves were observed, the plantlets generally survived transplanting to cell-packs and readily ecame adapted to the greenhouse environment. Rooted plantlets were hardened off by covering with 80% shade cloth for a week, followed by a week with 40% shade, after which all coverings were removed.

Colonization of micropropagated plantlets with the ericoid

nycorrhizae, Pezizella ericae, did not increase survival or growth

responses when plantlets were grown for 12 weeks in the greenhouse.







57


Differences between the media used apparently masked any differences due to colonization after 12 weeks of growth. Additional growth in 4-liter containers is presently under observation and there appears to be greater bud break and growth on colonized plantlets 2 weeks after new growth began. Final growth measurements will be made after a month of additional growth. Mycorrhizal colonization of Ericaceae species is known to increase the plants' ability to take up nitrogen and phosphorous, as well as, tolerance to heavy metal toxicity (Stribley and Read, 1974, 1975; Duddridge and Read, 1982)


5.2 Taxus floridana

Using cuttings from mixed aged trees, conventional propagation in a 1:1 (v:v) Canadian peat:perlite rooting medium was successful, resulting in 40-67% rooting in 120 days. Larger cuttings (15-20 cm vs 10 cm) rooted more quickly and vigorously. This agrees with the findings of commercial propagators (Keen, 1954). There was an obvious difference in rooting percentages between plants of different ages. Cuttings from the Champion tree rooted poorly (0-40%) while cuttings from much younger trees rooted 25-67%. According to Sargent (1947), 275 annual growth rings were counted from a 9.5 cm diameter T. floridana trunk section

suggesting that the 16 cm diameter Champion from which the "older" cuttings were collected was considerably older.

Cuttings from young trees root at high rates. Lower branches of are commonly observed in the wild to naturally layer when branches remain in contact with the ground. However, lateral cuttings retain their plagiotropic growth habit even after 2 years in containers, generally producing a 15-20 cm tall ground cover. Keen (1954) noted that for most Tayus species and cultivars, orthotropic shoots are







58


necessary for producing excurrent trees while lateral cuttings generally produce only spreading shrubs. Keen also indicated that even very large cuttings (up to 65 cm) can be successfully rooted, resulting in a salable plant in a shorter time.

Attempts to root in 100% coarse, builder's sand under intermittent mist resulted in basal rotting however, rooting often occurred above the media especially front abaxial side of lateral branches (data not given). At first it was assumed that the 20% alcohol in the IBA quick-dip solution damaged the tissue. However, in later experiments using better drained rooting medium with quick-dip solution, no basal rotting occurred. Apparently the builders sand used initially was not as well drained as the sandblasting sand normally used for rooting. The occurrence of high moisture conditions and poor drainage most likely caused the observed basal rotting.

Continued growth of cuttings occurred best in well drained media

(1:1:1 v:v:v) course sand:Canadian peat:loamy soil) and under 80% vs 40% or 60% shade.

Micropropagation of shoot-tips did not result in prolific

stimiulation of axillary buds. Using shoot-tip explants from lateral and orthotropic basal branches, only 2 to 4 axillary shoots elongated when placed in culture. These shoots appeared in specific positions along the shoot-tip suggesting that they were preformed and part of the normal branching pattern of the whole plant. McCown and Amos (1932) reported successful shoot-tip propagation of Taxus spp. but did not indicate the numbers of shoots obtained. They also noted in their findings that gymnosperms are generally more sensitive to low levels of cytokinins.







59


The use of Dore juvenile tissues such as embryos or seedlings as explants probably would result in increased shoot numbers. Dr. A. M. El-Nil (per. coin., 1982) recommended pretreatment of conifer seedlings with cytokinin sprays as a more effective means of inducing multiple shoots from conifers. This treatment was not investigated for Taxus

floridana due to the lack of seedling material although this was a promising technique with Torreya taxifolia.

Wounded mature Taxus floridana trees in the wild have been observed to produce masses of short shoots, most with spiral phyllotaxy and apparently orthotropic growth. Culture of explants from shoots arising from multiple shoots from "ourls" has been successful for Sequoia sempervirens (Ball, 1978; Boulay, 1979), and Pinus eldarica (Herrera and Phillips, 1984). Attempts should be made to obtain permission to collect seedlings and "burl" material from the wild.

Micropropagation of "embryonic shoots" from swelling buds according to the techniques of Bonga (1977,1981) was unsuccessful due to high rates of contamination and the difficulty in excising the small buds (1-3mm). A few aseptic cultures were established but buds did not continue to expand and eventually darkened and died.

Culture of Di crosporangia was also attempted since Dale plants were available. These were also very small (2-3mm) and difficult to excise. Uncontaminated cultures were easily obtained but did not grow in culture. No megasporangia were available locally and this approach was not pursued since norphogenesis from immature female cones has been reported only rarely with cultures of Pinus mugo and Picea abies (Bonga, 1981).






60


Attempts to overcome seed dormancy by excision of embryos and

culture in liquid medium according to the techniques of LePage-Degivry (1968,1970,1973abc) were unsuccessful. This technique failed even when embryos were leached in 2 cycles of liquid culture followed by placement on media with GA3 or when cultures were chilled for 2 months at 5 C. Seed availability is very sporatic with reports of other species of Taxus alternate bearing with +/- 7 year cycles (Schopmeyer, 1974) so further experiments will be limited.

Seeds of Taxus floridana were given 1 or 2 months warm

stratification before excision as well as treatment with 1 month warm followed by 2 months cold stratification before excision. None of the embryos of Taxus floridana germinated. Presumably, they require a longer warm stratification period than 2 months since embryos did not respond to leaching treatment. LePage (1968) indicates that the seeds were collected from mature trees of Taxus baccata but did not indicate the time period before excision. Schropmeyer (1974) mentioned differences in duration of warm stratification between species and that the International Seed Testing Association reconmnended, in general, stratification periods for 90-210 days at 16 C, followed by 60-120 days at 2-5 C.


5.3 Torreya taxifolia

Conventional propagation was successful with 20-56' rooting

from mixed cuttings from nature and juvenile plants. Cuttings from different aged plants were combined since only a limited number of cuttings were available. Optimal rooting occurred when 4000-8000 ,ng/l IBA was used. Rooted plagiotropic branches may be useful as seed or pollen sources so that intercrossing of the remaining, limited gene pool






61


could be accomplished by collecting cuttings from as nany plants as practical. Establishing seed nurseries of these cuttings would allow a practical means of preserving the remaining genetic diversity under controlled management where environmental stresses could be reduced and fungicides used more efficiently.

Micropropagation using shoot-tips did not result in mass

proliferation of shoots. Commonly 2 to 4 shoots could be stimulated to elongate in culture but these appeared to be preformed buds of the

normal branching patterns. These lateral shoots commonly grew horizonally and then geotropically into the medium. Occasionally these explants rooted in culture, especially after 4 to 6 months on the same medium. They however did not survive transfer to soil. The use of more juvenile tissues such as seedlings and dormant buds located at the base of the trunk may increase the possibility of stimulating development of orthotropic shoots. Ball (1978) was successful in obtaining multiple shoots from "burl" shoots of Sequoia sempervirens, however, there has only been limited investigation using similar explants from Torreya due to very limited availability of this material. The formation of numerous shoots from burls, such as occurs occasionally with wounded Taxus floridana, has not been observed although occasionally 3 or 4 ortotrophic shoots have been observed arising from a

common point at the base of a trunk.

Contamination of shoot-tip cultures continues to hamper

investigation. In cooperation with Mr. N. El-Ghol, Plant Pathologist with the Florida Division of Plant Industry, 3 systemic fungi were isolated (Phyllosticta spp., Colletotrichum gloeosporioides and Guigmnardia sp.). Establishment of stock plants in growth chambers






62


greatly reduced the amount of contamination. Presumably, the combination of low night temperatures and reduced humidity levels, may have been less favorable for growth of the pathogens. Recently, the weekly treatment over 4 weeks of these stock plants with the fungicide, Zyban, a broad spectrum systemic and contact fungicide, has been effective in eliminating one of these pathogens, Phyllasticta. Soaking of shoot-tip explants in fungicide solutions before clorox treatment was ineffective in reducing contamination. Soaking for 15, 30, 60, 120 and 240 minutes actually increased fungal contamination, possibly due to increased spread of hyphae. A 24 hour soak in Zyban reduced fungal contamination to 15%, however, this resulted in an increase in bacterial contami nation.

Cultures of expanding "embryonic shoots" [vernalized shoot buds with scale leaves removed, according to the techniques of Bonga (1977,1981)] did not produce multiple shoots. These "embryonic shoots" were large enough to be easily excised after the beginning of visible bud enlargement, especially 1 or 2 weeks before actual bud break. However, all bud cultures were either lost to contamination or were killed by disinfestation procedures. Fungicide treatment of mature trees may allow future investigation into this technique.

Culture of micro- and negasporangia was investigated. Although

these explants could be nore readily established in culture without high levels of contamination, none grew. Microsporangia are large and numerous on male trees, however, these are only available in early spring and only a few species have been reported to produce multiple shoots from these structures (Bonga 1977,1981).





63


The pretreatment of seedlings with cytokinin sprays before placing shoots in culture appears to offer one of the better chances of obtaining multiple shoots. Seedling stock plants produce multiple orthotropic basal shoots when sprayed weekly for 4 weeks with cytokinins, BA or 2-i-P. BA appears to be slighlty more effective. Following excision, additional orthotropic shoots continued to develop over at least a several month period. When these shoot-tips and nodal

sections are cultured on media with low cytokinins, numerous axillary buds and "bud-masses" developed. Further investigations will be necessary to optimize this technique and to develop entire plantlets.

















6.0 CONCLUSIONS


The primary objective of this research was to investigate clonal propagation techniques for 3 endangered native ornamental plants. Conventional cutting propagation of Torreya and Taxus was investigated and compared to micropropagation using shoot-tip cultures and embryo culture. Clonal propagation using cuttings was successful for Taxus floridana and Torreya taxifolia with maximum rooting of cuttings collected during winter and treated with IBA. Younger trees rooted at higher rates than larger and older trees. However, cutting material is limited by scarcity of plants and plagiotrophic growth when lateral branches are used. Rooting procedures for Rhododendron chapmanii cuttings remain undetermined due to limited experimental material.

A very successful micropropagation system with commercial potential has been developed for R. chapmanii. Large numbers of plantlets were

produced from a few shoot-tips and with minimal impact on the remaining natural populations. It has been estimated that over 11,000 rooted plantlets could be produced from a single shoot-tip in 1 year. Colonization of rooted plantlets with the ericoid mycorrhizae, Pezizella ericae, did not result in superior survival and growth when grown for 12 weeks in a greenhouse. Colonization of plantlets appears to increase bud break and growth of over-wintered containerized plantlets, however, additional time will be necessary to determine if this trend continues. The present micropropagation system was designed to maintain clonal characteristics.
64







65


Micropropagation techniques were only partially successful for

Taxus floridana and Torreya taxifolia. Shoot-tip cultures and cultured buds produced only limited numbers of shoots apparently from preformed axillary meristems. Culture of "embryonic shoots" and micro- and megasporangia were unsuccessful in stimulating organogenesis. Embryo culture for both these species was unsuccessful presumably due to insufficient chilling. Pretreatment of stock plants before in vitro culture with growth regulators appears to be a promising technique and deserves further investigation.






66


LIST OF REFERENCES

Abbott, A.J. 1977. Propagating temperate woody species in tissue
culture. Scientific Horticulture 28(4):155-162.

Aitken, J., K.J. Horgan and T.A. Thorpe. 1981. Influence of explant
selection on the shoot-forming capacity of juvenile tissue of
radiata pine. Can. J. For. Res. 11:112-117.

Amos, R.R. and B.H. McCown. 1981. Micropropagation of members of the
coniferae. HortScience 16(3):89(abstract).

Anderson, W.C. 1975. Propagation of rhododendrons by tissue culture.
Part 1. Development of a culture medium for multiplication of
shoots. Proc. Intern. Plant Propagators Soc. 25:129-135.

Anderson, W.C. 1978. Rooting of tissue cultured rhododendrons. Proc.
Intern. Plant Propagators Soc. 28:135-139.

Arnold, S. and D.T. Eriksson. 1978. Induction of adventitious buds
on embryos of Norway spruce grown in vitro. Physiol. Plant.
44:283-287.

Ayensu, E.S. and R.A. DeFilipps. 1978. Endangered and threatened
plants of the United States. Washington, D.C. Smithsonian
Institution and World Wildlife Fund Inc. 403pp.

Ball, E.A. 1978. Cloning in vitro of Sequoia sempervirens. In: K.W.
Hughes, R. Henke and M. Constatin, LTds_., Propagation of higher
plants through tissue culture, a bridge between research and
application, Tech. Inform. Center, U.S.D.E. Conference 7804111,
p. 259.

Baker, W. 1983. Unpublished. Torreya Management Symposium. Florida
Dept. Nat. Res. Tallahassee.

Bonga, J.M. 1977. Organogenesis in in vitro cultures of embryonic
shoots of Abies balsamea (BalsauFir)T.In Vitro 13(1):41-48.

Bonga, J.M. 1980. Plant propagation through tissue culture. In: F.
Sala, B. Parisi, R. Cella and 0. Cuerri, [Eds.], Plant Cell
Cultures: Results and Perspectives. Elsevier Press. pp. 253-254.

Bonga, J.M. 1981. Organogenesis in vitro of tissues from mature
conifers. In Vitro 17:511-518.

Boulay, M. 1979. Multiplication et clonage rapide du Sequoia
sempervirem par la culture in vitro. Etudes et Recherches, AFOCEZ,
Domaine de 1'Etancon, 77370~Nangis, France 12:49-55.






67


Briggs, B. and S.M. McCullock. 1983. Progress in micropropagation of
woody plants in the United States and Western Canada. Proc.
Intern. Plant Propagators Soc. 33:239-248.

Brown, C.L. and H.E. Sommer. 1974. An atlas of gymnosperms cultured
in vitro: 1924-1974. GA. For. Res. Council, 271p.

Brown, C.L. and H.E. Sommer. 1977. Bud and root differentiation in
conifer cultures. Tappi 60:72-73.

Chalupa, V. and D.J. Durzan. 1973. Growth and development of resting
buds of conifers in vitro. Can. J. For. Res. 3:196-208.

Chaturvedi, H.C. and G.C. Mitra. 1974. Clonal propagation of citrus
from somatic callus cultures. HortScience 9(2):118-120.

Cheng, T. 1975. Adventitious bud formation in culture of Douglas fir
(Pseudotsuga menziesii, Mirb.) France. Plant Sci. Lett. 5:97-102.

Cheng, T.Y. 1976. Vegetative propagation of Western hemlock (Tsuga
heterophylla) through tissue culture. Plant and Cell Physiol.
17:1347-135M.

Cheng, T.Y. 1978. Clonal propagation of woody plant species through
tissue culture techniques. Proc. Intern. Plant Propagators Soc.
28:139-155.

Chevre, A-Marie, S.S. Gill, A. Mouras and G. Salesses. 1983. In vitro
vegetative multiplication of chestnut. J. Hort. Sci. 58(l123-2T9.

Coleman, W.K. and T.A. Thorpe. 1977. In vitro culture of Western
red-cedar (Thuja plicata, Donn.) IT~Plantlet formation. Bot.
Gaz. 138:298-34.

David, A., H. David and T. Mateille. 1982. In vitro adventitious
budding on Pinus pinaster cotyledons and needles. Physiol. Plant.
56:102-107.

Debergh, P.C. and L.J. Maene. 1981. A scheme for commercial propagation
of ornamental plants by tissue culture. Scientia Hort. 14:335-345.

Duddridge, J. and D.J. Read. 1982. An ultrastructural analysis of
the development of mycorrhizas in Rhododendron ponticum. Can. J.
Bot. 60:2345-2356.

Durzan, D.J. 1980. Prospects for the mass propagation of economically
important conifers by cell and tissue cultures. In: Developments
in plant biology, Vol. 5, Elsevier/N. Holland, Biomed. Press.
pp. 283-288.

Durzan, D.J. and R.A. Campbell. 1974. Prospects for the mass
production of improved stock of forest trees by cell and tissue
culture. Can. J. Forest Res. 4:151-174.






68


Durzan, D.J. and S.M. Lopushanski. 1975. Propagation of American elm
via cell suspension cultures. Can. J. For. Res. 5:273-277.

Englander, L. 1981. Endomycorrhizae by septate fungi. In: N.C.
Schenck, [Ed.], Methods and principles of mycorrhizal research.
American Phytopath. Soc., St. Paul, Minn. pp. 11-12.

Federal Register. 1983. Endangered and threatened wildlife and plants:
Proposal to determine Torreya taxifolia (Florida torreya) as an
endangered species. Fed. Reg.48(68):15168-15171.

Florida Statutes. 1978. Preservation of native trees, shrubs and
plants. s. 581.185,581.186, 581.187. and 581.211. In:
I.F.A.S., Urban Gardener 3(8).

Giovanettii, M. and B. Mosse. 1980. An evaluation of techniques for
measuring vescicular-arbuscular mycorrhizal infection in roots.
New Phytol. 84:489-500.

Godfrey, R.K. and H. Kurz. 1962. The Florida Torreya destined for
extinction. Science 136:900-902.

Granger, R.L., C. Plenchette and J.A. Fortin. 1983. Effect of a
vesicular arbuscular (VA) endomycorrhizal fungus (Glomus epigaeum)
on the growth and leaf mineral content of two apple clones
propagated in vitro. Can. J. Pl. Sci. 63(2):551-555.

Gray, H. 1978. Chemical aids in rooting rhododendrons and Ilex
cuttings. Proc. Intern. Plant Propagators Soc. 28:5173T9.

Haines, R.J. and R.A. deFossard. 1977. Propagation of Hoop Pine
(Araucaria cunninghamii, Act.) by organ culture. In: G. Boesman, [Chrmanj, Symposium on Tissue Culture for Horticultural Purposes.
pp. 297-302.

Hartman, H.T. and D.E. Kester. 1975. Plant propagation principles
and practices. 3rd ed. Prentice Hall, Englewood Cliffs, N.J.
662pp.

Hearne, D.A. 1982. Preliminary report on a technique which provides a
"maturity factor" for trees grown in tissue culture. Proc.
Intern. Plant Propagators Soc. 32:109-113.

Herrera, H.G. and G.C. Phillips. 1984. Tissue culture of Pinus
eldorica, Med. Sym. Prop. Higher Plants Through Tissue Culture
III. (abstract/poster), Knoxville, Tenn.

House Document 95-41 (1975) Report on endangered and threatened plant
species of the United States. Washington, D.C., Smithsonian
Institute. 200pp.

Hu, C.Y. and I.M. Sussex. 1971. In vitro development of embryoids on
cotyledons of Ilex aquifoliumnf Phytomorphology 21:103-107.






69


Isikawa, H. 1974. In vitro formation of adventitious buds and roots on
the hypocatyl of Cryptomeria jeponica. Bot. Mag. Tokyo 87:73-77.

Jansson, E. and C.H. Bornman. 1983. Morphogenesis in dormant
embryonic shoots of Picea abies-influence of the crown and cold
treatment. Physiol. Plant. 79( ):l-8.

Keen, R.A. 1954. The propagation of taxus-a review. Proc. Intern.
Plant Propagators Soc. 4:63-69.

Keys, R.N. and F.C. Cech. 1981. Plantlet formation in American
chestnut embryonic tissue in vitro. Proc. 2nd North Central Tree
Improve Conf., Lincoln, Nebraska, Aug. 5-7, 1981, p. 189-194.

Keys, R.N. and F.C. Cech. 1982. Propagation of American chestnut in
vitro. Proc USDA For. Ser., Amer. Chest. Cooperators Meeting,
Jan 5-7, 1982, Morgantown, W. Va., pp. 106-110.

Konar, R.N. and V.P. Oberoi. 1965. In vitro development of embryoids
on the cotyledons of Biota orientEalis. Phytomorphology 15:137-140.

Lakshmi Sita, G., J. Shobha and C.S. Vaidyanathan. 1980. Regeneration
of whole plants by embryogenesis from cell suspention cultures of
sandal wood. Curr. Sci. 49:196-197.

Larkin, P. J. and W. R. Scowcroft. 1981. Somaclonal variation a novel
source of variability from cell cultures for plant improvement.
Theor. Appl. Genet. 60:197-214.

LaRue, C.D. 1943. Regeneration in the megagametophyte of Zamia
floridana. Bull. Torrey Bot. Club 75:597-503.

LaRue, C.D. 1954. Studies of growth and regeneration of gametophytes
and sporophytes of gymnosperms. Brookhaven Sym. Biol. 6:137-208.

Lazarte, J.E. 1981. Woody tissue culture research. Proc. Intern.
Plant Propagators Soc. 31:649-655.

LePage, M.T. 1968. Mise en vidence d'une dormance associ'e 'a une
immaturity' de 1'embryon chez Taxus baccata L. C.R. Acad. Sci.
Paris, 266D:1028-1030.

LePage-Degivry, M.T. 1970. Acid abcissique et dormance chez les
embryons le Taxus baccata L. C.R. Acad. Sci. Paris
2710:482-484.

LaPage-Degivry, M.T. 1973a. Etude en cluture in vitro de la dormance
embryonnaire chez Taxus baccata L. Biol. Plant. 15:264-269.

LePage-Degivry, M.T. 1973b. Influence de lacide abscissique sur le
development des embryous de Taxus baccata L. cutives in vitro Z.
Pflanzenphysiol. 70:406-413.







70


LePage-Degivry, M.T. 1973c. Intervention d'un inhibiteur lie' dans la
dormance embryonndire de Taxus baccata L. C.R. Acad. Sci. Paris
277D:177-180.

LePage-Degivry, M.T. and G. Garello. 1973. La dormance embryonnaire
chez Taxus baccata: Influence de la composition du milieu guide sur l'induction de la germination. Physiol. Plant. 29:204-207.

Linsmaier, E.M. and J. Skoog. 1965. Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant
18:100-127.

Litz, R.E. 1984. In vitro responses of adventitious embryos of two
polyembryonic E genia species. 19(5):720-722.

Liu, J.R., K.C. Sink and F.G. Dennis. 1983. Plant regeneration from
apple seedling explants and callus cultures. Plant Cell, and
Tissue Organ Culture 2:293-304.

Lloyd, G. and B. McCown. 1980. Commercially-feasible micropropagation
of mountain laurel, Kalmia latifolia, by use of shoot-tip culture.
Proc. Intern. Plant Propagators Soc. 30:421-427.

Loreti, F. and S. Mozini. 1982. Mass propagation of fruit trees in
Italy by tissue culture: present status and perspectives. Proc.
Intern. Plant Propagators Soc. 32:283-290.

Lyrene, P.M. 1981. Juvenility and production of fast-rooting cuttings
from blueberry shoot cultures. J. Amer. Soc. Hort. Sci.
106(3):396-398.

Ma, S.S. and S.O. Wang. 1977. Clonal multiplication of azaleas
through tissue culture. Acta Hortic. (The Hague) 78:209-215.

Marx, D.H. and D.S. Kenny. 1981. Production of ectonycorrhizal
fungus inoculum. In: N.C. Schenck, [Ed.], Methods and
principles of mycorrhizal research. American Phytopath. Soc.,
St. Paul, Minn. p.131-146.

McComb, J.A. 1978. Clonal propagation of woody plants using tissue
culture, with special reference to apples. Proc. Intern Plant
Propagators Soc. 28:413-426.

McCown, B.H. and R. Amos. 1982. A survey of the response of
gymnosperms to microculture. HortScience 17(3):489 (abstract).

McCown, B.H. and G. Lloyd. 1981. Woody plant medium (WPM)- A mineral
nutrient formulation for microculture of woody plant species.
HortScience 16(3):89 (abstract).

McCown, B.H. and G.B. Lloyd. 1983. A survey of the response of
Rhododendron to in vitro culture. Plant Cell, Tissue and Organ
Culture 2(1):77-IT.







71


Meyer, M.M. 1982. In vitro propagation of Rhododendron catawbiense
froiD floral buds. HortScience 17(6):891-892.

Moore-Parkhurst, S. and L. Englander. 1981. A method for the
synthesis of mycorrhizal association between Pezizella ericae and
Rhododendron maximum seedlings growing in a defined medium.
Mycologia 737(794-997.

Morandi, D., S. Gianininazzi and V. Gianininazzi-Pearson. 1979.
Interet de l'endomycorrhization dans la reprise et la croissence du
Framboisier issu de multiplication ve'ge'tative in vitro. Ann.
Amelior. Plantes 29(6):623-630.

Mott, R.L. and H.V. Amerson. 1981. A tissue culture process for the
clonal production of loblolly pine plantlets. N.C. Agr. Res.
Ser. Tech. Bull. 271. 14pp.

Mott, R.L., R.H. Seltzer, A. Mehra-Palta and B.J. Zobel. 1977.
Production of forest trees by tissue culture. Tappi 60(6):62-64.

Murashige, T. 1974. Plant propagation through tissue culture. Ann.
Rev. Plant Physiol. 25:135-166.

Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant.
15:473-497.

Norstog, K. 1965. Induction of apogamy in megagametophytes of Zamia
integrifolia. Amer. J. Bot. 52:993-999.

Norstog, K. and E. Rhamstine. 1967. Isolation and culture of haploid
and diploid cycad tissues. Phytomorphology 17:374-381.

Patel, R.V. and G.P. Berlyn. 1982. Genetic instability of multiple
buds of Pinus coulteri regenerated from tissue culture. Can. J.
For. Res. 12:93-101.

Pearson, V. and D.J. Read. 1973. The biology of mycorrhiza in the
Ericaceae. I. The isolation of the endophyte and synthesis of
iycorrhizae in aseptic culture. New Phytol. 72:371-379.

Pons, F., V. Gianinazzi-Pearson, S. Gianinazzi and J.C. Navatel.
1983. Studies of VA mycorrhizae in vitro: mycorrhizal synthesis
of axenically propagated wild cherry (Prunus avium L.) plants.
Plant and Soil 71:217-221.

Ramawat, K.G., H.C. Asya. 1976. Growth and morphogenesis in callus
cultures of Ephedra gerardiana. Phytomorphology 26:395-403.

Rancillac, M.M. Faye and A. David. 1982. In vitro rooting of
cloned shoots in Pinus pinaster. PhysTol.Plant. 56:97-101.

Read, D.J. 1983. The biology of mycorrhiza in the Ericales. Can. J.
Bot. 61:985-1004.






72


Reilly, K.J. and J. Washer. 1977. Vegetative propagation of radiata
pine by tissue culture: Plantlet formation from embryonic tissue.
N.Z.J. For. Sci. 7(2):199-206.

Rhodes, H.L.J. 1968. Juvenility in relation to adventitious bud and
root initiation in woody plants. Proc. Intern. Plant Propagators
Soc. 18:252-254.

Sargent, C.P. 1947. Silva of North America. Vol. X. Murry Print Co.
Cambridge, Mass.

Schopmeyer, C.S. 1974. Seeds of woody plants in the United States
Agriculture Handbook No. 450. Forest Service, U.S. Dept. of
Agriculture. 883pp.

Simon, R.W. 1983. Chapman's rhododendron recovery plan. U.S. Fish
and Wildlife Service. Atlanta, Ga., 41pp.

Sommer, H.E. and C.L. Brown. 1980. Embryogenesis in tissue cultures
of sweetgum. For. Sci. 26:257-260.

Sommer, H.E., C.L. Brown and P.P. Kormanik. 1975. Differentiation of
plantlets in long leaf pine (Pinus palustris, Mill.) tissue
cultured in vitro. Bot. Gaz. 16(2):196-2.

Stribley, D.P. and D.J. Read. 1974. The biology of mycorrhiza in th?5
Ericeae. IV. The effect of mycorrhizal infection on uptake of N
from labeled soil by Vaccinum macrocarpum Ail. New Phytol.
73:1149-1156.

Stribley, D.P. and D.J. Read. 1975. Some nutritional aspects of the
biology of ericaceous mycorrhizas. New Phytol. 73:1149-1163.

Strode, R. 1979. Commercial micropropagation of rhododendrons. Proc.
Intern Plant Propagators Soc. 29:439-442.

Styer, D.J. and C.K. Chin. 1983. Meristem and shoot-tip culture for
propagation, pathogen elimination and gernplasm preservation.
Hort. Rev. 4:221-277.

Tulecke, W. 1959. The pollen cultures of C.D. LaRue: A tissue from
the pollen of Taxus. Bull. Torrey Bot. Club. 86:283-289.

Vieitez, A.M. and E. Vieitez. 1980. Plantlet formaton froin
embryonic tissue of chestnut grown in vitro. Physiol. Plant.
50:127-130.

Vieitez, A.M. and M.L. Vieitez. 1980. Culture of chestnut shoots
from buds in vitro. J. Hort. Sci. 55(l):83-84.

Wdang, P.J. and C.Y. Hu. 1984. In vitro cloning of the decidious
timber tree Sassafras randaiense. Z. Pflanzenphysiol.
113 (4 ): 331 36.







73


Ward, D.B. 1978. Plants. In: P.C. Pritchard, [Ed.], Rare and
endangered biota of Florida, Vol. 5, University Presses of
Florida. Gainesville, FL. 175p.

Wilkins, C. P., J. L. Cabrera and J. H. Dodds. 1985. Tissue culture
propagation of trees. Outlook on Agriculture 14(1):2-13.

Winton, L. 1970. Shoot and tree production from aspen cultures. Amer.
J. Bot. 57:904-909.

Winton, L. 1978. Morphogenesis in clonal propagation of woody plants.
In: T.A. Thorpe, [Ed.], Frontiers of plant tissue culture.
University of Calgary Press, Calgary. pp.419-426.

Winton, L.L. and S.A. Verhagen. 1977. Shoots from Douglas fir
cultures. Can. J. Bot. 55:1246-1250.

Wochok, Z.S. 1981. The role of tissue culture in preserving threatened
and endangered plant species. Biological Conservation
20(1981 ):83-89.

Wochok, Z.S. and M.A. El-Nil. 1977. Conifer tissue culture. Proc.
Intern. Plant Propagators Soc. 27:131-139.

Wong, S. 1982. Direct rooting of tissue-cultured rhododendrons into an
artificial soil mix. Proc. Intern. Plant Prop. Soc. 31:36-39.

Zenkteler, M.A. and I. Guzowska. 1970. Cytological studies on
regenerating mature female gametophyte of Taxus baccata L. and
mature endosperm of Tilia platyphyllos Scop. in in vitro culture.
Acta. Soc. Bot. Poloniae 39:161-173.

Zimmerman, R.H. 1978. Tissue culture of fruit trees and other fruit
plants. Proc. Intern. Plant Propagators Soc. 28:539-545.

















BIOGRAPHICAL SKETCH


Lee Roy Barnes, Jr., was born in Durham, North Carolina. Following graduation from high school, he attended and graduated from North Carolina State University with a double major in biological science and botany in May 1976. Transferring to the Department of Horticultural Science, he completed the Master of Science degree in December, 1978. His thesis was titled "In vitro propagation of watermelon" (Drs. Fred Cochran and Ralph Mott, co-chairmen). Following a two year leave of work experience and travel, he entered the University of Florida, Department of Ornamental Horticulture, in the Fall of 1981 interested in conventional propagation and micropropagation of native plants, especially rare and endangered species.


74
















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.





Charles R. Johnsoqa, Chairman Professor or Oprnmental
Horticul ture




I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





Thomas J. S+ehan Professor of Ornamental
Horticul ture




I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.





Indra K. Vasil
Graduate Research Professor of Botany











I certify that I have read this study and that in iny opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.






Mike J. Young
Associate Proe9'ssor of// Fruit 'C-Gops




This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School, and was accepted as partial ful fillment of the requirements for the degree of Doctor of Philosophy.

August 1985




Dean, College of Agriculture


Dean, Graduate School




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CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS RHODODENDRON CHAPMANII GRAY, TAXUS FLORIDANA NUTT. AND TORREYA TAXIFOLIA ARN. by LEE ROY BARNES, JR. A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE RFQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1985

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ACKNOWLEDGEMENTS I would like to acknowledge the support and advice of my graduate committee, Drs. Charles Johnson, Thomas Sheehan, Mike Young, Indra Vasil, as well as, Norman Schenck. I also thank the Department of Ornamental Horticulture for financial support, Mr. Jim Stevenson and Dr. Greg Brock with the Florida Department of Natural Resources for their friendly cooperation, and the Park Manager and staff of Torreya State Park for their assistance. I would also like to thank the following for their patient and enthusiastic help, Ms. Nancy Philman, Ms. Shirley Anderson, Ms. Jackie Host, Mr. T.D. Townsend, Mr. Jan Weinbrecht, Ms. Barbara Prichard, Ms. Debbie Gaw and Ms. Robin Reddick. Also I thank Ms. Anne Whealy, Ms. Sara Rosenbaum, Mr. Chris Fooshee, Mr. Ed Duke and Ms. Beth Logan for human support. Special thanks are offered to Dr. Rita Hummel and Dr. Jasper Joiner for their humor, laughter, and personal concern. Finally, I would like to acknowledge the timeless support and love of my parents and sister.

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TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS 1* 1 LIST OF TABLES AND FIGURES vi KEY TO ABBREVIATIONS viii ABSTRACT 1 X 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 3 2.1 Endangered and Threatened Native Plants 3 2.2 Advantages of Micropropagation Techniques 4 2.3 Species Description and Distribution 5 2.3.1 Rhododendron chapinanii Gray 5 2.3.2 Taxus floridana Nutt. 5 2.3.3 Torreya taxi folia Arn 6 2.4 Woody Plant Tissue Culture 6 2.4.1 Organogenesis from Callus and Suspension Cultures 7 2.4.2 Organogenesis from Plant Organs and Organ Sections 8 2.4.3 Stimulation of Axillary Meri stems 15 2.4.4 Taxus Embryo Culture 18 2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets 19 3.0 MATERIALS AND METHODS 21 3.1 Rhododendron chapmanii Gray 21 3.1.1 Establishment of Stock Plants 21 3.1.2 Disinfestation 22 3.1.3 Madia Selection 22 3.1.4 Selection of Growth Regulator Levels 23 3.1.5 Stage II Subcultures 23 3.1.6 Rooting and Establishment 24 3.1.7 Colonization with Ericoid Mycorrhizae 24

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3.2 Taxus floridana Nutt 26 3.2.1 Conventional Propagation by Cuttings 26 3.2.2 Culture of Quiescent Shoot-tips 26 3.2.3 Culture of Expanding Vegetative Buds 27 3.2.4 Microsporangia Culture 28 3.2.5 Embryo Culture 28 3.3 Torreya taxi folia Arn, 29 3.3.1 Conventional Propagation by Cuttings 29 3.3.2 Culture of Quiescent Shoot-tips 30 3.3.3 Culture of Expanding Vegetative Buds 30 3.3.4 Culture of Microand Megasporangia 30 3.3.5 Embryo Culture 30 3.3.6 Effect of Cytokinin Sprays on Stock Plants 31 4.0 RESULTS 33 4.1 Rhododendron chapmanii Gray 33 4.1.1 Oisinfestation 33 4.1.2 Selection of Growth Regulators Levels 33 4.1.3 Stage II Subcultures 36 4.1.4 Rooting and Establishment 36 4.1.5 Colonization with Ericoid Mycorrhizae 39 4.2 Taxus floridana Nutt 43 4.2.1 Conventional Propagation by Cuttings 43 4.2.2 Micropropagation of Taxus Shoottips 43 4.2.3 Culture of Expanding Vegetative Buds 43 4.2.4 Culture of Microsporangia 46 4.2.5 Embryo Culture 46 4.3 Torreya taxi folia Arn 46 4.3.1 Conventional Propagation by Cuttings 46 4.3.2 Micropropagation of Shoot-tips 48 4.3.3 Culture of Expanding Vegetative Buds 48 4.3.4 Culture of Microsporangia and Megasporangia 48 4.3.5 Embryo Culture 48 4.3.6 Cytokinin Sprayed Seedlings 51 5.0 DISCUSSION 53 5.1 Rhododendron chapmanii 53 5.2 Taxus floridana 57 5.3 Torreya taxi folia 60 6.0 CONCLUSIONS 64

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LIST OF REFERENCES 66 BIOGRAPHICAL SKETCH 74

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LIST OF TABLES AND FIGURES Table 1*21 4.1.1 Percent contamination of j_n vitro cultured Rhododendron chapmanii shoot-tips grown in shade house and growth chambers 34 4.1.2 Growth response of J_n vitro cultured Rhododendron chapmanii shoot-tips cultured on Woody Plant Medium with 5 levels of the cytokinin, 2-i-P 35 4.1.3 Growth response of i_n vitro cultured Rhododendron chapmanii shoottips grown in 3x3 factorial combination of auxin (IAA) and cytokinin (2-i-P) 37 4.1.4 Survival and rooting response of microcuttings of Rhododendron chapmanii following treatment with auxins from 2 sources 4 4.1.5 Summary of micropropagation system developed for Rhododendron chapmanii 41 4.1.6 Response of Rhododendron chapmanii plantlets produced in vitro to 2 establishment media and inoculation with or without ericoid mycorrhizae 42 4.2.1 Rooting response of cuttings from 2 aged groups of Taxus floridana to 3 levels of the auxin, IBA 44 4.2.2 In vitro growth response of Taxus floridana shoot-tips on" 4 nutrient media 45 4.3.1 Rooting response of cuttings of Torreya taxi folia treated with 3 levels of the auxin, IBA 47 4.3.2 Growth response of cultured shoot-tips of Torreya taxifol ia grown i_n vitro on 4 nutrient media 49 4.3.3 Average number of axillary shoots of Torreya taxi folia developing in vitro on 3 nutrient media and 5 concentrations of the cytokinin, BA 50 Figure 4.1 Multiplication rates of in vitro cultured Rhododendron chapmanii during initial and additional SII subcultures.. 38 vi

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KEY TO ABBREVIATIONS GROWTH REGULATORS ABA Abscisic acid BA Benzyl adenine GA3 Gibberellic acid IAA Indole acetic acid IBA Indole butanoic acid 2-1 -P 2-isopentyl adenine NAA Naphthalene acetic acid NUTRIENT MEDIA H Heller's LS Linsmaier and Skoog 1/2LS 1/2 Strength Macronutrients Linsmaier and Skoog MS Murashige and Skoog WPM Woody Plant Medium 1/2WPM 1/2 Strength Macronutrients Woody Plant Medium

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS RHODODENDRON CHAPMAN 1 1 GRAY, TAXUS FLORIDANA NUTT. AND TORREYA TAXIFOLIA ARN. By Lee Roy Barnes, Jr. August 1985 Chairman: Dr. Charles R. Johnson Major Department: Horticultural Science Clonal propagation utilizing conventional and inicropropagation techniques was investigated for three endemic and endangered Florida plant species, Rhododendron chapmanii Gray, Taxus floridana Nutt. and Torreya taxi folia Arn. This is critical for their preservation since these species exist in limited numbers but have high ornamental value which has threatened their survival due to illegal collection. Micropropagation of R. chapmanii was investigated using shoot-tip cultures. An average of 7.6 fold increase in lateral shoots was achieved for three additional Stage II multiplication cycles on low salts Woody Plant Medium (WPM) with 2-i-P (10 mg/1 ) and adenine sulfate (30 mg/1). Microcuttings rooted in 4-6 weeks when direct-stuck into autoclaved 1:1:1 (v:v:v) Canadian peat : vermi cul ite: perl ite medium in covered flats. A 5 second quick-dip in 1000 mg/1 IBA (20% EtOH + 80% H 2 0) increased survival and rooting of microcuttings. Inoculation of plantlets with the mycorrhizal fungus, Pezizella ericae increased survival and growth when plantlets were established in a less than optimal soil -less medium (1:1 v:v Fired montmorillonite clay :Canadian peat) but not in MetroMix-500. vii i

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Torreya and Taxus cuttings could be easily propagated when treated with 2,000-4,000 mg/1 IBA. However, cuttings taken from lateral branches demonstrated strict plagiotropic growth. Best rooting and growth of Torreya cuttings (20%) occurred following treatment with 4000 mg/1 IBA. Taxus cuttings rooted best (67%) following treatment with 2000 mg/1 IBA. Shoot-tip cultures (3-5 cm long) of Taxus and Torreya developed only 2-3 lateral shoots, apparently from preformed buds. Cultured "embryonic shoots" from expanding vegetative buds failed to produce multiple shoots. Excised embryos did not germinate following liquid culture to leach inhibitors. Microand megasporangia did not grow or differentiate in culture. The most promising technique for Torreya involved application of cytokines (4 weekly sprays with 100-200 mg/1 BA or 2-i-P) to seedlings before culture. Newly stimulated axillary shoots developed additional shoots and multiple "bud-masses" when cultured on WPM with BA (1 mg/1) and NAA (0.01 mg/1). These were slow to develop but could be subcultured with additional growth of multiple shoots. No rooted plantlets were obtained.

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1.0 INTRODUCTION Preservation of indigenous endangered plant species is a critical issue requiring timely investigation of propagation techniques to increase the numbers of these rare species before they become extinct. Three endangered plant species of immediate concern are Rhododendron chapmanii Gray Taxus floridana Nutt and Torreya taxi folia Arn Al 1 are endemic to restricted areas of northern Florida. Presently only 3000 Rhododendron chapmanii (Chapman's rhododendron) plants are known jm situ and these are endangered due to habitat destruction or alteration, and over-collection for use in ornamental plantings. Taxus floridana (Florida yew) is endangered due to limited numbers and distribution but is highly desirable for landscape use and is subsequently subject to over-collection. Torreya taxi folia (Florida torreya) is endangered due not only to yery limited numbers but also due to being decimated by a fungal disease which has destroyed most seed bearing trees. Today only 1200 apparently diseased seedlings and weak stump sprouts remain. Presently, all three species are protected by state and federal laws. However, in spite of legal protection, the remaining specimens of each species is declining. Numerous R. chapmanii have been illegally dug from the wild. Torreya taxi folia does not appear to be reproducing in the wild and is all but extinct, except for a few apparently healthy plants located in botanical gardens elsewhere in the United States. The purpose of this investigation was to determine the feasibility of clonal propagation techniques for the mass production of 1

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these native ornamental species. Micropropagation techniques could potentially increase the number of these species with little impact on the present populations, as well as offer the possibility of eliminating the systemic pathogen(s) associated with Torreya die-back. If large numbers of these highly desirable plants could be produced, then they would be available for stabilizing natural populations, landscape plantings, and germ plasm preservation via meristem culture and cryopreservation.

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2.0 LITERATURE REVIEW 2.1 Endangered and Threatened Native Plants One group of native plants of particular interest are those which are rare, threatened or endangered in their present environments. Endangered and threatened plants are often legally classified at both federal and state levels. "Endangered" plants are defined as "species in imminent danger of extinction or extirpation and whose survival is unlikely if the causal factors presently at work continue operating" (Ward, 1978. p. xiii). "Threatened" plants are defined as "species which are believed likely to move into the above endangered catagory in the near future if the causal factors now at work continue operating" (Ward, 1978. p. xiii). A "rare" species classification includes those species which are not presently endangered or threatened, but are potentially at ri sk Congress first attempted to protect endangered plant species by enacting the Endangered Species Act of 1973 (House Document 95-41, 1975). Ayensu and de Filipps (1978) revised this initial listing and estimated that approximately 10% of the native vascular flora in the United States (approximately 20,000 spp.) or 2,000 species, subspecies and varieties are presently endangered or threatened. Ayensu and DeFilipps (1978) concluded there are numerous reasons for protecting these species including the advantages of maintaining a broad gene pool and genetic diversity within and among species,

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especially with important food, fiber and timber species. Many of these threatened plants show great potential for ornamental use. Within the state of Florida, there are an estimated 3,500 vascular plant species which are native or naturalized (Ward, 1978). Of these, 124 species are presently listed at the state level as being endangered or threatened. Ward (1978) concludes that this number represents only 3.5% of the total plant species but further estimates that the true number of endangered, threatened or rare species may vary from the listed number by a factor of 2 or as large as 7. In 1978, the State of Florida (s. 581.185 Florida Statutes) revised its listing of "endangered" plants to include over 30 species. They listed as "threatened" species all bromeliads, all cacti (except Opuntia ), all orchids, all native rhododendrons, most ferns, most native Ilex spp., all Zami a spp., all Zephranthes spp. and 48 other species. 2.2 Advantages of Micropropagation Techniques While many of the endangered and threatened plants are best protected by procurement and management of sensitive habitats, many of these species can be propagated and introduced into the horticultural trade with minimal impact on the remaining populations or their genetic diversity. Seedage and conventional vegetative propagation by cuttings can be used to increase the number of plants. However, these techniques are often of limited value due to the lack of propagation and cultural information for these rare species. Also the expense of collecting seed or vegetative propagules would be prohibitive due to limited numbers and travel expense. Micropropagation of endangered species offers many advantages over both seed and conventional vegetative propagation methods, such as

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(1) high initial multiplication rates from limited plant material in a small area; (2) clonal propagation of selected phenotypes via stimulation of existing axillary meri stems; (3) potential to force rejuvenation of selected mature plants resulting in increased rooting ability of shoots with juvenile rooting capacity (Rhodes, 1968; Lyrene, 1931); (4) ability to propagate infertile or low fertility species (Wochok, 1981); (5) high degree of environmental control to minimize variability with limited plant material and (6) possibility of obtaining disease-free material via meri stem culture (Abbott, 1977; Boxus and Druart, 1980; Styer and Chin, 1984). Wochok (1981) suggested that tissue culture can be ^ery useful in preserving threatened and endangered species and developed micropropagation techniques for 4 native species ( Atrip! ex canescens Mahonia aqui folium compacta Mahonia repens and Populus tremuloides ) 2.3 Species Description and Distribution 2.3.1. Rhododendron chapmanii Gray Rhododendron chapmanii Gray (Chapman's rhododendron) is a small evergreen shrub, 1-2 meters in height, which produces a showy display of terminal clusters of clear pink to rose-pink flowers in late spring (April -May). This species is apparently yery drought, heat, and pest resistant (Simon, 1983). Conventional propagation by seeds and cuttings has been successful (Salter, 1982 per.com.; Gensel 1983 per. com.), albeit with a low rate of multiplication.

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2.3.2 Taxus floridana Nutt. Taxus floridana Nutt. (Florida yew; Savin) is an evergreen, needle-bearing shrub or small tree 3-8 m (rarely 10 m) in height. It is highly adapted to heavy shade and tolerates severe pruning. Conventional clonal propagation by cuttings is possible; however, cuttings from lateral branches grow only plagiotrophically and few orthotropic shoots are available. Seed propagation is limited by a double dormancy within the seed which usually requires two seasons to germinate. Seed set is limited and heavy fruiting has been observed only once in the last ten years in Torreya State Park (Womble, 1982, per. com.). While over 200 seeds were collected in 1982, only 25 were collected in 1983 following an extensive, tree to tree search. 2.3.3 Torreya taxi folia Arn. Torreya taxi folia Arn. (Florida torreya; Stinking Cedar; Polecat Wood; Gopherwood; Savin; Tumi on) is a medium sized, pungent smelling (when bruised or crushed) evergreen tree which grows to 15 m. Conventional propagation by cuttings is possible; however, lateral cuttings have been found to continue growing only plagiotrophically. Seed propagation has been successful but is limited by extreme scarcity of seed and a poorly understood double dormancy which usually prevents germination until the second year. 2.4 Woody Plant Tissue Culture Tissue culture techniques have been applied to three general areas of interest, namely clonal propagation of (1) woody ornamentals (Murashige, 1974; Abbott, 1977; Oebergh and Maene, 1981; McComb, 1978; Lazarte, 1981; 3riggs and McCullock, 1983), (2) fruit trees (McComb,

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1978; Zimmerman, 1978; Lazarte, 1981; Loreti andMozini, 1982), and (3) superior genotypes of selected forest species (Durzan and Campbell, 1974; Abbott, 1977; Brown and Sommer, 1977; Cheng, 1978; Bonga, 1980; Durzan, 1980). Propagation of woody species has been investigated by three different approaches: (1) organogenesis from callus or suspension cultures, (2) organogenesis directly from plant organ or organ sections and (3) axillary shoot stimulation. Attempts have been made to propagate from both selected mature tissues and from the more competent or plastic juvenile tissues. Although it is more desirable to obtain plants from elite mature genotypes, these tissues have been difficult to root. Rejuvenation treatments and the use of juvenile zones within the plants have been successful in some cases (Lyrene, 1981). By far the greatest amount of success has occurred with the use of explants from juvenile tissues, especially embryos and young seedlings. Although morphogenically flexible and generally easy to root, these juvenile sources ^re genetically variable and untested as to their mature phenotype. 2.4.1 Organogenesis from Callus and Suspension Cultures Organogenesis from callus or suspension cultures established from elite trees would offer the greatest advantages. This would allow large scale and cost efficient production of superior trees. This would be especially important if somatic embryogenesis could be obtained and microencapsulation developed so that clonal "seeds" could be produced. However, at least two factors prevent the wide scale use of these techniques. First, organogenesis from callus and suspension cultures has not been successful for most important forest species (Wilkins et al

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1985). Secondly, the genetic stability of adventitious organogenesis is often questionable (Wilkins et al 1985; Larkin and Scowcroft, 1981). Successful regeneration of plantlets via somatic embryogenesis from suspension and callus cultures has been reported for Santa! urn album (Lakshmi Sita et al 1980), and Liquidambar styraciflua (Sommer and Brown, 1980). Regeneration of shoots and roots from callus has been reported for triploid Populus tremuloides (Winton, 1970), Citrus grandis and C_. sinensis (Chaturvedi and Mitra, 1974), Ulmus americana (Durzan and Lopushanski, 1975), Ephedra gerardiana (Ramawat and Arya, 1976), Larix decidua (Bonga, 1981), Mai us domestica (Liu et al., 1983), Pinus eldarica (Herrara and Phillips, 1984), Eugenia jambos and E. malaccenis (litz, 1984). In summary, organogenesis from true cell suspensions and callus cultures has only been successful for a limited number of woody species (especially with timber, tropical fruit, and arborescent monocotyledonous species), usually at low frequencies and often with the inability to root shoots from callus derived from mature trees (Wilkins et al 1985). 2.4.2 Organogenesis from Plant Organs and Organ Sections Direct organogenesis from plant organs and organ sections has been widely accomplished among woody species. In most cases, this has occurred from yery juvenile explants, especially embryos or young seedlings. Rejuvenation from mature tissues has been successful from inflorescenes or microand megasporangia, tissues temporally associated with meiosis (Bonga, 1980). Organogenesis has occurred in widely dispersed plant families. LaRue (1948) obtained low frequency shoot regeneration from the megagametophyte of Zamia floridana. He later reported (1954) regeneration of

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shoots and roots from megagametophytes of both Zami a floridana and Cycas revoluta. Norstog (1965) cultured megagametophytes of Zami a integri folia and was able to obtain diploid and haploid roots and leaves in 35% of his cultures. He was also able to obtain diploid and haploid embryo ids from cultures of excised embryos and haploid megagametophytes, respectively. Plants were successfully established in soil. Konar and Oberoi (1965) obtained embryo ids on cotyledons of Biota oriental is Endl (now Platycladus oriental is ). Further development of these embryo ids did not occur until transfer to a different medium. These "embryoids" may have been inappropriately named since they did not appear to have a root apex although normal appearing cotyledons and shoot apex developed into a young shoot. Morstog and Rhamstine (1967) induced proliferation of callus from haploid and diploid tissues of Zami a integri folia and haploid tissues of Cycas species. Hu and Sussex (1971) were able to stimulate embryogenesis from cultured cotyledons of Ilex aquifolium They noted that embryoids developed only on cotyledons where the shoot-tip failed to develop, suggesting an antagonism between shoot growth and initiation, possibly due to apical dominance of the active shoot-tip. Isikawa (1974) reported the formation of adventitious buds and roots on the hypocotyl s of Cryptomeria japonica primarily in illuminated cultures. This organogenesis required low levels of the growth regulators ABA, NAA and BA. He concluded that callus formation may be antagonistic to shoot formation of conifers. Sommer et al (1975) reported high frequency adventitious bud formation on cultured cotyledons of Pinus palustris when grown on

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10 modified Gresshoff and Doy medium. If maintained on media with high auxin/cytokinin ratios, excess callus began to form after 5-6 weeks. They also mentioned success in obtaining adventitious buds on cotyledons of P_. elliotii P_, taeda F\ virginiana P_. rigida P_. strobus and Pseudotsuga menziesii but did not mention if plantlets were obtained from these species. Cheng (1975) obtained adventitious bud formation on cotyledons and cotyledon slices of Pseudotsuga menziesii during primary culture, and was able to maintain the capacity to produce buds after 3 and 4 subcultures. This system required 3 separate steps: (1) culture initiation with equal amounts of auxins and cytokinins, (2) organ initiation with high cytokinin levels, and (3) further growth of adventitious buds on 1/2 strength modified MS medium and no growth regulators for further growth of adventitious buds. Plantlets were obtained by treating shoot cuttings with IBA (amounts not given). The necessity for separate steps suggests that conditions necessary for initiation of one step were inhibitory to further development. Cheng (1976) reported adventitious buds from cotyledons of Tsuga heterophylla using techniques similar to those employed for Pseudotsuga menziesii In addition, she was able to root shoots directly in soil mix thus saving one culture step. Winton and Verhagen (1976) induced adventitious shoots directly from cotyledons of Pseudotsuga memesii and also from subcultured callus, obtaining up to 100-200 clonal shoots from each seed. They also obtained callus on needles from 3 month old seedlings and from needles from a 25 year old tree. However, they were only able to stimulate adventitious shoots from the 3 month old needle callus.

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11 3onga (1977) initiated a new approach to obtain adventitious shoots from 15-20 year old Abies balsamea "Embryonic shoots," composed of dormant buds with scale leaves excised, were soaked either in sterile water for 24 hours, or in aqueous solutions of growth regulators. Soaked buds formed additional axillary shoots when transferred to a growth medium while non-soaked buds only expanded as a single shoot. Re i 11 y and Washer (1977) cultured embryos of Pinus radiata and obtained adventitious buds directly from cotyledons and hypocotyls and from meristematic callus which proliferated from these organs. This callus could be subcultured on cytokinin-free medium every 4 weeks. When shoot primordia were separated from these meristematic masses, they developed into well formed shoots. By serial subculture of callus, they were able to obtain over 200 shoots from 1 embryo in 6 months. These shoots could be rooted on medium with auxin (1-25 mg/1 IBA). Brown and Sommer (1977) reported that 9 Pinus species developed buds on cotyledons at a frequency of 26-100% depending on the species and medium. They were unable to root these elongating shoots except very rarely (less than 1% rooted). Mott et al (1977) summarized their work with Pinus taeda whereby they obtained more buds on excised cotyledons vs intact embryos. They found significant differences in organogenic response between seed families but these differences could generally be reduced by adjusting the growth regulator combinations. Coleman and Thorpe (1977) cultured cotyledons from seeds and shoot-tips from 4 to 10 year old trees of Thuja plicata Using lateral shoot-tips from 4 to 10 year old trees, they obtained adventitious shoots which grew only orthotrophically. The addition of high levels of

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12 GA. (0.01 mg/1 ) to the medium induced the development of male strobili from the vegetative apex. Fifty percent of the adventitious buds from juvenile tissues rooted when transferred to a medium with 1/2 strength MS and 1.6 mg/1 BA, while only 11% of those from the mature shoots rooted. They concluded that the successful formation of plantlets requires 3 distinct sequential treatments (1) meristematic induction, (2) bud elongation and (3) rooting and establishment. Arnold and Eriksson (1978) collected vegetative buds of Picea abies from 5 year old trees, 75 year old hedged trees (2 m tall) and from trees in 4 age classes: 5-10, 10-15, 15-20, and 20-50 year old trees. Mo differences were found in the organogenic ability of these explants with an average of 30% of cultures producing buds. They made no attempt to test the rooting ability of these buds. Aitken et al (1981) compared explant sources for regeneration of Pinus radiata buds. Excised whole embryos, and intact and excised cotyledons from 1 week old seedlings were cultured, resulting in an average of 9 rootable shoots from the embryos, 18 from intact cotyledons and 180 from the excised cotyledons from 1 week old seedlings. Induction of nodular, smooth-surfaced meristematic callus appeared necessary for large-scale propagation. After 24 weeks they had obtained over 1300 shoots from the excised cotyledons from 1 seed thus greatly increasing the efficiency of this technique. Bonga (1981) published a second paper on organogenesis from 15-20 year old mature trees including Abies balsamea Morphogenesis occurred rarely from female cones of Pinus mugo and Picea abies Vegetative shoots from A. balsamae P_. glauca and Pseudotsuga menziesii produced buds at the base of young expanding needles. His methods included

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13 collecting buds before bud break, excising underwater and soaking for 15 minutes in 1 g/1 malonic acid solution or for 24 hours under water. Mott and Ainerson (1981) updated their work with Pinus taeda summarizing the need for sequential transfer of explants. They stressed the point that 1 medium and combination of growth regulators initiated a response but further development was inhibited until they were transferred to a different medium. They stressed the need for judicious observation of the timing and duration of growth regulator exposure and termed the short term exposure to certain growth regulator combinations as "pulsing." Patel and Berlyn (1982) questioned the genetic stability of multiple buds regenerated from embryo explants of Pinus coulteri even when no cytokinins were used in the medium. Cells from callus and regenerated buds showed a progressive increase in DNA levels over time. After 6 weeks in culture, 30% of cells in regenerated buds had DNA levels between 4C and 12C as determined with a microspectrophotometer. They did not determine whether this resulted in mutated whole plants. David et al (1982) reported adventitious budding on Pinus pinaster cotyledons and from short-shoots and elongating needles collected from a 10 year old tree which had been sprayed weekly with BA. Meri stems of cultured short-shoots produced buds from 2 locations, from the apical meristem of the short-shoot and from mitotically active cells at the base of the expanding needles. When they altered the NH 4 /K ratio of the medium to 1, they obtained increased adventitious bud formation. They emphasized the importance of having mitotically active and morphogenically nondetermined cells.

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14 Jansson and Bornman (1983) reported improved morphogenesis of spring collected "embryonic shoots" (bud with budscales removed) of Picea abies by inclusion of the "crown," a layer of 4 to 7 extremely thickened collenchyma cells located at the base of the shoot. This anatomical feature is common to Abies Cedrus, Larix Pseudotsuga Picea Sequoia Taxus Torreya and Tsuga Following excision and culture of the embryonic shoots with attached crowns, an average of 10 buds per explant was produced while crown-minus explants produced only 3 buds per explant. They reiterated comments by Chalupa and Durzan (1973) that the crown may act as a filter or translocation barrier to nutrients and hormonal stimuli. They also suggest that the crown may be a barrier to basipetal transport. In summary, organogenesis from juvenile tissues, especially cotyledons and hypocotyls, is common among many conifers. Mitotically active cells of expanding needles also appear morphogenically flexible. However, the number of buds which can be produced by this method is limited (few to dozens) or rarely, hundreds from a single explant. By subculture of meristematic masses derived from these organs, up to 1300 plantlets have been obtained from a single seed of Pinus radiata (Aitken et al., 1981). The ability to obtain plantlets usually requires several different media and growth regulator regimes with careful attention paid to the duration of each exposure. Shoot initiation, shoot elongation, root initiation and root elongation require different conditions to maximize the propagation rates. For many species, rooting has occurred at low frequencies but evidence suggests that more careful attention to duration of auxin exposure and light levels may increase rooting.

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15 2.4.3 Stimulation of Axillary Meri stems The stimulation of axillary meri stems has been most successful with ornamental and fruit tree species. Styer and Chin (1983) list over 145 genera which have been cultured from excised meri stems and shoot-tips. 3riggs and McCulloch (1984) list 63 genera and 106 species and cultivars which are being propagated commercially or in research laboratories in the United States and Western Canada. Haines and de Fossard (1977) reported stimulation of axillary buds from orthotrophic stem sections of Araucaria cunninghamia Shoots which developed from bud traces of ortotrophic branches grew upright while shoots derived from plagiotrophic branches grew only laterally. Vieitez and Vieitez (1980a) propagated Castanea sativa by culture from lateral buds from 3 to 4 month old seedlings and from axillary buds of cultured embryos. Keys and Cech (1981,1982) propagated Castanea dentata by stimulating axillary meri stems from mature embryos and from 4 month old seedlings grown in a greenhouse. Chevre et al (1983) micropropagated C_. sativa and obtained increased axillary multiplication and elongation by doubling the calcium and magnesium concentrations in MS medium and by lowering the pH to 4.0. Rancillac et al (1982) developed methods to induce axillary shoot development from 1 month old seedlings of Pinus pinaster Rooting of these shoots was most successful when shoots were cultured for 17 days on media with 2 mg/1 NAA followed by transfer to an auxin-free media. Wang and Hu (1983) cultured shoot-tips of 5 year old Sassafras randaiense and obtained "multiple-bud-mass" proliferation on MS plus 60 mg/1 kinetin within 2 months of incubation. The rooting percentage

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16 remained below 20%, but a 95% survival rate was achieved following transfer to vermiculite in flats. While explants from mature trees are generally difficult to root, Hearne (1982) reported the advantages of using mature tissues. He suggested that for many species where mature tissues could be air layered, these mature tissues are desirable since they retain their mature features and often root easily in culture. He cited the advantages of earlier flowering, and that plantlets commonly have reduced canopy size which reduced the need for pruning and eased harvesting. He cited precocious flowering roses within 49 days following deflasking, and fruiting grapes and papaya within 12 weeks from deflasking. His observations suggest that axillary shoot explants from mature plants retain their mature qualities yet root as if they had been "phenotypically" rejuvenated while in culture. Amos and McCown (1981) reported their attempts to micropropagate conifers by shoottip and axillary meristem culture. They obtained shoot multiplication from species of Juniperus Sequoia Taxus Thuja, and Tsuga when cultured with very low levels of BA. Picea and Pinus did not show rapid shoot development. They concluded that as a group, conifers are more sensitive to intermediate levels of cytokinins than woody dicot species. They were able to root harvested shoots, but did not comment on the further growth of these cultures (i.e. whether orthotrophic or plagiotropic shoots developed). Micropropagation of rhododendrons has been a great commercial success. Anderson (1975) first reported micropropagation of Rhododendron using shoot-tip cultures on a modified, approximately 1/4 strength MS with a much reduced K content, obtaining an average of 6.2

PAGE 26

17 new shoots per culture within 8 weeks. He noted that BA proved phytotoxic while 2-i-P was not, and that the addition of 80 mg/1 adenine sulfate was necessary for superior shoot development. Anderson (1978) also reported successful direct rooting of micropropagated rhododendrons from Stage II culture thus saving a very labor intensive step. Several rooting media were successful as long as they were well drained and porous, for example 1:1 (v:v) perlite:peat. Strode (1979) reported on the commercial micropropagation of rhododendrons using a revised Anderson (1975) medium amended with 5 mg/1 2-i-P and 1 mg/1 IAA. After 4 subcultures at 6 week intervals, he obtained an average of 22.9 plants which could be attained when 4 shoot-tips were cultured in a single container. Harvested shoots rooted 90% within 5 weeks. Meyer (1982) reported the successful use of tissues from flower buds and inflorescences for rhododendron cultivars. Flower buds were chosen as they had been used successfully with several herbaceous species and resulted in lesser initial contamination. Meyer excised buds from dormant plants between October and April and obtained "granular masses" of tissues on Anderson's medium with 1-4 mg/1 IAA and 5-15 mg/1 2-i-P. McCown and LLoyd (1983) surveyed the response of 7 genotypes of rhododendron to micropropagation. They used Woody Plant Medium (WPMa modified Gresshoff and Doy medium), which was originally developed for micropropagation of Kalmia and other woody species (McCown and Lloyd, 1981). They observed that 2-i-P stimulated the greatest shoot multiplication, while BA was phytotoxic to all but elipidote and lepidote rhododendrons. Optimal cytokinin levels varied little with the

PAGE 27

18 genotype, with 1-3.5 mg/1 2-i-P generally optimal. They observed that adventitious shoot development occurred occasionally when 2-i-P greater than 2.75 mg/1 was used. Average multiplication rates were 7 to 21 depending on cultivar. It was estimated that with an average of 40 shoots per 6 week subculture period, over 75,000 shoots could be generated per square meter of culture space per year. Plantlets from callus cultures were also obtained; however, many aberrant types developed. With axillary shoot stimulation, plants remained true-to-type even after 6 years of culture, except for occasional albino/green chimeras which were readily observed and easily rouged. It was noted that the resulting rooted plantlets grew vigorously with a high degree of basal branching, similar to the growth habit of seedling rhododendrons. They stated their uncertainty as to whether this branching was induced by growth regulators or whether the seedling morphology was being mimicked. 2.4.4 Taxus Embryo Culture Taxus species have been cultured in vitro by several researchers. Tulecke (1959) cites LaRue's work with cultured pollen from Taxus brevifolia whereby he obtained an unusual "pincushion callus" with cells resembling pollen tubes sticking out at right angles to the surface of the callus. LaRue maintained long term cultures of this slow growing callus but never obtained regeneration of shoots. Zenkteler and Guzowska (1970) cultured mature female gametophytes of Taxus baccata on White's medium and obtained proliferation of callus from 2-5% of the explants. Variation in chromosome number from n to 3n was found in callus from the 14th and 15th 8-10 week subcultures. Regeneration of shoots or roots was not observed. Cultured embryos did

PAGE 28

19 not develop even when placed next to growing gametophytic tissues. LePage-Degivry (1968,), LePage-Degivry (1970), 1973abc) and LePage-Degivry and Garello (1973) initiated a series of experiments in which she cultured excised embryos from Taxus baccata She (LePage, 1968) found that by culturing mature excised embryos in liquid Heller's medium with sucrose she was able to leach out inhibitors to germination. Later, it was determined that the culture of excised embryos for 15-20 days in liquid media was necessary to remove inhibitors (LePage-Degivry, 1970). The inhibitor was identified as ABA by paper chromatography. After subculturing to solid medium with 1 mg/1 GA-, or after a chilling period, the embryos readily germinated. When embryos which had begun germination were treated with extracts of the conditioned liquid medium, growth ceased (LePage-Degivry, 1970). However, an additional GA 3 treatment resulted in resumption of germination. Paper chromatography of the extract revealed that ABA was leached from the explant during liquid culture. It was later determined that ABA was not leached out by distilled or mineral water alone, and that sucrose and Ca or K ions in the medium were required to remove inhibition (LePage-Degivry, 1973b). LePage-Degivry (1973c) reported renewal of inhibition of leached and germinating embryos with the inclusion of ABA in the solid medium. No adventitious organogenesis was reported. 2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets Several micropropagated woody species have been inoculated with mycorrhizal fungi in vitro and following rooting and establishment in soil. Inoculation of micropropagated plantlets with mycorrhizae could potentially increase survival, growth, and tolerance to environmental

PAGE 29

20 stresses. Morandi et al (1979) observed that the colonization of micropropagated raspberries ( Rubus idaeus ) with vesicular arbuscular mycorrhizae (VAM) resulted in more vigorous and uniform sized plants, with a 53-130% increase in shoot dry weight depending on the specific endophyte and cultivar used. Granger et al (1983) noted that colonization of micropropagated apple plantlets (Mailing clones) with VAM resulted in increased plant heights, total leaf areas, leaf dry weights and leaf content of Cu and P. This increase in growth occurred with the cultivar Mailing 7; however, colonization did not increase growth of Mailing Merton 111. Pons et al (1983) noted colonization of micropropagated sweet cherry plantlets ( Prunus avium L. ) with VAM in vitro but did not obtain any observed growth enhancement. In preliminary experiments where cherry plantlets were inoculated at time of transplanting into soil media, a "marked improvement in growth" occurred. Inoculation of rhododendron with ericoid mycorrhizae has been successful in situ (Duddridge and Read, 1982) and in vitro (Pearson and Read, 1973; Moore-Parkhurst and Englander, 1981). Ericoid plants appear to be require the specific endophyte, Pezizella ericae (Read, 1982). Colonization of rhododendron with Pezizella resulted in enhanced nitrogen and phosphorus supply to the plant as well as increased resistance to heavy metal toxicity (Duddridge and Read, 1982; Read, 1983). Moore-Parkhurst and Englander (1981) developed a technique for in vitro colonization of rhododendron and Pezizella ; however, this technique is impractical for commercial use. The most efficient time to inoculate plantlets would be at the time of transfer to soil.

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3.0 MATERIALS AND METHODS 3.1 Rhododendron chapinanii Gray 3.1.1 Establishment of Stock Plants Stock plants were established from seeds collected in late October, 1982, from cultivated plants located around the Doyle Conner Building, Division of Plant Industry, Gainesville, Florida. They were obtained as soon as the brown capsules began to dehisce, disinfested with 15% Clorox solution (0.79% by weight sodium hypochlorite) for 5 minutes, and rinsed with 3 changes of sterile deionized water. Seeds were spread out on sterile moistened filter papers in petri plates and incubated in light (16 hrs light/3 hrs dark at 25 C +/5 C with irradrance of 50-90 2 1 uMm s ). Germination occurred within 2 weeks and when seedlings reached a height of 5 cm, they were transferred individually to 4-liter -2 -1 pots maintained in a growth chamber illuminated with 150 ^iMm s cool white fluorescent lighting for 16 hours daily. Temperatures were maintained at 25 C +/1 C (day) and 20 C +/1 C (night). Plants were watered from below to avoid wetting the foliage. Succulent shoot-tips, approximately 3-5 cm in length (5-10 nodes), were pruned by hand and then washed in soapy water, then rinsed in cool running tap water for 1 hour. Pruned stock plants subsequently developed numerous additional axillary shoots, which developed into succulent shoot-tips of sufficient size to culture in 4-6 weeks. 21

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22 3.1.2 Pi si nfestati on An experiment was designed to compare contamination rates of shoot-tip explants from 2 sources. Explants from plants grown both outside under 40% polypropylene (Saran) shade cloth and in a growth chamber were used. Culture room conditions remained the same for all experiments. Cultures were illuminated with cool white fluorescent lights at 90 7 1 uMm" s for 16 hours daily, and a temperature of 25 C + /5 C. Unless otherwise indicated, 20 ml of medium was aliquoted into 25x150 mm Pyrex test tubes and capped with Kaputs. Media were autoclaved at 1.05 kg/cm, at 110 C for 20 minutes and then allowed to cool on a slant. Shoot-tips from both 60% shade (grown out of doors) and growth chamber grown stoc'< plants were disinfested for 15 minutes in 15$ or 20% Clorox then rinsed in sterile distilled water 3 times. Shoot-tips were further trimmed to remove the bleached petiole stubs, stem base and apex resulting in a 5 node stem section. Explants were inserted vertically into agar to approximately half their total length. Culture medium consisted of basal Woody Plant Medium (WPM McCown and Lloyd; 1981) plus 2% sucrose, 0.8% Bacto aar and pH adjusted to 5.2 prior to autoclaving. Ten replications per treatment were used. Data on contamination were collected after 2 weeks under culture conditions described previously. 3.1.3 Media Selection An experiment was inititated to determine the best medium for micropropagation of Rhododendron chapmanii Four nedia used for woody plants were selected : Woody Plant Medium (WPM-McCown and Lloyd, 1981), 1/2 strength macronutrients of WPM (1/2WPM), Linsmaier and Skoog medium

PAGE 32

23 (LS-Linsmaier and Skoog, 1964) and 1/2 strength macronutrients of LS (1/2LS). Culture vessels were 25x150 mm Pyrex test tubes containing 20 mis of medium. Each medium was amended with 1, 5, 10, 15, or 20 mg/1 2-i-P, and (in mg/1) myo-inositol (100), NaH 2 P0 4 (100), adenine sulfate (80), sucrose (30,000) and Bacto-agar (8,000). The pH of the medium was adjusted to 5.2 before autoclaving. Ten replications per treatment were used with individual 5 node shoot-tips used as experimental units. Data were collected after 6 weeks. 3.1.4 Selection of Growth Regulators Levels Two experiments were performed to determine effects of growth regulators on rapid multiplication. In the first experiment, the cytokinin, 2-i-P, was examined at 0, 5, 10, and 15 mg/1 with ten replications per treatment. Individual 5 node shoot-tips were used as experimental units. In the second experiment, the interaction of cytokinins with auxins was investigated in a 3X3 factorial with 2-i-P at 5, 10, and 15 mg/1 and IAA at 0, 1, and 2 mg/1. Each treatment was replicated 10 times. Data were collected after 60 days in culture. 3.1.5 Stage II Subcultures Following initial culture on multiplication medium (SII), explants were subcultured through 4 additional SII cycles on WPM with (in Mg/1) 2-i-P (10) and adenine sulfate (80). The initial SII culture utilized 25x150 mm test tubes, after which explants were transferred and cultured in small baby food jars (approximately 125 ml) containing 40 ml of medium and covered with aluminum foil and wrapped with Parafilm. Thirty to 50 mm tall microcuttings consisting of a single primary shoot with developing lateral shoots were subcultured. Uniform sized explants,

PAGE 33

24 consisting of a single primary shoot with developing lateral shoots, were subcultured. These explants were placed 3 to a baby food jar with modified WPM medium. The explants were oriented horizontally and pressed down slightly so that most nodes were in direct contact with agar. Ten or more replicates were made. Cultures were transferred onto fresh multiplication medium at 6 to 8 week intervals. Multiplication rates were evaluated over 5 subcultures on WPM. Data were collected at each subculture time e^ery 6-8 weeks. 3.1.6 Rooting and Establ ishment Stage II microcuttings were given one of the following treatments: (1) control, (2) 0.1% IBA in talc or (3) O.H IBA (in 10% ethonol ) for 5 seconds. They then were stuck directly into 54x28x6.8 cm plastic flats containing 5 cm deep of autoclaved rooting medium, 1:1:1 (v:v:v) Canadian peat : vermicul ite: perl ite. Ninety-six explants were used for each treatment. Microcuttings were protected from drying by covering the flat with an inverted translucent plastic flat. They were misted daily by hand to increase humidity and were watered as needed with deionized water. Flats were illuminated under cool white fluorescent lights at ? 1 approximately 90 uMm s~ with a 16 hour photoperiod at 25 C +/2 C. 3.1.7 Colonization with Ericoid Mycorrhizae An experiment was established to determine the growth response of rooted plantlets to inoculation with mycorrhizae and establishment in two different container media. Well -rooted plantlets were inoculated with the ericoid mycorrhizal fungus, Pezizella encae Read, and transplanted individually into 6 cm 2 plastic "cell-pack" (4 cells/pack). Two soilless media were used: (1) Metro-Mix 500 (MM500) and (2) 2:1 (v:v) fired

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25 inontmoril Unite clay: Canadian peat (FMC:CP). Fungal inoculum of P. ericae was prepared according to Marx and Kenny (1981). Pure cultures of P_. ericae were obtained from the American Type Tissue Collection (culture no. 32985). The cultures were increased in 250 ml flasks containing 100 ml of modified Melin-Norkrans fungal nedium with a 1 centimeter layer of broken glass in the bottom of the container. Cultures were maintained at 25 C in the dark for 4 weeks, but were shaken weekly to fragment the hyphae. A mixture of 28:1 (v:v) vermicul ite:f inely screened Canadian peat was moistened with 750 ml of Melin-Norkrans medium and autoclaved. After cooling, 100 ml of prepared P_. ericae inoculum was added and this mixture was cultured for 3 weeks in the dark at 25 C. This inoculum was then leached by wrapping in several layers of cheese cloth and rinsing for 3 to 4 minutes under cool, running tap water to remove excess glucose and nutrients which might support growth of intrusive saprophytes. The vermicul ite:peat inoculum was then air dried at 25 C for 3 days. Approximately 1 gm of inoculum was placed immediately below the transplanted plantlet's root zone. Non-inoculated plantlets had 1 g.n of autoclaved vermiculite added below the roots as a control. Colonization and growth responses were evaluated after 15 weeks, using the technique described by Englander (1982). Roots were stained with Lactophenol -Trypan blue stain for 10 minutes at 90 C. Percent colonization was estimated by use of the modified gridline intersection method of Giovanettii and Mosse (1980). Each treatment was replicated 12 times, with 4 plantlets per replication.

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26 3.2 Taxus floridana Nutt 3.2.1 Conventional Propagation by Cuttings An experiment was initiated to determine the optimal auxin treatment for root formation on cuttings. Dormant, evergreen cuttings of Taxus floridana were collected in early February, 1982, from native populations in Torreya State Park and from plantings on the University of Florida campus. Ten to 15 cm long terminal and subterminal cuttings were collected from upright and lateral branches. The bases were trimmed and quick dipped for 10 seconds in 2000, 4000, or 8000 mg/1 I3A (in 20% EtOH). Each treatment was replicated 10 times with individual cuttings as experimental units. Cuttings were stuck 5 cm deep in plastic flats containing 10 cm of a mixture of 1:1 (v:v) course sand:Canadian peat. They were misted intermittently (5 sec ewery 2 min) during daylight hours. Bottom heat (28 C) was supplied from electric cables placed 5 cm below the flats. Rooting was evaluated 120 days after sticking. Well rooted cuttings were transplanted into 1:1:1 (v:v:v) Canadian peat:perlite:sandy loam medium in 4-liter containers. After establishment, stock plants were placed in a growth chamber with the same cultural conditions mentioned for R. chapman ii (3.1.2). 3.2.2 Culture of Quiescent Shoot-tips Four different plant organs were investigated--(l ) quiescent shoot-tips (3.2.2), (2) developing vegetative buds just before bud break (3.2.3), (3) swelling microsporangium (3.2.4)and (4) excised embryos (3.2.5). Quiescent shoot-tips were collected in October, 1982, from the most recently matured branches of plants located on University of Florida campus. Three to 4 cm long shoot-tips were washed in soapy

PAGE 36

27 water, rinsed in running tap water for 1 hour and then disinfested with a 15-20% Clorox solution (with 5 drops Tween-20 per liter) for 15-20 minutes. Shoot-tips were rinsed in 3 separate changes of sterile deionized water. The bleached bases were trimmed and explants were inserted vertically to half their length into 20 ml of solidified WPM medium in 25x150 Pyrex test tubes. An experiment was initiated to determine the best medium for culture of Taxus floridana The 4 media selected were the same used for Rhododendron chapmanii (Sect. 3.1.3). Data were collected after 6 weeks in culture. Twenty explants per medium were cultured with individual shoot-tips as experimental units. Basal media were amended with (in mg/1) myo-inositol (100), NaH 2 P0 4 (100), sucrose (30,000), Bacto-agar (3,000). The pH was adjusted before autoclaving to 5.2 with the addition of 1.0 or 0.1 N solution of NaOH or 1.0 or 0.1 N HC1 prior to autoclaving. Data were collected on length of new growth, number of developing shoots and explant color. 3.2.3 Culture of Expanding Vegetative Buds Swelling vegetative buds were collected weekly for 3 successive weeks preceding bud break in late February and into the second week of March, 1983. Buds were washed and disinfested as described previously (Sect. 3.2.3) and outer bud-scales were carefully excised under water as described by Bonga (1977,1981) and Jansson and Bornman (1983). Four preculture treatments were used. Trimmed buds were allowed to soak for 24 hours in sterile distilled water or 3 hours in either 22.5, 28.0, or 34.0 mg/1 BA solutions before culturing on WPM amended with myo-inositol (100), NaH 2 P0 4 (100), sucrose (20,000), agar (8,000) and 5.2 pH. Ten

PAGE 37

28 replications per treatment were used. Data were collected on overall growth and specific morphogenic changes. 3.2.4 Microsporagium Culture Expanding microsporangia were collected and disinfested as described in Section 3.2.2. Microsporangia were trimmed of outer scales and soaked in the 4 previously described solutions (3.2.3). Explants were established on basal WPM with agar (8,000). Each treatment involved 10 replications with individual microsporangium and data were collected after 6 weeks in culture. 3.2.5 Embryo Culture Attempts were made to promote germination of excised embryos by leaching of inhibitors to seed germination. Seeds were surface disinfested using 20% Clorox for 20 minutes. The 1-2 mm long embryos were excised and further disinfested in 5% Clorox for 10 minutes. Embryos were rinsed 3 times with sterile deionized water, then placed into 20 ml of liquid Heller's medium with 2% sucrose. Medium pH was adjusted to 5.5 before autoclaving. The small embryos were most easily handled by catching them in a drop of liquid held in a 5 mm wide bacterial transfer loop. Cultures were maintained in the dark without shaking (LePage, 1968). Due to limited seed availability, only 5 excised embryos were used per treatment with individual embryos as experimental units. Embryos were subjected to 3 different treatments. Following an initial 2 week culture period, they were transferred to either (1) Heller's medium supplemented with 1 mg/1 GA 3 and solidified with 0.8% agar or (2) Heller's solidified nedium and stored for 1 month in a

PAGE 38

29 refrigerator at 4 C before returning to culture room conditions. Treatment (3) involved a second leaching cycle in liquid Heller's medium for 2 weeks before placement on solid Heller's medium with 1 mg/1 GA^. GA~ was filtered sterilized and added to the cooling but still liquid medium. Data were collected from the cultures grown for 40 days in the dark at 25 C +/2 C. 3.3 Torreya taxi folia Arn. 3.3.1 Conventional Propagation by Cuttings Evergreen cuttings were collected in early February, 1982, from trees growing in Maclay Gardens, Tallahassee, and from plantings on the University of Florida campus. Ten to 15 cm long terminal and subterminal cuttings were collected from lateral branches. The cuttings were wounded twice along opposite sides by scraping the basal 2.5 cm of the cuttings to expose the lighter green cambium. Cuttings were treated in the same manner as described for Taxus (Sect. 3.2.1) involving treatment with 3 concentrations of IBA (2,000, 4,000, and 8,000 mg/1). Individual cuttings were used as experimental units, with 5 replications per treatment. Cuttings were evaluated 120 days following sticking and well -rooted cuttings were transplanted into 1:1:1 (v:v:v) Canadian peat :perl ite: sandy loam medium in 4-liter containers. Data collection included percent rooting, number and total length of roots. 3.3.2 Culture of Quiescent Shoot-tips Five explant sources were investigated to determine the morphogenic capacity of various organs as reported in the literature— (1) quiescent shoot-tips (3.3.2), (2) expanding vegetative buds (3.3.3), (3)

PAGE 39

30 expanding megaand microsporangia (3.3.4), (4) excised embryos (3.3.5), and (5) seedling stock plants sprayed with cytokinins before culture (3.3.6). In the first experiment (3.3.2), quiescent shoot-tips were collected in October, 1982, from plantings located on the University of Florida campus. Three to 4 cm shoot-tips were washed and disinfested following the procedures previously described for quiescent shoot-tips of Taxus floridana (Sect. 3.2.2). They were cultured in each of four media (WPM, 1/2WPM, IS, and 1/2LS). 3.3.3 Culture of Expanding Vegetative Buds Expanding vegetative buds were collected weekly over a 3 week period preceeding budbreak in early spring (midto late March, 1984). Buds were trimmed of bud-scales and were handled following the procedures of Boulay (1977,1981) and Jansson and Bornman (1983) as described for Taxus in Sect. 3.2.4. Ten buds were used per treatment with individual buds as experimental units. Thus, 3 collection times were combined with 4 preculture soaks. 3.3.4 Culture of Microand Megasporangia Expanding microand megasporangia were collected as they swelled following budbreak in the middle of March, 1984. These were washed and disinfested as with Taxus floridana microsporangia (3.2.4). Ten explants of each were treated with individual sporangia as experimental units. 3.3.5 Embryo Culture An experiment was initiated to determine if germination inhibitors could be leached from excised embryos. They were excised following

PAGE 40

31 the techniques of LePage (1968), LePage-Degivry (1970, 1973ab) and LePage-Degivry and Garello (1973) for Taxus baccata Seeds, which had been warm stratified for 2 months followed by 2 months of cold stratification were surface sterilized in 20% Clorox followed by 3 sterile water rinses. Embryos, 2-5 mm long, were aseptically excised and further disinfested in 5% Clorox for 10 minutes, followed by 3 rinses in sterile deionizsd water. The small embryos could be most easily handled with a bacterial transfer loop. Excised embryos were cultured in 20 ml liquid Heller's medium as described for Taxus (Sect. 3.2.6). Excised embryos were exposed to the 3 treatment regimes described for Taxus floridana (3.2.5). Individual embryos were used as experimental units with 5 embryos per treatment. 3.3.6 Effect of Cytokinin Sprays on Stock Plants Torreya seedling stock plants were sprayed weekly for 4 weeks with 2 concentrations of 2 cytokinins (BA and 2-i-P at 100 mg/1 and 200 mg/1 plus 5 drops Tween-20 per liter). These treatments were suggested by Dr. M. A. El-Nil (1983, per. com.). Plants were sprayed until run off. Only 5 one-year old seedlings were available for experimentation, limiting the treatments to only 1 plant per treatment. A single control plant was sprayed weekly with the same 10% EtOH carrier solution as the cytokinins. All developing basal and axillary shoots were removed and placed in culture. Explants consisted of shoottips and stem sections 1-2 cm in length. They were cultured on solidified WPM (0.8% agar) in 25x150 ml Pyrex test tubes with 4 concentrations of growth regulators in the medium: (1) WPM with 1 mg/1 BA and 0.01 mg/1 NAA, (2) WPM with 10 mg/1 BA, (3) WPM with 1 mg/1 2-i-P, and (4) WPM with 10 mg/1 2-i-P.

PAGE 41

32 -2 -1 Expl ants were cultured at 25 C (+/5 C) and illuminated 50 uMm s for 16 hours a day.

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4.0 RESULTS 4.1 Rhododendron chapmanii 4.1.1 Disinfestation There was ar) average of 55* reduction in amount of contamination (30-40%) when explants were collected from stock plants grown in a growth chamber as compared to field grown explants (Table 4.1.1). There was no difference in contamination % between the two levels of Clorox used. Contamination of explants from shade house grown plants averaged 90% while those from growth chamber plants contaminated only 35%. Field grown material remained \iery difficult to disinfest. In a later experiment (Section 4.1.2), leaves were removed from growth chamber grown shoot-tips prior to Clorox treatment. Contamination in this case was reduced to only 4% (data not given), when leaves were removed from explants prior to treatment with 15% clorox for 15 minutes. For all subsequent experiments in this section, leaf removal prior to Clorox treatment virtually eliminated contamination. Subcultures from visually contamination-free cultures remained clear and presumably without contamination. 4.1.2 Selection of Growth Regulator Levels There was a difference between basal medium without cytokinins vs all levels of 2-i-P tested, but no differences between the 2-i-P levels (Table 4.1.2). Tnere was a significant reduction in the number of leaves formed when with 2-i-P levels greater than 10 :ng/l were used. 33

PAGE 43

34 Table 4.1.1 Percent contamination of in vitro cultured Rhododendron chapmanii shoot-tips grown in shade house and growth chambers Treatment Time (mi n CI orox Contamination % Chamber Field 15 15 15 20 30 40 100 80

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36 There were no differences between the total number of leaves and shoots produced for the 2-i-P and IAA combinations (Table 4.1.3). Differences between IAA means were significant at the 0.10 level, however, inclusion of IAA in the medium generally resulted in declining numbers of shoots and leaves. Optimal shoot and leaf production occurred between 5-10 mg/1 2-i-P. With higher 2-i-P levels, axillary shoots remained short and elongated much more slowly than plantlets cultured on 10 mg/1 2-i-P. 4.1.3 Stage II Subcultures Following initial SII culture in test tubes, the medium in all additional multiplication cycles contained adenine sulfate (80 mg/1) and explants were established in small baby food jars. Basal nodes and especially those embedded in the agar produced abundant axillary shoots. When explants were cultured upright in individual test tubes on WPM witn 10 mg/1 2-i-P and no adenine sulfate (Fig. 4.1) an average 5.8 shoots developed per explant in 6-8 weeks. For additional SII subcultures, increase in shoots was especially high when the explants were placed horizonally and pushed slightly into the medium (Fig. 4.1.5). This also resulted in more uniform elongation of shoots. Each additional multiplication cycle for the next three SII subcultures in baby food jars averaged a 7.6 fold increase in number of shoots, however, this multiplication rate decreased to 5.0 during the fourth subculture (Fig. 4.1). 4.1.4 Rooting and establishment Preliminary rooting attempts using an agar substrate were unsuccessful. In a few cases where cultures were left undisturbed for 4

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35 Table 4.1.2 Growth response of In vitro cultured Rhododendron chapmanii shoot-tips cultured on Woody Plant Medium with 5 levels of the cytokinin, 2-i-P 2-i-P concentration No. expanding Mean no. (mg/1) axillary buds leaves 1.4a Y 8.4b Z 5 3.0b 13.7a 10 3.6b 16.6a 15 2.2b 7.3b 20 2.2b 7.6b y Means in a colomn followed by the same letter are not significantly different at the 5% level by Duncan's Multiple Range Test. Means in a column followed by the same letter are not significantly different at the 10% level by Duncan's Multiple Range Test.

PAGE 46

37 Table 4.1.3 Growth response of j_n_ vitro cultured Rhododendron c h a p m a n i i grown on a 3x3 factorial combination of auxin ( I A A"] and cytokinin (2-1 P ) 2 i P mg/1) IAA mg/1 5 10 15 Tot a 1 No of Lea ves 38.9a Z 35.2a 37.2a 38.4a 38.2a 34.6a 30.6a 39.4a 30.0a Ave. No. Shoots Greater than 5 mm 5

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38 Fold increase in no. of subculturable shoot-clumps y

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39 to 6 months, roots appeared spontaneously above the agar surface. Also, the extremely fine "hair roots" characteristic of rhododendrons did not develop properly in poorly aerated substrates. Direct sticking of microcuttings into 1:1:1 (v:v:v) Canadian peat:vermicul ite:perl ite resulted in well-rooted plantlets within 4 to 6 weeks. Treatment of microcuttings before sticking with 1000 mg/1 I8A (dissolved in 10% EtOH) resulted in improved survival and rooting (Table 4.1.4). A quick dip treatment (5 seconds) with 1000 mg/1 IBA resulted in greatest survival and approximately 60% rooting (Table 4.1.4). Treatment with 1000 mg/1 of IBA in talc resulted in poorer rooting and survival than controls. Survival improved slightly with more careful observation and daily misting with deionized water. Best survival to date has been 75-80% (data not given). Table 4.1.5 summarizes the micropropagation staging system developed for R_. chapmanii With this rate of multiplication and rooting, it can be conservatively estimated that over 10,000 rooted plantlets could be produced from a single shoot-tip in one year (1 x 5.8 x 7.6 x 7.6 x 7.6 = 19350 x 60% rooting = 11610 plantlets in 9.5 months +2 1/2 months additional growth). 4.1.5 Colonization with Ericoid Mycorrhizae Inoculation of rooted plantlets with the ericoid mycorrhizae, Pezizella ericae resulted in colonization in two potting media, MM500 and FMC:CP. There were considerable differences (?F > 0.0001) between media for height of main stem, total stem lengths, number of leaves, and main stem caliper after 12 weeks of growth in the greenhouse (Table 4.1.5). Differences in number of stems was significant at the .05 level.

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40 Table 4.1.4 Survival and rooting response of microcuttings of Rhododendron chapmanii following treatment with auxins from 2 sources Treatment Survival and Rooting % none 26. Z 1000 mg/1 IBA 19.8 (in talc) 1000 mg/1 IBA 59.4 (1 iquid-dip) Percent survival and rooting were significantly different at the 1% level using analysis of catagorical data.

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41 Table 4.1.5 Summary of micropropagation system developed for Rhododendron chapmanii STAGE EXPLANT DURATION RATE MEDIA X GR0WTH Y CULTURE 2 (weeks) REG. CONDITIONS SI shoot-tips 6-8 5.8x WPM 10 mg/1 20 uMm~ 2s_1 3-5 cm 2-1 -P 25 + 5 C SI I axillary 6-8 7.6x WPM 10 mg/1 20 uMm" 2s_1 shoot-clumps 2-i-P 25 + 5 C SIV axillary 4-6 80% 1:1:1 Quick90 uMm~ 2s_1 shoots Rooting dip (>15mm) Medium 25 +_ 5 C 1000 mg/1 in high IBA humidity tent X Medium WPM = Woody Plant Medium Y Growth Regulators Z 2 1 Culture conditions—light intensity in uMm s light duration 16 hrs light/ 8 hrs dark temperature in degrees C

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42 Table 4.1.6 Response of Rhododendron chapmanii plantlets produced in vitro to 2 establishment media and inoculation with or without ericoid mycorrhizae Media V Trt. W Plt. X Ht. Y No. Z Total Y No. Y Main Y % X Surv. Main Stems Stem Lvs Stem Col % Stem Length Caliper (mm) (mm) (mm) M500 +M 91.7a 67.0a 1.77a 96.1a 23.6a 0.98a 61.4a M500 -M 95.8a 60.8a 2.14a 98.5a 26.7a 1.0a FMC:CP +M 80.9a 28.3b 1.40b 40.0b 14.9b 0.35b 41.1a FMC:CP -M 65.3a 23.0b 1.18b 34.1b 12.8b 0.28b V Media M500 = MetroMix-500 FMC:CP = Fired montmori 11 ini te clay :Canadian peat W Trt. = mycorrhizal treatment +M = with inoculation -M = without inoculation Pit. surv. % = Plantlet survival % X Means in a column followed by the same letter are not significantly different at the 5% level by analysis of catagorical data. Y Means in a column followed by the same letter are not significantly different at the 1% level by Duncan's Multiple Range Test. Means in a column followed by the same letter are not significantly different at the 5% level by Duncan's Multiple Range Test.

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43 Plantlets grown in the less fertile FMC:CP potting mix were consistantly smaller regardless of colonization. 4.2 Taxus floridana 4.2.1. Conventional Propagation by Cuttings Conventional vegetative propagation resulted in rooting within 120 days under mist (Table 4.2.1). Cuttings in the "mixed clones" group were collected from smaller and younger trees 2-4 m tall. Cuttings from the younger mixed clones rooted at better (best 67%) and with greater number of roots. The best rooting percentage from the older Champion tree was 40% but with fewer roots initiated and of shorter length than from the younger clones. 4.2.2 Micropropagation of Taxus Shoot-tips The response of shoot-tips grown for 5 months on 4 different media supplemental with 0.5 mg/1 IAA is shown in Table 4.2.2. There were no difference in shoot size, average number of shoots produced or length of new growth. WPM was chosen for additional experiments primarily due to the better color quality and general overall appearance of the explants. Exp! ants on WPM or 1/2WPM were light green, the normal color of new growth on whole plants. 4.2.3 Culture of Expanding Vegetative Shoot-tips Culture of expanding vegetative shoot-tips collected 3 successive weeks before bud break resulted in either death of the explant, contamination of cultures or no growth. Shoot-tips were very small (2-3 mm), were not easily dissected and may have been damaged by disinfestation treatments or by removal of outer bud scales.

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44 Table 4.2.1 Rooting response of Tax us floridana cuttings from 2 aged groups of plants to 3 levels of the auxin, IBA IBA mg/1 Rooting Mean root no Mean root length ( cm) Total root length (cm) 2000 4000 8000

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45 Table 4.2.2 In vitro growth response of T. f 1 o r i d a n a shoot-t i p

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46 4.2.4 Culture of Microsporangia Culture of expanding microsporangia was attempted successive for 3 weeks preceding budbreak. Microsporangia were very small (2-3mm) and difficult to excise. Clean cultures could be consistently obtained, but no further development was observed. Explants treated with chemicals or water soak did not develop further in culture and no differences were noted between treatments. 4.2.5 Embryo culture Attempts to culture excised embryos were unsuccessful. None of the 3 treatments were successful in promoting germination. Embryos which showed no signs of growth after 1 month on solidified Heller's medium with GA 3 or following removal from cold treatment were recultured into a second cycle of fresh liquid medium. After 3 weeks in liquid medium, these embryos were again transferred to solidified medium with GA^, but no growth occurred. 4.3 To rrey a taxifol ia 4.3.1 Conventional Propagation by Cuttings Rooting results are summarized in Table 4.3.1. The greatest percent rooting (56%) occurred with 8000 mg/1 IBA. The number and average length of roots with this treatment were substantually less than those produced with 4000 mg/1 IBA treatment. Cuttings treated with 4000 mg/1 IBA rooted at a low percentage (20%) but produced more numerous roots with greater average and total length.

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47 Table 4.3.1 Rooting response of cuttings of Tor rey a t a x i f o 1 i a

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4.3.2 Micropropagation of Shoot-tips The response of shoot-tips collected from lateral branches of mature trees (est. 15-20 years old) in culture is summarized in Table 4.3.2. The color and apparent health of explants cultured on WPM or 1/2WPM were superior to LS or 1/2LS, being darker green. In a second experiment, shoot-tips collected in October and grown on 3 nutrient media each with 5 levels of BA was recorded after 2 months in culture (Table 4.3.3). Little or no growth occurred on either LS or 1/2LS. A few axillary shoots developed on WPM at low to medium levels of BA (0.5-1.0 mg/1 ) but declined with higher levels. 4.3.3 Culture of Expanding Vegetative Buds All cultures of expanding buds were either contaminated or were killed by disinfestation procedures. Contamination continues to be a serious problem from field grown plants but is much less of a problem when stock plants are grown inside with lower light intensities and lower night temperatures. 4.3.4 Culture of Microsporangia and Megasporangia Host of these cultures became contaminated or were killed by disinfestation procedures. Microsporangia were sufficiently large enough to be handled and were plentiful in early spring. 4.3.5 Embryo culture Embryo culture of T. taxi fol ia also proved unsuccessful. Although embryos could be easily disinfested with treatment with 10% Clorox following aseptic excision, they did not generally respond to the treatment used successfully for Taxus baccata embryos (LaPage, 1968). In 1 case among 10 attempts, the cotyledons parted and appeared to

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49 Table 4.3.2 Growth response of shoot-tips of J^ t a x i f o 1 i a on 4 nutrient media Med i urn Mean size (mm ) Mean no shoots Col or Length new growth (mm ) WPM^ 1/2WPM LS 1/2LS 44.5ab 18.7b 56.3a 42. Sab 2.0a 2. la 1 .6a 2.0a DG A

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50 Table 4.3.3 Average number of axillary shoots of Tor reya t a x i f o 1 i a developing j_n vitro on 3 nutrient media and 5 concentrations of the cytokinin, BA BA (mg/1 WPM' No axi 1 1 ary shoots LS 1/2LS 0.5 1 .0 5.0 10.0 1 .0a

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51 begin to germinate. When transferred to solid medium with 1.0 mg/1 GA^, a shoot-tip appeared but degenerated shortly thereafter. 4.3.6 Cytokinin Sprayed Seedlings Only five seedlings were available for investigation so each treatment was applied to a single seedling. Treatments were based on suggestions from Dr. M. A. El -Nil (1982, per. com.). Stock plants produced numerous basal shoots following treatment with cytokinin sprays. The control plant produced a few basal shoots which could be cultured. Excised basal shoots developed axillary bud masses after 30 days when cultured on media with cytokinins. Best results occurred when explants were collected from plants sprayed weekly with 1000 mg/1 BA and then cultured on medium with 1 mg/1 BA and 0.01 mg/1 MAA. Higher concentrations resulted in much smaller masses which did not enlarge much until transferred to media with lower or no cytokinin. Culture on media with 2-i-P resulted in little development until these were transferred to WPM medium with 1.0 mg/1 BA and 0.01 mg/1 MAA after which bud elongation occurred and occasional multiple shoots were observed. Two morphologically different types of axillary bud development were observed. In the first case, normally shaped axillary shoots elongated with additional axillary shoot development from the leaf axils at the base of the cultured buds. The second type of development involved the development of compact "bud-masses" consisting of small, tightly appressed scale-like leaves with the ultimate emergence of multiple shoots after 3 months in culture. When buds from the same treatments were subcultured to the same or lower cytokinin medium, the buds on the lower cytokinin medium always began to show signs of growth

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52 sooner. These buds were transferred to fresh medium but have since degenerated. No rooted plantlets were obtained.

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5.0 DISCUSSION 5.1 Rhododendron chapmanii Conventional propagation using cuttings was attempted but was unsuccessful due to failure of misting equipment. The experiment was not repeated because of insufficient cutting material and the development of a highly successful micropropagation system using shoot-tips. Microcuttings could be rooted and established with 60% or greater survival. Conventional propagation of evergreen Rhododendron species usually involves softwood cuttings (Hartman and Kester, 1981) or leafly hardwood cuttings collected in the fall which are treated with concentrated NaOH and IBA solution (Gray, 1974). Micropropagation of R. chapmanii was highly successful using shoot-tip explants and modifying the techniques of Anderson (1975) and the medium of McCown and Lloyd (1981). Technique improvements included decapitation of shoot apex and the horizontal placement of the explants in the medium so that most nodes were slightly embedded in it. Horizontal placement of shoots into multiplication medium greatly increased the number and uniformity of shoots produced during each multiplication cycle (SII). The addition of 80-100 mg/1 adenine sulfate improved the number and vigor of shoots produced. BA proved to be phytotoxic to R. chapmanii as has been previously reported for most Rhododendron species (Anderson, 1975; Lloyd and McCown, 1980; and McCown and Lloyd, 1983). Decapitated shoot-clump bases could be recultured as suggested by Strode (1979), however; this technique did 53

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54 not result in as rapid an increase in shoots per cycle as when elongated shoots were laid horizontally on and pressed slightly into the medium. The use of higher levels of 2-1 -P (greater than 15-20 mg/1 ) resulted in a greater number of shoots, however, these grew slower and took 2 to 3 months vs 6 to 8 weeks to reach a subculturable size. The reduced stem elongation could have been due to high cytokinin levels which inhibited shoot elongation. It is also probable that the amounts of nutrients available to the numerous shoots were limiting and that culture on a greater volume of medium or more frequent transfers might hasten growth and multiplication. Ma and Wang (1977) suggested the use of short term, agitated liquid culture to increase survivability and mul tipl i cation. Using the micropropagation system shown in Table 4.1.7, one can calculate that over 11,000 plants could be obtained from 1 shoot-tip in 9.5 months. With 2.5 months additional growth in the greenhouse, these plantlets would be large enough to be transplanted to larger containers. Alternately, a square meter of lighted shelf space could produce 15,16,000 SII microcuttings in o to 8 weeks (225 bottles/M X 50-70 microcutting/bottle) Although large numbers of shoots could be produced and routinely subcultured, a 60-80 survival rate on transfer to soil is still inefficient. All attempts to root R. chapmanii in Stage III culture have failed even though several nedia were investigated along with a range of auxin treatments. This differs from reports of 80% rooting in SI 1 1 by Ma and Wang (1977) and Strode et al (1978). Rhododendron roots are extremely fine and thread-like and do not appear to grow easily into agar media.

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55 Best rooting and survival of microcuttings of R_. chapmanii occurred when explants consisting of 3 to 5 shoots were direct-stuck in rooting medium with at least 30% Canadian peat moss. This agrees with the findings of Anderson (1978), Lloyd and McCown (1980) and Wong (1981). Direct rooting of microcuttings vs use of S 1 1 1 cultures results in considerable savings in labor since at this stage explants must be handled individually only once for direct rooting vs twice when SI 1 1 cultures are used (Strode et al 1979; Wong, 1981). Microcuttings were rooted in flats with clear plastic covers or covered with inverted translucent flats to prevent drying. If covered flats were maintained under light levels greater that 100 -2 -1 uMm sec excess heating occurred and plantlets declined and fungal contamination was greater. If microcuttings were initially maintained -2 -1 under lower light intensities (e.g. 25 jjMm s ) for the first 2 weeks -2 -1 then maintained at 90 uMm s better survival and rooting occurred. Placement of microcuttings directly under intermittent mist resulted in poor rooting and survival and the development of chl orotic leaves due to either excessive leaching or possibly due to poor root-zone aeration. The use of fog systems or high humidity tents should be further investigated. In this investigation, the use of rooting compounds on microcuttings resulted in better rooting and survival when a 1000 mg/1 solution of IBA was applied as a 5 second quick-dip. Treatment with the same concentration of IBA (in talc) resulted in lower survival and rooting. Wong (1981) mentioned that the use of hormone powder increased proliferation of roots but not time to root. He also mentioned that the economic advantages of such treatments were yet to be determined.

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56 Poor rooting and survival may be attributed to the small size and succulence of the microcuttings. Commercial producers of micropropagated rhododendrons (3riggs, per. com.) use lower cytokinin levels which result in reduced multiplication rates but these microcuttings are much larger and hardier. Determination of optimal multiplication rates should not necessarily be based on optimal shoot numbers, but must include duration of cycle times, labor and space requirements, rooting survival rates and duration, as well as post-transplanting growth rates. When shoot-clumps containing 3 to 5 shoots are used for rooting, better survival and rooting occurs, possibly due to reduced handling shock, greater carbohydrate (or minerals, etc.) reserves within each propagule, and due to the fact that if one shoot dies, another shoot is available for replacement. Once rooted and transplanted to containers, the plants from shoot-clumps form a multi-stem liner which is more desirable than a single-stem plant. Root emergence in rooting medium became visible after 3 to 4 weeks, and plants were well rooted by 6 weeks. After root intiation, the explants began to produce new leaves which were slightly larger and les^ succulent. After these new leaves were observed, the plantlets generally survived transplanting to cell-packs and readily became adapted to the greenhouse environment. Rooted plantlets were hardened off by covering with 80% shade cloth for a week, followed by a week with 40% shade, after which all coverings were removed. Colonization of micropropagated plantlets with the ericoid mycorrhizae, Pezizella ericae did not increase survival or growth responses when plantlets were grown for 12 weeks in the greenhouse.

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57 Differences between the media used apparently masked any differences due to colonization after 12 weeks of growth. Additional growth in 4-1 iter containers is presently under observation and there appears to be greater bud break and growth on colonized plantlets 2 weeks after new growth began. Final growth measurements will be made after a month of additional growth. Mycorrhizal colonization of Ericaceae species is known to increase the plants' ability to take up nitrogen and phosphorous, as well as, tolerance to heavy metal toxicity (Stribley and Read, 1974, 1975; Duddridge and Read, 1982) 5.2 Taxus floridana Using cuttings from mixed aged trees, conventional propagation in a 1:1 (v:v) Canadian peat : perl ite rooting medium was successful, resulting in 40-67% rooting in 120 days. Larger cuttings (15-20 cm vs 10 cm) rooted more quickly and vigorously. This agrees with the findings of commercial propagators (Keen, 1954). There was an obvious difference in rooting percentages between plants of different ages. Cuttings from the Champion tree rooted poorly (0-40%) while cuttings from much younger trees rooted 25-67%. According to Sargent (1947), 275 annual growth rings were counted from a 9.5 cm diameter J_. floridana trunk section suggesting that the 16 cm diameter Champion from which the "older" cuttings were collected was considerably older. Cuttings from young trees root at high rates. Lower branches of are commonly observed in the wild to naturally layer when branches remain in contact with the ground. However, lateral cuttings retain their plagiotropic growth habit even after 2 years in containers, generally producing a 15-20 cm tall ground cover. Keen (1954) noted that for most Ta*us species and cultivars, orthotropic shoots dre

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58 necessary for producing excurrent trees while lateral cuttings generally produce only spreading shrubs. Keen also indicated that even very large cuttings (up to 65 cm) can be successfully rooted, resulting in a salable plant in a shorter time. Attempts to root in 100% coarse, builder's sand under intermittent mist resulted in basal rotting however, rooting often occurred above the media especially from abaxial side of lateral branches (data not given). At first it was assumed that the 20% alcohol in the IBA quick-dip solution damaged the tissue. However, in later experiments using better drained rooting medium with quick-dip solution, no basal rotting occurred. Apparently the builders sand used initially was not as well drained as the sandblasting sand normally used for rooting. The occurrence of high moisture conditions and poor drainage most likely caused the observed basal rotting. Continued growth of cuttings occurred best in well drained media (1:1:1 v:v:v) course sand:Canadian peat:loamy soil) and under 80% vs 40% or 60% shade. Micropropagation of shoot-tips did not result in prolific stimulation of axillary buds. Using shoot-tip explants from lateral and orthotropic basal branches, only 2 to 4 axillary shoots elongated when placed in culture. These shoots appeared in specific positions along the shoot-tip suggesting that they were preformed and part of the normal branching pattern of the whole plant. McCown and Amos (1932) reported successful shoot-tip propagation of Taxus spp. but did not indicate the numbers of shoots obtained. They also noted in their findings that gymnosperms are generally more sensitive to low levels of cytokinins.

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59 The use of more juvenile tissues such as embryos or seedlings as explants probably would result in increased shoot numbers. Dr. A. M. El -Nil (per. com., 1982) recommended pretreatment of conifer seedlings with cytokinin sprays as a more effective means of inducing multiple shoots from conifers. This treatment was not investigated for Taxus floridana due to the lack of seedling material although this was a promising technique with Torreya taxifol ia Wounded mature Taxus floridana trees in the wild have been observed to produce masses of short shoots, most with spiral phyllotaxy and apparently orthotropic growth. Culture of explants from shoots arising from multiple shoots from "ourls" has been successful for Sequoia sempervirens (Ball, 1978; Boulay, 1979), and Pinus eldarica (Herrera and Phillips, 1984). Attempts should be made to obtain permission to collect seedlings and "burl" material from the wild. Micropropagation of "embryonic shoots" from swelling buds according to the techniques of Bonga (1977,1981) was unsuccessful due to high rates of contamination and the difficulty in excising the small buds (l-3mm). A few aseptic cultures were established but buds did not continue to expand and eventually darkened and died. Culture of microsporangia was also attempted since male plants were available. These were also very small (2 -3mm) and difficult to excise. Uncontaminated cultures were easily obtained but did not grow in culture. No megasporangia were available locally and this approach was not pursued since morphogenesis from immature female cones has been reported only rarely with cultures of Pinus mugo and Picea abies (Bonga, 1981).

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60 Attempts to overcome seed dormancy by excision of embryos and culture in liquid medium according to the techniques of LePage-Degivry (1968, 1970, 1973abc) were unsuccessful. This technique failed even when embryos were leached in 2 cycles of liquid culture followed by placement on media with GA-, or when cultures were chilled for 2 months at 5 C. Seed availability is very sporatic with reports of other species of Taxus alternate bearing with +/7 year cycles (Schopmeyer, 1974) so further experiments will be limited. Seeds of Taxus floridana were given 1 or 2 months warm stratification before excision as well as treatment with 1 month warm followed by 2 months cold stratification before excision. None of the embryos of Taxus floridana germinated. Presumably, they require a longer warm stratification period than 2 months since embryos did not respond to leaching treatment. LePage (1968) indicates that the seeds were collected from mature trees of Taxus baccata but did not indicate the time period before excision. Schropmeyer (1974) mentioned differences in duration of warm stratification between species and that the International Seed Testing Association recommended, in general, stratification periods for 90-210 days at 16 C, followed by 60-120 days at 2-5 C. 5.3 Torreya taxi folia Conventional propagation was successful with 20-56% rooting from mixed cuttings from mature and juvenile plants. Cuttings from different aged plants were combined since only a limited number of cuttings were available. Optimal rooting occurred when 4000-8000 mg/1 IBA was used. Rooted plagiotropic branches may be useful as seed or pollen sources so that intercrossing of the remaining, limited gene pool

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61 could be accomplished by collecting cuttings from as many plants as practical. Establishing seed nurseries of these cuttings would allow a practical means of preserving the remaining genetic diversity under controlled management where environmental stresses could be reduced and fungicides used more efficiently. Micropropagation using shoot-tips did not result in mass proliferation of shoots. Commonly 2 to 4 shoots could be stimulated to elongate in culture but these appeared to be preformed buds of the normal branching patterns. These lateral shoots commonly grew horizonally and then geotropical ly into the medium. Occasionally these explants rooted in culture, especially after 4 to 6 months on the same medium. They however did not survive transfer to soil. The use of more juvenile tissues such as seedlings and dormant buds located at the base of the trunk may increase the possibility of stimulating development of orthotropic shoots. Ball (1978) was successful in obtaining multiple shoots from "burl" shoots of Sequoia sempervirens however, there has only been limited investigation using similar explants from To rrey a due to very limited availability of this material. The formation of numerous shoots from burls, such as occurs occasionally with wounded Taxus floridana has not been observed although occasionally 3 or 4 ortotrophic shoots have been observed arising from a common point at the base of a trunk. Contamination of shoot-tip cultures continues to hamper investigation. In cooperation with Mr. N. El-Ghol, Plant Pathologist with the Florida Division of Plant Industry, 3 systemic fungi were isolated ( Phyllosticta spp., Coll etotrichum gloeosporioides and Guigmardia sp.). Establishment of stock plants in growth chambers

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62 greatly reduced the amount of contamination. Presumably, the combination of low night temperatures and reduced humidity levels, may have been less favorable for growth of the pathogens. Recently, the weekly treatment over 4 weeks of these stock plants with the fungicide, Zyban, a broad spectrum systemic and contact fungicide, has been effective in eliminating one of these pathogens, Phyllasticta Soaking of shoot-tip explants in fungicide solutions before clorox treatment was ineffective in reducing contamination. Soaking for 15, 30, 60, 120 and 240 minutes actually increased fungal contamination, possibly due to increased spread of hyphae. A 24 hour soak in Zyban reduced fungal contamination to 15%, however, this resulted in an increase in bacterial contamination. Cultures of expanding "embryonic shoots" [vernalized shoot buds with scale leaves removed, according to the techniques of Bonga (1977,1981)] did not produce multiple shoots. These "embryonic shoots" were large enough to be easily excised after the beginning of visible bud enlargement, especially 1 or 2 weeks before actual bud break. However, all bud cultures were either lost to contamination or were killed by di sinfestation procedures. Fungicide treatment of mature trees may allow future investigation into this technique. Culture of microand megasporangia was investigated. Although these explants could be more readily established in culture without high levels of contamination, none grew. Microsporangia are large and numerous on male trees, however, these are only available in early spring and only a few species have been reported to produce multiple shoots from these structures (Bonga 1977,1981).

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63 The pretreatment of seedlings with cytokinin sprays before placing shoots in culture appears to offer one of the better chances of obtaining multiple shoots. Seedling stock plants produce multiple orthotropic basal shoots when sprayed weekly for 4 weeks with cytokinins, BA or 2-i-P. BA appears to be slighlty more effective. Following excision, additional orthotropic shoots continued to develop over at least a several month period. When these shoot-tips and nodal sections are cultured on media with low cytokinins, numerous axillary buds and "bud-masses" developed. Further investigations will be necessary to optimize this technique and to develop entire plantlets.

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6.0 CONCLUSIONS The primary objective of this research was to investigate clonal propagation techniques for 3 endangered native ornamental plants. Conventional cutting propagation of Torreya and Taxus was investigated and compared to micropropagation using shoot-tip cultures and embryo culture. Clonal propagation using cuttings was successful for Taxus floridana and Torreya taxifol ia with maximum rooting of cuttings collected during winter and treated with ISA. Younger trees rooted at higher rates than larger and older trees. However, cutting material is limited by scarcity of plants and plagiotrophic growth when lateral branches are used. Rooting procedures for Rhododendron chapmanii cuttings remain undetermined due to limited experimental material. A yery successful micropropagation system with commercial potential has been developed for R. chapmanii Large numbers of plantlets were produced from a few shoottips and with minimal impact on the remaining natural populations. It has been estimated that over 11,000 rooted plantlets could be produced from a single shoot-tip in 1 year. Colonization of rooted plantlets with the ericoid mycorrhizae, Pezizella ericae did not result in superior survival and growth when grown for 12 weeks in a greenhouse. Colonization of plantlets appears to increase bud break and growth of over-wintered containerized plantlets, however, additional time will be necessary to determine if this trend continues. The present micropropagation system was designed to maintain clonal characteristics. 64

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65 Micropropagation techniques were only partially successful for Taxus floridana and Torreya taxi folia Shoot-tip cultures and cultured buds produced only limited numbers of shoots apparently from preformed axillary meri stems. Culture of "embryonic shoots" and microand megasporangia were unsuccessful in stimulating organogenesis. Embryo culture for both these species was unsuccessful presumably due to insufficient chilling. Pretreatment of stock plants before in vitro culture with growth regulators appears to be a promising technique and deserves further investigation.

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66 LIST OF REFERENCES Abbott, A.J. 1977. Propagating temperate woody species in tissue culture. Scientific Horticulture 28(4 ): 1 55-1 62. Aitken, J., K.J. Horgan and T.A. Thorpe. 1981. Influence of explant selection on the shoot-forming capacity of juvenile tissue of radiata pine. Can. J. For. Res. 11:112-117. Amos, R.R. and B.H. McCown. 1981. Micropropagation of members of the coniferae. HortScience 16(3):89(abstract) Anderson, W.C. 1975. Propagation of rhododendrons by tissue culture. Part 1. Development of a culture medium for multiplication of shoots. Proc. Intern. Plant Propagators Soc. 25:129-135. Anderson, W.C. 1978. Rooting of tissue cultured rhododendrons. Proc. Intern. Plant Propagators Soc. 28:135-139. Arnold, S. and D.T. Eriksson. 1978. Induction of adventitious buds on embryos of Norway spruce grown in vitro Physiol. Plant. 44:283-287. Ayensu, E.S. and R.A. DeFilipps. 1978. Endangered and threatened plants of the United States. Washington, D.C. Smithsonian Institution and World Wildlife Fund Inc. 403pp. Ball, E.A. 1978. Cloning in vitro of Sequoia sempervirens In: K.W. Hughes, R. Henke and M. Constatin, LEds.J, Propagation of higher plants through tissue culture, a bridge between research and application, Tech. Inform. Center, U.S.D.E. Conference 7804111, p. 259. Baker, W. 1983. Unpublished. Torreya Management Symposium. Florida Dept. Nat. Res. Tallahassee. Bonga, J.M. 1977. Organogenesis in in vitro cultures of embryonic shoots of Abies balsamea ( Bal samTi 7V. In Vitro 13(1) :41-48. Bonga, J.M. 1980. Plant propagation through tissue culture. In: F. Sala, B. Parisi, R. Cella and 0. Cuerri [Eds.], Plant Cell Cultures: Results and Perspectives. Elsevier Press, pp. 253-254. Bonga, J.M. 1981. Organogenesis in vitro of tissues from mature conifers. In Vitro 17:511-518. Boulay, M. 1979. Multiplication et clonage rapide du Sequoia sempervirem par la culture in vitro Etudes et Recherches, AFOCEZ. Domaine de 1 'Etancon, 77370 Nangis, France 12:49-55.

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67 Bn'ggs, 8. and S.M. McCullock. 1983. Progress in micropropagation of woody plants in the United States and Western Canada. Proc. Intern. Plant Propagators Soc. 33:239-248. Brown, C.L. and H.E. Sommer. 1974. An atlas of gymnosperms cultured in vitro : 1924-1974. GA. For. Res. Council, 271p. Brown, C.L. and H.E. Sommer. 1977. Bud and root differentiation in conifer cultures. Tappi 60:72-73. Chalupa, V. and D.J. Durzan. 1973. Growth and development of resting buds of conifers in vitro Can. J. For. Res. 3:196-208. Chaturvedi H.C. and G.C. Mitra. 1974. Clonal propagation of citrus from somatic callus cultures. HortScience 9(2):1 18-120. Cheng, T. 1975. Adventitious bud formation in culture of Douglas fir ( Pseudotsuga menziesii Mirb.) France. Plant Sci. Lett. 5:97-102. Cheng, T.Y. 1976. Vegetative propagation of Western hemlock ( Tsuga heterophyll a) through tissue culture. Plant and Cell Physiol. 17:1347-1350. Cheng, T.Y. 1978. Clonal propagation of woody plant species through tissue culture techniques. Proc. Intern. Plant Propagators Soc. 28:139-155. Chevre, A-Marie, S.S. Gill, A. Mouras and G. Salesses. 1983. In vitro vegetative multiplication of chestnut. J. Hort. Sci. 53(lTT23-29. Coleman, W.K. and T.A. Thorpe. 1977. In_ vitro culture of Western red-cedar ( Thuja plicata Donn.) I. Plantlet formation. Bot. Gaz. 138:298^334". David, A., H. David and T. Mateille. 1982. In vitro adventitious budding on Pinus pinaster cotyledons and needles. Physiol. Plant. 56:102-107. Debergh, P.C. and L.J. Maene. 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Scientia Hort. 14:335-345. Duddridge, J. and D.J. Read. 1982. An ultrastructural analysis of the development of mycorrhizas in Rhododendron ponticum Can. J. Bot. 60:2345-2356. Durzan, D.J. 1980. Prospects for the mass propagation of economically important conifers by cell and tissue cultures. In: Developments in plant biology, Vol. 5, Elsevier/N. Holland, Biomed. Press, pp. 283-288. Durzan, D.J. and R.A. Campbell. 1974. Prospects for the mass production of improved stock of forest trees by cell and tissue culture. Can. J. Forest Res. 4:151-174.

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68 Durzan, D.J. and S.M. Lopushanski. 1975. Propagation of American elm via cell suspension cultures. Can. J. For. Res. 5:273-277. Englander, L. 1981. Endomycorrhizae by septate fungi. In: N.C. Schenck, [Ed.], Methods and principles of mycorrhizal research. American Phytopath. Soc, St. Paul, Minn. pp. 11-12. Federal Register. 1983. Endangered and threatened wildlife and plants: Proposal to determine Torreya taxi folia (Florida torreya) as an endangered species. Fed. Reg. 48(68 ): 15168-15171. Florida Statutes. 1978. Preservation of native trees, shrubs and plants, s. 581.185,581.186, 581.187. and 581.211. In: I.F.A.S., Urban Gardener 3(8). Giovanettii, M. and B. Mosse. 1980. An evaluation of techniques for measuring vescicular-arbuscular mycorrhizal infection in roots. New Phytol. 84:489-500. Godfrey, R.K. and H. Kurz. 1962. The Florida Torreya destined for extinction. Science 136:900-902. Granger, R.L., C. Plenchette and J. A. Fortin. 1983. Effect of a vesicular arbuscular (VA) endomycorrhizal fungus ( Glomus epigaeum ) on the growth and leaf mineral content of two apple clones propagated in vitro Can. 'J. PI. Sci 63 (2): 551-555. Gray, H. 1978. Chemical aids in rooting rhododendrons and Ilex cuttings. Proc. Intern. Plant Propagators Soc. 28:517-518. Haines, R.J. and R.A. deFossard. 1977. Propagation of Hoop Pine ( Araucaria cunninghamii Act.) by organ culture. In: G. Boesman, [ChrmanJ, Symposium on Tissue Culture for Horticultural Purposes, pp. 297-302. Hartman, H.T. and D.E. Kester. 1975. Plant propagation principles and practices. 3rd ed. Prentice Hall, Englewood Cliffs, N.J. 662pp. Hearne, D.A. 1982. Preliminary report on a technique which provides a "maturity factor" for trees grown in tissue culture. Proc. Intern. Plant Propagators Soc. 32:109-113. Herrera, H.G. and G.C. Phillips. 1984. Tissue culture of Pinus eldorica Med. Sym. Prop. Higher Plants Through Tissue Culture III. ("abstract/poster) Knoxville, Tenn. House Document 95-41 (1975) Report on endangered and threatened plant species of the United States. Washington, D.C., Smithsonian Institute. 200pp. Hu, C.Y. and I.M. Sussex. 1971. In vitro development of embryoids on cotyledons of Ilex aqui folium. Phytomorphology 21:103-107.

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69 Isikawa, H. 1974. ln_ vitro formation of adventitious buds and roots on the hypocatyl of Cryptomeria jeponica Bot. Mag. Tokyo 87:73-77. Jansson, E. and C.H. Bornman. 1983. Morphogenesis in dormant embryonic shoots of FMcea abiesinfluence of the crown and cold treatment. Physiol. Plant"! 5T(l):l-8. Keen, R.A. 1954. The propagation of taxus-a review. Proc. Intern. Plant Propagators Soc. 4:63-69. Keys, R.N. and F.C. Cech. 1981. Plantlet formation in American chestnut embryonic tissue in vitro Proc. 2nd North Central Tree Improve Conf., Lincoln, Nebraska, Aug. 5-7, 1981, p. 189-194. Keys, R.N. and F.C. Cech. 1982. Propagation of American chestnut in vitro Proc USDA For. Ser., Amer. Chest. Cooperators Meeting, Jan 5-7, 1982, Morgantown, W. Va., pp. 106-110. Konar, R.N. and V.P. Oberoi 1965. In vitro development of embryoids on the cotyledons of Biota oriental is Phytomorphology 15:137-140. Lakshmi Sita, G., J. Shobha and C.S. Vaidyanathan. 1980. Regeneration of whole plants by embryogenesis from cell suspention cultures of sandal wood. Curr. Sci 49:196-197. Larkin, P. J. and W. R. Scowcroft. 1981. Somaclonal variation a novel source of variability from cell cultures for plant improvement. Theor. Appl Genet. 60:197-214. LaRue, CD. 1948. Regeneration in the megagametophyte of Zamia floridana Bull. Torrey Bot. Club 75:597-603. LaRue, CD. 1954. Studies of growth and regeneration of gametophytes and sporophytes of gymnosperms. Brookhaven Sym. Biol. 6:187-208. Lazarte, J.E. 1981. Woody tissue culture research. Proc. Intern. Plant Propagators Soc. 31:649-655. LePage, M.T. 1968. Mise en evidence d'une dormance associee a une immaturite' de l'embryon chez Taxus baccata L. C.R. Acad. Sci. Paris, 2660:1028-1030. LePage-Degivry, M.T. 1970. Acid abcissique et dormance chez les embryons le Taxus baccata L. C.R. Acad. Sci. Paris 2710:482-484. LaPage-Degivry, M.T. 1973a. E'tude en cluture in vitro de la dormance embryonnaire chez Taxus baccata L. Biol. Plant. 15:264-269. LePage-Degivry, M.T. 1973b. Influence de l'acide abscissique sur le development des embryous de Taxus baccata L. cutives in vitro Z. Pflanzenphysiol 70:406-413.

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70 LePage-Degivry, M.T. 1973c. Intervention d'un inhibiteur lie dans la dormance embryonndire de Taxus baccata L. C.R. Acad. Sci. Paris 277D:177-180. LePage-Degivry, M.T. and G. Garello. 1973. La dormance embryonnaire chez Taxus baccata : Influence de la composition du milieu guide sur 1 induction de la germination. Physiol. Plant. 29:204-207. Linsmaier, E.M. and J. Skoog. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18:100-127. Litz, R.E. 1984. In vitro responses of adventitious embryos of two polyernbryonic Eugenia species. 19(5): 720-722. Liu, J.R., K.C. Sink and F.G. Dennis. 1983. Plant regeneration from apple seedling explants and callus cultures. Plant Cell, and Tissue Organ Culture 2:293-304. Lloyd, G. and B. McCown. 1980. Commercially-feasible micropropagation of mountain laurel, Kalmia lati folia by use of shoot-tip culture. Proc. Intern. Plant Propagators Soc. 30:421-427. Loreti F. and S. Mozini. 1982. Mass propagation of fruit trees in Italy by tissue culture: present status and perspectives. Proc. Intern. Plant Propagators Soc. 32:283-290. Lyrene, P.M. 1981. Juvenility and production of fast-rooting cuttings from blueberry shoot cultures. J. Amer. Soc. Hort. Sci. 106(3):396-398. Ma, S.S. and S.O. Wang. 1977. Clonal multiplication of azaleas through tissue culture. Acta Hortic. (The Hague) 78:209-215. Marx, D.H. and D.S. Kenny. 1981. Production of ectomycorrhizal fungus inoculum. In: N.C. Schenck, [Ed.], Methods and principles of mycorrhizal research. American Phytopath. Soc, St. Paul Minn. p. 131-146. McComb, J. A. 1978. Clonal propagation of woody plants using tissue culture, with special reference to apples. Proc. Intern Plant Propagators Soc. 28:413-426. McCown, B.H. and R. Amos. 1982. A survey of the response of gymnosperms to microcul ture. HortScience 17(3): 489 (abstract). McCown, B.H. and G. Lloyd. 1981. Woody plant nedium (WPM)A mineral nutrient formulation for microcul ture of woody plant species. HortScience 16(3 ) :89 (abstract). McCown, B.H. and G.B. Lloyd. 1983. A survey of the response of Rhododendron to in vitro culture. Plant Cell Tissue and Organ Culture 2(1): 77-35".

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71 Meyer, M.M. 1982. In vitro propagation of Rhododendron catawbiense from floral buds? HortScience 17 (6 ): 891-892. Moore-Parkhurst, S. and L. Englander. 1981. A method for the synthesis of mycorrhizal association between Pezizella ericae and Rhododendron maximum seedlings growing in a defined medium. Mycologia /3 (5): 994-997 Morandi D. S. Gianininazzi and V. Gianininazzi-Pearson. 1979. Inte'ret de 1'endomycorrhization dans la, reprise et la croissence du Framboisier issu de multiplication vege'tative in vitro Ann. Amelior. PI antes 29 (6): 623-630. Mott, R.L. and H.V. Amerson. 1981. A tissue culture process for the clonal production of loblolly pine plantlets. N.C. Agr. Res. Ser. Tech. Bull 271 14pp. Mott, R.L., R.H. Smeltzer, A. Mehra-Palta and B.J. Zobel 1977. Production of forest trees by tissue culture. Tappi 60(6):62-64. Murashige, T. 1974. Plant propagation through tissue culture. Ann. Rev. Plant Physiol. 25:135-166. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Norstog, K. 1965. Induction of apogamy in megagametophytes of Zami a integri folia Amer. J. Bot. 52:993-999. Norstog, K. and E. Rhamstine. 1967. Isolation and culture of haploid and diploid cycad tissues. Phytomorphology 17:374-381. Patel R.V. and G.P. Serlyn. 1982. Genetic instability of multiple buds of Pinus coulteri regenerated from tissue culture. Can. J. For. Res. 12:93-101. Pearson, V. and D.J. Read. 1973. The biology of mycorrhiza in the Ericaceae. I. The isolation of the endophyte and synthesis of mycorrhizae in aseptic culture. New Phytol 72:371-379. Pons, F., V. Gianinazzi-Pearson, S. Gianinazzi andJ.C. Navatel 1983. Studies of VA mycorrhizae j_n vitro: mycorrhizal synthesis of axenically propagated wild cherry~ TP~runus avium L.) plants. Plant and Soil 71:217-221. Ramawat, K.G., H.C. Asya. 1976. Growth and morphogenesis in callus cultures of Ephedra gerardiana Phytomorphology 26:395-403. Rancillac, M.M. Faye and A. David. 1982. In vitro rooting of cloned shoots in Pinus pinaster Physiol Plant. 56:97-101. Read, D.J. 1983. The biology of mycorrhiza in the Ericales. Can. J. Bot. 61:985-1004.

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72 Reilly, K.J. and J Washer. 1977. Vegetative propagation of radiata pine by tissue culture: Plantlet formation from embryonic tissue. N.Z.J. For. Sci. 7(2):199-206. Rhodes, H.L.J. 1968. Juvenility in relation to adventitious bud and root initiation in woody plants. Proc. Intern. Plant Propagators Soc. 18:252-254. Sargent, C.P. 1947. Silva of North America. Vol. X. Murry Print Co. Cambridge, Mass. Schopmeyer, C.S. 1974. Seeds of woody plants in the United States Agriculture Handbook No. 450. Forest Service, U.S. Dept. of Agriculture. 883pp. Simon, R.W. 1983. Chapman's rhododendron recovery plan. U.S. Fish and Wildlife Service. Atlanta, Ga., 41pp. Sommer, H.E. and C.L. Brown. 1980. Embryogenesis in tissue cultures of sweetgum. For. Sci. 26:257-250. Sommer, H.E., C.L. Brown and P.P. Kormanik. 1975. Differentiation of plantlets in long leaf pine ( Pinus palustris Mill.) tissue cultured in vitro Bot. Gaz. HF(2 ):1 96-200. Stribley, D.P. and D.J. Read. 1974. The biology of mycorrhiza in the 5 Ericeae. IV. The effect of mycorrhizal infection on uptake of N from labeled soil by Vaccinum macrocarpum Ail. New Phytol 73:1149-1156. Stribley, D.P. and D.J. Read. 1975. Some nutritional aspects of the biology of ericaceous mycorrhizas. New Phytol. 73:1149-1163. Strode, R. 1979. Commercial micropropagation of rhododendrons. Proc. Intern Plant Propagators Soc. 29:439-442. Styer, D.J. and C.K. Chin. 1983. Meristem and shoot-tip culture for propagation, pathogen elimination and gernplasm preservation. Hort. Rev. 4:221-277. Tulecke, W. 1959. The pollen cultures of CD. LaRue: A tissue from the pollen of Taxus Bull. Torrey Bot. Club. 86:283-289. Vieitez, A.M. and E. Vieitez. 1980. Plantlet formation from embryonic tissue of chestnut grown in vitro Physiol. Plant. 50:127-130. Vieitez, A.M. and M.L. Vieitez. 1980. Culture of chestnut shoots from buds in vitro J. Hort. Sci. 55(l):83-84. Wang, P.J. and C.Y. Hu. 1984. In vitro cloning of the decidious timber tree Sassafras randaiense Z. Pfl anzenphysiol 113 (4): 331-315":

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73 Ward, O.B. 1978. Plants. In: P.C. Pritchard, [Ed.], Rare and endangered biota of Florida, Vol. 5, University Presses of Florida. Gainesville, FL. 175p. Wilkins, C. P., J. L. Cabrera and J. H. Dodds. 1985. Tissue culture propagation of trees. Outlook on Agriculture 14( 1 ) : 2-13. Winton, L. 1970. Shoot and tree production from aspen cultures. Amer. J. Bot. 57:904-909. Winton, L. 1978. Morphogenesis in clonal propagation of woody plants. In: T.A. Thorpe, [Ed.], Frontiers of plant tissue culture. University of Calgary Press, Calgary, pp. 419-426. Winton, L.L. and S.A. Verhagen. 1977. Shoots from Douglas fir cultures. Can. J. Bot. 55:1246-1250. Wochok, Z.S. 1981. The role of tissue culture in preserving threatened and endangered plant species. Biological Conservation 20(1981 ):83-89. Wochok, Z.S. and M.A. El-Nil. 1977. Conifer tissue culture. Proc. Intern. Plant Propagators Soc. 27:131-139. Wong, S. 1982. Direct rooting of tissue-cultured rhododendrons into an artificial soil mix. Proc. Intern. Plant Prop. Soc. 31:36-39. Zenkteler, M.A. and I. Guzowska. 1970. Cytological studies on regenerating mature female gametophyte of Taxus baccata L. and mature endosperm of Ti 1 i a platyphyl los Scop, in Tni vitro culture. Acta. Soc. Bot. Poloniae 39:161-173. Zimmerman, R.H. 1978. Tissue culture of fruit trees and other fruit plants. Proc. Intern. Plant Propagators Soc. 28:539-545.

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BIOGRAPHICAL SKETCH Lee Roy Barnes, Jr., was born in Durham, North Carolina. Following graduation from high school, he attended and graduated from North Carolina State University with a double major in biological science and botany in May 1976. Transferring to the Department of Horticultural Science, he completed the Master of Science degree in December, 1978. His thesis was titled "In vitro propagation of watermelon" (Drs. Fred Cochran and Ralph Mott, co-chairmen). Following a two year leave of work experience and travel, he entered the University of Florida, Department of Ornamental Horticulture, in the Fall of 1981 interested in conventional propagation and micropropagation of native plants, especially rare and endangered species. 74

PAGE 84

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Charles R. JohnsoX, Chairman Professor or Oprramental Horticulture I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Thomas J. SJre'ehan Professor of Ornamental Horticulture I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Indra K. Vasil Graduate Research Professor of Botany

PAGE 85

I certify that I have read this study and that in iny opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Mike J. Young p* / Associate Professor of// FruitN^ops This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 1985 Dean, College of Agriculture Dean, graduate School

PAGE 86

UNIVERSITY OF FLORIDA 3 1262 08553 4245


CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS
RHODODENDRON CHAPMAN 11 GRAY, TAXUS FLORIDANA NUTT.,
AND TORREYA TAXIFOLIA ARN.
by
LEE ROY BARNES, JR.
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1985

ACKNOWLEDGEMENTS
I would like to acknowledge the support and advice of my graduate
committee, Drs. Charles Johnson, Thomas Sheehan, Mike Young, Indra Vasil,
as well as, Norman Schenck. I also thank the Department of Ornamental
Horticulture for financial support, Mr. Jim Stevenson and Dr. Greg Brock
with the Florida Department of Natural Resources for their friendly
cooperation, and the Park Manager and staff of Torreya State Park for
their assistance.
I would also like to thank the following for their patient and
enthusiastic help, Ms. Nancy Philman, Ms. Shirley Anderson, Ms. Jackie
Host, Mr. T.D. Townsend, Mr. Jan Weinbrecht, Ms. Barbara Prichard, Ms.
Debbie Gaw and Ms. Robin Reddick. Also I thank Ms. Anne Whealy, Ms. Sara
Rosenbaum, Mr. Chris Fooshee, Mr. Ed Duke and Ms. Beth Logan for human
support.
Special thanks are offered to Dr. Rita Hummel and Dr. Jasper
Joiner for their humor, laughter, and personal concern.
Finally, I would like to acknowledge the timeless support and love
of my parents and sister.

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ii
LIST OF TABLES AND FIGURES vi
KEY TO ABBREVIATIONS viii
ABSTRACT ix
1.0 INTRODUCTION 1
2.0 LITERATURE REVIEW 3
2.1 Endangered and Threatened Native Plants 3
2.2 Advantages of Micropropagation Techniques 4
2.3 Species Description and Distribution 5
2.3.1 Rhododendron chapinanii Gray 5
2.3.2 Taxus floridana Nutt. 5
2.3.3 Torreya taxi folia Arn 6
2.4 Woody Plant Tissue Culture 6
2.4.1 Organogenesis from Callus and Suspension Cultures 7
2.4.2 Organogenesis from Plant Organs and Organ Sections 8
2.4.3 Stimulation of Axillary Meristems 15
2.4.4 Taxus Embryo Culture 18
2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets 19
3.0 MATERIALS AND METHODS 21
3.1 Rhododendron chapmanii Gray 21
3.1.1 Establishment of Stock Plants 21
3.1.2 Disinfestation 22
3.1.3 Media Selection 22
3.1.4 Selection of Growth Regulator Levels 23
3.1.5 Stage II Subcultures 23
3.1.6 Rooting and Establishment 24
3.1.7 Colonization with Ericoid Mycorrhizae 24

r
3.2Taxus floridana Nutt 26
3.2.1 Conventional Propagation by Cuttings 26
3.2.2 Culture of Quiescent Shoot-tips 26
3.2.3 Culture of Expanding Vegetative Buds 27
3.2.4 Microsporangia Culture 28
3.2.5 Embryo Culture 28
3.3Torreya taxifolia Arn 29
3.3.1 Conventional Propagation by Cuttings 29
3.3.2 Culture of Quiescent Shoot-tips 30
3.3.3 Culture of Expanding Vegetative Buds 30
3.3.4 Culture of Micro- and Megasporangia 30
3.3.5 Embryo Culture 30
3.3.6 Effect of Cytokinin Sprays on Stock Plants 31
4.0 RESULTS 33
4.1 Rhododendron chapmanii Gray 33
4.1.1 Disinfestation 33
4.1.2 Selection of Growth Regulators Levels 33
4.1.3 Stage II Subcultures 36
4.1.4 Rooting and Establishment 36
4.1.5 Colonization with Ericoid Mycorrhizae 39
4.2Taxus floridana Nutt 43
4.2.1 Conventional Propagation by Cuttings 43
4.2.2 Micropropagation of Taxus Shoot-tips 43
4.2.3 Culture of Expanding Vegetative Buds 43
4.2.4 Culture of Microsporangia 46
4.2.5 Embryo Culture 46
4.3 Torreya taxifolia Arn 46
4.3.1 Conventional Propagation by Cuttings 46
4.3.2 Micropropagation of Shoot-tips 48
4.3.3 Culture of Expanding Vegetative Buds 48
4.3.4 Culture of Microsporangia and Megasporangia 48
4.3.5 Embryo Culture 48
4.3.6 Cytokinin Sprayed Seedlings 51
5.0 DISCUSSION 53
5.1 Rhododendron chapmanii 53
5.2 Taxus floridana 57
5.3 Torreya taxi fol ia 60
6.0 CONCLUSIONS 64
i v

LIST OF REFERENCES
66
BIOGRAPHICAL SKETCH
v

LIST OF TABLES AND FIGURES
Table Page
4.1.1 Percent contamination of j_n vitro cultured Rhododendron
chapmanii shoot-tips grown in shade house and growth
chambers 34
4.1.2 Growth response of in vitro cultured Rhododendron
chapmanii shoot-tips cultured on Woody Plant Medium
with 5 levels of the cytokinin, 2-i-P 35
4.1.3 Growth response of j_n vi tro cultured Rhododendron
chapmanii shoot-tips grown in 3x3 factorial combination
of auxin (IAA) and cytokinin (2-i-P) 37
4.1.4 Survival and rooting response of microcuttings of
Rhododendron chapmanii following treatment with auxins
from 2 sources 40
4.1.5 Summary of micropropagation system developed for
Rhododendron chapmani i 41
4.1.6Response of Rhododendron chapmanii plantlets produced
in vitro to 2 establishment media and inoculation with
or without ericoid mycorrhizae 42
4.2.1Rooting response of cuttings from 2 aged groups of
Taxus floridana to 3 levels of the auxin, IBA 44
4.2.2_In_ vi tro growth response of Taxus fl ori dana shoot-tips
on 4 nutrient media 45
4.3.1 Rooting response of cuttings of Torreya taxi folia
treated with 3 levels of the auxin, IBA 47
4.3.2 Growth response of cultured shoot-tips of Torreya
taxifol ia grown in vitro on 4 nutrient media 49
4.3.3Average number of axillary shoots of Torreya taxi folia
developing in vitro on 3 nutrient media and 5
concentrations of the cytokinin, BA 5U
Figure
4.1 Multiplication rates of in vitro cultured Rhododendron
chapmanii during initial and additional SII subcultures.. 38
vi

KEY TO ABBREVIATIONS
GROWTH
REGULATORS
ABA
Absc isle acid
BA
Benzyl adenine
GA3
Gibberellic acid
IAA
Indole acetic acid
IBA
Indole butanoic acid
2-1 -P
2-isopentyl adenine
NAA
Naphthalene acetic acid
NUTRIENT MEDIA
H
Hel1er's
LS
linsmaier and Skoog
1/2LS
1/2 Strength Macronutrients
tinsmaier and Skoog
MS
Murashige and Skoog
WPM
Woody Plant Medium
1/2WPM
1/2 Strength Macronutrients
Woody Plant Medium
vi i

Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CLONAL PROPAGATION OF ENDANGERED NATIVE PLANTS RHODODENDRON
CHAPMAN 11 GRAY, TAXUS FLORIDANA NUTT. AND TORREYA TAXIFOLIA ARN.
By
Lee Roy Barnes, Jr.
August 1985
Chairman: Dr. Charles R. Johnson
Major Department: Horticultural Science
Clonal propagation utilizing conventional and micropropagation
techniques was investigated for three endemic and endangered Florida
plant species, Rhododendron chapmanii Gray, Taxus floridana Nutt, and
Torreya taxi folia Arn. This is critical for their preservation since
these species exist in limited numbers but have high ornamental value
which has threatened their survival due to illegal collection.
Micropropagation of IR. chapmanii was investigated using shoot-tip
cultures. An average of 7.6 fold increase in lateral shoots was achieved
for three additional Stage II multiplication cycles on low salts Woody
Plant Medium (WPM) with 2-i-P (10 mg/1) and adenine sulfate (80 mg/1).
Microcuttings rooted in 4-6 weeks when direct-stuck into autoclaved 1:1:1
(v:v:v) Canadian peat:vermiculite:perl ite medium in covered flats. A 5
second quick-dip in 1000 mg/1 IBA (20% EtOH + 80% H^O) increased survival
and rooting of microcuttings. Inoculation of plantlets with the
mycorrhizal fungus, Pezizella ericae increased survival and growth when
plantlets were established in a less than optimal soil-less medium (1:1
v:v Fired montmori11onite cl ay:Canadian peat) but not in MetroMix-500.
v i i i

Torreya and Taxus cuttings could be easily propagated when treated
with 2,000-4,000 mg/1 IBA. However, cuttings taken from lateral branches
demonstrated strict plagiotropic growth. Best rooting and growth of
Torreya cuttings (20%) occurred following treatment with 4000 mg/1 IBA.
Taxus cuttings rooted best (67%) following treatment with 2000 mg/1 IBA.
Shoot-tip cultures (3-5 cm long) of Taxus and Torreya developed only
2-3 lateral shoots, apparently from preformed buds. Cultured "embryonic
shoots" from expanding vegetative buds failed to produce multiple shoots.
Excised embryos did not germinate following liquid culture to leach
inhibitors. Micro- and megasporangia did not grow or differentiate in
culture.
The most promising technique for Torreya involved application of
cytokinins (4 weekly sprays with 100-200 mg/1 BA or 2-i-P) to seedlings
before culture. Newly stimulated axillary shoots developed additional
shoots and multiple "bud-masses" when cultured on WPM with BA (1 mg/1)
and NAA (0.01 mg/1). These were slow to develop but could be subcultured
with additional growth of multiple shoots. No rooted plantlets were
obtained.

1.0 INTRODUCTION
Preservation of indigenous endangered plant species is a critical
issue requiring timely investigation of propagation techniques to
increase the numbers of these rare species before they become extinct.
Three endangered plant species of immediate concern are Rhododendron
chapmanii Gray, Taxus floridana Nutt, and Torreya taxifolia Arn. All
are endemic to restricted areas of northern Florida. Presently only
3000 Rhododendron chapmanii (Chapman's rhododendron) plants are known in
situ and these are endangered due to habitat destruction or alteration,
and over-collection for use in ornamental plantings. Taxus floridana
(Florida yew) is endangered due to limited numbers and distribution but
is highly desirable for landscape use and is subsequently subject to
over-collection. Torreya taxi folia (Florida torreya) is endangered due
not only to very limited numbers but also due to being decimated by a
fungal disease which has destroyed most seed bearing trees. Today only
1200 apparently diseased seedlings and weak stump sprouts remain.
Presently, all three species are protected by state and federal
laws. However, in spite of legal protection, the remaining specimens of
each species is declining. Numerous R_. chapmanii have been illegally dug
from the wild. Torreya taxi folia does not appear to be reproducing in
the wild and is all but extinct, except for a few apparently healthy
plants located in botanical gardens elsewhere in the United States.
The purpose of this investigation was to determine the
feasibility of clonal propagation techniques for the mass production of
1

2
these native ornamental species. Micropropagation techniques could
potentially increase the number of these species with little impact on
the present populations, as well as offer the possibility of
eliminating the systemic pathogen(s) associated with Torreya die-back.
If large numbers of these highly desirable plants could be produced,
then they would be available for stabilizing natural populations,
landscape plantings, and germ plasm preservation via meristem culture and
cryopreservation.

2.0 LITERATURE REVIEW
2.1 Endangered and Threatened Native Plants
One group of native plants of particular interest are those which
are rare, threatened or endangered in their present environments.
Endangered and threatened plants are often legally classified at both
federal and state levels. "Endangered" plants are defined as "species
in imminent danger of extinction or extirpation and whose survival is
unlikely if the causal factors presently at work continue operating"
(Ward, 1978. p. xiii). "Threatened" plants are defined as "species which
are believed likely to move into the above endangered catagory in the
near future if the causal factors now at work continue operating" (Ward,
1978. p. xiii). A "rare" species classification includes those species
which are not presently endangered or threatened, but are potentially at
ri sk.
Congress first attempted to protect endangered plant species by
enacting the Endangered Species Act of 1973 (House Document 95-41, 1975).
Ayensu and de Filipps (1978) revised this initial listing and
estimated that approximately 10% of the native vascular flora in the
United States (approximately 20,000 spp.) or 2,000 species, subspecies
and varieties are presently endangered or threatened.
Ayensu and DeFilipps (1978) concluded there are numerous reasons
for protecting these species including the advantages of maintaining a
broad gene pool and genetic diversity within and among species,
3

4
especially with important food, fiber and timber species. Many of these
threatened plants show great potential for ornamental use.
Within the state of Florida, there are an estimated 3,500 vascular
plant species which are native or naturalized (Ward, 1978). Of these,
124 species are presently listed at the state level as being endangered
or threatened. Ward (1978) concludes that this number represents only
3.5% of the total plant species but further estimates that the true
number of endangered, threatened or rare species may vary from the
listed number by a factor of 2 or as large as 7.
In 1978, the State of Florida (s. 581.185 Florida Statutes)
revised its listing of "endangered" plants to include over 30 species.
They listed as "threatened" species all bromeliads, all cacti (except
Opuntia), all orchids, all native rhododendrons, most ferns, most native
IIex spp., all Zamia spp., all Zephranthes spp. and 48 other species.
2.2 Advantages of Micropropagation Techniques
While many of the endangered and threatened plants are best
protected by procurement and management of sensitive habitats, many
of these species can be propagated and introduced into the horticultural
trade with minimal impact on the remaining populations or their genetic
diversity. Seedage and conventional vegetative propagation by cuttings
can be used to increase the number of plants. However, these techniques
are often of limited value due to the lack of propagation and cultural
information for these rare species. Also the expense of collecting seed
or vegetative propagules would be prohibitive due to limited numbers and
travel expense.
Micropropagation of endangered species offers many advantages
over both seed and conventional vegetative propagation methods, such as

5
(1) high initial multiplication rates from limited plant material in a
small area; (2) clonal propagation of selected phenotypes via
stimulation of existing axillary meristems; (3) potential to force
rejuvenation of selected mature plants resulting in increased rooting
ability of shoots with juvenile rooting capacity (Rhodes, 1968; Lyrene,
1981); (4) ability to propagate infertile or low fertility species
(Wochok, 1981); (5) high degree of environmental control to minimize
variability with limited plant material and (6) possibility of obtaining
disease-free material via meristem culture (Abbott, 1977; Boxus and
Druart, 1980; Styer and Chin, 1984).
Wochok (1981) suggested that tissue culture can be very useful
in preserving threatened and endangered species and developed
micropropagation techniques for 4 native species (Atriplex canescens,
Mahonia aquifoliurn 'compacta', Mahonia repens, and Populus tremuloides).
2.3 Species Description and Distribution
2.3.1. Rhododendron chapmanii Gray
Rhododendron chapmanii Gray (Chapman's rhododendron) is a small
evergreen shrub, 1-2 meters in height, which produces a showy display of
terminal clusters of clear pink to rose-pink flowers in late spring
(April-May). This species is apparently very drought, heat, and pest
resistant (Simon, 1983).
Conventional propagation by seeds and cuttings has been successful
(Salter, 1982 per.com.; Gensel, 1983 per. com.), albeit with a low
rate of multiplication.

6
2.3.2 Taxus floridana Nutt.
Taxus floridana Nutt. (Florida yew; Savin) is an evergreen,
needle-bearing shrub or small tree 3-8 m (rarely 10 m) in height. It is
highly adapted to heavy shade and tolerates severe pruning.
Conventional clonal propagation by cuttings is possible; however,
cuttings from lateral branches grow only piagiotrophically and few
orthotropic shoots are available. Seed propagation is limited by a
double dormancy within the seed which usually requires two seasons to
germinate. Seed set is limited and heavy fruiting has been observed
only once in the last ten years in Torreya State Park (Womble, 1982,
per. com.). While over 200 seeds were collected in 1982, only 25 were
collected in 1983 following an extensive, tree to tree search.
2.3.3 Torreya taxi folia Arn.
Torreya taxi folia Arn. (Florida torreya; Stinking Cedar; Polecat
Wood; Gopherwood; Savin; Tumion) is a medium sized, pungent smelling
(when bruised or crushed) evergreen tree which grows to 15 m.
Conventional propagation by cuttings is possible; however, lateral
cuttings have been found to continue growing only piagiotrophically.
Seed propagation has been successful but is limited by extreme scarcity
of seed and a poorly understood double dormancy which usually prevents
germination until the second year.
2.4 Woody Plant Tissue Culture
Tissue culture techniques have been applied to three general areas
of interest, namely clonal propagation of (1) woody ornamentals
(Murashige, 1974; Abbott, 1977; Oebergh and Maene, 1981; McComb, 1978;
Lazarte, 1981; Briggs and McCullock, 1983), (2) fruit trees (McComb,

7
1978; Zimmerman, 1978; Lazarte, 1981; Loreti and Mozini, 1982), and
(3) superior genotypes of selected forest species (Durzan and Campbell,
1974; Abbott, 1977; Brown and Sommer, 1977; Cheng, 1978; Bonga,
1980; Durzan, 1980).
Propagation of woody species has been investigated by three
different approaches: (1) organogenesis from callus or suspension
cultures, (2) organogenesis directly from plant organ or organ sections
and (3) axillary shoot stimulation. Attempts have been made to
propagate from both selected mature tissues and from the more competent
or plastic juvenile tissues. Although it is more desirable to obtain
plants from elite mature genotypes, these tissues have been difficult to
root. Rejuvenation treatments and the use of juvenile zones within the
plants have been successful in some cases (Lyrene, 1981). By far the
greatest amount of success has occurred with the use of explants from
juvenile tissues, especially embryos and young seedlings. Although
morphogenically flexible and generally easy to root, these juvenile
sources are genetically variable and untested as to their mature
phenotype.
2.4.1 Organogenesis from Callus and Suspension Cultures
Organogenesis from callus or suspension cultures established from
elite trees would offer the greatest advantages. This would allow large
scale and cost efficient production of superior trees. This would be
especially important if somatic embryogenesis could be obtained and
microencapsulation developed so that clonal "seeds" could be produced.
However, at least two factors prevent the wide scale use of these
techniques. First, organogenesis from callus and suspension cultures has
not been successful for most important forest species (Wilkins et al.

8
1985). Secondly, the genetic stability of adventitious organogenesis is
often questionable (Wilkins et al., 1985; Larkin and Scowcroft, 1981).
Successful regeneration of plantlets via somatic embryogenesis from
suspension and callus cultures has been reported for Santa! urn album
(Lakshmi Sita et al., 1980), and Liquidambar styraciflua (Sommer and
Brown, 1980). Regeneration of shoots and roots from callus has been
reported for triploid Populus tremuloides (Winton, 1970), Citrus grandis
and C_. sinensis (Chaturvedi and Mitra, 1974), Ulmus americana (Durzan
and Lopushanski, 1975), Ephedra gerardiana (Ramawat and Arya, 1976),
Larix decidua (Bonga, 1981), Mal us domestica (Liu et al., 1983), Pinus
eldarica (Herrara and Phillips, 1984), Eugenia jambos and E. malaccenis
(Litz, 1984).
In summary, organogenesis from true cell suspensions and callus
cultures has only been successful for a limited number of woody species
(especially with timber, tropical fruit, and arborescent monocotyledonous
species), usually at low frequencies and often with the inability to root
shoots from callus derived from mature trees (Wilkins et al., 1985).
2.4.2 Organogenesis from Plant Organs and Organ Sections
Direct organogenesis from plant organs and organ sections has been
widely accomplished among woody species. In most cases, this has
occurred from very juvenile explants, especially embryos or young
seedlings. Rejuvenation from mature tissues has been successful from
inflorescenes or micro- and megasporangia, tissues temporally
associated with meiosis (Bonga, 1980).
Organogenesis has occurred in widely dispersed plant families.
LaRue (1948) obtained low frequency shoot regeneration from the mega-
gametophyte of Zamia floridana. He later reported (1954) regeneration of

9
shoots and roots from megagametophytes of both Zamia floridana and Cycas
revoluta. Norstog (1965) cultured megagametophytes of Zamia integrifolia
and was able to obtain diploid and haploid roots and leaves in 35% of his
cultures. He was also able to obtain diploid and haploid embryoids from
cultures of excised embryos and haploid megagametophytes, respectively.
Plants were successfully established in soil.
Konar and Oberoi (1965) obtained embryoids on cotyledons of Biota
orientalis Endl. (now Platycladus oriental is). Further development of
these embryoids did not occur until transfer to a different medium.
These "embryoids" may have been inappropriately named since they did
not appear to have a root apex although normal appearing cotyledons and
shoot apex developed into a young shoot.
Norstog and Rhamstine (1967) induced proliferation of callus from
haploid and diploid tissues of Zamia integrifolia and haploid tissues of
Cycas species.
Hu and Sussex (1971) were able to stimulate embryogenesis from
cultured cotyledons of Ilex aquifoliurn. They noted that embryoids
developed only on cotyledons where the shoot-tip failed to develop,
suggesting an antagonism between shoot growth and initiation, possibly
due to apical dominance of the active shoot-tip.
Isikawa (1974) reported the formation of adventitious buds and
roots on the hypocotyls of Cryptomeria japónica, primarily in
illuminated cultures. This organogenesis required low levels of the
growth regulators ABA, NAA and BA. He concluded that callus formation
may be antagonistic to shoot formation of conifers.
Sommer et al. (1975) reported high frequency adventitious bud
formation on cultured cotyledons of Pinus palustris when grown on

10
modified Gresshoff and Doy medium. If maintained on media with high
auxin/cytokinin ratios, excess callus began to form after 5-6 weeks.
They also mentioned success in obtaining adventitious buds on cotyledons
of P_. el 1 ioti i, P_. taeda, P_. vi rgi ni ana, P_. rigi da, P_. strobus and
Pseudotsuga menziesii but did not mention if plantlets were obtained
from these species.
Cheng (1975) obtained adventitious bud formation on cotyledons and
cotyledon slices of Pseudotsuga menziesii during primary culture, and
was able to maintain the capacity to produce buds after 3 and 4
subcultures. This system required 3 separate steps: (1) culture
initiation with equal amounts of auxins and cytokinins, (2) organ
initiation with high cytokinin levels, and (3) further growth of
adventitious buds on 1/2 strength modified MS medium and no growth
regulators for further growth of adventitious buds. Plantlets were
obtained by treating shoot cuttings with IBA (amounts not given). The
necessity for separate steps suggests that conditions necessary for
initiation of one step were inhibitory to further development.
Cheng (1976) reported adventitious buds from cotyledons of Tsuga
heterophyl1 a using techniques similar to those employed for Pseudotsuga
menziesii. In addition, she was able to root shoots directly in soil
mix thus saving one culture step.
Winton and Verhagen (1976) induced adventitious shoots directly
from cotyledons of Pseudotsuga memesii and also from subcultured
callus, obtaining up to 100-200 clonal shoots from each seed. They also
obtained callus on needles from 3 month old seedlings and from needles
from a 25 year old tree. However, they were only able to stimulate
adventitious shoots from the 3 month old needle callus.

11
3onga (1977) initiated a new approach to obtain adventitious shoots
from 15-20 year old Abies balsamea. "Embryonic shoots," composed of
dormant buds with scale leaves excised, were soaked either in sterile
water for 24 hours, or in aqueous solutions of growth regulators.
Soaked buds formed additional axillary shoots when transferred to a
growth medium while non-soaked buds only expanded as a single shoot.
Reilly and Washer (1977 ) cultured embryos of Pi ñus radiata and
obtained adventitious buds directly from cotyledons and hypocotyls and
from meristematic callus which proliferated from these organs. This
callus could be subcultured on cytokinin-free medium every 4 weeks.
When shoot primordia were separated from these meristematic masses, they
developed into well formed shoots. By serial subculture of callus, they
were able to obtain over 200 shoots from 1 embryo in 6 months. These
shoots could be rooted on medium with auxin (1-25 mg/1 IBA).
Brown and Sommer (1977) reported that 9 Pinus species developed
buds on cotyledons at a frequency of 26-100% depending on the species
and medium. They were unable to root these elongating shoots except
very rarely (less than 1% rooted).
Mott et al. (1977) summarized their work with Pinus taeda whereby
they obtained more buds on excised cotyledons vs intact embryos. They
found significant differences in organogenic response between seed
families but these differences could generally be reduced by adjusting
the growth regulator combinations.
Coleman and Thorpe (1977) cultured cotyledons from seeds and
shoot-tips from 4 to 10 year old trees of Thuja pi i cata. Using lateral
shoot-tips from 4 to 10 year old trees, they obtained adventitious
shoots which grew only orthotrophically. The addition of high levels of

12
GA^ (0.01 mg/1) to the medium induced the development of male strobili
from the vegetative apex. Fifty percent of the adventitious buds from
juvenile tissues rooted when transferred to a medium with 1/2 strength
MS and 1.6 mg/1 BA, while only 11% of those from the mature shoots
rooted. They concluded that the successful formation of plantlets
requires 3 distinct sequential treatments (1) meristematic induction,
(2) bud elongation and (3) rooting and establishment.
Arnold and Eriksson (1978) collected vegetative buds of Picea abies
from 5 year old trees, 75 year old hedged trees (2 m tall) and from
trees in 4 age classes: 5-10, 10-15, 15-20, and 20-50 year old trees.
No differences were found in the organogenic ability of these explants
with an average of 30% of cultures producing buds. They made no
attempt to test the rooting ability of these buds.
Aitken et al. (1981) compared explant sources for regeneration of
Pinus radiata buds. Excised whole embryos, and intact and excised
cotyledons from 1 week old seedlings were cultured, resulting in an
average of 9 rootable shoots from the embryos, 18 from intact cotyledons
and 180 from the excised cotyledons from 1 week old seedlings.
Induction of nodular, smooth-surfaced meristematic callus appeared
necessary for large-scale propagation. After 24 weeks they had obtained
over 1300 shoots from the excised cotyledons from 1 seed thus greatly
increasing the efficiency of this technique.
Bonga (1981) published a second paper on organogenesis from 15-20
year old mature trees including Abies balsamea. Morphogenesis occurred
rarely from female cones of Pi ñus mugo and Pi cea abies. Vegetative
shoots from A. bal samae, P_. gl auca and Pseudotsuga menziesi i produced
buds at the base of young expanding needles. His methods included

13
collecting buds before bud break, excising underwater and soaking for 15
minutes in 1 g/1 malonic acid solution or for 24 hours under water.
Mott and Amerson (1981) updated their work with Pinus taeda,
summarizing the need for sequential transfer of explants. They stressed
the point that 1 medium and combination of growth regulators initiated a
response but further development was inhibited until they were
transferred to a different medium. They stressed the need for judicious
observation of the timing and duration of growth regulator exposure and
termed the short term exposure to certain growth regulator combinations
as "pulsing."
Patel and Berlyn (1982) questioned the genetic stability of
multiple buds regenerated from embryo explants of Pinus coulteri even
when no cytokinins were used in the medium. Cells from callus and
regenerated buds showed a progressive increase in DMA levels over time.
After 6 weeks in culture, 80% of cells in regenerated buds had DNA
levels between 4C and 12C as determined with a microspectrophotometer.
They did not determine whether this resulted in mutated whole plants.
David et al. (1982) reported adventitious budding on Pinus
pinaster cotyledons and from short-shoots and elongating needles
collected from a 10 year old tree which had been sprayed weekly with BA.
Meristems of cultured short-shoots produced buds from 2 locations, from
the apical meristem of the short-shoot and from mitotically active cells
at the base of the expanding needles. When they altered the NH4+/K+
ratio of the medium to 1, they obtained increased adventitious bud
formation. They emphasized the importance of having mitotically active
and morphogenically nondetermined cells.

14
Jansson and Bornman (1983) reported improved morphogenesis of
spring collected "embryonic shoots" (bud with budscales removed) of
Picea abies by inclusion of the "crown," a layer of 4 to 7 extremely
thickened collenchyma cells located at the base of the shoot. This
anatomical feature is common to Abies, Cedrus, Larix, Pseudotsuga,
Picea, Sequoia, Taxus, Torreya and Tsuga. Following excision and
culture of the embryonic shoots with attached crowns, an average of 10
buds per explant was produced while crown-minus explants produced only 3
buds per explant. They reiterated comments by Chalupa and Durzan (1973)
that the crown may act as a filter or translocation barrier to nutrients
and hormonal stimuli. They also suggest that the crown may be a barrier
to basipetal transport.
In summary, organogenesis from juvenile tissues, especially
cotyledons and hypocotyls, is common among many conifers. Mitotically
active cells of expanding needles also appear morphogenically flexible.
However, the number of buds which can be produced by this method is
limited (few to dozens) or rarely, hundreds from a single explant. By
subculture of meristematic masses derived from these organs, up to 1300
plantlets have been obtained from a single seed of Pinus radiata (Aitken
et al., 1981). The ability to obtain plantlets usually requires several
different media and growth regulator regimes with careful attention paid
to the duration of each exposure. Shoot initiation, shoot elongation,
root initiation and root elongation require different conditions to
maximize the propagation rates. For many species, rooting has occurred
at low frequencies but evidence suggests that more careful attention to
duration of auxin exposure and light levels may increase rooting.

15
2.4.3 Stimulation of Axillary Meristems
The stimulation of axillary meristems has been most successful with
ornamental and fruit tree species. Styer and Chin (1983) list over 145
genera which have been cultured from excised meristems and shoot-tips.
3riggs and McCulloch (1984) list 63 genera and 106 species and cultivars
which are being propagated commercially or in research laboratories in
the United States and Western Canada.
Haines and de Fossard (1977) reported stimulation of axillary buds
from orthotrophic stem sections of Araucaria cunninghamia. Shoots which
developed from bud traces of ortotrophic branches grew upright while
shoots derived from piagiotrophic branches grew only laterally.
Vieitez and Vieitez (1980a) propagated Castanea sativa by culture
from lateral buds from 3 to 4 month old seedlings and from axillary buds
of cultured embryos. Keys and Cech (1981,1982) propagated Castanea
dentata by stimulating axillary meristems from mature embryos and from 4
month old seedlings grown in a greenhouse. Chevre et al. (1983)
micropropagated C_. sativa and obtained increased axillary
multiplication and elongation by doubling the calcium and magnesium
concentrations in MS medium and by lowering the pH to 4.0.
Rancillac et al . (1982) developed methods to induce axillary shoot
development from 1 month old seedlings of Pinus pinaster. Rooting of
these shoots was most successful when shoots were cultured for 17 days
on media with 2 mg/1 NIAA followed by transfer to an auxin-free media.
Wang and Hu (1983) cultured shoot-tips of 5 year old Sassafras
randaiense and obtained "multiple-bud-mass" proliferation on MS plus 60
mg/1 kinetin within 2 months of incubation. The rooting percentage

16
remained below 20%, but a 95% survival rate was achieved following
transfer to venniculite in flats.
While explants from mature trees are generally difficult to root,
Hearne (1982) reported the advantages of using mature tissues. He
suggested that for many species where mature tissues could be air
layered, these mature tissues are desirable since they retain their
mature features and often root easily in culture. He cited the
advantages of earlier flowering, and that plantlets commonly have
reduced canopy size which reduced the need for pruning and eased
harvesting. He cited precocious flowering roses within 49 days
following deflasking, and fruiting grapes and papaya within 12 weeks
from deflasking. His observations suggest that axillary shoot explants
from mature plants retain their mature qualities yet root as if they
had been "phenotypically" rejuvenated while in culture.
Amos and McCown (1981) reported their attempts to micropropagate
conifers by shoot-tip and axillary meristem culture. They obtained
shoot multiplication from species of Juniperus, Sequoia, Taxus, Thuja,
and Tsuga when cultured with very low levels of BA. Picea and Pinus did
not show rapid shoot development. They concluded that as a group,
conifers are more sensitive to intermediate levels of cytokinins than
woody dicot species. They were able to root harvested shoots, but did
not comment on the further growth of these cultures (i.e. whether
orthotrophic or plagiotropic shoots developed).
Micropropagation of rhododendrons has been a great commercial
success. Anderson (1975) first reported micropropagation of
Rhododendron using shoot-tip cultures on a modified, approximately 1/4
strength MS with a much reduced K content, obtaining an average of 6.2

17
new shoots per culture within 8 weeks. He noted that BA proved
phytotoxic while 2-i-P was not, and that the addition of 80 mg/1 adenine
sulfate was necessary for superior shoot development. Anderson (1978)
also reported successful direct rooting of micropropagated rhododendrons
from Stage II culture thus saving a very labor intensive step. Several
rooting media were successful as long as they were well drained and
porous, for example 1:1 (v:v) perlite:peat.
Strode (1979) reported on the commercial micropropagation of
rhododendrons using a revised Anderson (1975) medium amended with 5 mg/1
2-i-P and 1 mg/1 IAA. After 4 subcultures at 6 week intervals, he
obtained an average of 22.9 plants which could be attained when 4
shoot-tips were cultured in a single container. Harvested shoots rooted
90% within 5 weeks.
Meyer (1982) reported the successful use of tissues from flower
buds and inflorescences for rhododendron cultivars. Flower buds were
chosen as they had been used successfully with several herbaceous
species and resulted in lesser initial contamination. Meyer excised
buds from dormant plants between October and April and obtained
"granular masses" of tissues on Anderson's medium with 1-4 mg/1 IAA and
5-15 mg/1 2-i-P.
McCown and LLoyd (1983) surveyed the response of 7 genotypes of
rhododendron to micropropagation. They used Woody Plant Medium (WPM- a
modified Gresshoff and Doy medium), which was originally developed for
micropropagation of Kalmia and other woody species (McCown and Lloyd,
1981). They observed that 2-i-P stimulated the greatest shoot
multiplication, while BA was phytotoxic to all but elipi dote and
lepidote rhododendrons. Optimal cytokinin levels varied little with the

18
genotype, with 1-3.5 mg/1 2-i-P generally optimal. They observed that
adventitious shoot development occurred occasionally when 2-i-P greater
than 2.75 mg/1 was used. Average multiplication rates were 7 to 21
depending on cultivar. It was estimated that with an average of 40
shoots per 6 week subculture period, over 75,000 shoots could be
generated per square meter of culture space per year. Plantlets from
callus cultures were also obtained; however, many aberrant types
developed. With axillary shoot stimulation, plants remained
true-to-type even after 6 years of culture, except for occasional
albino/green chimeras which were readily observed and easily rouged. It
was noted that the resulting rooted plantlets grew vigorously with a
high degree of basal branching, similar to the growth habit of seedling
rhododendrons. They stated their uncertainty as to whether this
branching was induced by growth regulators or whether the seedling
morphology was being mimicked.
2.4.4 Taxus Embryo Culture
Taxus species have been cultured i_n vi tro by several researchers.
Tulecke (1959) cites LaRue's work with cultured pollen from Taxus
brevifolia whereby he obtained an unusual "pincushion callus" with cells
resembling pollen tubes sticking out at right angles to the surface of
the callus. LaRue maintained long term cultures of this slow growing
callus but never obtained regeneration of shoots.
Zenkteler and Guzowska (1970) cultured mature female gametophytes
of Taxus baccata on White's medium and obtained proliferation of callus
from 2-5* of the explants. Variation in chromosome number from n to 3n
was found in callus from the 14th and 15th 8-10 week subcultures.
Regeneration of shoots or roots was not observed. Cultured embryos did

19
not develop even when placed next to growing gametophytic tissues.
LePage-Degivry (1968,), LePage-Degivry (1970), 1973abc) and
LePage-Degivry and Garello (1973) initiated a series of experiments in
which she cultured excised embryos from Taxus baccata. She (LePage,
1968) found that by culturing mature excised embryos in liquid Heller's
medium with sucrose she was able to leach out inhibitors to germination.
Later, it was determined that the culture of excised
embryos for 15-20 days in liquid media was necessary to remove
inhibitors (LePage-Degivry, 1970). The inhibitor was identified as ABA
by paper chromatography. After subculturing to solid medium with 1 mg/1
GA^ or after a chilling period, the embryos readily germinated. When
embryos which had begun germination were treated with extracts of the
conditioned liquid medium, growth ceased (LePage-Degivry, 1970).
However, an additional GA^ treatment resulted in resumption of
germination. Paper chromatography of the extract revealed that ABA was
leached from the explant during liquid culture.
It was later determined that ABA was not leached out by distilled
or mineral water alone, and that sucrose and Ca++ or K+ ions in the
medium were required to remove inhibition (LePage-Degivry, 1973b).
LePage-Degivry (1973c) reported renewal of inhibition of leached and
germinating embryos with the inclusion of ABA in the solid medium. No
adventitious organogenesis was reported.
2.4.5 Mycorrhizal Synthesis of Micropropagated Plantlets
Several micropropagated woody species have been inoculated with
mycorrhizal fungi in vitro and following rooting and establishment in
soil. Inoculation of micropropagated plantlets with mycorrhizae could
potentially increase survival, growth, and tolerance to environmental

20
stresses. Morandi et al. ( 1979 ) observed that the colonization of
micropropagated raspberries (Rubus idaeus) with vesicular arbuscular
mycorrhizae (VAM) resulted in more vigorous and uniform sized plants,
with a 53-130% increase in shoot dry weight depending on the specific
endophyte and cultivar used. Granger et al. (1983) noted that
colonization of micropropagated apple plantlets (Mailing clones) with
VAM resulted in increased plant heights, total leaf areas, leaf dry
weights and leaf content of Cu and P. This increase in growth occurred
with the cultivar Mailing 7; however, colonization did not increase
growth of Mailing Merton 111. Pons et al. (1983) noted colonization of
micropropagated sweet cherry plantlets (Prunus avium L.) with VAM in
vi tro but did not obtain any observed growth enhancement. In
preliminary experiments where cherry plantlets were inoculated at time
of transplanting into soil media, a "marked improvement in growth"
occurred.
Inoculation of rhododendron with ericoid mycorrhizae has been
successful in situ (Duddridge and Read, 1982) and in vitro (Pearson and
Read, 1973; Moore-Parkhurst and Englander, 1981). Ericoid plants
appear to be require the specific endophyte, Pezizella ericae (Read,
1982). Colonization of rhododendron with Pezizel1 a resulted in enhanced
nitrogen and phosphorus supply to the plant as well as increased
resistance to heavy metal toxicity (Duddridge and Read, 1982; Read,
1983). Moore-Parkhurst and Englander (1981) developed a technique for
in vitro colonization of rhododendron and Pezizel 1 a; however, this
technique is impractical for commercial use. The most efficient time to
inoculate plantlets would be at the time of transfer to soil.

3.0 MATERIALS AND METHODS
3.1 Rhododendron chapmanii Gray
3.1.1 Establishment of Stock Plants
Stock plants were established from seeds collected in late October,
1982, from cultivated plants located around the Doyle Conner Building,
Division of Plant Industry, Gainesville, Florida. They were obtained as
soon as the brown capsules began to dehisce, disinfested with 15% Clorox
solution (0.79% by weight sodium hypochlorite) for 5 minutes, and rinsed
with 3 changes of sterile deionized water. Seeds were spread out on
sterile moistened filter papers in petri plates and incubated in light
(16 hrs light/3 hrs dark at 25 C +/- 5 C with irradfance of 50-90
-2 -1
pMm s ). Germination occurred within 2 weeks and when seedlings
reached a height of 5 cm, they were transferred individually to 4-liter
-2 -1
pots maintained in a growth chamber illuminated with 150 ^iMm s cool
white fluorescent lighting for 16 hours daily. Temperatures were
maintained at 25 C +/- 1 C (day) and 20 C +/- 1 C (night). Plants were
watered from below to avoid wetting the foliage.
Succulent shoot-tips, approximately 3-5 cm in length (5-10 nodes),
were pruned by hand and then washed in soapy water, then rinsed in cool
running tap water for 1 hour. Pruned stock plants subsequently developed
numerous additional axillary shoots, which developed into succulent
shoot-tips of sufficient size to culture in 4-6 weeks.
21

22
3.1.2 Di sinfestation
An experiment was designed to compare contamination rates of
shoot-tip explants from 2 sources. Explants from plants grown both
outside under 40% polypropylene (Saran) shade cloth and in a growth
chamber were used.
Culture room conditions remained the same for all experiments.
Cultures were illuminated with cool white fluorescent lights at 90
-2 -1
pMm s for 16 hours daily, and a temperature of 25 C + /- 5 C. Unless
otherwise indicated, 20 ml of medium was aliquoted into 25x150 mm
Pyrex test tubes and capped with Kaputs. Media were autoclaved at
2
1.05 kg/cm , at 110 C for 20 minutes and then allowed to cool on a
si ant.
Shoot-tips from both 60% shade (grown out of doors) and growth
chamber grown stock plants were disinfested for 15 minutes in 15% or 20%
Clorox then rinsed in sterile distilled water 3 times. Shoot-tips were
further trimmed to remove the bleached petiole stubs, stem base and apex
resulting in a 5 node stem section. Explants were inserted vertically
into agar to approximately half their total length. Culture medium
consisted of basal Woody Plant Medium (WPM - McCown and Lloyd; 1981) plus
2% sucrose, 0.8% Bacto aar and pH adjusted to 5.2 prior to autoclaving.
Ten replications per treatment were used. Data on contamination were
collected after 2 weeks under culture conditions described previously.
3.1.3 Media Selection
An experiment was inititated to determine the best medium for
micropropagation of Rhododendron chapmanii. Four media used for woody
plants were selected : Woody Plant Medium (WPM-McCown and Lloyd, 1981),
1/2 strength macronutrients of WPM (1/2WPM), Linsmaier and Skoog medium

23
(LS-Linsinaier and Skoog, 1964) and 1/2 strength macronutrients of LS
(1/2LS). Culture vessels were 25x150 mm Pyrex test tubes containing 20
mis of medium. Each medium was amended with 1, 5, 10, 15, or 20 mg/1
2-i-P, and (in mg/1) myo-inositol (100), Nah^PO^ (100), adenine sulfate
(80), sucrose (30,000) and Bacto-agar (8,000). The pH of the medium was
adjusted to 5.2 before autoclaving. Ten replications per treatment were
used with individual 5 node shoot-tips used as experimental units. Data
were collected after 6 weeks.
3.1.4 Selection of Growth Regulators Levels
Two experiments were performed to determine effects of growth
regulators on rapid multiplication. In the first experiment, the
cytokinin, 2-i-P, was examined at 0, 5, 10, and 15 mg/1 with ten
replications per treatment. Individual 5 node shoot-tips were used as
experimental units. In the second experiment, the interaction of
cytokinins with auxins was investigated in a 3X3 factorial with 2-i-P at
5, 10, and 15 mg/1 and IAA at 0, 1, and 2 mg/1. Each treatment was
replicated 10 times. Data were collected after 60 days in culture.
3.1.5 Stage II Subcultures
Following initial culture on multiplication medium (S11), explants
were subcultured through 4 additional SI I cycles on WPM with (in Mg/1)
2-i-P (10) and adenine sulfate (80). The initial SI I culture utilized
25x150 mm test tubes, after which explants were transferred and cultured
in small baby food jars (approximately 125 ml) containing 40 ml of
medium and covered with aluminum foil and wrapped with Parafilm. Thirty
to 50 mm tall microcuttings consisting of a single primary shoot with
developing lateral shoots were subcultured. Uniform sized explants,

24
consisting of a single primary shoot with developing lateral shoots, were
subcultured. These explants were placed 3 to a baby food jar with
modified WPM medium. The explants were oriented horizontally and pressed
down slightly so that most nodes were in direct contact with agar. Ten
or more replicates were made. Cultures were transferred onto fresh
multiplication medium at 6 to 8 week intervals. Multiplication rates
were evaluated over 5 subcultures on WPM. Data were collected at each
subculture time every 6-8 weeks.
3.1.6 Rooting and Establishment
Stage II microcuttings were given one of the following treatments:
(1) control, (2) 0.1% IBA in talc or (3) 0.1% IBA (in 10% ethonol) for 5
seconds. They then were stuck directly into 54x28x6.8 cm plastic flats
containing 5 cm deep of autoclaved rooting medium, 1:1:1 (v:v:v) Canadian
peat:vermiculite:perlite. Ninety-six explants were used for each
treatment. Microcuttings were protected from drying by covering the flat
with an inverted translucent plastic flat. They were misted daily by
hand to increase humidity and were watered as needed with deionized
water. Flats were illuminated under cool white fluorescent lights at
-2 -1
approximately 90 pMm s with a 16 hour photoperiod at 25 C +/- 2 C.
3.1.7 Colonization with Ericoid Mycorrhizae
An experiment was established to determine the growth response of
rooted plantlets to inoculation with mycorrhizae and establishment in two
different container media. Well-rooted plantlets were inoculated with
the ericoid mycorrhizal fungus, Pezizella encae Read, and transplanted
o
individually into 6 cm plastic "cell-pack" (4 cells/pack). Two soilless
media were used: (1) Metro-Mix 500 (MM500) and (2) 2:1 (v:v) fired

25
montmori11 i ni te clay: Canadian peat (FMC:CP). Fungal inoculum of P.
ericae was prepared according to Marx and Kenny (1981). Pure cultures of
P_. ericae were obtained from the American Type Tissue Collection (culture
no. 32985). The cultures were increased in 250 ml flasks containing 100
ml of modified Melin-Norkrans fungal medium with a 1 centimeter layer of
broken glass in the bottom of the container. Cultures were maintained
at 25 C in the dark for 4 weeks, but were shaken weekly to fragment the
hyphae. A mixture of 28:1 (v:v) vermiculite:finely screened Canadian
peat was moistened with 750 ml of Melin-Norkrans medium and autoclaved.
After cooling, 100 ml of prepared P_. ericae inoculum was added and this
mixture was cultured for 3 weeks in the dark at 25 C. This inoculum was
then leached by wrapping in several layers of cheese cloth and rinsing
for 3 to 4 minutes under cool, running tap water to remove excess
glucose and nutrients which might support growth of intrusive
saprophytes. The vermiculite:peat inoculum was then air dried at 25 C
for 3 days. Approximately 1 gm of inoculum was placed immediately below
the transplanted plantlet's root zone. Non-inoculated plantlets had 1 gin
of autoclaved vermiculite added below the roots as a control.
Colonization and growth responses were evaluated after 16 weeks, using
the technique described by Englander (1982). Roots were stained with
Lactophenol-Trypan blue stain for 10 minutes at 90 C. Percent
colonization was estimated by use of the modified gridline intersection
method of Giovanettii and Mosse (1980). Each treatment was replicated
12 times, with 4 plantlets per replication.

26
3.2 Taxus floridana Nutt.
3.2.1 Conventional Propagation by Cuttings
An experiment was initiated to determine the optimal auxin
treatment for root formation on cuttings. Dormant, evergreen cuttings
of Taxus floridana were collected in early February, 1982, from native
populations in Torreya State Park and from plantings on the University
of Florida campus. Ten to 15 cm long terminal and subterminal cuttings
were collected from upright and lateral branches. The bases were
trimmed and quick dipped for 10 seconds in 2000, 4000, or 8000 mg/1 I3A
(in 20% EtOH). Each treatment was replicated 10 times with individual
cuttings as experimental units. Cuttings were stuck 5 cm deep in plastic
flats containing 10 cm of a mixture of 1:1 (v:v) course sand:Canadian
peat. They were misted intermittently (5 sec every 2 min) during
daylight hours. Bottom heat (28 C) was supplied from electric cables
placed 5 cm below the flats. Rooting was evaluated 120 days after
sticking. Well rooted cuttings were transplanted into 1:1:1 (v:v:v)
Canadian peat:perlite:sandy loam medium in 4-liter containers.
After establishment, stock plants were placed in a growth chamber with
the same cultural conditions mentioned for R^. chapmani i (3.1.2).
3.2.2 Culture of Quiescent Shoot-tips
Four different plant organs were investigated--(l) quiescent
shoot-tips (3.2.2), (2) developing vegetative buds just before bud break
(3.2.3), (3) swelling microsporangium (3.2.4)and (4) excised embryos
(3.2.5).
Quiescent shoot-tips were collected in October, 1982, from
the most recently matured branches of plants located on University of
Florida campus. Three to 4 cm long shoot-tips were washed in soapy

27
water, rinsed in running tap water for 1 hour and then disinfested with
a 15-20% Clorox solution (with 5 drops Tween-20 per liter) for 15-20
minutes. Shoot-tips were rinsed in 3 separate changes of sterile
deionized water. The bleached bases were trimmed and explants were
inserted vertically to half their length into 20 ml of solidified WPM
nedium in 25x150 Pyrex test tubes.
An experiment was initiated to determine the best medium for
culture of Taxus floridana. The 4 media selected were the same used for
Rhododendron chapmanii (Sect. 3.1.3). Data were collected after 6 weeks
in culture. Twenty explants per medium were cultured with individual
shoot-tips as experimental units. Basal media were amended with
(in mg/1) myo-inositol (100), MaH^PO^ (100), sucrose (30,000), Bacto-agar
(3,000). The pH was adjusted before autoclaving to 5.2 with the addition
of 1.0 or 0.1 N solution of NaOH or 1.0 or 0.1 N HC1 prior to
autoclaving. Data were collected on length of new growth, number of
developing shoots and explant color.
3.2.3 Culture of Expanding Vegetative Buds
Swelling vegetative buds were collected weekly for 3 successive
weeks preceding bud break in late February and into the second week of
March, 1983. Buds were washed and disinfested as described previously
(Sect. 3.2.3) and outer bud-scales were carefully excised under water as
described by Bonga (1977,1981) and Jansson and Bornman (1983). Four
preculture treatments were used. Trimmed buds were allowed to soak for
24 hours in sterile distilled water or 3 hours in either 22.5, 28.0, or
34.0 mg/1 BA solutions before culturing on WPM amended with myo-inositol
(100), NaH^PO^ (100), sucrose (20,000), agar (8,000) and 5.2 pH. Ten

28
replications per treatment were used. Data were collected on overall
growth and specific morphogenic changes.
3.2.4 Microsporagium Culture
Expanding inicrosporangia were collected and disinfested as
described in Section 3.2.2. Microsporangia were trimmed of outer
scales and soaked in the 4 previously described solutions (3.2.3).
Explants were established on basal WPM with agar (8,000). Each
treatment involved 10 replications with individual microsporangium and
data were collected after 6 weeks in culture.
3.2.5 Embryo Culture
Attempts were made to promote germination of excised embryos by
leaching of inhibitors to seed germination. Seeds were surface
disinfested using 20% Clorox for 20 minutes. The 1-2 mm long embryos
were excised and further disinfested in 5% Clorox for 10 minutes.
Embryos were rinsed 3 times with sterile deionized water, then placed
into 20 ml of liquid Heller's medium with 2% sucrose. Medium pH was
adjusted to 5.5 before autoclaving. The small embryos were most easily
handled by catching them in a drop of liquid held in a 5 mm wide
bacterial transfer loop. Cultures were maintained in the dark without
shaking (LePage, 1968). Due to limited seed availability, only 5
excised embryos were used per treatment with individual embryos as
experimental units.
Embryos were subjected to 3 different treatments. Following an
initial 2 week culture period, they were transferred to either (1)
Heller's medium supplemented with 1 mg/1 GA^ and solidified with 0.8%
agar or (2) Heller's solidified nedium and stored for 1 month in a

29
refrigerator at 4 C before returning to culture room conditions.
Treatment (3) involved a second leaching cycle in liquid Heller's medium
for 2 weeks before placement on solid Heller's medium with 1 mg/1 GA^.
GA^ was filtered sterilized and added to the cooling but still
liquid medium. Data were collected from the cultures grown for 40 days
in the dark at 25 C +/- 2 C.
3.3 Torreya taxi folia Arn.
3.3.1 Conventional Propagation by Cuttings
Evergreen cuttings were collected in early February, 1982, from
trees growing in Maclay Gardens, Tallahassee, and from plantings on the
University of Florida campus. Ten to 15 cm long terminal and subterminal
cuttings were collected from lateral branches. The cuttings were wounded
twice along opposite sides by scraping the basal 2.5 cm of the cuttings
to expose the lighter green cambium. Cuttings were treated in the same
manner as described for Taxus (Sect. 3.2.1) involving treatment with 3
concentrations of IBA (2,000, 4,000, and 8,000 mg/1). Individual
cuttings were used as experimental units, with 5 replications per
treatment.
Cuttings were evaluated 120 days following sticking and well-rooted
cuttings were transplanted into 1:1:1 (v:v:v) Canadian
peat:perlite:sandy loam medium in 4-liter containers. Data collection
included percent rooting, number and total length of roots.
3.3.2 Culture of Quiescent Shoot-tips
Five explant sources were investigated to determine the morphogenic
capacity of various organs as reported in the 1iterature--(l)
quiescent shoot-tips (3.3.2), (2) expanding vegetative buds (3.3.3), (3)

30
expanding mega- and microsporangia (3.3.4), (4) excised embryos (3.3.5),
and (5) seedling stock plants sprayed with cytokinins before culture
(3.3.6).
In the first experiment (3.3.2), quiescent shoot-tips were
collected in October, 1982, from plantings located on the University of
Florida campus. Three to 4 cm shoot-tips were washed and disinfested
following the procedures previously described for quiescent shoot-tips of
Taxus floridana (Sect. 3.2.2). They were cultured in each of four media
(WPM, 1/2WPM, IS, and 1/2LS).
3.3.3 Culture of Expanding Vegetative Buds
Expanding vegetative buds were collected weekly over a 3 week
period preceeding budbreak in early spring (mid- to late March, 1984).
Buds were trimmed of bud-scales and were handled following the
procedures of Boulay (1977,1981) and Jansson and Bornman (1983) as
described for Taxus in Sect. 3.2.4. Ten buds were used per treatment
with individual buds as experimental units. Thus, 3 collection times
were combined with 4 preculture soaks.
3.3.4 Culture of Micro- and Megasporangia
Expanding micro- and megasporangia were collected as they swelled
following budbreak in the middle of March, 1984. These were washed
and disinfested as with Taxus floridana microsporangia (3.2.4). Ten
explanes of each were treated with individual sporangia as experimental
units.
3.3.5 Embryo Culture
An experiment was initiated to determine if germination inhibitors
could be leached from excised embryos. They were excised following

31
the techniques of LePage (1968), LePage-Degivry (1970, 1973ab) and
LePage-Degivry and Garello (1973) for Taxus baccata. Seeds, which had
been warm stratified for 2 months followed by 2 months of cold
stratification were surface sterilized in 20% Clorox followed by 3
sterile water rinses. Embryos, 2-5 mm long, were aseptically excised and
further disinfested in 5% Clorox for 10 minutes, followed by 3 rinses in
sterile deionized water. The small embryos could be most easily handled
with a bacterial transfer loop. Excised embryos were cultured in 20 ml
liquid Heller's medium as described for Taxus (Sect. 3.2.6). Excised
embryos were exposed to the 3 treatment regimes described for Taxus
floridana (3.2.5). Individual embryos were used as experimental units
with 5 embryos per treatment.
3.3.6 Effect of Cytokinin Sprays on Stock Plants
Torreya seedling stock plants were sprayed weekly for 4 weeks with
2 concentrations of 2 cytokinins (BA and 2-i-P at 100 mg/1 and 200 mg/1
plus 5 drops Tween-20 per liter). These treatments were suggested by Dr.
M. A. El-Nil (1983, per. com.). Plants were sprayed until run off.
Only 5 one-year old seedlings were available for experimentation,
limiting the treatments to only 1 plant per treatment. A single control
plant was sprayed weekly with the same 10% EtOH carrier solution as the
cytokinins. All developing basal and axillary shoots were removed and
placed in culture. Explants consisted of shoot-tips and stem sections
1-2 cm in length. They were cultured on solidified WPM (0.8% agar)
in 25x150 ml Pyrex test tubes with 4 concentrations of growth regulators
in the medium: (1) WPM with 1 mg/1 BA and 0.01 mg/1 NAA, (2) WPM with 10
mg/1 BA, (3) WPM with 1 mg/1 2-i-P, and (4) WPM with 10 mg/1 2-i-P.

32
-2 -1
Explants were cultured at 25 C (+/- 5 C), and illuminated 5Ü pMm s
for 16 hours a day.

4.0 RESULTS
4.1Rhododendron chapmanii
4.1.1 Pi sinfestation
There was an average of 55% reduction in amount of con tain i nation
(30-40%) when explants were collected from stock plants grown in a
growth chamber as compared to field grown explants (Table 4.1.1). There
was no difference in contamination % between the two levels of Clorox
used. Contamination of explants from shade house grown plants averaged
90% while those from growth chamber plants contaminated only 35%.
Field grown material remained very difficult to disinfest. In a
later experiment (Section 4.1.2), leaves were removed from growth
chamber grown shoot-tips prior to Clorox treatment. Contamination in
this case was reduced to only 4% (data not given), when leaves were
removed from explants prior to treatment with 15% clorox for 15 minutes.
For all subsequent experiments in this section, leaf removal prior to
Clorox treatment virtually eliminated contamination. Subcultures from
visually contamination-free cultures remained clear and presumably
without contamination.
4.1.2 Selection of Growth Regulator Levels
There was a difference between basal medium without cytokinins vs
all levels of 2-i-P tested, but no differences between the 2-i-P levels
(Table 4.1.2). Tnere was a significant reduction in the number of leaves
formed when with 2-i-P levels greater than 10 mg/1 were used.
33

34
Table 4.1.1 Percent contamination of in vitro cultured
Rhododendron chapman ii shoot-tips grown in shade house
and growth chambers
Treatment Contamination
l
Time
C1o rox
Chamber
Field
(min)
(%)
15
15
30
100
15
20
40
80

36
There were no differences between the total number of leaves and
shoots produced for the 2-i-P and IAA combinations (Table 4.1.3).
Differences between IAA means were significant at the 0.10 level,
however, inclusion of IAA in the medium generally resulted in declining
numbers of shoots and leaves.
Optimal shoot and leaf production occurred between 5-10 mg/1 2-i-P.
With higher 2-i-P levels, axillary shoots remained short and elongated
much more slowly than plantlets cultured on 10 mg/1 2-i-P.
4.1.3 Stage II Subcultures
Following initial SI I culture in test tubes, the medium in all
additional multiplication cycles contained adenine sulfate (30 mg/1) and
explants were established in small baby food jars. Basal nodes and
especially those embedded in the agar produced abundant axillary shoots.
When explants were cultured upright in individual test tubes on WPM witn
10 mg/1 2-i-P and no adenine sulfate (Fig. 4.1) an average 5.8 shoots
developed per explant in 6-8 weeks. For additional SI I subcultures,
increase in shoots was especially high when the explants were placed
horizonally and pushed slightly into the medium (Fig. 4.1.5). This also
resulted in more uniform elongation of shoots.
Each additional multiplication cycle for the next three SII
subcultures in baby food jars averaged a 7.6 fold increase in number of
shoots, however, this multiplication rate decreased to 5.0 during the
fourth subculture (Fig. 4.1).
4.1.4 Rooting and establishment
Preliminary rooting attempts using an agar substrate were
unsuccessful. In a few cases where cultures were left undisturbed for 4

35
Table 4.1.2 Growth response of in vitro cultured Rhododendron
chapmanii shoot-tips cultured on Woody Plant Medium with 5 levels
of the cytokinin, 2-i-P
2-i-P concentration
(mg/1)
No. expanding
axillary buds
Mean no.
leaves
0
Y
1.4ai
8.4bZ
5
3.0b
13.7a
10
3.6b
16.6a
15
2.2b
7.3b
20
2.2b
7.6b
y
Means in a colomn followed by the same letter are not significantly
different at the 5% level by Duncan's Multiple Range Test.
ZMeans in a column followed by the same letter are not significantly
different at the 10% level by Duncan's Multiple Range Test.

37
Table 4.1.3 Growth response of j_n_ vitro cultured
Rhododendron chapmanii grown on a 3x3 factorial combination
of auxin (IAAl and cytokinin (2-i-P)
2-i-P
(mg/1 )
IAA
(mg/1 )
0
1
2
Total No
. of Lea ves
5
38.9aZ
35 .2a
30.6a
10
37 .2a
38.4a
39.4a
15
38.2a
34 .6a
30.0a
Ave. No. Shoots
Greater than
5 mm
5
3 . 2 a Z
2.8a
2.8a
10
2.8a
2.6a
2.6a
15
3.6a
2.5a
1 .6a
Means in a column and row followed by the same letter are
not significantly different at the 1% level using Duncan's
Multiple Range Test.

38
9 T
Fold increase
in no. of
subculturable
shoot-clumps
8
5 -
\
\
\
\
\
\
\
5°0
4
3
—i 1 1 1 1—
Sill SI 12 SI 13 SI 14 SI 15
Stage II Multiplication Cycles
Figure 4.1 Multiplication rates of in vitro cultured Rhododendron
chapmanii during initial and 4 additional SII subcultures.

39
to 6 months, roots appeared spontaneously above the agar surface. Also,
the extremely fine "hair roots" characteristic of rhododendrons did not
develop properly in poorly aerated substrates.
Direct sticking of microcuttings into 1:1:1 (v:v:v) Canadian
peat:venniculite:perlite resulted in well-rooted plantlets within 4 to 6
weeks. Treatment of microcuttings before sticking with 1000 mg/1 I3A
(dissolved in 10% EtOH) resulted in improved survival and rooting (Table
4.1.4).
A quick dip treatment (5 seconds) with 1000 mg/1 IBA resulted in
greatest survival and approximately 60% rooting (Table 4.1.4).
Treatment with 1000 mg/1 of IBA in talc resulted in poorer rooting and
survival than controls. Survival improved slightly with more careful
observation and daily misting with deionized water. Best survival to
date has been 75-80% (data not given).
Table 4.1.5 summarizes the micropropagation staging system
developed for R_. chapmani i. With this rate of multiplication and
rooting, it can be conservatively estimated that over 10,000 rooted
plantlets could be produced from a single shoot-tip in one year (1 x 5.8
x 7.6 x 7.6 x 7.6 = 19350 x 60% rooting = 11610 plantlets in 9.5 months
+ 2 1/2 months additional growth).
4.1.5 Colonization with Ericoid Mycorrhizae
Inoculation of rooted plantlets with the ericoid mycorrhizae,
Pezizella ericae, resulted in colonization in two potting media, MM500
and FMC:CP. There were considerable differences (PF > 0.0001) between
madia for height of main stem, total stem lengths, number of leavas, and
main stem caliper after 12 weeks of growth in the greenhouse (Table
4.1.5). Differences in number of stems was significant at the .05 level.

40
Table 4.1.4 Survival and rooting response of microcuttings of
Rhododendron chapmanii following treatment with auxins from 2
sources
Treatment
Survival and Rooting
%
none
26.0Z
1000 mg/1 IBA
19.8
(in talc)
1000 mg/1 IBA
59.4
(1iquid-dip)
^Percent survival and rooting were significantly different at
the 1% level using analysis of catagorical data.

41
Table 4.1.5 Summary of micropropagation system developed for
Rhododendron chapmanii
STAGE EXPLANT DURATION RATE MEDIAX GROWTH7 CULTURE2
(weeks) REG. CONDITIONS
SI
shoot-tips
6-8
5 .8x
WPM
10 mg/1
20
pMm'
-2s
3-5 cm
2-i-P
25
+ 5
C
SII
axi11 ary
6-8
7.6x
WPM
10 mg/1
20
pMm"
-2s
shoot-clumps
2-i-P
25
+ 5
C
SIV
axi11 ary
4-5
80%
1:1:1
Quick-
90
pMm"
-2s
shoots
Rooting
di p
(>15mm)
Mediurn
25
+ 5
C
1000 mg/1 in high
IBA humidity
tent
^Medium WPM = Woody Plant Medium
Y
Growth Regulators
Z 2 1
Culture condi ti ons--l i ght intensity in pMm s
light duration 16 hrs light/ 8 hrs dark
temperature in degrees C

42
Table 4.1.6 Response of Rhododendron chapmanii plantlets
produced in vitro to 2 establishment media and inoculation
with or without ericoid mycorrhizae
MediaY
Trt.W
Plt.X
Surv.
%
Ht.Y
Mai n
Stem
(mm)
No .Z
Stems
TotalY
Stem
Length
(mm)
Y
No.'
Lvs
Main
Stem
Caliper
(mm)
%X
Col .
M500
+M
91.7a
67.0a
1.77a
96.1a
23.6a
0.98a
61.4a
M500
-M
95.8a
60.8a
2.14a
98.5a
26.7a
1.0a
0
FMC:CP
+M
80.9a
28.3b
1.40b
40.0b
14.9b
0.35b
41.1a
FMC:CP
-M
65,3a
23.0b
1.18b
34.1b
12.8b
0.28b
0
YMedia M500 = MetroMix-500
FMC:CP = Fired montmori11 i nite clay:Canadian peat
W
Trt. = mycorrhizal treatment +M = with inoculation
-M = without inoculation
Pit. surv. % = Plantlet survival %
X
Means in a column followed by the same letter are not significantly
different at the 5% level by analysis of catagorical data.
Y
Means in a column followed by the same letter are not significantly
different at the 1% level by Duncan's Multiple Range Test.
ZMeans in a column followed by the same letter are not significantly
different at the 5% level by Duncan's Multiple Range Test.

43
Plantlets grown in the less fertile FMC:CP potting mix were consistantly
smaller regardless of colonization.
4.2Taxus floridana
4.2.1. Conventional Propagation by Cuttings
Conventional vegetative propagation resulted in rooting within 120
days under mist (Table 4.2.1). Cuttings in the "mixed clones" group
were collected from smaller and younger trees 2-4 m tall. Cuttings from
the younger mixed clones rooted at better (best 67%) and with greater
number of roots. The best rooting percentage from the older Champion
tree was 40% but with fewer roots initiated and of shorter length than
from the younger clones.
4.2.2 Micropropagation of Taxus Shoot-tips
The response of shoot-tips grown for 5 months on 4 different media
supplemental with 0.5 mg/1 IAA is shown in Table 4.2.2. There were no
difference in shoot size, average number of shoots produced or length of
new growth. WPM was chosen for additional experiments primarily due to
the better color quality and general overall appearance of the explants.
Explants on WPM or 1/2WPM were light green, the normal color of new
growth on whole plants.
4.2.3 Culture of Expanding Vegetative Shoot-tips
Culture of expanding vegetative shoot-tips collected 3 successive
weeks before bud break resulted in either death of the explant,
contamination of cultures or no growth. Shoot-tips were very small (2-3
mm), were not easily dissected and may have been damaged by
disinfestation treatments or by removal of outer bud scales.

44
Table 4.2.1 Rooting response of Tax us floridana cuttings
from 2 aged groups of plants to 3 levels of the auxin, IBA
IBA
(mg/1 )
Rooting Mean root Mean root
% no. length
(cm)
Total root
length
( cm)
C h a m pion
Tree
2000
0
0
0
0
4000
40
4
1 .0
4.0
8000
40
5
Mixed Clones
1 .5
3.3
2000
67
4
1.0
4.0
4000
33
4
PO
o
O
CO
8000
25
14
o
no
42.0

45
Table 4.2.2 J_n_ vitro growth response of f 1 or i dana
shoot-tips on 4 nutrient media
Medium
Size
(mm)
Mean no.
shoots
Color
Length
new growth
(mm)
WPMZ
70.0aY
1 .2a
LG X
17.5a
1/2WPM
42.0a
2.4a
LG
5.7a
LS
61.2a
2.8a
BR
21 .2a
1/2LS
63.0a
1 .4a
LG/BR
ÃœO
O
O
Qj
WPM = Woody Plant Medium
1/2WPM = 1/2 strength macronutrients of Woody Plant Media
LS = Linsmaier and Skoog Medium
1/2LS = 1/2 strength macronutrients of Linsmaier and Skoog
Y
Means in a column followed by the same letter are not
significantly different at the 1% level by Duncan's Multiple
Range Test.
XColor Key LG = Light Green BR =
Brown

46
4.2.4 Culture of Microsporangia
Culture of expanding microsporangia was attempted successive for 3
weeks preceding budbreak. Microsporangia were very small (2-3mm) and
difficult to excise. Clean cultures could be consistently obtained, but
no further development was observed. Explants treated with chemicals or
water soak did not develop further in culture and no differences were
noted between treatments.
4.2.5 Embryo culture
Attempts to culture excised embryos were unsuccessful. None of the
3 treatments were successful in promoting germination. Embryos which
showed no signs of growth after 1 month on solidified Heller's medium
with GA^ or following removal from cold treatment were recultured into a
second cycle of fresh liquid medium. After 3 weeks in liquid medium,
these embryos were again transferred to solidified medium with GA^, but
no growth occurred.
4.3 Torreya taxifolia
4.3.1 Conventional Propagation by Cuttings
Rooting results are summarized in Table 4.3.1. The greatest
percent rooting (56%) occurred with 8000 mg/1 IBA. The number and
average length of roots with this treatment were substantually less than
those produced with 4000 mg/1 ISA treatment. Cuttings treated with 4000
mg/1 IBA rooted at a low percentage (20%) but produced more numerous
roots with greater average and total length.

47
Table 4.3.1 Rooting response of
taxifolia treated with 3 levels
cuttings of
of the auxin
Torreya
, IBA
IBA T reatment
(mg/1 )
Rooting
%
Mean no.
roots
Mean length
roots
(cm)
2000
14
3.5
1 .0
4000
20
13.0
2.5
8000
56
4.5
1.5

48
4.3.2 Micropropagation of Shoot-tips
The response of shoot-tips collected from lateral branches of
mature trees (est. 15-20 years old) in culture is summarized in Table
4.3.2. The color and apparent health of explants cultured on WPM or
1/2WPM were superior to LS or 1/2LS, being darker green.
In a second experiment, shoot-tips collected in October and grown
on 3 nutrient nedia each with 5 levels of BA was recorded after 2 months
in culture (Table 4.3.3). Little or no growth occurred on either LS or
1/2LS. A few axillary shoots developed on WPM at low to medium levels
of 8A (0.5-1.0 mg/1) but declined with higher levels.
4.3.3 Culture of Expanding Vegetative Buds
All cultures of expanding buds were either contaminated or were
killed by disinfestation procedures. Contamination continues to be a
serious problem from field grown plants but is much less of a problem
when stock plants are grown inside with lower light intensities and lower
night temperatures.
4.3.4 Culture of Microsporangia and Megasporangia
Most of these cultures became contaminated or were killed by
disinfestation procedures. Microsporangia were sufficiently large enough
to be handled and were plentiful in early spring.
4.3.5 Embryo culture
Embryo culture of T_. taxi fol ia also proved unsuccessful. Although
embryos could be easily disinfested with treatment with 10% Clorox
following aseptic excision, they did not generally respond to the
treatment used successfully for Taxus baccata embryos (LaPage, 1968).
In 1 case among 10 attempts, the cotyledons parted and appeared to

49
Table 4.3.2 Growth response of shoot-tips of T. taxifolia
on 4 nutrient media
Medium
Mean size
(mm)
Mean no.
shoots
Color
Length
new growth
(mm)
WPMZ
44.5abY
2.0a
DG X
20 . 5a
1/2WPM
18.7b
2.1a
LG
9.4a
LS
56 .3a
1 .6a
YG
38.2a
1/2LS
42.8ab
2.0a
LG to YG 28.9a
WPM = Woody Plant Medium
1/2WPM = 1/2 strength macronutrients Woody Plant Medium
LS = Linsmaier and Skoog Medium
1/2LS = 1/2 strength macronutrients Linsmaier and Skoog
Medium
Y
Means in a column followed by the same letter are not
significantly different at the 5% level using Duncan's
Multiple Range Test.
XColor Key DG = Dark Green, LG = Light Green, YG = Yellow
Green

50
Table 4.3.3 Average number of axillary shoots of Torreya
taxi f ol i a developing _i_£ vitro on 3 nutrient media and 5
concentrations of the cytokinin, BA
BA
(mg/1 )
No .
WPMZ
axillary shoots
LS
1/2LS
0
1 .0a Y
1.0
1 .0
0.5
1 .3a
0
0
1.0
1 .7a
0
0
5.0
1.0a
0
0
10.0
0.3a
0
0
WPM = Woody Plant Medium
LS = Linsmaier and Skoog Medium
1/2LS = 1/2 strength macronutrients of Linsmaier and Skoog
Medium
Y
Means in a column followed by the same letter are not
significantly different at the 5% level by analysis of
catagorica1 data .

51
begin to germinate. When transferred to solid medium with 1.0 mg/1 GA^,
a shoot-tip appeared but degenerated shortly thereafter.
4.3.6 Cytokinin Sprayed Seedlings
Only five seedlings were available for investigation so each
treatment was applied to a single seedling. Treatments were based on
suggestions from Dr. M. A. El-Nil (1982, per. com.). Stock plants
produced numerous basal shoots following treatment with cytokinin sprays.
The control plant produced a few basal shoots which could be cultured.
Excised basal shoots developed axillary bud masses after 30 days when
cultured on media with cytokinins. Best results occurred when explants
were collected from plants sprayed weekly with 1000 mg/1 BA and then
cultured on medium with 1 mg/1 BA and 0.01 mg/1 NAA. Higher
concentrations resulted in much smaller masses which did not enlarge much
until transferred to media with lower or no cytokinin. Culture on media
with 2-i-P resulted in little development until these were transferred to
WPM medium with 1.0 mg/1 BA and 0.01 mg/1 NAA after which bud elongation
occurred and occasional multiple shoots were observed.
Two morphologically different types of axillary bud development
were observed. In the first case, normally shaped axillary shoots
elongated with additional axillary shoot development from the leaf axils
at the base of the cultured buds. The second type of development
involved the development of compact "bud-masses" consisting of small,
tightly appressed scale-like leaves with the ultimate emergence of
multiple shoots after 3 months in culture. When buds from the same
treatments were subcultured to the same or lower cytokinin medium, the
buds on the lower cytokinin medium always began to show signs of growth

52
sooner. These buds were transferred to fresh medium but have since
degenerated. No rooted plantlets were obtained.

5.0 DISCUSSION
5.1 Rhododendron chapmanii
Conventional propagation using cuttings was attempted but was
unsuccessful due to failure of misting equipment. The experiment was not
repeated because of insufficient cutting material and the development of
a highly successful micropropagation system using shoot-tips.
Microcuttings could be rooted and established with 60% or greater
survival. Conventional propagation of evergreen Rhododendron species
usually involves softwood cuttings (Hartman and Kester, 1981) or leafly
hardwood cuttings collected in the fall which are treated with
concentrated NaOH and IBA solution (Gray, 1974).
Micropropagation of R_. chapmanii was highly successful using
shoot-tip explants and modifying the techniques of Anderson (1975) and
the medium of McCown and Lloyd (1981). Technique improvements included
decapitation of shoot apex and the horizontal placement of the explants
in the medium so that most nodes were slightly embedded in it.
Horizontal placement of shoots into multiplication medium greatly
increased the number and uniformity of shoots produced during each
multiplication cycle (S11). The addition of 80-100 mg/1 adenine
sulfate improved the number and vigor of shoots produced. BA proved to
be phytotoxic to R_. chapmani i as has been previously reported for most
Rhododendron species (Anderson, 1975; Lloyd and McCown, 1980; and
McCown and Lloyd, 1983). Decapitated shoot-clump bases could be
recultured as suggested by Strode (1979), however; this technique did
53

54
not result in as rapid an increase in shoots per cycle as when elongated
shoots were laid horizontally on and pressed slightly into the medium.
The use of higher levels of 2-i-P (greater than 15-20 mg/1)
resulted in a greater number of shoots, however, these grew slower and
took 2 to 3 months vs 6 to 8 weeks to reach a subculturable size. The
reduced stem elongation could have been due to high cytokinin levels
which inhibited shoot elongation. It is also probable that the amounts
of nutrients available to the numerous shoots were limiting and that
culture on a greater volume of medium or more frequent transfers might
hasten growth and multiplication. Ma and Wang (1977) suggested the use
of short term, agitated liquid culture to increase survivabi1ity and
muí ti piication.
Using the micropropagation system shown in Table 4.1.7, one can
calculate that over 11,000 plants could be obtained from 1 shoot-tip in
9.5 months. With 2.5 months additional growth in the greenhouse, these
plantlets would be large enough to be transplanted to larger containers.
A1ternately, a square meter of lighted shelf space could produce 15,-
16,000 SI I microcuttings in 5 to 8 weeks (225 bottles/M X 60-70
microcutting/bottl e).
Although large numbers of shoots could be produced and routinely
subcultured, a 60-80'S survival rate on transfer to soil is still
inefficient. All attempts to root R_. chapmanii in Stage III culture
have failed even though several media were investigated along with a
range of auxin treatments. This differs from reports of 80S rooting in
Sill by Ma and Wang (1977) and Strode et al. (1978). Rhododendron
roots are extremely fine and thread-like and do not appear to grow
easily into agar media.

55
Best rooting and survival of microcuttings of chapmanii
occurred when explants consisting of 3 to 5 shoots were direct-stuck in
rooting medium with at least 30% Canadian peat moss. This agrees with
the findings of Anderson (1978), Lloyd and McCown (1980) and Wong
(1981). Direct rooting of microcuttings vs use of Sill cultures results
in considerable savings in labor since at this stage explants must be
handled individually only once for direct rooting vs twice when Sill
cultures are used (Strode et al., 1979; Wong, 1981).
Microcuttings were rooted in flats with clear plastic covers or
covered with inverted translucent flats to prevent drying. If
covered flats were maintained under light levels greater that 100
-2 -1
pMm sec , excess heating occurred and plantlets declined and fungal
contamination was greater. If microcuttings were initially maintained
-2 -1
under lower light intensities (e.g. 25 pMm s ) for the first 2 weeks
-2 -1
then maintained at 90 pMm s , better survival and rooting occurred.
Placement of microcuttings directly under intermittent mist resulted in
poor rooting and survival and the development of chlorotic leaves due to
either excessive leaching or possibly due to poor root-zone aeration.
The use of fog systems or high humidity tents should be further
investigated.
In this investigation, the use of rooting compounds on
microcuttings resulted in better rooting and survival when a 1000 mg/1
solution of IBA was applied as a 5 second quick-dip. Treatment with the
same concentration of IBA (in talc) resulted in lower survival and
rooting. Wong (1981) mentioned that the use of hormone powder increased
proliferation of roots but not time to root. He also mentioned that the
economic advantages of such treatments were yet to be determined.

56
Poor rooting and survival may be attributed to the small size and
succulence of the microcuttings. Commercial producers of
micropropagated rhododendrons (3riggs, per. com.) use lower cytokinin
levels which result in reduced multiplication rates but these
microcuttings are much larger and hardier. Determination of optimal
multiplication rates should not necessarily be based on optimal shoot
numbers, but must include duration of cycle times, labor and space
requirements, rooting survival rates and duration, as well as
post-transplanting growth rates.
When shoot-clumps containing 3 to 5 shoots are used for rooting,
better survival and rooting occurs, possibly due to reduced handling
shock, greater carbohydrate (or minerals, etc.) reserves within each
propagule, and due to the fact that if one shoot dies, another shoot is
available for replacement. Once rooted and transplanted to containers,
the plants from shoot-clumps form a multi-stem liner which is more
desirable than a single-stem plant.
Root emergence in rooting medium became visible after 3 to 4 weeks,
and plants were well rooted by 6 weeks. After root intiation, the
explants began to produce new leaves which were slightly larger and lesb
succulent. After these new leaves were observed, the plantlets
generally survived transplanting to cell-packs and readily became
adapted to the greenhouse environment. Rooted plantlets were hardened
off by covering with 80% shade cloth for a week, followed by a week with
40% shade, after which all coverings were removed.
Colonization of micropropagated plantlets with the ericoid
mycorrhizae, Pezizella ericae, did not increase survival or growth
responses when plantlets were grown for 12 weeks in the greenhouse.

57
Differences between the media used apparently masked any differences due
to colonization after 12 weeks of growth. Additional growth in 4-1 iter
containers is presently under observation and there appears to be
greater bud break and growth on colonized plantlets 2 weeks after new
growth began. Final growth measurements will be made after a month of
additional growth. Mycorrhizal colonization of Ericaceae species is
known to increase the plants' ability to take up nitrogen and
phosphorous, as well as, tolerance to heavy metal toxicity (Stribley and
Read, 1974, 1975; Duddridge and Read, 1982)
5.2 Taxus floridana
Using cuttings from mixed aged trees, conventional propagation in a
1:1 (v:v) Canadian peatrperlite rooting medium was successful, resulting
in 40-57% rooting in 120 days. Larger cuttings (15-20 cm vs 10 cm)
rooted more quickly and vigorously. This agrees with the findings of
commercial propagators (Keen, 1954). There was an obvious difference in
rooting percentages between plants of different ages. Cuttings from the
Champion tree rooted poorly (0-40%) while cuttings from much younger
trees rooted 25-67%. According to Sargent (1947), 275 annual growth
rings were counted from a 9.5 cm diameter J. floridana trunk section
suggesting that the 16 cm diameter Champion from which the "older"
cuttings were collected was considerably older.
Cuttings from young trees root at high rates. Lower branches of
are commonly observed in the wild to naturally layer when branches
remain in contact with the ground. However, lateral cuttings retain
their plagiotropic growth habit even after 2 years in containers,
generally producing a 15-20 cm tall ground cover. Keen (1954) noted
that for most Taxus species and cultivars, orthotropic shoots are

58
necessary for producing excurrent trees while lateral cuttings generally
produce only spreading shrubs. Keen also indicated that even very large
cuttings (up to 65 cm) can be successfully rooted, resulting in a
salable plant in a shorter time.
Attempts to root in 100% coarse, builder's sand under intermittent
mist resulted in basal rotting , however, rooting often occurred above
the media especially from abaxial side of lateral branches (data not
given). At first it was assumed that the 20% alcohol in the I3A
quick-dip solution damaged the tissue. However, in later experiments
using better drained rooting medium with quick-dip solution, no basal
rotting occurred. Apparently the builders sand used initially was not
as well drained as the sandblasting sand normally used for rooting. The
occurrence of high moisture conditions and poor drainage most likely
caused the observed basal rotting.
Continued growth of cuttings occurred best in well drained media
(1:1:1 v:v:v) course sand:Canadian peat:loamy soil) and under 80% vs 40%
or 60% shade.
Micropropagation of shoot-tips did not result in prolific
stimulation of axillary buds. Using shoot-tip explants from lateral and
orthotropic basal branches, only 2 to 4 axillary shoots elongated when
placed in culture. These shoots appeared in specific positions along
the shoot-tip suggesting that they were preformed and part of the normal
branching pattern of the whole plant. McCown and Amos (1932) reported
successful shoot-tip propagation of Taxus spp. but did not indicate the
numbers of shoots obtained. They also noted in their findings that
gymnosperms are generally more sensitive to low levels of cytokinins.

59
The use of more juvenile tissues such as embryos or seedlings as
explants probably would result in increased shoot numbers. Or. A. M.
El-Nil (per. com., 1982) recommended pretreatment of conifer seedlings
with cytokinin sprays as a more effective means of inducing multiple
shoots from conifers. This treatment was not investigated for Taxus
floridana due to the lack of seedling material although this was a
promising technique with Torreya taxi folia.
Wounded mature Taxus floridana trees in the wild have been observed
to produce masses of short shoots, most with spiral phyllotaxy and
apparently orthotropic growth. Culture of explants from shoots arising
from multiple shoots from "ourls" has been successful for Sequoia
sempervirens (Ball, 1978; Boulay, 1979), and Pinus eldarica (Herrera
and Phillips, 1984). Attempts should be made to obtain permission to
collect seedlings and "burl" material from the wild.
Micropropagation of "embryonic shoots" from swelling buds according
to the techniques of Bonga (1977,1981) was unsuccessful due to high
rates of contamination and the difficulty in excising the small buds
(l-3mm). A few aseptic cultures were established but buds did not
continue to expand and eventually darkened and died.
Culture of microsporangia was also attempted since male plants were
available. These were also very small (2-3mm) and difficult to excise.
Uncontaminated cultures were easily obtained but did not grow in
culture. No megasporangia were available locally and this approach was
not pursued since morphogenesis from immature female cones has been
reported only rarely with cultures of Pi ñus mugo and Pi cea abies (Bonga,
1931).

60
Attempts to overcome seed dormancy by excision of embryos and
culture in liquid medium according to the techniques of LePage-Degivry
(1968,1970,1973abc) were unsuccessful. This technique failed even when
embryos were leached in 2 cycles of liquid culture followed by placement
on media with GA^ or when cultures were chilled for 2 months at 5 C.
Seed availability is very sporatic with reports of other species of
Taxus alternate bearing with +/- 7 year cycles (Schopmeyer, 1974) so
further experiments will be limited.
Seeds of Taxus floridana were given 1 or 2 months warm
stratification before excision as well as treatment with 1 month warm
followed by 2 months cold stratification before excision. None of the
embryos of Taxus floridana germinated. Presumably, they require a
longer warm stratification period than 2 months since embryos did not
respond to leaching treatment. LePage (1968) indicates that the seeds
were collected from mature trees of Taxus baccata but did not indicate
the time period before excision. Schropmeyer (1974) mentioned
differences in duration of warm stratification between species and that
the International Seed Testing Association recommended, in general,
stratification periods for 90-210 days at 16 C, followed by 60-120 days
at 2-5 C.
5.3 Torreya taxi folia
Conventional propagation was successful with 20-563 rooting
from mixed cuttings from mature and juvenile plants. Cuttings from
different aged plants were combined since only a limited number of
cuttings were available. Optimal rooting occurred when 4000-8000 mg/1
I8A was used. Rooted plagiotropic branches may be useful as seed or
pollen sources so that intercrossing of the remaining, limited gene pool

61
could be accomplished by collecting cuttings from as many plants as
practical. Establishing seed nurseries of these cuttings would allow a
practical means of preserving the remaining genetic diversity under
controlled management where environmental stresses could be reduced and
fungicides used more efficiently.
Micropropagation using shoot-tips did not result in mass
proliferation of shoots. Commonly 2 to 4 shoots could be stimulated to
elongate in culture but these appeared to be preformed buds of the
normal branching patterns. These lateral shoots commonly grew
horizonally and then geotropically into the medium. Occasionally these
explants rooted in culture, especially after 4 to 6 months on the same
medium. They however did not survive transfer to soil. The use of
more juvenile tissues such as seedlings and dormant buds located at the
base of the trunk may increase the possibility of stimulating
development of orthotropic shoots. Ball (1978) was successful in
obtaining multiple shoots from "burl" shoots of Sequoia sempervirens,
however, there has only been limited investigation using similar
explants from Torreya due to very limited availability of this material.
The formation of numerous shoots from burls, such as occurs occasionally
with wounded Taxus floridana, has not been observed although
occasionally 3 or 4 ortotrophic shoots have been observed arising from a
common point at the base of a trunk.
Contamination of shoot-tip cultures continues to hamper
investigation. In cooperation with Mr. N. El-Ghol, Plant Pathologist
with the Florida Division of PI ant Industry, 3 systemic fungi were
isolated (Phyl1osticta spp., Colletotrichum gloeosporioides and
Guigmardia sp.). Establishment of stock plants in growth chambers

62
greatly reduced the amount of contamination. Presumably, the
combination of low night temperatures and reduced humidity levels, may
have been less favorable for growth of the pathogens. Recently, the
weekly treatment over 4 weeks of these stock plants with the fungicide,
Zyban, a broad spectrum systemic and contact fungicide, has been
effective in eliminating one of these pathogens, Phyllasticta. Soaking
of shoot-tip explants in fungicide solutions before clorox treatment was
ineffective in reducing contamination. Soaking for 15, 30, 60, 120 and
240 minutes actually increased fungal contamination, possibly due to
increased spread of hyphae. A 24 hour soak in Zyban reduced fungal
contamination to 15%, however, this resulted in an increase in bacterial
contamination.
Cultures of expanding "embryonic shoots" [vernalized shoot buds
with scale leaves removed, according to the techniques of Bonga
(1977,1981)] did not produce multiple shoots. These "embryonic shoots"
were large enough to be easily excised after the beginning of visible
bud enlargement, especially 1 or 2 weeks before actual bud break.
However, all bud cultures were either lost to contamination or were
killed by disinfestation procedures. Fungicide treatment of mature
trees may allow future investigation into this technique.
Culture of micro- and megasporangia was investigated. Although
these explants could be more readily established in culture without high
levels of contamination, none grew. Microsporangia are large and
numerous on male trees, however, these are only available in early
spring and only a few species have been reported to produce multiple
shoots from these structures (Bonga 1977,1981).

63
The pretreatment of seedlings with cytokinin sprays before placing
shoots in culture appears to offer one of the better chances of
obtaining multiple shoots. Seedling stock plants produce multiple
orthotropic basal shoots when sprayed weekly for 4 weeks with
cytokinins, BA or 2-i-P. BA appears to be slighlty more effective.
Following excision, additional orthotropic shoots continued to develop
over at least a several month period. When these shoot-tips and nodal
sections are cultured on media with low cytokinins, numerous axillary
buds and "bud-masses" developed. Further investigations will be
necessary to optimize this technique and to develop entire plantlets.
V

6.0 CONCLUSIONS
The primary objective of this research was to investigate clonal
propagation techniques for 3 endangered native ornamental plants.
Conventional cutting propagation of Torreya and Taxus was investigated
and compared to rnicropropagation using shoot-tip cultures and embryo
culture. Clonal propagation using cuttings was successful for Taxus
floridana and Torreya taxifol ia with maximum rooting of cuttings
collected during winter and treated with ISA. Younger trees rooted at
higher rates than larger and older trees. However, cutting material is
limited by scarcity of plants and plagiotrophic growth when lateral
branches are used. Rooting procedures for Rhododendron chapinanii
cuttings remain undetermined due to limited experimental material.
A very successful rnicropropagation system with commercial potential
has been developed for R. chapmanii. Large numbers of plantlets were
produced from a few shoot-tips and with minimal impact on the remaining
natural populations. It has been estimated that over 11,000 rooted
plantlets could be produced from a single shoot-tip in 1 year.
Colonization of rooted plantlets with the ericoid mycorrhizae, Pezizel 1 a
ericae, did not result in superior survival and growth when grown for 12
weeks in a greenhouse. Colonization of plantlets appears to increase bud
break and growth of over-wintered containerized plantlets, however,
additional time will be necessary to determine if this trend continues.
The present rnicropropagation system was designed to maintain clonal
characteristics.
64

65
Micropropagation techniques were only partially successful for
Taxus floridana and Torreya taxi folia. Shoot-tip cultures and cultured
buds produced only limited numbers of shoots apparently from preformed
axillary meristems. Culture of "embryonic shoots" and micro- and
megasporangia were unsuccessful in stimulating organogenesis. Embryo
culture for both these species was unsuccessful presumably due to
insufficient chilling. Pretreatment of stock plants before in vitro
culture with growth regulators appears to be a promising technique and
deserves further investigation.

66
LIST OF REFERENCES
Abbott, A.J. 1977. Propagating temperate woody species in tissue
culture. Scientific Horticulture 28(4):155-162.
Aitken, J., K.J. Horgan and T.A. Thorpe. 1981. Influence of explant
selection on the shoot-forming capacity of juvenile tissue of
radiata pine. Can. J. For. Res. 11:112-117.
Amos, R.R. and B.H. McCown. 1981. Micropropagation of members of the
coniferae. HortScience 16(3):89(abstract).
Anderson, W.C. 1975. Propagation of rhododendrons by tissue culture.
Part 1. Development of a culture medium for multiplication of
shoots. Proc. Intern. Plant Propagators Soc. 25:129-135.
Anderson, W.C. 1978. Rooting of tissue cultured rhododendrons. Proc.
Intern. Plant Propagators Soc. 28:135-139.
Arnold, S. and D.T. Eriksson. 1978. Induction of adventitious buds
on embryos of Norway spruce grown in vitro. Physiol. Plant.
44:283-287.
Ayensu, E.S. and R.A. DeFilipps. 1978. Endangered and threatened
plants of the United States. Washington, D.C. Smithsonian
Institution and World Wildlife Fund Inc. 403pp.
Ball, E.A. 1978. Cloning in vitro of Sequoia sempervirens. In: K.W.
Hughes, R. Henke and M. Constatin, LEds.J, Propagation of higher
plants through tissue culture, a bridge between research and
application, Tech. Inform. Center, U.S.D.E. Conference 7804111,
p. 259.
Baker, W. 1983. Unpublished. Torreya Management Symposium. Florida
Dept. Nat. Res. Tallahassee.
Bonga, J.M. 1977. Organogenesis in in vitro cultures of embryonic
shoots of Abies balsamea (Balsam~Ti rj . In Vitro 13(1):41-48.
Bonga, J.M. 1980. Plant propagation through tissue culture. In: F.
Sala, B. Parisi, R. Celia and 0. Cuerri, [Eds.], Plant Cell
Cultures: Results and Perspectives. Elsevier Press, pp. 253-264.
Bonga, J.M. 1981. Organogenesis in vitro of tissues from mature
conifers. In Vitro 17:511-518.
Boulay, M. 1979. Multiplication et clonage rapide du Sequoia
sempervirem par la culture in vitro. Etudes et Recherches, AFOCEZ,
Domaine de l'Etancon, 77370 Nangis, France 12:49-55.

67
Briggs, B. and S.M. McCullock. 1983. Progress in micropropagation of
woody plants in the United States and Western Canada. Proc.
Intern. Plant Propagators Soc. 33:239-248.
Brown, C.L. and H.E. Sommer. 1974. An atlas of gymnosperms cultured
in vitro: 1924-1974. GA. For. Res. Council, 271p.
Brown, C.L. and H.E. Sommer. 1977. Bud and root differentiation in
conifer cultures. Tappi 60:72-73.
Chalupa, V. and D.J. Durzan. 1973. Growth and development of resting
buds of conifers in vitro. Can. J. For. Res. 3:196-208.
Chaturvedi, H.C. and G.C. Mitra. 1974. Clonal propagation of citrus
from somatic callus cultures. HortScience 9(2):118-120.
Cheng, T. 1975. Adventitious bud formation in culture of Douglas fir
(Pseudotsuga menziesii, Mirb.) France. Plant Sci. Lett. 5:97-102.
Cheng, T.Y. 1976. Vegetative propagation of Western hemlock (Tsuga
heterophyl1 a) through tissue culture. Plant and Cell Physiol.
Cheng, T.Y. 1978. Clonal propagation of woody plant species through
tissue culture techniques. Proc. Intern. Plant Propagators Soc.
28:139-155.
Chevre, A-Marie, S.S. Gill, A. Mouras and G. Salesses. 1983. In vi tro
vegetative multiplication of chestnut. J. Hort. Sci. 53(17T23-29.
Coleman, W.K. and T.A. Thorpe. 1977. In yitro culture of Western
red-cedar (Thuja plicata, Donn.) I. Pi antiet formation. Bot.
Gaz. 138:29ST1OT.
David, A., H. David and T. Mateille. 1982. In vitro adventitious
budding on Pinus pinaster cotyledons and needles. Physiol. Plant.
56:102-107.
Debergh, P.C. and L.J. Maene. 1981. A scheme for commercial propagation
of ornamental plants by tissue culture. Scientia Hort. 14:335-345.
Duddridge, J. and D.J. Read. 1982. An ultrastructural analysis of
the development of mycorrhizas in Rhododendron ponticum. Can. J.
Bot. 60:2345-2356.
Durzan, D.J. 1980. Prospects for the mass propagation of economically
important conifers by cell and tissue cultures. In: Developments
in plant biology, Vol . 5, Elsevier/N. Holland, Biomed. Press,
pp. 283-288.
Durzan, D.J. and R.A. Campbell. 1974. Prospects for the mass
production of improved stock of forest trees by cell and tissue
culture. Can. J. Forest Res. 4:151-174.

68
Durzan, D.J. and S.M. Lopushanski. 1975. Propagation of American elm
via cell suspension cultures. Can. J. For. Res. 5:273-277.
Englander, L. 1981. Endomycorrhizae by septate fungi. In: N.C.
Schenck, [Ed.], Methods and principles of mycorrhizal research.
American Phytopath. Soc., St. Paul, Minn. pp. 11-12.
Federal Register. 1983. Endangered and threatened wildlife and plants
Proposal to determine Torreya taxi fol ia (Florida torreya) as an
endangered species. Fed. Reg. 48(68):15168-15171.
Florida Statutes. 1978. Preservation of native trees, shrubs and
plants, s. 581.185,581.186, 581.187. and 581.211. In:
I.F.A.S., Urban Gardener 3(3).
Giovanettii, M. and B. Mosse. 1980. An evaluation of techniques for
measuring vescicular-arbuscular mycorrhizal infection in roots.
Mew Phytol. 84:489-500.
Godfrey, R.K. and H. Kurz. 1962. The Florida Torreya destined for
extinction. Science 136:900-902.
Granger, R.L., C. Plenchette and J.A. Fortin. 1983. Effect of a
vesicular arbuscular (VA) endomycorrhizal fungus (Glomus epigaeum)
on the growth and leaf mineral content of two apple clones
propagated in vitro. Can. 'J . PI. Sci. 63 (2): 551-555.
Gray, H. 1978. Chemical aids in rooting rhododendrons and IIex
cuttings. Proc. Intern. Plant Propagators Soc. 28:517-518.
Haines, R.J. and R.A. deFossard. 1977. Propagation of Hoop Pine
(Araucaria cunninghamii, Act.) by organ culture. In: G. Boesman,
[ChrmanJ, Symposium on Tissue Culture for Horticultural Purposes,
pp. 297-302.
Hartman, H.T. and D.E. Kester. 1975. Plant propagation principles
and practices. 3rd ed. Prentice Hall, Englewood Cliffs, N.J.
662pp.
Hearne, D.A. 1982. Preliminary report on a technique which provides a
"maturity factor" for trees grown in tissue culture. Proc.
Intern. Plant Propagators Soc. 32:109-113.
Herrera, H.G. and G.C. Phillips. 1984. Tissue culture of Pinus
cldorica, Med. Sym. Prop. Higher Plants Through Tissue Culture
III. Tabstract/poster), Knoxville, Tenn.
House Document 95-41 (1975) Report on endangered and threatened plant
species of the United States. Washington, D.C., Smithsonian
Institute. 200pp.
Hu, C.Y. and I.M. Sussex. 1971. In vitro development of embryoids on
cotyledons of Ilex aquifolium. Phytomorphology 21:103-107.

69
Isikawa, H. 1974. In vitro formation of adventitious buds and roots on
the hypocatyl of Cryptomeria jeponica. Bot. Mag. Tokyo 87:73-77.
Jansson, E. and C.H. Bornman. 1983. Morphogenesis in dormant
embryonic shoots of Picea abies-influence of the crown and cold
treatment. Physiol. Plant"! !T9(l):l-8.
Keen, R.A. 1954. The propagation of taxus-a review. Proc. Intern.
Plant Propagators Soc. 4:63-69.
Keys, R.N. and F.C. Cech. 1981. Plantlet formation in American
chestnut embryonic tissue in vitro. Proc. 2nd North Central Tree
Improve Conf., Lincoln, Nebraska, Aug. 5-7, 1981, p. 189-194.
Keys, R.N. and F.C. Cech. 1982. Propagation of American chestnut in
vitro. Proc USDA For. Ser., Amer. Chest. Cooperators Meeting,
Jan 5-7, 1982, Morgantown, W. Va., pp. 106-110.
Konar, R.N. and V.P. Oberoi. 1965. In vitro development of embryoids
on the cotyledons of Biota orientalis. Phytomorphology 15:137-140.
lakshmi Sita, G., J. Shobha and C.S. Vaidyanathan. 1980. Regeneration
of whole plants by embryogenesis from cell suspention cultures of
sandal wood. Curr. Sci. 49:196-197.
Larkin, P. J. and W. R. Scowcroft. 1981. Somaclonal variation - a novel
source of variability from cell cultures for plant improvement.
Theor. Appl. Genet. 60:197-214.
LaRue, C.D. 1948. Regeneration in the megagametophyte of Zamia
floridana. Bull. Torrey Bot. Club 75:597-603.
LaRue, C.D. 1954. Studies of growth and regeneration of gametophytes
and sporophytes of gymnosperms. Brookhaven Sym. Biol. 6:187-208.
Lazarte, J.E. 1981. Woody tissue culture research. Proc. Intern.
Plant Propagators Soc. 31:649-655.
LePage, M.T. 1968. Mise en evidence d'une dormance associee a une
immaturite de l'embryon chez Taxus baccata L. C.R. Acad. Sci.
Paris, 266D:1028-1030.
LePage-Dsgivry, M.T. 1970. Acid abcissique et dormance chez las
embryons le Taxus baccata L. C.R. Acad. Sci. Paris
2710:482-484.
LaPage-Degivry, M.T. 1973a. E'tude en cluture in vitro de la dormance
embryonnaire chez Taxus baccata L. Biol. Plant. 15:264-269.
LePage-Degivry, M.T. 1973b. Influence de l'acide abscissique sur le
development des ernbryous de Taxus baccata L. cutives j_n vi tro Z.
Pflanzenphysiol. 70:406-413.

70
LePage-Degivry, M.T. 1973c. Intervention d'un inhibiteur 1 ie dans la
dormance embryonndire de Taxus baccata L. C.R. Acad. Sci. Paris
27 7D:177-180.
LePage-Degivry, M.T. and G. Garello. 1973. La dormance embryonnaire
chez Taxus baccata: Influence de la composition du milieu guide
sur 11induction de la germination. Physiol. Plant. 29:204-207.
Linsmaier, E.M. andJ. Skoog. 1965. Organic growth factor
requirements of tobacco tissue cultures. Physiol. Plant
13:100-127.
Litz, R.E. 1984. In vitro responses of adventitious embryos of two
polyernbryonic Eugeni a species. 19 (5 ): 720-722.
Liu, J.R., K.C. Sink and F.G. Dennis. 1983. Plant regeneration from
apple seedling explants and callus cultures. Plant Cell, and
Tissue Organ Culture 2:293-304.
Lloyd, G. and B. McCown. 1980. Commercially-feasible micropropagation
of mountain laurel, Kalmia latifolia, by use of shoot-tip culture.
Proc. Intern. Plant Propagators Soc. 30:421-427.
Loreti, F. and S. Mozini. 1982. Mass propagation of fruit trees in
Italy by tissue culture: present status and perspectives. Proc.
Intern. Plant Propagators Soc. 32:283-290.
Lyrene, P.M. 1981. Juvenility and production of fast-rooting cuttings
from blueberry shoot cultures. J. Amer. Soc. Hort. Sci.
106(3):396-398.
Ma, S.S. and S.0. Wang. 1977. Clonal multiplication of azaleas
through tissue culture. Acta Hortic. (The Hague) 78:209-215.
Marx, D.H. and D.S. Kenny. 1981. Production of ectomycorrhizal
fungus inoculum. In: N.C. Schenck, [Ed.], Methods and
principles of mycorrhizal research. American Phytopath. Soc.,
St. Paul, Minn, p.131-146.
McComb, J.A. 1978. Clonal propagation of woody plants using tissue
culture, with special reference to apples. Proc. Intern Plant
Propagators Soc. 28:413-426.
McCown, B.H. and R. Amos. 1982. A survey of the response of
gymnosperms to microculture. HortScience 17(3):489 (abstract).
McCown, B.H. and G. Lloyd. 1981. Woody plant medium (WPM)- A mineral
nutrient formulation for microculture of woody plant species.
HortScience 16(3):89 (abstract).
McCown, B.H. and G.8. Lloyd. 1983. A survey of the response of
Rhododendron to in vi tro culture. Plant Cell , Tissue and Organ
Cul ture 2(1): 77-HIT.

71
Meyer, M.M. 1982. I_n vi tro propagation of Rhododendron catawbiense
from floral buds. HortScience 17(6) :891-892.
Moore-Parkhurst, S. and L. Englander. 1981. A method for the
synthesis of mycorrhizal association between Pezizella ericae and
Rhododendron maximum seedlings growing in a defined medium.
Mycologia 73(5 ):994-997.
Morandi, D., S. Gianininazzi and V. Gianininazzi-Pearson. 1979.
Interet de 1'endomycorrhization dans la^ reprise et la croissence du
Framboisier issu de multiplication ve'ge'tati ve in vitro. Ann.
Amelior. Plantes 29(6):623-630.
Mott, R.L. and H.V. Amerson. 1981. A tissue culture process for the
clonal production of loblolly pine plantlets. N.C. Agr. Res.
Ser. Tech. Bull. 271. 14pp.
Mott, R.L., R.H. Smeltzer, A. Mehra-Palta and B.J. Zobel. 1977.
Production of forest trees by tissue culture. Tappi 60(6):62-64.
Murashige, T. 1974. Plant propagation through tissue culture. Ann.
Rev. Plant Physiol. 25:135-166.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant.
15:473-497.
Norstog, K. 1965. Induction of apogamy in megagametophytes of Zamia
integrifolia. Amer. J. Bot. 52:993-999.
Norstog, K. and E. Rhamstine. 1967. Isolation and culture of haploid
and diploid cycad tissues. Phytomorphology 17:374-381.
Patel, R.V. and G.P. Berlyn. 1982. Genetic instability of multiple
buds of Pinus coulteri regenerated from tissue culture. Can. J.
For. Res. 12:93-101.
Pearson, V. and D.J. Read. 1973. The biology of mycorrhiza in the
Ericaceae. I. The isolation of the endophyte and synthesis of
mycorrhizae in aseptic culture. New Phytol. 72:371-379.
Pons, F., V. Gianinazzi-Pearson, S. Gianinazzi andJ.C. Navatel.
1983. Studies of VA mycorrhizae in vitro: mycorrhizal synthesis
of axenically propagated wild cherry (Prunus avium L.) plants.
Plant and Soil 71:217-221.
Ramawat, K.G., H.C. Asya. 1976. Growth and morphogenesis in callus
cultures of Ephedra gerardiana. Phytomorphology 26:395-403.
Rancillac, M.M. Faye and A. David. 1982. In vitro rooting of
cloned shoots in Pinus pinaster. Physiol. Plant. 56:97-101.
Read, D.J. 1983. The biology of mycorrhiza in the Ericales. Can. J.
Bot. 61:985-1004.

72
Reilly, K.J. and J. Washer. 1977. Vegetative propagation of radiata
pine by tissue culture: Plantlet formation from embryonic tissue.
N.Z.J. For. Sci. 7(2 ):199-206.
Rhodes, H.L.J. 1968. Juvenility in relation to adventitious bud and
root initiation in woody plants. Proc. Intern. Plant Propagators
Soc. 18:252-254.
Sargent, C.P. 1947. Silva of North America. Vol. X. Murry Print Co.
Cambridge, Mass.
Schopmeyer, C.S. 1974. Seeds of woody plants in the United States
Agriculture Handbook No. 450. Forest Service, U.S. Dept, of
Agriculture. 383pp.
Simon, R.W. 1983. Chapman's rhododendron recovery plan. U.S. Fish
and Wildlife Service. Atlanta, Ga., 41pp.
Sommer, H.E. and C.L. Brown. 1980. Embryogenesis in tissue cultures
of sweetgum. For. Sci. 26:257-260.
Sommer, H.E., C.L. Brown and P.P. Kormanik. 1975. Differentiation of
plantlets in long leaf pine (Pinus palustris, Mill.) tissue
cultured in vitro. Bot. Gaz. 136(21:196-200.
Stribley, D.P. and D.J. Read. 1974. The biology of mycorrhiza in th^
Ericeae. IV. The effect of mycorrhiza! infection on uptake of N
from labeled soil by Vaccinum macrocarpum Ail. New Phytol.
73:1149-1156.
Stribley, D.P. and D.J. Read. 1975. Some nutritional aspects of the
biology of ericaceous mycorrhizas. New Phytol. 73:1149-1163.
Strode, R. 1979. Commercial micropropagation of rhododendrons. Proc.
Intern Plant Propagators Soc. 29:439-442.
Styer, D.J. and C.K. Chin. 1983. Meristem and shoot-tip culture for
propagation, pathogen elimination and germplasm preservation.
Hort. Rev. 4:221-277.
Tulecke, W. 1959. The pollen cultures of C.D. LaRue: A tissue from
the pollen of Taxus. Bull. Torrey Bot. Club. 86:283-289.
Vieitez, A.M. and E. Vieitez. 1980, Plantlet formation from
embryonic tissue of chestnut grown in vitro. Physiol. Plant.
50:127-130.
Vieitez, A.M. and M.L. Vieitez. 1980. Culture of chestnut shoots
from buds i n vi tro. J. Hort. Sci. 55(1 ):83-84.
Wang, P.J. and C.Y. Hu. 1984. In vitro cloning of the decidious
timber tree Sassafras randaiense. Z. Pflanzenphysiol.
113(4):331-375T

73
Ward, D.B. 1978. Plants. In: P.C. Pritchard, [Ed.], Rare and
endangered biota of Florida, Vol. 5, University Presses of
Florida. Gainesville, FL. 175p.
Wilkins, C. P., J. L. Cabrera and J. H. Dodds. 1985. Tissue culture
propagation of trees. Outlook on Agriculture 14(1):2-13.
Winton, L. 1970. Shoot and tree production from aspen cultures. Amer.
J. Bot. 57:904-909.
Winton, L. 1978. Morphogenesis in clonal propagation of woody plants.
In: T.A. Thorpe, [Ed.], Frontiers of plant tissue culture.
University of Calgary Press, Calgary, pp.419-426.
Winton, L.L. and S.A. Verhagen. 1977. Shoots from Douglas fir
cultures. Can. J. Bot. 55:1246-1250.
Wochok, Z.S. 1981. The role of tissue culture in preserving threatened
and endangered plant species. Biological Conservation
20(1981 ):83-89.
Wochok, Z.S. and M.A. El-Nil. 1977. Conifer tissue culture. Proc.
Intern. Plant Propagators Soc. 27:131-139.
Wong, S. 1982. Direct rooting of tissue-cultured rhododendrons into an
artificial soil mix. Proc. Intern. Plant Prop. Soc. 31:36-39.
Zenkteler, M.A. and I. Guzowska. 1970. Cytological studies on
regenerating mature female gametophyte of Taxus baccata L. and
mature endosperm of Tilia piatyphyl los Scop, in i_n vitro culture.
Acta. Soc. Bot. Poloniae 39:161-173.
Zimmerman, R.H. 1978. Tissue culture of fruit trees and other fruit
plants. Proc. Intern. Plant Propagators Soc. 28:539-545.

BIOGRAPHICAL SKETCH
Lee Roy Barnes, Jr., was born in Durham, North Carolina. Following
graduation from high school, he attended and graduated from North
Carolina State University with a double major in biological science and
botany in May 1976. Transferring to the Department of Horticultural
Science, he completed the Master of Science degree in December, 1978.
His thesis was titled “In vitro propagation of watermelon" (Drs. Fred
Cochran and Ralph Mott, co-chairmen). Following a two year leave of
work experience and travel, he entered the University of Florida,
Department of Ornamental Horticulture, in the Fall of 1981 interested in
conventional propagation and micropropagation of native plants,
especially rare and endangered species.
74

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Charles R. Johnsopf, Chairman
Professor or Ornamental
Horticulture
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Thomas J. Sheehan
Professor of Ornamental
Horticulture
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
V
Indra K. Vasil
Graduate Research Professor
of Botany

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
KJBsl/
//
Mike J. Young
Associate Pro
Frui t 'GVops
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Phi 1osophy.
August 1985
Dean, College of Agriculture
Dean, Graduate School



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