SOMATIC EMBRYOS AS A SYNTHETIC SEEDING SYSTEM
USING FLUID DRILLING TECHNIQUES FOR DIRECT SOWING OF
SWEET POTATO IPOMOEAA BATATAS POIR.)
JONATHAN RICHARD SCHULTHEIS
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
I wish to thank Dr. Daniel J. Cantliffe for his full support during
my studies at the University of Florida. His help, encouragement, and
guidance during my graduate program were greatly appreciated. Beyond our
professional relationship, I value him as my good friend.
Special thanks is also extended to Dr. Indra K. Vasil for his
instruction and helpful suggestions during my program. I would also like
to thank Dr. Thomas E. Humphreys and Dr. Francis W. Zettler for their
encouragement and valued advice during this study. I gratefully
acknowledge Dr. Herbert H. Bryan for his help and support, particularly
in the Homestead field study.
Thanks are conveyed to faculty, staff, and students in the vegetable
crops department for their friendship and help throughout my graduate
studies. Specifically, I want to thank Marie Bieniek for her technical
assistance. Special thanks are conveyed to Dr. Raymond P. Chee for his
valuable input and suggestions during my research program.
My gratitude for the financial support during my program is extended
to the Gas Research Institute.
Finally, I want to thank my family for their constant love, support,
and prayers during my entire graduate program. Appreciation and a special
thanks is expressed to my wife, Nancy, and my daughter, Katharine Anne
"Katie," who endured many sacrifices yet provided me with their continuous
help, love, and encouragement during my graduate program.
TABLE OF CONTENTS
ABSTRACT .......................................................... v
1 INTRODUCTION................................................ 1
2 LITERATURE REVIEW............................................ 5
Botanical Classification..................................... 5
Seed Characteristics......................................... 5
Environmental Requirements for Optimum Sweet Potato Growth.. 7
Root Characterization and Development....................... 8
Cultural Management Practices and Their Effects on Yield..... 10
Tissue Culture............................................... 16
Somatic Embryogenesis ....................................... 17
Somaclonal Variation........................................ 39
Sweet Potato Tissue Culture.................................. 41
Delivery Methods for Somatic Embryos......................... 43
Fluid Drilling............................................... 49
Soil Covers.................................................. 54
Summary ..................................................... 55
3 OPTIMIZING SWEET POTATO ROOT AND PLANT FORMATION BY
SELECTION OF PROPER DEVELOPMENTAL STAGE, EMBRYO SIZE,
AND GEL TYPE FOR FLUIDIZED SOWING........................... 57
Introduction.............................. .................. 57
Materials and Methods ....................................... 58
Results................................... .................. 62
Discussion................................ .................. 70
Summary ................. .................................... 74
4 OPTIMIZING ROOT AND PLANT PRODUCTION FROM SOMATIC
EMBRYOS OF SWEET POTATO WITH NUTRIENT, CARBOHYDRATE,
AND HORMONE AMENDMENTS TO HYDROXYETHYL CELLULOSE GEL
FOR FLUIDIZED SOWING......................................... 77
Introduction.............................. .................. 77
Materials and Methods ....................................... 80
Results and Discussion....................................... 87
Summary ..................................................... 109
5 EARLY PLANT GROWTH AND YIELDS OF SWEET POTATO GROWN
FROM SEED, CUTTINGS, AND SOMATIC EMBRYOS...........
Materials and Methods ..............................
A APPENDIX OF DATA TABLES ...................................... 157
B APPENDIX OF FIGURES.......................................... 164
LITERATURE CITED................................................... 175
BIOGRAPHICAL SKETCH................................................ 194
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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
SOMATIC EMBRYOS AS A SYNTHETIC SEEDING SYSTEM
USING FLUID DRILLING TECHNIQUES FOR DIRECT SOWING OF
SWEET POTATO IPOMOEAA BATATAS POIR.)
JONATHAN RICHARD SCHULTHEIS
Chairman: Daniel J. Cantliffe
Major Department: Horticultural Science
Lack of seed production is a major limiting factor for large-scale
production of many promising energy crops such as sweet potato. Somatic
embryos have great potential use as propagules in a synthetic seeding
system such as fluid drilling. The critical factors which affected the
rate, uniformity, and percentage embryo-to-plant formation in a
fluidized gel were examined.
Hydroxyethyl cellulose gel amended with Murashige and Skoog (MS)
basal medium was a compatible gel carrier for fluid drilling somatic
embryos and supported plant formation. Embryos at the elongated torpedo
developmental stage formed roots earlier and consistently produced a
greater percentage of plants than embryos at the cotyledonary and
torpedo stages. Embryo conversion to plants was doubled regardless of
embryo developmental stage when matured on agar-solidified basal medium
for 16 days rather than 25.
In gel amended with Hoagland, Gamborg, White, or MS media, the
most plant formation was obtained with Gamborg or MS medium. The
optimum concentrations of MS media for plant formation (40%) were one-
half and full strength. Micro and macro salts were essential for
optimum plant development, while vitamins were not.
No root or plant formation occurred in gel without carbohydrate or
with galactose. The best plant formation was obtained with fructose,
glucose or maltose. The optimum fructose and sucrose concentrations for
both root and plant development ranged from 23 to 93 mM. Plant dry
weight and nodes per plant increased with increasing carbohydrate
Vegetative growth and yield of storage roots of propagules derived
from somatic embryos, stock plants and/or zygotic seed were compared in
field experiments. More nodes per plant and greater vine length were
consistently obtained from stock plants and zygotic seeds versus somatic
embryos. Total yield and root size were greater from plants derived
from stock plants than somatic embryos. Under favorable environmental
conditions and extended growing seasons, plants from somatic embryos
produced normal large-sized roots.
The determination of a gel and those additives conducive for plant
growth is a key step towards commercial synthetic seed technology via
fluid drilling for vegetatively propagated species such as sweet potato.
Sweet potato (Ipomoea batatas (L.) Poir.) ranks seventh among
crops grown for food and tenth among all crops grown world-wide (FAO
Production Yearbook, 1986). Besides food production, sweet potato has
good potential as a biomass crop for ethanol and methane (Smith and
Frank, 1984.) The accumulated carbohydrate in the roots can be
efficiently digested and converted into ethanol or methane gas and used
for energy (Smith et al., 1987).
Methane yield conversion efficiencies from various groups of plant
species were compared; freshwater aquatics, forage and grasses, roots
and tubers, and saltwater species (Smith et al., 1987.) Roots and
tubers produced at least 36% greater yields than the other three groups
when equivalent plant quantities for each group were compared. Sweet
potato roots are not only efficiently converted to energy, but the crop
has added benefits. It can be grown with minimal energy inputs, has few
disease or insect pests (Bouwkamp, 1985), and can be produced in a short
130 day growing season (O'Hair et al., 1987). Two sweet potato crops
can be harvested annually in tropical growing regions, while in
temperate zones, cool season biomass crops such as turnip can follow a
sweet potato crop.
Lack of seed production is a major limiting factor for large-scale
production for many of the promising energy crop species such as sweet
potato. Thus, vegetative propagation is the plant establishment method
currently available for commercial plant production. Problems with this
planting method include the following: 1) length of time needed for
plants to survive stressful conditions, 2) maintenance and production of
disease-free plants, 3) spacial requirements for large-scale production,
and 4) labor costs to maintain and plant propagules (Cantliffe et al.,
1987). Direct seeding of asexually propagated biomass crops would make
mass plantings of these specie types much more cost-effective.
Unfortunately, in sweet potato, few or no seeds are ever produced since
they are self-incompatible, and those seeds which are produced result in
plants which genetically differ from the mother plant due to their
Micropropagation of high-value crops [i.e., ornamentals (Holdgate,
1977), citrus (Speigel-Roy and Vardi, 1984), pineapple (Rangan, 1984),
strawberry (Boxus et al., 1984), etc.] has resulted in successful
regeneration of plants. The use of a micropropagation system has the
advantage of producing plantlets which are fairly genetically uniform;
however, this system is limited to small-scale use since the
multiplication of propagules is relatively low and the plantlets must be
acclimated prior to planting. This leads to a relatively high cost per
plantlet which would be prohibitive for large-acreage crops.
In order for a tissue culture propagation method to be applicable
for large-acreage plantings, the following requirements would have to be
met. The propagation method must rapidly produce millions of propagules
and propagule cost must be competitive economically with conventional
propagation methods (Murashige, 1977).
Somatic embryogenesis is one such tissue culture method that could
be used to clone greater numbers of propagules in less space and shorter
times compared to normal seed or vegetative stock production. Some
problems must be overcome if somatic embryogenesis is to be implemented
as a synthetic seeding system. These problems include obtaining somatic
embryos which are singulated, which are uniformly mature, uniform in
size, and which grow rapidly into plants. Handling and planting of
these tissue-cultured propagules could then be patterned after
conventional seeding techniques.
Several delivery methods have been proposed for direct-field
synthetic seeding of somatic embryos. They are as follows: 1)
encapsulation of singulated embryos in an alginate gel capsule and
planting with a conventional drill (Redenbaugh et al., 1984, 1986; Jeon
et al., 1986), 2) embryo desiccation, then seeding with a conventional
drill (Gray et al., 1987), 3) simultaneous desiccation of embryos in a
water-soluble resin and planted as a wafer or on a seed tape (Kitto and
Janick, 1984ab), and 4) gel seeding embryos with fluid drilling
equipment (Drew, 1979, Baker, 1985).
Encapsulation research and development is in its infancy, cost per
propagule is high, and the emergence of the seedling is inhibited by the
capsule (Redenbaugh et al., 1987b). Desiccation has also been a problem
with this method since embryo injury and reduced plant formation has
occurred once the embryos are imbibed after being desiccated. Fluid
drilling, in comparison, is a developed planting technology that has
been used for planting zygotic seed for over 20 years (Gray, 1981.)
With fluid drilling, somatic embryos could be removed from tissue
culture without growth arrest or desiccation, and planted 'as is' or at
an advanced germinated stage. The gel carrier would not only protect
the somatic embryo but could act as an endosperm, since it can be
amended with nutrients, hormones, beneficial microbes, and other growth
factors. Various pesticides could also be incorporated into the gel for
added protection against disease or insects. Few studies have dealt
with fluid drilling of somatic embryos (Baker, 1985). Beyond this, few
field studies have compared yields of plants vegetatively propagated or
obtained from somatic embryos.
The objectives of this research were twofold. One part involved
determining the gel and additives that would facilitate the most rapid,
uniform, and complete embryo-to-plant formation when seeding with a
fluidized gel. Secondly, early plant growth and root yield comparisons
were made when plants were derived from original plant stock or derived
through somatic embryogenesis. Sweet potato was chosen for these
investigations because of its high potential for biomass production.
Sweet potato (Ipomoea batatas (L.) Poir.) is a member of the
Convolvulaceae (morning glory) family and is usually grown for its
edible roots. Sweet potato is often confused with yam since both
storage organs look and taste similar. Yams, however, belong to the
genus Dioscorea, (family Dioscoreaceae) are monocots, diploid (2n=20),
and storage organs are tubers whereas sweet potatoes are dicots,
hexaploid (2n-90), and the storage organs are botanically classified as
roots (Wilson and Collins, 1988). The literature often confuses the
issue by incorrectly calling the sweet potato's storage organ a tuber
Sweet potato plants generally do not produce seeds since most
cultivars are self-incompatible (Hernandez and Miller, 1964; Wang, 1964;
Onwueme, 1978). Those plants which are produced from seeds are highly
heterozygous and differ genetically from the mother plant. Seeds mature
approximately 40-60 days after anthesis (Matsuo et al., 1984), and are
about 3 mm in length with one surface flattened and the other convex
(Hayward, 1938; Onwueme, 1978). They also have hard, nearly water-
impervious seed coats. Within the seed coat lies the embryo composed of
a hypocotyl connected to two large, tightly folded cotyledons. Seeds
are albuminous and the endosperm envelops the embryo. Scarification
either with sulfuric acid for about 45 minutes or by mechanically
clipping part of the seed coat is necessary for optimum germination,
which may reach approximately 90 percent (Jones and Dukes, 1982).
Germination usually occurs within one to two days, while plant emergence
takes an additional day or two under favorable growing conditions
The analysis of metabolites in seeds can give clues as to the
importance of certain substances critical for the growth, development,
and germination of a seed. Several endogenous GAs were identified by
Matsuo et al. (1984) in both immature (15-30 days post-anthesis) and
mature (40-60 days post-anthesis) sweet potato seeds. In immature seeds
(15-30 days post-anthesis), GA19 and GA23 were the major GAs identified.
Also, abscisic acid (ABA) was found in immature seeds using gas-liquid
chromatography and mass spectrometry analysis. In mature seeds (40-60
days post-anthesis), GA3, 5, 8, 17, 19, 20, 23, and 44 were identified in
the seed tissue, with GA19 and GA23 the most concentrated at 200 and 160
pg/kg fresh weight, respectively. The authors determined that GA levels
increased in the seed up to 15 to 20 days after anthesis, then decreased
as the seed matured and dried. They suggested that the rapid cotyledon
growth 10 to 15 days after pollination might be attributed to the
increased GA levels found during this phase of seed development.
Gibberellin-like substances have been measured in mature seeds of
several families and species (Murakami, 1959). In particular, the
Convolvulaceae family had high levels of GA3, while specifically,
Ipomoea batatas had the highest concentrations (90 pg gibberellin
equivalents/100 g fresh weight in mature seed) of those species
evaluated in the family. Thus, GA appears to be of particular
importance in the development of sweet potato seed.
Environmental Requirements for Optimum Sweet Potato Growth
Sweet potatoes require a growing season that has at least four to
five frost-free months to produce ample-sized storage roots. Warm
conditions are required since temperatures at or below 15*C resulted in
little or no plant growth (Pierce, 1987). Above 15*C, Harter and
Whitney (1927) determined plant growth rates continued to increase up to
a temperature of 35*C, while Pierce (1987) reported optimum growth
occurred at air temperatures of 29*C.
The crop grows best in well-aerated, sandy-type soils, with a pH
ranging between 5 and 7.8 (O'Hair, 1984). However, good yields of 17.9
and 17.0 MT/ha have been obtained at soil pH values of 4.3 and 4.6
(Abruna et al., 1978). Soil aeration, when limiting, can inhibit
storage root formation and development. Wilson (1970) reported lack of
storage root production when sweet potatoes were grown under waterlogged
Sweet potatoes will grow and yield well under dry growing
conditions. Yields of 12.5 MT/ha, 15.0 MT/ha, and 19.7 MT/ha in seasons
which had only 10.1, 6.9, and 9.7 cm rainfall, respectively were
obtained in three separate investigations (Lamberth, 1957 cited in
Bouwkamp, 1985; Carpena et al., 1977; Villareal et al., 1979). In
several studies, supplemental irrigation improved yields and/or root
quality (root shape, soluble solids etc.) (Hernandez et al., 1965;
Jones, 1961; Constantin et al., 1974). Onwueme (1978) reported that
sweet potatoes grow best with an average rainfall of 50 cm over a
growing season. Moisture levels are particularly critical during plant
establishment (Bouwkamp, 1985; Godfrey-Sam-Aggrey, 1974; Anon., 1940).
Hernandez et. al. (1965) and Edmond and Amerman (1971) reported that
yields were most reduced when drought conditions occurred the first 40
days after transplanting.
Root Characterization and Development
Although sweet potato is grown extensively throughout the world,
the controlling mechanisms in storage root formation are poorly
understood. Most information dealing with this topic is based on
environmental and anatomical studies.
Root Origin. Type, and Size
Some extensive sweet potato root classification studies were
conducted by Wilson (1970,1982) and Kays (1985). Wilson has classified
roots into ten different groups based on morphology and origin. A
simpler classification scheme was proposed by Kays and will be briefly
described. Based on origin, Kays placed roots into two groups: 1)
adventitious roots, which emanate from underground stems, transplants,
or root pieces, and 2) lateral roots, which arise from preexisting
roots. Early in development young adventitious roots can be separated
into "thin" or "thick" roots, the former typically arising from the stem
internode, the latter from the stem node.
Anatomically, roots can be identified early in their development
by vascular arrangement. Pentarch and hexarch vascular arrangements are
destined to become storage roots while non-storage roots, which
typically are from "thin" roots, have a tetrarch arrangement. According
to Hahn and Hoyzo (1984), Togari reported that the number of storage
roots is determined the first 30 days after planting, which suggests
that environmental conditions during this period are critical for
obtaining maximum yields.
Young "thick" roots principally produce storage and pencil roots
(Kays, 1985). Storage roots are greater than 15 mm in diameter while
pencil roots, sometimes derived from "thin" roots, are 5 to 15 mm in
diameter. Primary fibrous roots typically arise from "thin" roots and
are less than 5 mm in diameter. The lateral roots of sweet potato arise
from preexisting roots and function as a nutrient and water-absorbing
system for the plant.
The vascular cambium and anomalous cambium are the two major
regions in the root which promote root enlargement through meristem
activity (Wilson and Lowe, 1973). Root enlargement from "thick" roots
occurs if environmental conditions are favorable; otherwise,
lignification of the growing region occurs, halting storage root
development with subsequent formation of non-storage root types.
Environmental Effects on Root Formation
Light. Several environmental factors influence storage root
formation and yields of sweet potato. Light can affect storage root
formation in two primary ways. Short day lengths promote storage root
formation (Kim, 1961) Roots exposed to light do not enlarge unless
covered with soil (Hahn and Hoyzo, 1984).
Oxeyen. Oxygen concentrations in the soil impact on the type of
roots which develop. Chua and Kays (1981) determined that a 2.5% oxygen
concentration in the root zone resulted in 12% dry weight in storage
roots, while a 21% oxygen concentration had 89% dry weight distributed
in storage roots. Dry, compacted soils also were reported to inhibit
storage root formation (Togari, 1950, cited in Kays, 1985). This can
likely be attributed to reduced oxygen concentrations in the soil as was
reported above by Chua and Kays (1981).
Fertilization. Fertilization, particularly with nitrogen (N) and
potassium (K), can influence storage root formation. High levels of N
increased vine growth at the expense of storage roots (Stino and Lashin,
1953). Large quantities of soil N reduced meristematic activity and
increased lignification which favored production of non-storage roots
(Togari, 1950, cited in Hahn and Hozyo, 1984). Potassium has
consistently increased yields and storage root formation (Bouwkamp,
1985) and will be discussed at length later.
Temperature. Sweet potato storage root yields are highly
temperature-dependent. The effect of temperature on root formation was
tested by varying the temperature in the root zone at 10*, 15*, 20,
25*, 30*, and 35C (Spence and Humphries, 1972). Storage root formation
was greatest at 25*C, while fibrous root formation was similar
regardless of temperature when tested above 15C. Thermoperiodism may
also be important in storage root formation. Daytime temperatures of
29C and nighttime temperatures of 21C increased storage root
development compared with a constant temperature of 29*C, which resulted
in greater vine than storage root growth (Kim, 1961).
Cultural Management Practices and Their Effects on Yield
Sweet potatoes will yield under conditions of low soil fertility;
however, root yields generally are improved by supplemental fertilizer
applicationss, particularly with K (Bouwkamp, 1985). Speights and
Paterson (1969) reported yields were increased 4.2 MT/ha with an
application of 70 kg/ha of K, while yields were further increased 2.2
MT/ha with an additional 70 kg/ha. Jones et al. (1979) determined that
yields increased linearly with K applications, and yield responses were
negatively correlated with extractable soil K. Yield increases were
also reported by Stino and Lashin (1953) with each 15 kg/ha addition of
K. Vine growth was determined to account for most of the K used during
the first three growing months, while K was accumulated and utilized
predominately by storage roots the last two months of the season (Scott,
1950). Potassium is of primary importance in sweet potato in starch
metabolism (Murata and Akazawa, 1968), translocation and photosynthesis
Several field experiments have been conducted to study the effect
of N on sweet potato yields. Results have been variable and less
consistent than with K. Addition of 100 versus 50 kg N/ha increased
sweet potato yields 17% (Speights and Paterson, 1969). Knavel (1971)
determined that root yields were increased after nine weeks with the
addition of N at 140 and 280 ppm. Conversely, Yong (1969) reported that
90 or 180 kg N/ha did not increase storage root yields compared with 45
kg N/ha, but a significant increase in vegetative weight was obtained.
Spence and Ahmad (1967) found that storage root formation occurred in N-
deficient plants in spite of severely restricted growth. Bouwkamp
(1985) suggested that variable responses to N may depend on several
factors such as N-supplying capacity of the soil or type of cultivar
Bouwkamp (1985) summarized nutrient studies of sweet potatoes
including manganese, phosphorus, magnesium, sulfur, and calcium and
which led to improved yields; however, as mentioned earlier, yield
improvement seemed most responsive to K applications. Storage root
formation was absent in magnesium, calcium, and K deficient plants
(Spence and Ahmad, 1967). Deficiency symptoms were also noted on
leaves, roots, and stems when dried tissue samples were below 0.08%
sulfur, 0.12% phosphorus, 0.75% K, 0.16% magnesium, and 0.2% calcium.
Length of Growing Season
Storage root development and yields were increased with longer
growing seasons. Thirty sweet potato lines and/or varieties were
harvested 108, 130 149 and 169 days after planting (Cordner and
Galeotti, 1961). An average yield of 151 bu/acre harvested 108 days
after planting represented 46% of the full season yield of 590 bu/acre
gathered 169 days after planting. The yields of some varieties and
cultivars were very low when harvested earlier. Yields of all varieties
and cultivars were improved with later harvests. Yong (1969) also
reported increased total yields with a longer growing season, and
corresponding decline in vine yields. Similarly, decreases of 50
bu/acre were recorded for every two weeks that planting was delayed in
South Carolina with the variety 'Porto Rico' (Beattie et al., 1938).
Spacing and Competition
Bouwkamp (1985) summarized the effects plant spacing had on yields
by evaluating several studies where spacings ranged from 15-110 cm
within the row for various cultivars. He concluded total yields were
improved at high plant densities (40,000 to 50,000 plants/ha) regardless
Root size could be controlled by plant density. Roots were
smaller the closer the plant spacing (Bouwkamp and Scott, 1980). Also,
the percentage of smaller roots was cultivar-dependent. Walker and
Randle (1987) determined that cultivars with different growth habits
planted in adjacent rows competed with one another. They proposed
harvesting the middle row of three-row plots to reduce the competitive
effects of adjacent row cultivars. However, Bouwkamp (1986) asserted
the competitive effect of cultivars did not alter yields enough to
implement usage of multi-row plots. Recommendations for plant spacing
in Florida are 25-30 cm between plants or approximately 22,000 30,000
plants/ha. (Stall et al., 1984).
Plastic mulch was used to improve yields of northern (Hochmuth and
Howell, 1983) and southern (Bryan, 1966) grown sweet potatoes. Black
plastic mulch increased leaf area, number of leaves, and total
vegetative weight compared with unmulched plots (Hochmuth and Howell,
1983). Marketable root yields were increased 7.1 MT/ha on mulched flat
beds and 8.4 MT/ha on mulched raised beds. Marketable sweet potato
yields were increased 53, 211, and 168% on black, clear, and white
plastic mulch, respectively, compared with unmulched plots (Bryan,
Cost of transplants is a major expense in the commercial
production of sweet potato (Kushman et al., 1970). As mentioned
earlier, commercial plantings are all vegetatively propagated. In
growing regions where sweet potatoes cannot be grown continuously, roots
must be saved, planted in beds, and sprouts or 'slips' which develop
from the roots used for propagation. In warmer climates where a crop
can be maintained perennially, or several weeks after slips have been
transplanted, vine cuttings may be used for crop establishment.
Effect of Establishment Practices on Storage Root Yields.
Several investigations have been conducted to determine the
effects of various propagule types and establishment practices on
storage root yields. Root plants (slips) and vine cuttings were
compared for their effect on storage root yields in four sweet potato
breeding lines or varieties (Cordner and Galeotti, 1961). Yields from
cuttings were similar to that obtained from slips. In another study
which compared vine cuttings to slips, yields were also similar when
total yields and the USDA size grades were evaluated (El-Kattan and
Propagule length and its effect on yields has also been
investigated. Three studies were conducted in succeeding years in which
slips were removed from the mother root and separated into two size
categories, 23 to 25 cm (large) and 15 to 18 cm (small)(Beattie et al.,
1938). In one study, five weeks after transplanting, the large-plant
plots had greater vine growth than small-plant plots. However, the
better vegetative growth obtained from the large-plant plots did not
improve yields versus the small-plant plots. Mean yields were similar
throughout the three one-year studies and were not dependent on slip
length. A similar evaluation was made between vine cutting lengths of
23, 31, 46, and 61 cm in four studies for two cultivars (Godfrey-Sam-
Aggrey, 1974). Three months after planting, the 46 and 61 cm cuttings
of both cultivars,'Missus' and 'Madam,' increased root yields compared
to the smaller 23 and 31 cm cuttings. Vine production per hectare,
however, was similar regardless of propagule length. These results were
the opposite of that found in the previously mentioned study.
Hall (1986) examined vine cutting transplant lengths, number of
nodes planted underground, and orientation of transplants on sweet
potato yields. Total marketable root yields of 'Red Jewel' were 2.7
MT/ha greater with the 40-45 cm than the 20-25 cm vine cuttings. Yields
were equivalent when similar comparisons were made with 'Georgia Jet.'
Total marketable yield of either cultivar was unaffected by the number
of nodes planted underground or transplant orientation. Generally,
longer propagules improved yields, while variations in the number of
underground nodes and transplant orientation appeared to be more
cultivar dependent. Although longer propagules have been reported to
increase yields, they require more labor and expense.
Six methods of propagating sweet potatoes were investigated
(Templeton-Somers and Collins, 1986). Three used in vitro techniques:
1) shoots obtained from leaf explants, then rooted; 2) buds obtained
from roots of leaf explants, then rooted; and 3) lateral buds excised
and tissue cultured. The other three utilized in vivo techniques: 1)
nodes excised in 10 mm pieces and rooted in transplant trays; and 15 to
20 cm long 2) slips; and 3) vine cuttings. In the above study, the
plants propagated in vivo by either slips or vine cuttings had greater
total and grade one root yields per plant than those obtained via in
vitro techniques. A carryover study determined if differences due to
propagation method in the first planting continued the subsequent year.
To test this, roots were saved from each propagation treatment and
bedded, then slips were harvested and transplanted the following growing
season. No significant differences in yields were measured from slips
obtained from different propagules the previous year. The low yields
obtained from the in vitro propagules the previous year could not be
attributed to alteration in genetic composition.
To reduce propagation costs, the use of root pieces, as practiced
with potato, (Solanum tuberosum L.), was investigated as an alternative
for plant establishment (Bouwkamp, 1982). Twenty to 25 gm root pieces
in over 220 lines were screened throughout a nine-year period. The type
of root development was cultivar dependent. Some cultivars had several
good quality roots arise from the root piece, while in some others, the
root pieces enlarged with no additional root formation. Root pieces
were disease-susceptible in many cases, making plant establishment a
major problem. Use of root pieces led to inconsistent root yields, is
still in an experimental stage, and needs more work before its
Commercial plantings of sweet potatoes is primarily for food
production, but attention in recent years has focused on this crop as a
biomass crop for energy production. Southern Japan, in some instances,
has used sweet potato for energy production (Yen, 1974). Mass
production, however, is limited by the high cost of vegetative
propagation (Cantliffe et al., 1987). The use of tissue culture has
been suggested as a means to economically produce crops normally
The birth of plant tissue culture can be traced back to 1902 when
Haberlandt first attempted to culture isolated plant cells and
demonstrate totipotency. His efforts failed, but some of the theories
he put forth were later proven, among them, totipotency (Vasil and
Hildebrandt, 1965). Today, tissue culture continues to advance science
and new technologies.
Discovery. Definition, and Potential Advantages
A significant finding in tissue culture occurred when two
independent research groups discovered in 1958 that somatic embryos
would abundantly form from carrot callus tissue, then grow directly into
plants (Reinert, 1958; Steward et al., 1958). In the years that
followed, it was demonstrated that numerous plant species and families
were capable of somatic embryogenesis (Tisserat et al., 1979; Ammirato,
Somatic embryogenesis can be defined as the developmental process
which produces embryos from somatic cells. Sharp et al. (1980)
distinguished between two developmental patterns: 1) direct
embryogenesis, in which embryos originate directly from tissues without
callus proliferation; and 2) indirect embryogenesis, in which callus is
maintained and proliferated before embryo development. In either case,
the formation of somatic embryos closely follows the developmental
sequence in zygotic embryos which are globular, heart, and torpedo
stages, then plant formation.
Somatic embryogenesis differs from micropropagation through shoot
primordia and adventitious bud differentiation. With the latter two,
the shoot or bud is unipolar, while somatic embryos are bipolar
structures with closed vascular systems. Several advantages are gained
using somatic embryogenesis rather than micropropagation techniques: the
capacity to produce large numbers of embryos in limited space; no need
for physical separation of individual propagules; and direct embryo
conversion into plants (Lawrence, 1981).
Explant Selection for Somatic Embryogenesis
The first criterion to consider for induction of somatic
embryogenesis is explant source. Nearly all plant parts have been used
successfully to induce somatic embryogenesis: hypocotyls (Halperin,
1966), stems (Banks, 1979), embryos (Bayliss and Dunn, 1979), ovules
(Kochba et al., 1974), shoot tips (Lakshmi Sita et al., 1979), young
inflorescences (Chu et al., 1984), roots (Okamura et al., 1973), and
protoplasts (Vasil and Vasil, 1980). Somatic embryogenesis can be
induced from a variety of explants in certain species like carrot, i.e.,
hypocotyl (Halperin, 1966), petioles (Wetherall and Dougall, 1976),
taproots (Okamura et al., 1973) and young roots (Smith and Street,
1974); however, some species, particularly those in the Gramineae, may
respond in more limited regions in the plant, specifically the actively
growing regions (Ammirato, 1983.) For example, somatic embryo formation
was obtained from immature embryos of Triticum (Ozias-Akins and Vasil,
1982), immature inflorescences of Setaria itlica (Zhi-hong et al.,
1984), and apical meristems and leaf primordia of corn (Zea mays) (Fahey
et al., 1986). Environmental conditions (Ammirato, 1983) and
developmental age.at which explant sources are obtained also influence
the capacity for embryogenesis (Vasil and Vasil, 1984).
Induction of Embryogenic Callus
Once an adequate explant source is determined, it must be
sterilized and placed on solid or in liquid medium to induce the
formation of embryogenic callus. Typically this medium, often termed
primary or induction medium, consists of inorganic salts, carbohydrates,
organic supplements such as vitamins, and hormones necessary for growth.
Specific requirements for optimum growth must be determined empirically
for a species, while requirements may even differ between cultivars of
of a species. Chen et al.(1987) tested 50 genotypes of three alfalfa
(Medicago spp.) species and determined that one particular combination
of medium and growth regulators resulted in formation of somatic embryos
in one genotype, while another combination was required for a different
Auxin is the critical growth regulator needed to induce
embryogenic callus formation (Tisserat et al., 1979). Auxins such as
2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA), and
napthlalene acetic acid (NAA) have been used successfully. The auxin
2,4-D was most commonly used among species (approximately 60% of the
time) for embryogenic callus induction (Ammirato, 1983; Evans et al.,
1981). Cytokinins may be useful for embryogenic callus production (Chee
and Cantliffe, 1988b), but these hormones may not be needed in some
families such as Umbelliferae (Ammirato, 1983). Schenck and Hildebrandt
(1972), however, reported low levels of cytokinins were essential for
most dicot cell cultures.
Embryo Induction and Maturation
Introduction. Conditions in the primary medium that favor callus
growth are not necessarily the same as those associated with embryo
formation and maturation. Embryo development is typically induced by
transfer of embryogenic callus to another medium containing with no
hormones or reduced hormone concentrations. Although embryo formation
may be easily induced, production of large quantities of embryos which
rapidly convert to plants may not. A number of studies are described
which have considered the effects of nutrient, carbohydrate, and growth
regulator formulations on the induction, development, and number of
somatic embryos produced.
The nutrient salt medium of Murashige and Skoog (MS) is most often
used for somatic embryogenesis. Evans et al. (1981) reported 70% usage
of this medium in somatic embryogenesis protocols. Other media, such as
Gamborg's (Gamborg et al., 1968), Schenk and Hildebrandt's (SH) (1972)
and White's (1943) have been successfully used or were superior for some
species (Evans et al., 1981). Sucrose is the carbon source most
frequently incorporated into tissue culture media (Ozias-Akins and
Vasil, 1984). Other sugar types such as galactose, glucose, maltose,
and fructose have been used with success (Verma and Dougall, 1977).
Lower levels of auxin, and various concentrations of cytokinin, abscisic
acid (ABA), and gibberellic acid (GA) have been reported in different
instances to improve either somatic embryo formation or maturation and
will be discussed later.
Inorganic media composition and comparisons. White (1943)
developed a medium for the culture of excised tomato roots. Murashige
and Skoog (1962) used White's medium as a reference to develop a medium
designed to increase production of tobacco callus. These researchers
determined that concentrations of N, K, phosphorus, magnesium, and
calcium were particularly important if optimal growth was to be
achieved. Gamborg's (1968) medium has been used for protoplast culture
and was specifically created for culture of soybean root cells. Schenck
and Hildebrandt (1972) developed a medium which promoted the growth of
callus in a variety of monocotyledonous and dicotyledonous plants.
Other nutrient media, usually less concentrated, such as Hoagland
solution (Hoagland and Arnon, 1950) have been used specifically for
hydroponic culture of plants.
Murashige and Skoog medium is used in many tissue culture
laboratories when the objective is plant formation; however, White's,
SH, Gamborg and other media will work as well. Compared with many
media, MS medium contains very high concentrations of inorganic salts.
Murashige and Skoog medium contains nearly two times more N and 20 times
more phosphorus than either Gamborg or SH media, with concentrations
being even greater than Whites medium (Table A-l). Another key
distinction between media is MS contains 20 mM ammonium compared to 1,
2.6 and 0 mM for Gamborg, SH, and White's media, respectively. The N
source, particularly ammonium or a reduced form of N has been reported
especially critical in somatic embryogenesis (Halperin and Wetherell,
1965; Stuart and Strickland, 1984ab).
Effect of nitrogen source. Halperin and Wetherall (1965), in one
of the earliest studies, reported ammonium was critical for
embryogenesis in wild carrot. The reduced N form, glutamine, could also
be substituted for ammonium and enhance embryogenesis (Halperin and
Wetherell, 1965; Halperin, 1966). Callus liquid cultures grown on
medium lacking reduced N formed adventitious roots while, in contrast,
cultures in medium with reduced N yielded large numbers of embryos.
Similarly, Wetherall and Dougall (1976) reported that reduced N
was critical for somatic embryogenesis in wild carrot. They determined
ammonium was the best reduced N source when cultures were grown in the
presence of 20 mM of nitrate, but also found that glutamate was the best
for embryogenesis when used as the sole nitrogen source at either the 15
or 30 mM levels. Kamada and Harada (1979) determined 10 mM alanine
stimulated embryo production in wild carrot nearly 40-fold more than the
optimum ammonium concentrations in the presence of 20 mM nitrate. To a
lesser extent, the amino acids glutamine, glutamate, aspartate,
arginine, proline and asparagine increased embryo production compared
with ammonium. Although greater numbers of embryos were produced with
the above amino acids, more mature embryos (heart and torpedo-shaped)
were produced only with alanine, glutamine, glutamate, and aspartate.
Besides the N form, Reinert (1967) reported that total N was a critical
factor for optimization of embryogenesis in wild carrot.
During the 1980s, the effect of N source on embryo development and
production was scrutinized more carefully on species other than wild
carrot. Walker and Sato (1981) determined that Medicago sativa L. cv
Regens (alfalfa) was similar to carrot in response to the ammonium ion.
Root formation was inhibited by ammonium levels 50 mM and above, while
somatic embryos formed at the minimal ammonium concentration tested of
12.5 mM up to a high concentration of 100 mM. Stuart and Strickland
(1984a) evaluated the effect of various amino acids on embryogenesis
when added to SH medium. Addition of 250 to 300 mM proline to SH medium
increased the number of somatic embryos nearly three times compared to
basal medium without proline. Glutamine, lysine, serine, asparagine,
alanine, and arginine, to a lesser extent, also increased embryo
Stuart and Strickland (1984a) correlated improved embryo quality
with increased embryo size, which led to higher percentages of embryos
converting to plants. Using these criteria, inclusion of glutamine,
arginine, alanine, and proline produced greater-sized embryos and led to
improved plant formation compared with embryos which developed on the
medium with the addition of only ammonium. Somatic embryogenesis was
dependent on the inclusion of ammonium in the medium, while the
improved somatic embryo number and quality obtained with proline was
dependent on ammonium (Stuart and Strickland, 1984b). The optimum
concentrations for embryogenesis was 100 mM proline and 25 mM ammonium.
The authors suggested that proline would improve somatic embryogenesis
in a number of species.
Optimum ammonium levels were 5 mM for embryo induction, while 20
mM was best for embryo development, increased somatic embryo yields, and
plant conversion of Medicago sativa cv falcata (Meijer and Brown, 1987).
Somatic embryos produced with 30 mM ammonium and nitrate often led to
formation of small, abnormal embryos. Contrary to reports by Stuart and
Strickland (1984ab), incorporation of amino acids such as proline did
not improve embryo production, but was often inhibitory.
The nitrate:ammonium ratio of 2:1 was determined best for embryo
production in Solanum melongena (eggplant) (Gleddie et al., 1983). When
the 2:1 ratio was maintained, significantly more embryos were produced
between 60 to 90 mM total nitrogen. No embryo formation occurred when
either nitrate or ammonium were absent from the medium. The authors
suggested the optimum 2:1 ratio for embryo production might be
attributed in part to pH.
Effect of potassium. Potassium stimulated embryo formation in
wild carrot (Reinert et al., 1967). Percentage of cultures with embryos
was increased from 3 to 45% when K concentrations were increased from 3
to 20 mM. Brown et al. (1976) determined 1 mM K was required for
optimum growth of callus, while 20 mM caused embryo induction. The
effects of other nutrients on embryogenesis have received little or no
Effect of carbohydrates. Sucrose was reported to be the most
commonly used and effective carbon source for somatic embryogenesis
(Ammirato, 1983). Maretzki et al. (1974) concluded either sucrose and
glucose were best as carbon sources for growth of tissue cultures;
however, they suggested that other carbohydrates could substitute for
glucose or sucrose depending on the species and growth phase. The
following are carbohydrate studies involved with somatic embryogenesis
and include some examples of carbohydrate substitution for the
traditionally used sucrose.
Inclusion of galactose in the medium stimulated somatic embryo
development in citrus culture (Spiegal-Roy and Vardi, 1984). Glucose
levels at 6 to 10% improved somatic embryo development in carrots
(Homes, 1967 cited in Tisseret et al., 1979). Embryogenic callus was
initiated in cotton (Gossypium hirsutum) on medium containing 3%
glucose; however, sucrose replaced glucose for callus maintenance,
embryo maturation, and plant regeneration (Shoemaker et al., 1986).
Ozias-Akins and Vasil (1982) reported growth responses of wheat
scutellar callus were similar when grown on 2% glucose or sucrose, while
7% galactose inhibited growth. More somatic embryos formed in soybean
when carbohydrate concentrations were reduced from 12 to 1.5% when using
either glucose and sucrose (Lazzeri et al., 1987). In eggplant, at 60
mM a sucrose source led to the greatest embryo formation, followed by
glucose, then fructose (Gleddie et al., 1983). No embryos formed when
galactose, xylose, or raffinose were added to the medium.
Various carbohydrate sources for somatic embryogenesis of alfalfa
were compared at the 1 and 3% levels (Strickland et al., 1987).
Maltose, malt extract, and fructose increased embryo yields compared
with 3% sucrose. Maltose was evaluated in the remaining experiments
since embryo yields and germination were improved most with this
treatment. Maltose improved embryo development (embryo length,
diameter, and percentage embryos with cotyledonary lateral appendages)
compared with sucrose. Embryo formation was influenced by ammonium
carbohydrate interactions. The greatest formation was with the
combination of 15 mM ammonium and 3% maltose.
Stuart et al. (1988) determined that protein accumulation in
alfalfa somatic embryos was similar to that found in seed (Stuart and
Nelson, 1988). The 11S protein was the best protein marker found in
both somatic and zygotic embryos. A comparison between 11S protein
measured in mature seeds versus somatic embryos might be a good
biochemical marker for determining the physiological stage of a somatic
embryo. The amount of 11S protein expressed in the somatic embryo was
approximately 10% of that found in seed, indicating that somatic embryo
development was much less than that found in a seed. The 11S protein
levels of embryos produced with maltose or sucrose were compared.
Embryos obtained with maltose rather than sucrose had greater 11S
protein levels. Maltose, when used in media for regeneration, yielded
larger embryos which germinated better than sucrose-treated cultures.
Various sucrose concentrations (0 to 400 mM) were tested to
determine their effect on embryo initiation and differentiation on a
diploid and tetraploid alfalfa genotype (Meijer and Brown, 1987).
Embryo induction was high (number of embryos, developmental stage not
delineated) over a sucrose concentration ranging from 25 to 100 mM in
both genotypes. Embryo differentiation was best at either 100 or 200 mM
sucrose. The 100 mM range corresponded closely with the 88 mM sucrose
(3%) used in most standard tissue culture media.
Several carbohydrates were tested at 60 mM for their effects on
embryo growth and development, and germination in wild carrot (Daucus
carota) suspension cultures (Verma and Dougall, 1977). Dry weights of
embryos 14 days after incubation were much greater with either
filter-sterilized or autoclaved sucrose compared with glucose, fructose,
mannose, glucose + fructose, or galactose. In addition, a greater
number of more fully developed embryos, and more germinated embryos were
obtained with sucrose compared with the monosaccharides. Fructose,
after autoclaving, was the only sugar which inhibited growth and
embryogenesis. In a study over time, sucrose produced more embryos than
the other sugars after 13 days; however, after 27 days, embryo
production was similar with sucrose, fructose, glucose, or maltose. The
authors suggested that an initial delay in growth and embryogenesis
which occurred with galactose, raffinose, and stachyose might be
explained by slow uptake rates and/or metabolism of these sugars. They
proposed the rate of embryo growth and development might be controlled
by the rate of uptake and/or metabolism of the carbohydrate to a common
Several in vitro studies with zygotic embryos illustrate the
importance of carbohydrate type and concentration on embryo growth and
development. For example, Cobb and Hannah (1986) determined that
embryos of the wild type and shrunken-2 corn (Zea mays) species matured
and germinated when placed in medium containing glucose, fructose, or
sucrose. However, greater germination, starch content, and embryo
weight was obtained with sucrose rather than fructose or glucose. The
dynamic changes necessary for a maturing embryo is illustrated with
jimson weed (Datura stramonium) (Rietsema et al., 1953). Greater
sucrose concentrations (8-12%) were needed for less mature embryos
(pre-heart stage), while little or no sucrose (0-0.2%) was needed for
torpedo-stage or mature embryos.
Effect of hormones. After embryogenic callus has been formed or
proembyos initiated, the hormone levels) and/or type(s) often must be
altered to permit the maturation of embryos. Elimination of all
hormones from the medium promotes embryo maturation in species such as
carrot (Smith and Street, 1974; McWilliam et al., 1974), corn (Lowe et
al., 1985), and chicory (Heirwegh et al., 1985). Also, a reduction in
hormone concentrations can promote embryo development. For example,
Echinochloa muricata somatic embryos matured when callus was transferred
from medium containing 10 to 15 mg/L to one with less than 5 mg/L 2,4-D
(Cobb et al., 1985). In celery, torpedo embryos formed when both 2,4-D
and kinetin levels were reduced (Al-Abta and Collins, 1978).
Other studies revealed cytokinins were specifically required for
embryo maturation and/or increased production. Addition of kinetin
(KIN) (100 pM) or benzylaminopurine (BAP)(100 or 10 pM) to basal medium
improved embryo maturation of winter oil rapeseed (Loh et al., 1983).
MacKinnon et al. (1987) determined that a change in auxin source, from
2,4-D to IAA, and the addition of BAP improved embryo formation,
maturation and plant formation in wheat and sorghum. Similarly, Walker
and Sato (1981) replaced 2,4-D with NAA and added KIN to promote alfalfa
embryo maturation. Fujimura and Komaine (1975) determined somatic
embryogenesis of citrus was improved when 2,4-D was removed and 0.1 /M
zeatin was placed in the basal medium. Use of a specific cytokinin was
necessary since KIN and BAP were also tested, but unlike zeatin, these
cytokinins inhibited embryo development.
In Norway spruce (Picea babies a series of transfers to media
containing different hormone concentrations and types were required for
somatic embryo initiation and development (Gupta and Durzan, 1986).
Callus was removed from basal medium containing 2 AM of both BAP and KIN
and 5 pM 2,4-D to a medium containing less 2,4-D (1 /M) in which
globular embryos formed. A subsequent transfer to a medium containing
no auxin was necessary for embryo maturation.
Generally, gibberellic acid is not included in the maturation
medium for somatic embryo development. However, Ammirato (1977)
determined a balance of GA, ABA, and zeatin was necessary for normal
development and maturation of caraway (Carum carvi) somatic embryos.
Addition of 1 mg/L GA stimulated differentiation of sandalwood (Santalum
album L.) somatic embryos (Lakshmi Sita et al., 1979).
Gibberellic acid plays an important role in zygotic seed
development and its active metabolism during embryo development has been
related to embryo growth. In Japanese morning glory (Pharbitis nil)
zygotic embryos, the maximum growth of the seed occurred just after GA
levels peaked (Murakami, 1961). Gibberellic acid concentration was more
concentrated in the endosperm than any other plant organ in Sechium
edule and its concentration remained nearly constant throughout seed
development (Lorenzi and Ceccarelli, 1983).
Abscisic acid has been reported to impose dormancy and inhibit
germination of zygotic embryos (Black, 1981). In addition, it has been
shown in several instances to have a role in preventing precocious
germination of zygotic seed (Crouch and Sussex, 1981; Hendrix and Radin,
1984; Choinski et al., 1981 etc.). The effects of ABA on somatic embryo
development have also been investigated.
Ammirato (1974) determined that optimum levels of ABA (0.1 yM)
resulted in normal somatic embryo development in caraway. ABA reduced
formation of abnormal structures, such as adventitious embryos, and
repressed precocious germination (Ammirato, 1974, 1977). Similar
findings were reported by Kamada and Harada (1981) and Baker (1985) with
carrot. Ranch et al. (1985) determined the addition of 1 or 10 pM ABA
to Gamborg's medium decreased precocious germination and increased the
frequency of embryo maturation in soybean, while Vasil and Vasil (1981)
found similar responses with pearl millet (Penisetum americanum). Low
concentrations (0.1 and 0.05 mg/L) ABA promoted maturation of barley
somatic embryos (Kott and Kasha, 1983). ABA increased embryo production
of orange (Citrus sinensis) at 2.0 MM ABA (Kochba et al., 1978).
Finklestein and Crouch (1986) compared the affects of ABA and
osmotic potential on somatic and zygotic embryos of Brassica napus as
they matured. Cruciferin was produced as long as the zygotic embryo
continued maturation. Thus, it was designated a marker protein and
could be used to determine embryo maturation in somatic embryos.
Precocious germination, common in somatic embryogenesis, was inhibited
with the addition of either ABA or high osmoticum. The rate of
cruciferin synthesis was slowed in a precociously germinating embryo;
however, under conditions which suppressed precocious germination (ABA
and high osmoticum), the accumulation of the marker protein was similar
to that obtained by embryos grown in situ. Comparison of the kinetics
of ABA and osmotic effects on gene expression revealed osmotic effects
were more rapid than ABA.
Organic additives. Other undefined organic additives have been
beneficial for somatic embryo maturation. Litz et al. (1982) reported
coconut water was an important additive for improved embryogenesis of
mango. Casein hydrolysate has also enhanced somatic embryogenesis
(Ammirato and Steward, 1971). Activated charcoal proved useful for
better development of carrot somatic embryos, and led to embryo
development in cultures which failed to produce embryos when auxin was
eliminated (Fridborg et al., 1978). Embryo maturation was also improved
in onion in the same study. The charcoal was suggested to absorb
residual hormones in the medium or inhibitory substances which prevented
embryo maturation. Similar results were reported for ivy as 0.1%
charcoal maintained embryo integrity and development, and prevented
callusing (Banks, 1979).
Light. Other factors influencing embryo maturation are light,
osmotic potential, physical environment, pH, and temperature. High
light intensities have been reported necessary for somatic embryogenesis
in tobacco (Haccius and Lakshmanan, 1965 cited in Ammirato, 1983).
Induction of embryogenesis in eggplant was also dependent on light
(Gleddie et al., 1983). Gupta and Durzan (1986) reported maturation of
globular embryos to cotyledonary embryos with light at specific
wavelengths (2.8, 2.0, and 0.5 uW cm-2 nm-1 in blue, red, and far red).
In contrast, Ammirato (1974) reported caraway somatic embryo maturation
was more normal when grown without light.
Osmoticum. Carrot somatic embryo development has been controlled
by subjecting developing embryos to high carbohydrate levels thereby
increasing osmotic potential. Reduced precocious germination and
smaller embryos with cotyledons were obtained in cultures containing 12%
sucrose (Ammirato and Steward, 1971). Gleddie et al. (1983)
supplemented MS medium with 0 to 0.6 M mannitol and determined that as
osmotic potential increased, embryo frequency decreased, with no
embryogenesis above 0.4 mannitol. Crouch and Sussex (1981) determined
high osmoticum prevented premature germination of somatic embryos and
permitted continued protein accumulation like an embryo maturing in
pH. Wetherell and Dougall (1976) reported carrot embryo formation
best at a pH of 5.4, and was two times greater than if the pH was lower
than 5 or greater than 6. In a later study, Dougall and Verma (1978)
examined the effect of N sources on pH and proposed media containing
ammonium as a sole source of N resulted in variation in pH that would be
deleterious to cell growth and embryo formation. A broader pH range was
reported to facilitate the growth of Ipomoea suspension cultures (Martin
and Rose, 1975).
Temperature. Temperature can also be critical for obtaining
better embryo quality and an increased number of somatic embryos.
Gleddie et al. (1983) determined more embryos formed earlier at 30C
versus 25C. Redenbaugh et al. (1986) reported somatic embryo production
in celery was extremely temperature dependent. At 240C, 10 high quality
embryos formed per Petri dish. At 270C, 50 embryos formed and if
temperatures fluctuated between the two extremes, no embryos formed.
Summary of Embryo Initiation. Development, and Maturation
Much of the literature reviewed has dealt with the various factors
affecting somatic embryo formation and maturation. The gain of
knowledge in this developmental phase can have a significant impact on
the subsequent success of plantlet formation. As evidence, in alfalfa,
treatments which improved embryo maturation and quality resulted in
increased embryo to plant conversion from 0.5% in 1982 to 60% in 1986
(Redenbaugh et al., 1987a). The environmental conditions during embryo
development and maturation affect the overall quality and vigor of the
seed (Delouche, 1980) and most probably affect that of somatic embryos.
Development of Embryos to Plants
Overall survey. Once mature embryos are obtained, the next
developmental phase is their conversion to plants. Most studies in
somatic embryogenesis have not focused on protocols that result in plant
formation. Studies have been more limited when planting and rooting of
somatic embryos are considered; however, scattered reports, particularly
in the last five years, have documented the percentages of embryos
forming plants under varying conditions (i.e., Redenbaugh et al., 1986,
1987,; Stamp and Henshaw, 1982; Lazzeri et al., 1987). Some of the most
extensive work on embryo conversion to plants has been by Plant Genetics
Inc. in Davis, California, where the primary objective was to
economically produce somatic embryos as propagules for commercial
Hormones. In some cases, various hormones have been included in
the regeneration medium to enhance plant formation. Typically, auxin
levels are lowered, and often some type of cytokinin is included in the
regeneration medium. MacKinnon et al. (1987) reduced and changed the
auxin used in the medium from 5 and 2 mg/L 2,4-D to 0.1 and 1.0 mg/L IAA
for wheat and sorghum, respectively. In addition, 0.5 mg/L BAP was
included in the plant regeneration media of both species. The best
regenerating lines of wheat and sorghum produced 193 and 87 plants/gm
callus, respectively. The addition of an auxin (0.5 mg/L NAA) and
cytokinin (2 mg/L BAP) was also used for plant regeneration of millet
(Setaria italica) (Zhi-hong et al., 1984). With zygotic seed, usually
auxins inhibit or are ineffective as germination promoters, but can help
in some instances (Nickell, 1982).
In some cases, plant regeneration has been improved by the
addition of cytokinin to the medium. Regeneration of Vigna aconitifolia
was best in basal medium containing 2.2 jM BAP an average of 12.4 plants
from 50 mg callus (Kumar et al., 1988). Only 2.8 plants formed when BAP
was not added to the medium. The percentage of calli with embryos
forming plants was increased from 7 to 43% in rice (Oryza sativa L.)
when 1 mg/L KIN was added to MS medium (Abe and Futsuhara, 1985).
However, the addition of 0.02 mg/L 2,4-D reduced plant formation, even
when KIN was included in the medium. Root and shoot formation from
filbert (Corylus avellana) somatic embryos was similar regardless of the
addition or deletion of BAP (Perez et al., 1983). Cytokinins generally
have been found less effective and more limited in their role in
affecting seed germination othe than overcoming dormancy (Nickell,
1982). Kahn et al. (1971), however, reported that cytokinin removed the
block to germination imposed by inhibitors of germination in several
species. They suggested cytokinin permitted the completion of
gibberellin-mediated germinative processes and thereby acted in the
permissive role for germination.
Root and shoot formation can be controlled by changing the auxin
cytokinin ratio. Various combinations of these hormones with different
species or cultivars within a species have promoted plant formation.
For example, in Norway spruce, 12% of the calli with embryos formed
plants when placed on a modified MS medium supplemented with 5 pM 2,4-D
and 5 pM KIN (Hakman and Arnold, 1985). Embryos of cassava (Manihot
esculenta), 4 mm in length, rapidly formed roots and leaves when placed
in MS basal medium with 0.1 mg/L BAP and 0.01 mg/L 2,4-D (Stamp and
Henshaw, 1982). However, with soybean, Lazzeri et al. (1987) determined
that no additional plant formation occurred when different hormone
concentrations and ratios were tested using NAA, BAP, KIN, and zeatin.
The addition of GA has been beneficial for somatic embryo to plant
conversion in some cases. Gibberellic acid at 0.3 pM plus 0.6 pM IBA
improved plant regeneration of soybean (Ranch et al., 1985). After
embryos were in culture two to three weeks, Ghazi et al. (1986)
determined that the addition of 0.104 mg/L GA3 to basal medium resulted
in 20% plant formation compared to 0% when no hormones were added. More
root formation was obtained in wheat when 1 mg/L GA3 was included in the
basal medium (Ozias-Akins and Vasil, 1982). However, no increase in
shoot formation was observed. Chuang and Chang (1987) placed Dysoma
pleiantha somatic embryos on either Gamborg or MS medium containing 1
mg/L BA and 1 mg/L GA. More than 90% of these embryos formed plants.
Rooting of horse chestnut somatic embryos was stimulated by auxin (IAA
or IBA) and GA3, the highest germination being 65%, while cytokinins
(KIN,BAP, and zeatin) inhibited rooting (Radojevic, 1988).
Concentrations of GA3 were varied at 0.5 and 1.0 mg/L, with and
without BAP in combination to improve germination of grape somatic
embryos (Stamp and Meredith, 1988). Plant formation was improved up to
42% with 1.0 mg/L GA; while 0 or 0.5 mg/L GA, or the addition of BAP led
to reduced plant formation (0 to 9%). Filbert shoot elongation was
increased at 30 uM GA; however, 150 and 300 pM levels induced abnormal
development resulting in weaker shoots and less root formation (Perez et
Other growth substances can also enhance plant formation. Barley
(Hordeum vulgare) plant regeneration was better when MS medium was
supplemented with 3% 2,3,5-Triiodobenzoic acid (47%) compared either
with MS medium containing 1 pM IAA and 1 pM BAP (6%) or medium without
growth substances (29%) (Rengel and Jelaski, 1986).
Often the removal of all hormones in the medium facilitates plant
regeneration. For Norway spruce, the removal of all hormones was
necessary for plant formation (Dunstan et al., 1982). Similarly, about
30% of Digitalis obscura L. embryos developed into plants when placed on
basal medium containing no growth regulators (Arrillaga et al., 1986).
Carbohydrates. Carbohydrate type and level can affect somatic
embryo induction and maturation, but it can also influence subsequent
embryo germination and plant formation. Drew (1979) investigated the
possibility of stocking carrot somatic embryos with sucrose after three
subcultures in media with various sucrose levels. To accomplish this,
sucrose levels were increased two, five, and ten times or left
unchanged. The idea was for the embryos to accumulate an energy source
so that germination and plant formation could occur at sowing without
additional sucrose. However, under non-sterile conditions, only three
plants formed after eight weeks, while most stems that had elongated
became chlorotic and ceased growth. In concurrent studies, embryos with
cotyledons were placed on filter paper bridges supplemented with White's
nutrient medium at various sucrose levels. Without sucrose in the
medium, only 15% of the embryos produced plants under in vitro
conditions, while 1.5% strength sucrose led to 95% plant regeneration in
Ahwoowalia and Maretzki (1983) determined that 6% sucrose
stimulated root formation of sugar cane somatic embryos, while 3%
sucrose promoted rooting and plant conversion from mature somatic
embryos. Rietsema et al. (1953) had similar results with excised jimson
weed (Datura stramonium) zygotic embryos in that as the embryo matured,
the optimum sucrose concentration needed for growth and development was
A sucrose hormone interaction on somatic embryo rooting was found
with orange (Citrus sinensis cv Shamouti) (Kochba et al., 1974).
Rooting was best at 5 and 6% sucrose concentrations when adenine sulfate
(ADS) and GA were added to the medium, while rooting without ADS or GA
resulted over a broader sucrose concentration range of 2 to 6%. Only
15% of the embryos rooted on 10% sucrose concentration. In soybean,
nearly 40% of somatic embryos placed on one-half strength MS medium
containing 1.5% sucrose produced plants (Shoemaker et al, 1986).
Chicory plant regeneration was similar when sucrose concentrations were
lowered from 2 to 1% (Heirwegh et al., 1985).
Glucose and sucrose concentrations were adjusted to 1.5, 3, 6, and
12% and their effects on percentage rooting of soybean somatic embryos
tested (Lazzeri et al., 1987). Roots were produced on 73% of the
embryos when placed in medium with 1.5% sucrose, while 30, 15, and 0%
roots formed at 3, 6, and 12% sucrose, respectively. Root formation
(45%) occurred only at the lowest glucose concentration (1.5%). Root
formation was similar when either fructose or sucrose were used at 2%.
Arnold and Hakman (1988) tested the effect of 30, 60, and 90 mM sucrose
on shoot formation of Norway spruce somatic embryos. They found
one-half strength basal medium containing 60 mM sucrose increased shoot
formation 10 to 20% compared with the other treatments.
Redenbaugh et al. (1987) found that in alfalfa, glucose improved
embryo to plant conversion most when compared with sucrose. To a lesser
extent, maltose, cellubiose, lactose, and fructose also improved embryo
conversion in alfalfa when compared to sucrose. No plants were obtained
with galactose. All carbohydrates were autoclaved in the above study.
The effect of carbohydrate breakdown due to autoclaving was also
investigated. While autoclaved maltose resulted in 37% conversion,
filter-sterilization of maltose reduced conversion to 23%. Autoclaved
maltose led to a similar percentage plant formation compared with
autoclaved or filter-sterilized glucose. The authors suggested the
improved embryo conversion was due to a glucose moiety obtained by
hydrolyzation of maltose when autoclaved.
Nutrient media. The role of various nutrient media on soybean
plant quality was investigated by Lazzeri et al. (1987). They reported
inorganic salts affected plant quality more than any other component in
the regeneration medium. In order of best to worst, media for
regeneration of red clover (Trifolium pratense) were: hydroponic
nutrient medium > 1/2 MS > White > Gamborg > 1/2 Gamborg > MS. Gamborg
and MS media were compared for their effect on apical development and
germination of soybean embryos at two, four, and six weeks (Ranch et
al., 1985). The Gamborg basal medium hastened and increased plant
formation when results were pooled from four genotypes. All embryos
which exhibited apical development formed plants within 30 days when
transferred to Gamborg or MS medium containing 0.5 pM IBA.
The percentage of somatic embryos with shoots, that rooted on
media containing either half-strength MS macrosalts or full strength MS
macrosalts and supplemented with 5.7 pM IAA was the same (Rengal and
Jelaska, 1986). Some embryos formed only shoots (10-20%). The authors
suggested this to be a "prolonged effect" response to BAP or auxins of
the induction media.
Stuart and Strickland (1984a) matured alfalfa somatic embryos on
SH basal medium containing various amino acids. They determined that
plant formation (production of trifoliate leaves) depended on the N
source. The percentage plant regeneration ranged between 33 to 67%
after 30 days. Although the addition of proline was mentioned earlier
as a stimulator of embryo production, superior plant formation was
obtained with the addition of 30 mM glutamine instead of proline. The
next best amino acid sources for plant formation were alanine and
arginine. The authors also reported that embryo size was positively
correlated with embryo conversion, that is, larger embryos consistently
resulted in a greater number of more vigorous plants.
Light. Arnold and Hakman (1988) investigated the effect of light
(20 hours) versus darkness on germinating Norway spruce embryos.
Germination (not defined) occurred both in light and dark incubation
conditions. In the dark, 35% of the embryos formed plants, while in the
light, 25% of the embryos formed plants after three weeks. However,
embryos exhibited hypertrophy when left in the dark for two weeks.
Therefore, the authors suggested the embryos be transferred to light as
soon as the roots began to elongate.
Yield Comparisons Between Plants Derived from Somatic Embryos or
One field study compared yields from regenerated plants of napier
grass (Pennisetum purpureum Schum.) somatic embryos with yields from
vegetatively propagated plants (Rajasekaran et al., 1987). Plants
derived from tissue culture yielded 2,400 and 3,510 kg ha-1 more biomass
than vegetatively propagated plants in two separate studies. The
authors attributed the greater yields from tissue culture-derived plants
to earlier establishment and formation of significantly more tillers
than vegetatively propagated plants.
One of the major advantages gained by propagating via somatic
embryogenesis is clonal fidelity. Clonal fidelity must be maintained or
synthetic seeding would not be a viable option. Several investigators
have reported variability in plant tissue culture (Bayliss, 1980; Larkin
and Snowcraft, 1981). In fact, some researchers have suggested tissue
cultures would serve as an excellent source for creating new variability
resulting in new genotypes (Shepard et al., 1980; Larkin et al., 1984;
Zong-xiu et al., 1983; etc.). To date, however, no commercially grown
variety has been developed through somaclonal variation (Vasil, 1987).
Clearly, in some cases, plants obtained from tissue culture have
varied from the donor plant. Some of the most convincing evidence of
genetic variation from tissue culture was documented through cytological
examination. For example, McCoy et al. (1982) observed chromosome
breakages, partial chromosome loss, and various aneuploid types in oat
cell cultures. Similarly, McCoy and Phillips (1982) observed aneuploidy
in a number of corn cell cultures, while Sacristan (1971) found
chromosome rearrangements and polyploids in Crepis capillaris cell
Vasil (1987) points out that the type of cell culture
(differentiated vs undifferentiated) from which the cytological and
phenotypical variations of cells or plants were obtained often are not
well characterized, and Vasil concludes that genetic changes do not
occur in embryogenic cell cultures. He goes on to say that somatic
embryos do not develop beyond an early developmental stage akin to what
occurs in nature when an embryo aborts and loses its regenerative
capacity. This view is supported by Browers and Orton (1982) who found
celery plants were regenerated when cell cultures contained primarily
diploid cells whereas regeneration was not obtained with nondiploid
cells. Although the nutrient medium, transposition events, and the loss
of spacial and temporal control of cell division and differentiation
that operate in intact tissues and plants may lead to genetic
variability in culture, similar genetic changes can occur in vivo, but
often escape detection (Vasil, 1987). Thus, a natural variability is
already conferred into the differentiated tissues of a plant.
Observation of pearl millet (Pennesetum americanum) embryogenic cells
when cultured over a extended time period revealed that most cells
remained cytologically stable and that selection pressures favored the
formation of somatic embryos and plants from normal cells (Swedlund and
Vasil, 1985). From these reports, it seems apparent that plants
obtained from somatic embryos from embryogenic cells are likely to
maintain their genetic integrity.
Sweet Potato Tissue Culture
Although sweet potato ranks seventh among agricultural crops
world-wide, relatively few studies have dealt with cell and tissue
cultures of this species. Root and adventitious shoot formation was
obtained from callus derived from root plugs (Gunkel et al., 1972;
Yamaguchi and Nakajima, 1973). Responses to various hormone levels and
their effects on the rate of callus proliferation and shoot regeneration
were tested in two cultivars (Litz and Conover, 1978). Murashige and
Skoog medium supplemented with IAA stimulated callus production in
'White Star' explants. The addition of activated charcoal inhibited
callus growth, and 1 mg/L BAP promoted shoot proliferation averaging 8.5
shoots five weeks after culture establishment. Cultivar PI 315343
averaged 3.4 less shoots, while rooting and subsequent plant formation
was enhanced with 1 mg/L NAA.
Root and shoot formation was obtained from callus derived from
leaf explants on MS medium supplemented with 10-20 mg/L adenine or
0.1-0.5 mg/L KIN (Sehgel, 1975). Sehgal (1978) also reported nodulation
followed by plant formation from anther callus obtained on MS medium
containing adenine (10 or 20 ppm) plus 2,4-D (Ippm). Adventitious
shoots were generated from root disks in one of three species tested,
while shoots readily formed in all three species from petiole sections
(Hwang et al., 1983). Callus formed best from stem, leaf, and storage
root explants when placed on MS medium supplemented with 1.0 mg/L NAA
and 10 mg/L IBA. Roots and shoots also formed when roots were
subcultured on medium containing 1.0 mg/L NAA and 0.1 mg/L IBA. Plant
formation was improved by light.
Callus was derived from anthers (Tsay and Tseng, 1979). Optimum
callus production was obtained on MS medium supplemented with 2,4-D and
KIN. Somatic embryos formed in one cultivar when basal medium contained
0.1-1.0 mg/L ABA; however, no somatic embryos formed in the other two
cultivars. Plants were regenerated when embryos were placed on basal
medium supplemented with 1 mg/L IAA and 4 mg/L KIN.
Somatic embryos differentiated from callus derived from auxiliary
bud shoot tips in six of nine genotypes (Jarret et al., 1984).
Auxiliary shoot tips removed nearest the shoot apex resulted in the
greatest formation of embryogenic callus. Embryogenesis was facilitated
by 2,4-D concentrations between 0.1 to 3.0 mg/L for most genetypes.
Plant formation was obtained when embryos were removed and placed on
basal medium with half-strength MS salts and no hormones.
Embryogenic callus of sweet potato was generated when leaves,
shoot-tips, stems, and roots of the cultivars 'White Star' and 'GaTG 3'
were placed on MS medium with 1 mg/L 2,4-D (Liu and Cantliffe, 1984a).
However, embryogenic callus was most consistently obtained from leaves
(23%). Callus was readily produced between 2,4-D concentrations of 0.5
to 2.0 mg/L, while above 2.0 mg/L, callus formation was inhibited.
Embryos germinated and formed plants when removed from a medium with
hormones to one without. Plants flowered, five months after being grown
in growth chambers and some storage roots formed which were 15 cm or
larger in diameter.
Liu and Cantliffe (1984b) optimized 2,4-D and sucrose
concentrations for induction of embryogenic callus. Embryogenic callus
formed most frequently with 1 mg/L 2,4-D and 3% sucrose, while no
embryogenic callus formed without 2,4-D. Apical meristem domes alone or
with the youngest pair of leaf primordia most frequently formed
embryogenic callus (80-85%) than domes with two pairs or more leaf
primordia (30% or less).
Embryo formation was best when callus was fractionated and sieved,
such that the 500 to 2000 pM sizes were collected and plated on MS
medium containing one-half the concentration of ammonium nitrate (Chee
and Cantliffe, 1988a). Chee and Cantliffe (1988b) determined three
developmental stages in which somatic embryos become plants:l) late
torpedo; 2) cotyledonary; and 3) expanded torpedo stage. Larger embryo
sizes reportedly did not equate to more mature embryos and somatic
embryos generally had small cotyledons and large hypocotyls compared to
zygotic embryos. Conversion to plants from the above three embryo types
was approximately 50% when removed and subcultured on MS medium without
Delivery Methods for Somatic Embryos
Current Planting Scheme
Until recently, the ability to obtain at least a few plants from
somatic embryos was considered a major success. However, during the
1980s, more emphasis has been placed on improving embryo vigor and early
seedling growth, and increasing the percentage embryo to plant
conversion. With progress being made toward these goals, increased
attention has also been placed on development of delivery methods
suitable for economic propagation of mass quantities of somatic embryos.
To date, most plant conversion systems require several
intermediate steps prior to successful plant establishment in the field.
For example, Haydu and Vasil (1981) germinated napier grass (Pennisetum
purpureum) somatic embryos in tissue cultures until they were one to
five centimeters long. These small plants were then transferred to a
hormone-free medium in culture tubes to establish a more vigorous root
system. The next step was potting the plantlets into soil-vermiculite
in a growth chamber, then acclimating the plants to lower relative
humidities. Finally, plants were transplanted in the greenhouse and/or
For some species, plantlet formation is a very slow process.
Celery somatic embryos required five weeks before forming roots and
leaves (Dunstan et al., 1982). After adequate growth was obtained, the
plants were transferred to soil in propagation boxes in the greenhouse,
then to individual pots. Speigal-Roy and Vardi (1984) developed a
procedure for plant regeneration of citrus which required several
transfers from solid and liquid media. After plants reached a certain
size, they were cultured in tubes on paper bridges for further
development prior to transfer to soil. Sixteen to 18 weeks were
required before plants were obtained from somatic embryos and were
growing in the greenhouse.
Extensive transfer steps were also required to establish plants
from papaya Carica papaya L. somatic embryos (Litz and Conover, 1982).
Embryos were germinated and formed plants on White's medium supplemented
with 0.1-2.0 mg/L NAA and 0.05-0.20 mg/BAP. Plants were then moved to a
soilless potting mix and hardened off under intermittent mist for 2-2.5
weeks. These few examples illustrate the laborious and time consuming
task before plants from somatic embryos can be transplanted in the
Proposed Handling and Planting Methods of Somatic Embryos
Several delivery methods for propagating somatic embryos directly
from in vitro conditions to the field or greenhouse have been proposed.
They include: 1) simultaneous desiccation of somatic embryos using a
water soluble resin and planting in a wafer or seed tape (Kitto and
Janick, 1985ab); 2) desiccation, then planting dried somatic embryos
with a conventional drill (Gray et al., 1987); 3) encapsulation of
singulated somatic embryos in an alginate gel capsule, then planting
using a conventional drill (Redenbaugh et al., 1984,1986; Jeon et al.,
1986); 4) gel seeding somatic embryos with fluid drilling equipment
(Drew, 1979; Baker, 1985; Schultheis et al., 1986; Cantliffe et al.,
1987); and 5) an automated system for direct transfer of somatic embryos
to the greenhouse (Levin et al., 1988).
Desiccation in polyethyleneoxide. Desiccation is part of the
normal process during seed development and maturation (Bewley and Black,
1978). Desiccation of somatic embryos has been investigated with the
intent of arresting embryo growth, providing storage capability, and the
ability to plant as normal dry seeds (Kitto and Janick, 1985abc; Gray et
al., 1987; Gray, 1987).
Kitto and Janick (1985ab) were the first to successfully desiccate
somatic embryos of carrot and have them survive upon rehydration.
Addition of unspecified numbers of carrot embryos from suspension were
incorporated with polyethyleneoxide (Polyox), a water-soluble plastic
resin, and dried to form wafers under sterile conditions in a laminar
flow hood at ambient temperatures and humidities (Kitto and Janick,
1985b). A constant weight was achieved after 6.5 hours drying. Three
percent of the embryos survived encapsulation once rehydrated, although
it was unclear in the report as to whether plants formed. Embryo
survival decreased rapidly over drying time. No survival was obtained
when embryos were dried to a constant weight and not coated; however,
some embryos survived after 32 hours when they were coated with Polyox.
Hardening treatments, including 12% sucrose, chilling at 4C, high
inoculum density, and/or 1 pM ABA prior to desiccation reportedly
increased embryo survival compared with the nontreated control (Kitto
and Janick, 1985c). The use of a Polyox seed coat seemed to damage the
embryos during the coating and drying process as supported by low
percentage survival data. Desiccation of somatic embryos in Polyox does
not appear to be a practical seeding method, especially since coating
somatic embryos singly was difficult and that no plants formed after the
Desiccation. Desiccation of somatic embryos, as a separate step,
prior to encapsulation with a coating material has been proposed (Gray,
1987; Gray et al., 1987). Somatic embryos of orchardgrass became
quiescent when dried from 75 to 13% moisture (Gray et al., 1987). After
21 days of desiccated storage at 230C, 12% of embryos formed roots
(germination), while 4% grew into plants. Developmental stage before
desiccation was critical for embryo germination and plant formation.
Only those embryos with well differentiated scutellar and coleoptilar
regions prior to desiccation were able to germinate and/or form plants
Similar studies were conducted with grape somatic embryos (Gray,
1987). More germination (58%) and plant formation (20%) were obtained
with grape compared with orchardgrass (12% and 4% respectively) after 21
days of desiccated storage. Only 5% of grape somatic embryos
regenerated plants when embryos were not desiccated. Gray suggested
that desiccation may be an additional dormancy breaking mechanism in
grape. Breaking dormancy of grape somatic embryos by low temperatures
or with GA has already been documented (Rajasekaran and Mullins, 1979).
Parrott et al. (1988) reported that plant formation from soybean
somatic embryos typically was spread over a period of nine months.
However, after two weeks, when results from seven genotypes were pooled,
plant regeneration averaged 60% when embryos were desiccated compared
with 1% for embryos not desiccated. This study differed from the
proceeding desiccation studies in that it included a maturation period
of 28 days after which embryos were removed from their explant tissue
prior to desiccation.
Desiccation treatments have been useful for plant regeneration of
soybean and grape somatic embryos; however, the percentage plant
formation is still unacceptably low. Also, as storage times of the
desiccated embryos were increased, plant vigor and regeneration quickly
decreased (Gray et al., 1987). Relatively little is known about the
biological effects of desiccation. Thus, better control of the drying
process and more understanding of the desiccation process is necessary
before drying of somatic embryos can be incorporated in a synthetic seed
Encapsulation in soft gel capsule. A third delivery method
proposed was encapsulation of somatic embryos in a soft gel capsule
(Redenbaugh et al., 1984, 1986; Jeng et al., 1986). Several compounds
were tested for suitability for encapsulation, but calcium alginate, a
food thickener, derived from brown algae was selected (Redenbaugh et
al., 1986). This was selected due to its low embryo toxicity and ease
in forming capsules around the somatic embryo.
The gel capsules surrounding the embryo can be amended with
nutrients, hormones, and carbohydrates to facilitate the rapid growth
and survival of embryos. Most encapsulation research has dealt with
alfalfa somatic embryos which to date have consistently averaged 60%
plant formation under in vitro conditions and 20% when grown in the
greenhouse (Redenbaugh et al., cited in Fujii et al., 1987). The best
conversion frequency was 65% with celery somatic embryos when grown
under in vitro conditions with carefully selected embryos (Redenbaugh et
al., 1986). Other crops encapsulated and forming plants in vitro were
Brassica species, cotton, lettuce, and corn (Redenbaugh et al., 1987a).
Although encapsulated somatic embryos have been successfully converted
to plants in several species, there are problems with the alginate
capsules. Water-soluble nutrients have been reported to rapidly leach
out of the capsule (Redenbaugh et al., 1987b). Root and shoot emergence
have also been inhibited. In addition, Redenbaugh (1987b) stated there
is "lack of automation technology to allow medium to large-scale
production of single-embryo capsules." Finally, storage may be a
problem since embryo viability may decline over time due to inhibition
of embryo respiration in the capsule (Fujii et al., 1987). This seeding
technology is relatively new and will require much more intensive study
before it can be applied commercially.
Fluid drilling. Fluid drilling is the sowing of seeds in a
protective, flowable gel. The system was developed in England during
the 1970s (Gray, 1981). The concept of fluid drilling zygotic seed was
first conceived by Currah et al. (1974), while Drew (1979) was one of
the first to suggest the idea of fluid drilling somatic embryos.
Fluid drilling has improved plant establishment, and led to
earlier, improved yields in a number of small seed species such as
carrot (Currah et al., 1974), tomato (Gray et al., 1979), parsnip (Gray
and Steckel, 1977), celery (Biddington et al., 1975), lettuce (Gray,
1978) and pepper (Ghate and Phatak, 1982). Actively growing or
pregerminated seeds can be sown in the gel without damage. The gel can
also be amended with nutrients, carbohydrates, beneficial microorganisms
(mycorhizae, bacteria), and pesticides to stimulate embryo growth and
improve plantlet survival. The microenvironment created around the
seeds when fluid drilled can be modified and optimized such that the
above advantages can be realized.
Due to their various chemical compositions, certain gels may be
better seed carriers for fluid drilling. For example, some gels may
facilitate better oxygen movement, some may contain nutrients which
enhance plant growth, and others may hold more moisture. Several gels
have been used for fluid drilling zygotic seed. Some of the more
commonly used gels are: Laponite (magnesium silicate clay,), Liqua-gel
(potassium starch acrylamide manufactured), Planta-gel (copolymer of
potassium acrylate and acrylamide), Terrasorb (starch or synthetic
copolymer), Hydrozorb 30 (potassium acrylate), and N-gel (various
cellulose based materials, formerly called Natrosol).
A laboratory study by Brocklehurst (1979) determined the
polyacrylate mineral colloid gel was superior to the natural gum gels
since the later caused senescence of lettuce seedlings four days after
planting. Sosa-Coronel and Motes (1979) evaluated seven commercial gels
for planting carrot, onion, and pepper seed. Pregerminated seeds were
suspended in gels for four and 24 hours prior to seeding in the
greenhouse. Superior seedling growth was obtained for all species sown
with Viterra 2, Natrosol 250, and Bacto-Agar gels, while Gum Blend and
CLD were toxic.
Oxygen movement is dependent on gel type and temperature. Frazier
et al. (1982) determined that oxygen movement in gels was slower as
temperature decreased. Slower oxygen movement in the gel could be
critical since low temperatures at planting might restrict the
metabolism of the pregerminated seed. Seed viability was related to
oxygen diffusion in the gel. Of all the gels tested, Natrosol had the
highest oxygen diffusion rate and led to the most radicle emergence and
growth of snapdragon seedlings when suspended in gel. Pill and
Fieldhouse (1982) ran a similar comparison and also found that Natrosol
was the only gel that tomato seed could be stored in for more than four
days without reducing the percentage and rate of emergence. Bryan et
al. (1982) observed slower plant emergence and reduced pepper stands
when germinated seeds were sown in Laponite gel than without gel during
wet growing conditions. Laponite has been reported to reduce 02 uptake
by the seed (Brocklehurst, 1979).
Protection from Damage and Desiccation
Gels will protect actively growing seeds with or without roots.
In addition, gels absorb water to many times their weight. This keeps
moisture around the seeds and reduces the possibility of desiccation.
Gels may retain moisture around the seeds several days after planting
(Bryan et al., 1978).
Ghate (1982) studied the theological properties of gels when
amended with chemical additives. Little change occurred in the gel
without additives after one day when temperatures varied between 10 and
37*C. The addition of fertilizer caused gels to become thin while,
relative consistency was maintained after the addition of more gel.
Nutrient salts have been added to gels at planting to enhance
germination and early seedling growth from zygotic embryos. Nitrogen,
phosphorus, and K were added to guar gum gel with little effect on
lettuce or cabbage growth (Costigan and Locascio, 1982). Finch-Savage
and Cox (1982) reported improved early seedling growth of carrots with
the addition of monosodium phosphate at concentrations up to 30 g/L, but
nitrate (5-20 g/L) reduced seedling growth. In a similar study,
Finch-Savage and Cox (1983) obtained earlier maturity in lettuce and
onion when pregerminated seeds were fluid drilled in gel supplemented
with either sodium phosphate or ammonium nitrate compared with
nontreated, dry sown seed. No benefit was obtained when less than 10
g/L ammonium nitrate was incorporated in the gel.
Faster, more complete germination of pepper seeds was reported
when cytokinins, humic acids, or alpha-keto acids were added to the gel
and planted under wet environmental conditions (Bryan et al., 1982).
The addition of GA3 and GA4/7 to gel at 2 and 2.5 mg/L increased the
growth of tomato seedlings compared with gel without GA (Ohep and
Cantliffe, 1980). Tomato plants were taller when the gel was
supplemented with either Cytex or Cytozyme, while increased plant
height, dry weight, and leaf area under field conditions were obtained
when humic acids were incorporated. Gibberellic acid and diphenamid
hastened the emergence rates of pepper by one to three days (Ghate and
Various pesticides have improved plant establishment. Ohep et al.
(1984) reduced damping off of tomato seedlings by incorporating ethazol
plus thiophanate-methyl, fenaminosulf, and ethazol alone or combined
with benomyl, chloroneb, or captain into the gel. White (1979)
controlled white rot of onions more efficiently by placing 25 mg a.i./L
metalaxyl in the gel, while much higher concentrations of metalaxyl were
required to overcome the problem when incorporated in the soil.
Similarly, iprodione, when placed in gel, effectively controlled white
rot of salad onion at one-quarter the rate used for dry seeds
Hadar et al. (1984) added the biocontrol agents Trichoderma
harsianum and T. kininoii to gel to prevent seed rot in peas (Pisum
sativum L.) and navy beans (Phaseolus vulgaris L.) when sown in pythium-
infested soil. They determined that 95% of the plants emerged when
seeded in gel amended with T. harsianum and kininoii compared with 15%
when planted with dry seeds. Inclusion of Rhizobium in the gel resulted
in more than three times more root nodules on navy bean compared to
inoculation of dry seed (Hardwick and Hardaker, 1977).
The growth of Trichoderma hamatum, a biocontrol and antagonist
used to control Rhizoctonia solani, was compared in Viterra, Natrosol,
and Laponite gel carriers (Mihuta-Grimm and Rowe, 1986). Superior
growth of Trichoderma isolates was obtained in Natrosol versus Viterra
and Laponite gels. Due to better growth of the antagonist in Natrosol,
this gel was supplemented with Trichoderma to control damping off when
fluid sowing radish seeds. Four delivery systems for application of
Trichoderma isolates were compared; 1) fluid drilling in Natrosol, 2)
coated seed, 3) application of wheat-bran grown isolated applied in the
furrow before planting and 4) soil drenching with five day broth grown
cultures. The best disease control was consistently obtained with fluid
A peat-vermiculite mix was combined with Liqua-gel (gel mix) and
resulted in earlier, more uniform, and complete plant emergence of
pepper compared with gel seeding in either Laponite or Liqua-gel gels,
plug-mix, or dry sown seed (Schultheis et al., 1988). The increased
availability of moisture and nutrients were suggested as a reason for
improved plant stands.
Gel Studies with Somatic Embryos
Baker (1985) determined that a mixture of peat and vermiculite was
superior to vermiculite alone as a soil planting medium for carrot
somatic embryos since the former alleviated water stress. However,
vermiculite was the best soil cover since it did not crust and inhibit
emergence. Baker also compared Natrosol and Planta gel for suitability
as carriers for fluid drilling somatic embryos. No difference was
measured between gels with respect to embryo emergence, which was
approximately 5 to 30% under in vitro conditions. The polyacrylamide
material was used in subsequent greenhouse studies. In addition to
Baker, Schultheis et al.(1986) reported that somatic embryos of sweet
potato grew quite rapidly in N-gel when compared to three other gel
Naphthalene acetic acid and/or GA were placed in the gel to hasten
and improve survival of carrot somatic embryos; however, no advantage
was gained from the various hormone combinations used (Baker, 1985).
Survival ranged from 1 to 7% under greenhouse conditions after seven
days, but no plants developed into plants. Embryos survived in the gel
for several days, but stopped growth after a few days.
Soil covers have been used to provide an environment favorable for
plant establishment. A large number of materials have been tested as
anticrustants, from synthetic organic polymers to plain sand. Few
materials, unfortunately, have been shown to have broad applications.
Their effectiveness apparently is closely tied to soil type, species,
and environmental conditions, particularly moisture and temperature.
Hemphill (1982) evaluated the effect of several anticrustants on plant
establishment for a number of small seeded
species. He found the most consistent establishment was obtained when
using vermiculite followed by phosphoric acid.
In more recent studies, several soil amendments were evaluated for
their effects on cabbage (Perkins-Veazie and Cantliffe, 1987), tomato
(Odell and Cantliffe, 1987), and lettuce (Seale and Cantliffe, 1986)
stand establishment under a range of growing conditions. A calcine clay
(GrowSorb, MidFlorida Mining), high temperature fired (LVM) 24/48,
consistently improved percentage plant emergence. Seale and Cantliffe
(1986) suggested the advantage obtained with GrowSorb might be
attributed to its ability to maintain favorable moisture conditions
during germination. Another promising soil amendment was gel mix
(mixture of peat and gel) (Schultheis et al., 1988). This hastened
emergence and usually increased seedling growth both in tomato (Odell
and Cantliffe, 1987) and lettuce (Seale and Cantliffe, 1986).
In summary, there are many factors such as explant source, light,
temperature, nutrition, hormones, carbohydrates which influence the
quantity and quality of somatic embryos produced. These same factors,
as mentioned previously, affect the formation of roots, shoots and
plants obtained from somatic embryos. Although somatic embryos are
similar to zygotic embryos, the former lack many of the stored reserves
zygotic embryos have available for their early seedling growth. Thus,
it is important to find which "growth factors" are necessary to achieve
rapid and complete plant formation from somatic embryos. It is also
necessary to use a synthetic seeding method which could be easily
adapted for field use. The incorporation of additives around the
embryos to sustain and improve their growth and development into plants
would be required for the successful implementation of synthetic
The research which follows has focused on the fluid drilling
method. Fluid drilling offers several advantages over the other
planting methods mentioned earlier. They include the ease of amending
the gel with additives around the embryo, the ability to plant the
embryo in an advanced growth stage with no need for desiccation, the
high moisture retention of the gel which could protect the embryo from
desiccation and injury at planting, and the available technology and
equipment available for large-acreage planting.
The primary goal of the following studies was to find a gel
carrier and amendments suited for fluid drilling somatic embryos. Once
the gel carrier was determined, a series of experiments were conducted
to determine the gel additives that would promote rapid, uniform, and
complete plant conversion from somatic embryos. Early plant growth and
root yield comparisons were also made between plants obtained from
somatic embryos and vegetatively propagated.
OPTIMIZING SWEET POTATO ROOT AND PLANT FORMATION
BY SELECTION OF PROPER DEVELOPMENTAL STAGE,
EMBRYO SIZE, AND GEL TYPE FOR FLUIDIZED SOWING
Patterns of somatic embryony in sweet potato (Ipomoea batatas
Poir.) were recently determined (Chee and Cantliffe, 1988). The authors
identified embryos at three developmental stages which had the potential
of becoming plants: 1) torpedo, 2) cotyledonary, and 3) elongated
torpedo. Embryos capable of plant development usually ranged from 1 to
4 mm long.
Selection of the proper embryo developmental stage and size could
improve germination and subsequent plant formation of sweet potato.
Gray et al. (1987) determined that orchardgrass (Dactylis glomerata)
somatic embryos must have well-developed scutellar and coleoptilar
regions for germination to occur. Morphological features of the embryos
were suggested by Stuart et al. (1985) to be an identifiable marker in
determining which embryos would more readily convert to plants. Embryo
length has been positively correlated with germination and/or plant
formation in zygotic and somatic embryos (Gray et al., 1987; Austin et
al., 1969; Soffer and Smith, 1974; Stuart et al., 1985).
Several delivery methods have been proposed for direct-field
synthetic seeding of somatic embryos: 1) encapsulation of singulated
embryos in an alginate gel capsule (Redenbaugh et al., 1984, 1986); 2)
embryo desiccation in a hardened material (Gray et al., 1987); 3) embryo
encapsulation in a soft gel capsule and their subsequent desiccation
(Jeon et al., 1986); 4) simultaneous desiccation of embryos in water
soluble resin (Kitto and Janick, 1985ab); 5) an automated system for
direct transfer of embryos to the greenhouse (Levin et al., 1988) and 6)
fluid drilling (Drew, 1979:Baker, 1985; Schultheis and Cantliffe, 1988).
Drew (1979) was one of the first to suggest fluid drilling as a delivery
method but few researchers have investigated synthetic seeding using
this method (Baker, 1985; Schultheis and Cantliffe, 1988).
The purposes of these studies were two-fold. One goal was to find
a gel carrier which was compatible for fluid drilling somatic embryos.
Upon identification of the gel, the second goal was to determine the
effect of embryo developmental stage and embryo size on the timing and
percentage of root and plant development to improve synthetic seeding
via fluid drilling.
Materials and Methods
Sweet potato plants, cv. White Star, were vegetatively propagated
and maintained in a growth chamber at 27C and 16 hours light/12 hours
dark photoperiod. Vigorous vegetative growth was maintained by frequent
fertilizer applications, watering, and pruning of the stems. Shoot tips
2-cm long were excised and surface sterilized in 1.2% hypochlorite and
Tween-20 for five minutes. Apical domes with up to two leaf primordia
pairs were plated on agar-solidified medium containing 10 pM 2,4-D, and
basal medium containing inorganic salts of Murashige and Skoog (MS)
(1962), 500 pM myo-inositol, 5 pM thiamine.HC1, 10 pM nicotinic acid, 5
pM pyridoxine.HC1 (Table A-1), 3% wt/vol sucrose, and 0.7% wt/vol
'Phytagar' (GIBCO Lab., Grand Island, NY). The pH was adjusted to 5.8
using 1.0 N NaOH solution, then autoclaved for 20 minutes. Twenty ml of
medium was poured into presterilized 100 x 15 mm plastic Petri plates.
Incubation was in the dark at 27*C with unmonitored light interruptions
necessary for routine observation.
After eight weeks, embryogenic callus was transferred to Petri
plates containing basal medium plus 10 pM 2,4-D and 1 pM 6-
benzylaminopurine. Six to eight weeks later, embryogenic callus was
bulked from several plates and placed in beakers. Cell aggregates were
gently broken apart into natural units with a glass slide, then
separated into a 355 to 710 micron size fraction, and a 710 micron and
larger fraction with copper sieves. The largest-sized fraction was used
for embryo production and plated on basal medium without growth
regulators, 10 mM ammonium nitrate (half strength), and 0.8% wt/vol
Phytagar. The other fraction was used to maintain and proliferate
embryogenic callus. Incubation was one week in the dark with
unmonitored light interruptions, then in light in a 10/14 light/dark
cycle at 27*C until embryos were selected.
A solution containing MS inorganic salts, 500 pM myo-inositol, 5
pM thiamine.HCl and 3% wt/vol sucrose was adjusted to a pH of 6.5, then
mixed with each gel at a rate of 2.0% wt/vol. Four gel types were
compared: 1) copolymer of potassium acrylate and acrylamide (PC)
(Planta-gel, Nepera, Inc. Harriman, N.Y.); 2) potassium starch
polyacrylamide (PSA) (Liqua-gel, Miller Chemical Co., Hanover, Pa.); 3)
potassium acrylate (PA) (Hydrozorb 30, American Colloid, Arlington
Heights, Ill.); and 4) hydroxyethyl cellulose (HEC) (N-gel H, Aqualon
Inc., Wilmington, Del.). The gels were autoclaved for 20 min and about
8 ml of each gel dispensed into quadrants of 100 x 15 mm Petri dishes.
Although the pH was adjusted to 6.5 prior to autoclaving, the pH was 0.5
to 1.0 unit lower after HEC and PA gels were autoclaved, while the pH of
the PSA gel remained near 6.5 and the pH of the PC gel was increased to
One embryo was placed into the separated quadrants from a group of
randomly selected embryos. Ten embryo samples were used in each of
three replications. Embryo survival was rated by color; green (healthy,
viable embryo), some browning (embryo unhealthy), or completely brown or
black (embryo death) three and six days after being placed in gel.
Embryos were observed after 21 days for root and plant development.
Data were analyzed using analysis of variance and means separated with
Duncan's multiple range test.
Influence of Embryo Size. Maturation Time. and Stage of Development on
the Percentage of Embryos which Grew into Roots and Plants
Embryos at the torpedo, cotyledonary (recognized by formation of
lobed cotyledonary-like lateral appendages), and elongated torpedo stage
(hypocotyl expanded) of development were selected (Fig. 3-1). One of
each embryo type was placed in a 25 x 100 mm Petri plate (Lab-Tek, Nunc,
Inc., Naperville, Il) containing 20 ml gel. The gel was amended with
the inorganic nutrients of MS, and the same organic constituents used
for callus production. Length and diameter of all embryos were measured
using a stereomicroscope with a calibrated ocular lens. Embryos were
observed for root and plant development six, 12, 21, and 31 days after
plating. A radicle at least 2 mm long was defined as a "root" whereas a
well-developed shoot apex (with leaf primordia) and root was defined as
a "plant." Two experiments were conducted. Before embryos were placed
Fig. 3-1. Embryo developmental stages capable of plant conversion.
Torpedo embryo stage with rudimentary cotyledons (left),
cotyledonary embryo stage with small lobed
cotyledonary-like appendages (middle), and elongated
torpedo stage with elongated hypocotyl (right).
in the gel, they were incubated (allowed to mature) on agar-solidified
basal medium for 25 days in experiment 1 and 16 days in experiment 2.
Each experimental treatment was replicated three times, each replicate
had 10 embryo samples. Significance between means were determined using
standard error analysis.
Disregarding embryo development stage, somatic embryos were
separated into three size categories by length: 1) 1.0 to 1.95, 2) 2.0
to 2.95, and 3) 3.0 to 3.95 mm. Differences in root and plant formation
were determined at 31 days using least significant differences analysis.
The suitability of a gel carrier for growth and development of
somatic embryos was easily observed by the embryo color change shortly
after placement in the gels. All embryos placed in the HEC gel were
green and healthy after three and six days (Table 3-1). However,
placement of embryos in the acrylate or acrylamide type gels resulted in
at least 23% of the embryos with partial necrosis after three days.
After six days, the PSA gel was the best of the acrylamide gel types
with 40% green embryos. Nearly all embryos were dead after six days
when placed in the other two acrylate or acrylamide gels.
Some embryos grew into plants after being cultured 14 days in HEC
gel (Fig. 3-2). No root, shoot, or plant formation was observed when
embryos were placed in the other gels. Thus, in subsequent experiments,
the HEC gel carrier was used. A rate of 2.5% wt/vol was used instead of
the 2.0% because the thicker 2.5% consistency was most suited for gel
Table 3-1. Embryo survival three and six days after placement in gel.
Gelz 3 days 6 days
Green Necrosis Dead Green Necrosis Dead
cellulose 100ay Ob Oc 100a Ob Oc
Copolymer 17c 57a 27b Oc 13b 87a
polyacrylamide 70b 23ab 7bc 40b 36a 23b
acrylate 3c 40a 57a Oc 3b 97a
zMurashige and Skoog inorganic salts, 500 pM myoinositol, 5 pM
thiamine.HC1, and 3% wt/vol sucrose were incorporated in gels.
YMeans separated within columns using Duncan's multiple range test, 5%
Fig 3-2. Effect of gels on embryo survival and plantlet development.
Lack of embryo growth in the acrylamide and/or acrylate gels
(upper left and right quadrants, and lower left quadrant)
and plant formation obtained in hydroxyethyl cellulose gel
(lower right quadrant).
A American Colloid Company's Hydrozorb 30 gel.
L Liqua-gel by Miller Chemical Co.
V Viterra (Planta-gel) by Nepera Inc.
N N-gel by Aqualon, Inc.
Influence of Embryo Size. Maturation Time. and Stage of Development on
the Percentage of Embryos which Formed Roots or Developed into Plants
Elongated torpedo embryos formed roots earlier than the other
embryo types (Fig. 3-3A). After six days, 37% of the elongated embryos
had roots compared with 7% for the torpedo and cotyledonary embryos.
After 31 days in culture, the total percentage root formation was 58%
for the torpedo and 52% for the cotyledonary embryos, while 88% of the
elongated embryos produced roots.
Plant formation was similar regardless of embryo developmental
stage at 6, 12 and 21 days (Fig. 3-3B). However, the percentage of
embryos which converted to plants from elongated torpedo embryos was
greater than torpedo or cotyledonary embryos at 31 days (38 vs. 21 and
Using embryos at different stages of development had less effect
on the percentage of embryos which formed roots and plants in experiment
2 (Fig. 3-4) than in experiment 1 (Fig.3-3). However, root development
again occurred earlier in the elongated torpedo than either the
cotyledonary or torpedo embryos after 12 days (Fig. 3-4A). The final
percentage root formation was greater with embryos at the elongated
torpedo development stage than those at the cotyledonary development
stage. As in experiment 1, the percentage of embryos which grew into
plants were similar regardless of embryo development stage through day
21 (Fig 3-4B). However, more embryos at the elongated and torpedo
development stage grew into plants compared with cotyledonary stage
embryos (63 and 59 vs. 41%, respectively).
Embryos at three developmental stages removed from maturation
medium after 25 days and their effects on the percentage
root and plant formation after being placed in hydroxyethyl
cellulose gel with the inorganic salts of Murashige and Skoog
medium, 500 MM myo-inositol, 5 pM thiamine.HCl, and 3% wt/vol
sucrose, experiment 1.
A. Percentage root development of embryos at the torpedo,
cotyledonary, and elongated torpedo developmental stage
at 0, 6, 12, 21, and 31 days.
B. Percentage plant development of embryos at the torpedo,
cotyledonary, elongated torpedo developmental stage at
0, 6, 12, 21, and 31 days.
0 6 12 21 31
0 6 12 21 31
Embryos at three developmental stages removed from maturation
medium after 16 days and their effects on the percentage
root and plant formation after being placed in hydroxyethyl
cellulose gel with the inorganic salts of Murashige and Skoog
medium, 500 iM myo-inositol, 5 pM thiamine.HCl, and 3% wt/vol
sucrose, experiment 2.
A. Percentage root development of embryos at the torpedo,
cotyledonary, and elongated torpedo developmental stage
at 0, 6, 12, 21, and 31 days.
B. Percentage plant development of embryos at the torpedo,
cotyledonary, elongated torpedo developmental stage at
0, 6, 12, 21, and 31 days.
0 6 12 21 31
______ da, ............~....~ii ----- I -------\ --- \ --
In both experiments, embryos at the elongated torpedo
developmental stage had the greatest average embryo length. The next
longest embryos were those at the cotyledonary stage and the shortest
embryos were at the torpedo stage (Table 3-2). Selection of the
elongated torpedo embryos, which were the largest-size, consistently led
to improved root and plant development compared with embryos at the
other two developmental stages (Fig. 3-3 and 3-4). However, at least as
many of the smaller torpedo embryos grew into plants compared with the
larger cotyledonary embryos. Thus, embryo growth into plants was more
closely associated with developmental stage than to increased embryo
Embryos at all developmental stages were separated according to
length into three categories from both experiments. The percentage of
embryos which formed roots and plants were similar regardless of embryo
size (Table 3-3).
The superior embryo growth and plant formation obtained in the HEC
gel can be attributed to a number of factors. Considering that the
capacity for most gels to absorb water is great and that oxygen is not
very soluble in water, the effects of total quantity and rate of oxygen
movement in the gel was likely to have been extremely influential in the
growth and survival of the embryos in HEC gels. Frazier et al. (1982)
determined that seed viability across gel types was related to the
oxygen diffusion rate (ODR). They reported HEC had better ODR than
either Laponite (magnesium silicate clay) and Permasorb gels. Eastin
(personal communication, Kamterter Inc., Lincoln, Neb.) confirmed these
Table 3-2. Relationship of
Developmental stage obs.
embryo developmental stage to embryo size.z
Embryo size (mm)
Expanded torpedo 25
ZAverage mean standard error.
Table 3-3. Effect of embryo length on percentage root and plant
formation after 31 days in culture. Embryos were sorted
by size regardless of developmental stage.z
length embryo Embryo size (mm) % Response
(mm) samples Length Width Root Plant
1.00 1.95 63 1.71 .02Y 0.92 .02 67x 39"
2.00 2.95 76 2.38 .03 1.04 .02 74 38
3.00 3.95 41 3.39 .04 1.00 .03 79 43
ZData pooled from 2 experiments.
YAverage mean standard error.
XLSD(0.05) for comparing root formation means 16%.
WLSD(0.05) for comparing plant formation means 15%.
reports by placing seeds in HEC gel and subjecting them to anaerobic
conditions. He found more oxygen was present in the gel immediately
after than prior to treatment.
The pH of the gels was probably not a major factor affecting
embryo survival and growth. Although gel pHs ranged between 5.5 and
7.2, tissue cultures can grow within a wide range of pHs (Skirvin et
The HEC gel has other positive features which make it attractive
for planting somatic embryos. In addition to the high ODR, the pH was
measured at 6.5 prior to autoclaving and 5.8 after autoclaving which
were at levels where nutrients were available for plantlet growth.
Also, the HEC gel maintained its viscosity when amended with salts,
while many gels break down. Ghate (1982) reported that low levels of
fertilizer salts reduced the viscosity of all gels tested and their
usefulness as carriers of seed; however, the HEC gel was not included in
his study. It has been reported that HEC gels maintain their viscosity
better than polyacrylamide gels (Ward, 1980). The maintenance of gel
viscosity will be critical for synthetic seeding since additives must be
incorporated into the gel to replace the missing "growth factors" needed
by the naked somatic embryo.
The occurrence of faster root formation from elongated embryos was
probably due to these embryos being at an more advanced developmental
stage and the possible initiation of germination as evidenced by their
elongated hypocotyls. The elongated torpedo embryos, in addition, may
have had better shoot meristem development which increased plant
production compared with embryos at the cotyledonary and torpedo
For all embryo developmental stages, the percentage that formed
plants was much less than the percentage that formed roots. This
indicated that the shoot meristem was not as well organized or developed
as the root meristem. Chee et al. (1989) reported the lack of shoot
meristem often coincided with root formation in sweet potato somatic
embryo conversion studies. Precocious germination has been reported to
commonly occur in somatic embryos resulting in stunted and abnormal
embryos in species such as carrot and caraway (Ammirato, 1985). Better
apical meristem development is necessary during embryo maturation for
improved and more consistent plant formation.
More roots and plants were produced from embryos in experiment 2
than 1 as nearly two times more plants were produced from embryos at all
developmental stages in the former than latter experiment. The improved
root and plant production obtained in experiment 2 was probably related
to the relatively short incubation time that embryos were cultured on
maturation medium (16 days in experiment 2 vs. 25 days in experiment 1).
The time factor/variation may have affected the availability of either
nutrients and/or carbohydrates obtained from supporting tissues or
medium during embryo maturation. In these experiments, the shorter
incubation time period apparently resulted in better embryo quality.
This may have been due to more availability of nutrients or organic
reserves, while these reserves may have been depleted over time and led
to reduced embryo quality in experiment 1. Delouche (1980) reported
that environmental conditions during zygotic seed development played a
key role in the overall quality of the seeds produced. The same
conclusion most likely holds true for somatic embryos.
Embryo size has been correlated to improved seed vigor carrot
(Daucus carota)(Austin et al., 1969) and lettuce (Lactuca sativa)(Soffer
and Smith, 1974) or somatic embryo quality in alfalfa (Medicago
sativa)(Stuart et al., 1985), but this was not found to be the case in
these studies. The quality of somatic embryos was apparently more
influenced by length of maturation time than embryo size. Redenbaugh et
al.(1987) reported embryo quality as the most important factor for
somatic embryo to plant conversion. They defined quality as the ability
of the embryo to convert to a plant.
Improved seed or embryo quality is the key to the successful
establishment of any crop, whether by direct or synthetic seeding. Use
of protein markers (such as those that accumulate during seed
maturation) rather than morphological markers would appear to be more
effective in determining the overall embryo quality since the latter
does not reveal the physiological status of embryo development. Work in
this regard has been reported with somatic embryos in rapeseed (Sussex
and Crouch, 1982) and soybean (Stuart and Nelson, 1988) However, this
method is destructive and time consuming, while use of a morphological
marker is not. A computer sorting system has been developed for sorting
embryos by size and shape (Grand d'Esnon et al., 1988). Gray (1987)
effectively used morphology to discern which orchardgrass somatic
embryos would convert to plants.
Mass planting of many potentially important biomass crops is not
cost-effective because many of these crops cannot be propagated by seed.
Large quantities of somatic embryos can be cloned rapidly via
embryogenesis. However, the planting of these propagules has been
limited to small-scale use because a number of intermediate transfer
steps are required to insure embryo and plant survival. Direct planting
of somatic embryos produced from tissue culture could be accomplished on
a large-scale by using a sowing method such as fluid drilling. Studies
were conducted to find a gel carrier amenable for fluid sowing somatic
embryos and to identify an embryo maturation time, developmental stage,
and size which led to the fastest and/or most complete root and plant
To determine the compatibility of somatic embryos with gel,
embryos were placed in gel carriers (2% wt/vol) amended with basal
medium containing MS inorganic salts, vitamins and sucrose 3% (wt/vol).
Most embryos died after six days when placed in gels composed of
acrylamide and/or acrylate. All embryos placed in HEC gel were healthy
after six days, while some embryos developed into plants after two
weeks. Thus, HEC was determined to be suited for fluid drilling of
somatic embryos and was used in all subsequent experiments.
Embryos were incubated on agar-solidified basal medium for 25 days
in one experiment and 16 days in another. After incubation, embryos at
the torpedo, cotyledonary, and elongated torpedo stages of development
were selected and placed in the gel. More roots formed earlier from
embryos at the elongated torpedo stage than the cotyledonary and torpedo
stages of development, regardless of incubation time. When embryos were
incubated for 25 days before placement in gel, root development for the
elongated torpedo stage embryos was greater and plant conversion was two
times more at 31 days compared to the other stage embryos. However,
when embryos were incubated for only 16 days before placement in gel,
root and plant development were greater than when incubated for 25 days.
Also, when embryos were incubated for 16 days, the percentage root and
plant formation were similar regardless of embryo developmental stage.
The exception was the lower plant formation obtained with cotyledonary
Thus, the shorter 16 day incubation time is critical for more
plant conversion regardless of embryo developmental stage. The
selection of elongated torpedo embryos consistently resulted in the
greatest root and plant formation regardless of incubation time.
Embryos were also sorted according to size regardless of
developmental stage. In spite of size differences, the percentage of
embryos which formed roots and plants were similar. Embryos could be
sorted by size since elongated torpedo embryos are usually larger;
however, to insure the highest percentage plant conversion, elongated
torpedo embryos should first be selected before sorting.
The compatibility of HEC gel with somatic embryos, the use of
shorter incubation times during embryo development, and the selection of
embryos at the elongated torpedo stage of development are an important
steps towards facilitating the development of synthetic seeding via
OPTIMIZING ROOT AND PLANT PRODUCTION FROM SOMATIC EMBRYOS
OF SWEET POTATO WITH NUTRIENT, CARBOHYDRATE, AND HORMONE
AMENDMENTS TO HYDROXYETHYL CELLULOSE GEL FOR FLUIDIZED SOWING
Sweet potato ranks seventh among food crops grown world-wide (FAO
Production Yearbook, 1986). Although sweet potato is used primarily as
a food source, some cultivars store large quantities of starch and have
good biomass potential (Smith and Frank, 1984). The starches can be
readily converted in these cultivars to ethanol or methane (Smith et
Commercial plantings of sweet potato are planted by vegetative
propagation. Mass plantings of sweet potatoes for biomass would be too
costly since planting costs would exceed the returns from methane
(Cantliffe et al., 1987). This cost limitation could be partially
overcome by direct field sowing of synthetic seed propagules produced
via somatic embryogenesis.
Use of somatic embryogenesis in a synthetic seeding system offers
several potential advantages. They are: large quantities of propagules
produced in limited space, maintenance of genetic uniformity, rapid
propagule multiplication, and direct planting of somatic embryos into
the field, thus eliminating costly transplanting (Fujii et al., 1987).
Somatic embryos have been routinely obtained in sweet potato (Liu
and Cantliffe, 1984; Chee and Cantliffe, 1988). Although somatic
embryos are nearly identical to zygotic embryos (Steward, 1963;
Wetherall and Halperin,, 1963), the former lack protective seed coats
and the storage reserves that are available to zygotic embryos during
germination. A synthetic seeding method must be developed so that the
somatic embryo will be protected and the necessary growth requirements
strategically placed so that rapid and complete plant establishment will
occur when embryos are sown in the field.
The suspension of somatic embryos in a viscous gel (fluid
drilling) has been suggested as a synthetic seeding method (Drew, 1979).
A hydroxyethyl cellulose gel (N-gel) was proposed, as reported in the
previous chapter, as an ideal gel carrier for fluid drilling somatic
Most reviews dealing with somatic embryogenesis have concentrated
on developmental embryogenesis and have given little attention to the
efficient rooting and shoot formation of somatic embryos. (Ammirato,
1983; Tisserat et al., 1979). The response of somatic embryos to
different nutrient types and concentrations has varied among species
examined. For example, the greatest number of vigorous soybean plants
were obtained with half-strength Murashige and Skoog (MS) medium
(Lazzeri et al., 1987), whereas in barley, the percentage of embryos
which grew into plants was similar regardless whether half or full-
strength MS was used (Rengel and Jelaski, 1986). Stuart and Strickland
(1984a) found that the production of alfalfa plants was dependent on the
source of nitrogen (N), glutamine (30mM) being the best reduced N
A few investigators have determined the effect of different
carbohydrates on root and/or plant production. Redenbaugh et al. (1987)
determined that glucose led to superior alfalfa plant production
compared with maltose, cellobiose, lactose, fructose, and sucrose. Root
production reportedly increased in soybean with 44 mM sucrose compared
with 87 mM glucose (Lazzeri et al., 1987).
The effect of carbohydrate concentration on rooting and plant
development has varied among species. Soybean somatic embryos produced
roots in 73% of the embryos when placed in medium with 44 mM sucrose,
while 30, 15, and OX roots were produced from embryos when basal medium
was supplemented with 87, 154, and 349 mM sucrose, respectively (Lazzeri
et al., 1987). In sugarcane, rooting was increased with 154 mM sucrose;
however, shoot production was reduced (Ahwoowalia and Maretzki, 1983).
When the sucrose concentration was lowered to 87 mM, both root and shoot
production in sugarcane were improved. Chicory plant production from
somatic embryos was not affected by reducing the sucrose concentration
from 58 to 29 mM (Heirwegh et al., 1985). In Norway spruce, 60 mM
sucrose promoted shoot production when compared with 30 or 90 mM (Arnold
and Hakman, 1988).
The addition of auxin to basal medium has been beneficial for
improved plantlet formation in some cases (MacKinnon et al., 1987; Zhi
hou et al., 1984), while the addition of GA reportedly improved root and
plant development from somatic embryos in species such as soybean (Ranch
et al., 1985), horsechestnut (Radojevic, 1988), citrus (Kochba et al.,
1974) and grape (Stamp and Meridith, 1988).
The percentage of somatic embryos which develop into plants and
the rate in which the embryo grows into a plant must be improved if
field plantings are to be successful, especially when sown under hostile
conditions of environmental stress. The following studies were
conducted to optimize nutrient, carbohydrate, and hormone formulations
for root and plant production in sweet potato in a fluidized gel. The
determination of additives that improve plant formation in a gel carrier
would be an important step in the development of the fluid drilling
method for synthetic seeding.
Materials and Methods
Sweet potato plants, cv. White Star, were grown and maintained in
growth chambers at 270C. The photoperiod was 16 hours light and eight
hours dark and light intensity was 980 pEm-2s-1. Healthy shoot tips 2-cm
long were removed from the plants, and surface sterilized in 1.2%
hypochlorite and Tween-20 for five minutes. Apical domes with up to two
leaf primordia were plated on agar-solidified medium containing 10 AM
2,4-D, and basal medium containing the inorganic salts of MS (1962), 500
pM myoinositol, 5 pM thiamine.HCl, 5 pM pyridoxine.HCl, 10 pM nicotinic
acid (Table A-l), 87 mM (3% wt/vol) sucrose, and 0.7% wt/vol 'Phytagar'
(GIBCO Lab., Grand Island, NY). Prior to autoclaving, the pH was
adjusted to 5.8 using 1.0 N NaOH solution. Twenty ml of medium was
dispensed into presterilized 100 x 15 mm plastic Petri dishes.
Incubation was in the dark at 27*C with unmonitored light interruptions
that were necessary for routine observations.
Embryogenic callus was removed from plates after eight weeks and
placed in basal medium containing 10 pM 2,4-D and 1 pM 6-
benzylaminopurine. Six to eight weeks later, embryogenic callus was
removed from plates and collected in beakers. Cell aggregates were
broken apart at their natural cleavage lines using a glass slide, then
separated using copper sieves into a 355 to 710 micron fraction and a
710 micron and larger fraction. The largest-sized fraction was plated
for embryo production on basal medium containing no growth regulators,
10 mM ammonium nitrate (half-strength) and 0.8% Phytagar. Incubation
was one week in the dark, then for the remaining duration in a 10/14
light/dark cycle at 27C.
Gel Additive Studies
All gel additive studies were conducted in vitro. The treatment
media solutions were mixed and the pH adjusted to 6.5 prior to the
addition of 2.5 X wt/vol hydroxyethyl cellulose gel (N-gel, Aqualon,
Wilmington, Del.). Gel solutions were autoclaved for 20 minutes, then
25 ml of each gel treatment was poured into a presterilized 25 x 100 mm
Petri plate (Lab-Tek, Nunc, Inc., Naperville, Il). Embryos were used
when growth was arrested at either the torpedo, cotyledonary, or
elongated torpedo stages of development after 16 to 25 days (Fig. 3-1).
In each experiment, embryos at the same developmental stage were chosen
for each embryo sample and distributed equally among treatments.
Embryos were incubated in growth chambers at 27C in a 14 hour light and
10 hour dark photoperiod. Light intensity was 250 /iE m-2s-1.
Three types of nutritional studies were conducted to determine
their effects on the rate and/or percentage of root, shoot, and plant
production. One study compared the effects of various concentrations
and types of inorganic salts in previously published nutrient media,
including Hoagland's solution (1950), Gamborg's B5 medium (1968),
White's medium as reported by Singh (1981), and MS (1962) medium (Table
A-2). A gel with no inorganic salts served as a control treatment. The
second study tested the concentration of MS basal medium (Table A-1) at
0, 1/8, 1/4, 1/2, and 1 (normal) strength. A third nutrient study
examined the constituents contained in MS basal medium and their effects
on root and plant development. The experimental treatments included: 1)
macro and micro inorganic salts plus the organic components, 2) macro
and micro inorganic salts, 3) macro inorganic salts, 4) N, phosphorus
(P), and potassium (K), and 5) no nutrients (control).
Experiments were repeated twice and results were combined in each
of the three nutritional studies. All gel treatments used in the
nutrition studies were supplemented with 87 mM sucrose. The final root
and plant formation was determined at 28 days in the MS basal medium
concentration experiments and at 21 days in the other two nutrition
studies. A root of at least 2 mm was counted as "root formation", while
a root plus a well organized shoot tip, which later formed leaves (Fig.
4-1), was counted as a "differentiated plant" for all experiments which
follow. The rate and uniformity of embryo development was determined by
a modification of the method of Gerson and Honma (1978). Root or
plantlet formation Sum (Days to development)(No. developed)/Total
number developed. Root and plant formation were determined each day
through day 10, then at 12, 14, 16, 20, and 21 and/or 28 days after
placing the embryos in the gel.
The nutrient components and concentrations were examined by
comparing different media (media comparison study) on four plates, each
with ten embryos. In the studies which examined the concentrations of
MS basal medium, four replicates with 10 and 11 embryo samples each were
used in experiments 1 and 2, respectively. In the studies which
determined the effect of constituents in MS medium, every treatment in
the first experiment had four plates containing 12 embryos each, while
Fig. 4-1. Plant conversion of embryos.
Examples of embryos with roots and developing shoot apexes.
These were considered as able to form plants at this stage
of development and counted thusly.
in the second experiment, each treatment had two replicates with 12
Fructose, galactose, glucose, maltose, and sucrose sugars were
compared in two experiments and data were combined to determine which
carbohydrates) led to faster and/or greater root, shoot, and plant
production. Each carbohydrate source was tested at 47 and 93 mM
concentrations. The results from both carbohydrate concentrations were
combined and analyzed to distinguish differences between the five
carbohydrates. Murashige and Skoog basal medium was supplemented with
vitamins and incorporated in the gel for all the carbohydrate treatments
(Table A-l). Data for each experiment were an average of four blocks of
Another study was conducted two times and data were combined to
define the fructose and sucrose concentrations most conducive for
optimum early seedling growth, and for rapid and complete embryo to
plant conversion. The concentrations in both experiments were tested at
0, 23, 47, 93, 186, and 374 mM sucrose or fructose. An additional
treatment concentration of 748 mM fructose was included in the first
experiment, while in the second experiment, a 12 mM sucrose
concentration was included. The first experiment had four replications,
the second five replications. Thirteen embryo samples were evaluated
per replication for the first experiment and ten in the second for each
treatment. The rate and uniformity of root and plant formation were
determined. In addition, seedling growth was measured by counting the
nodes on each plant, as indicated by an expanded leaf, and plant fresh
and dry weights.
The basal medium of the gel used in these studies contained half
strength MS with no organic additives except for 47 mM (1.6% wt/vol)
sucrose. One study evaluated the effects of gibberellic acid (GA)
(GA4,/7 and GA3) at 0, 0.01, 0.1, 1.0, 10.0, and 100.0 pM on the
percentage and rate which roots and plants formed. The gel was
initially mixed with basal medium at 5% wt/vol. The GA compounds were
filter-sterilized with a 45 micron Nalgene syringe filter. Next, the GA
treatment solutions (GA plus sterilized distilled water) were added and
mixed with the gel basal medium to obtain the final GA concentrations
and 2.5% wt/vol gel concentration. The experiment was replicated four
times with ten embryos per replication.
The effect of naphthalene acetic acid (NAA) on the rate and
percentage root and plant conversion was tested at the 0. 0.1, 0.2, 0.5,
0.75, 1.0, 2.0 and 5.0 pM levels. The gel additives (basal medium plus
sucrose) used in this study were the same as those incorporated in the
gel in the GA study. Each treatment had four replications with ten
All studies were analyzed as a completely randomized design. When
experiments were combined, they were analyzed as separate blocks. All
data that are reported as a percentage were arc sine transformed. Least
significant differences and regression analyses were used to detect
differences between treatments which evaluated quantitative
measurements. Studies which compared means from qualitative treatments
were separated using Duncan's multiple range test.
Results and Discussion
In the studies which compared various nutrient media (Table A-2),
the greatest root, shoot, and plant formation was obtained with both
Gamborg (B5) and MS media (Table 4-1), compared to either White's medium
or Hoagland's solution. A major difference in the composition of the
four media tested was the N content with White's, Gamborg's, and MS's
media, and Hoagland's solution containing 2, 30, 60 and 15 mM total N,
respectively (Table A-2).
Thus, the N quantity appeared to be of prime importance for sweet
potato plant production from somatic embryos. Between 30 and 60 mM N
was adequate for plant conversion since Gamborg medium contained
approximately 30 mM and MS medium contained 60 mM, and both facilitated
plant production (Fig. 4-2). In comparison, Hoagland solution contained
15 mM and White's medium 2 mM N which resulted in the production of only
a few weak plants. Although the final percentage root, shoot, and plant
production from embryos were affected by nutrient formulation, the speed
and uniformity were not (Table 4-1).
The following studies were conducted to evaluate concentration
levels and the nutrient components of MS medium to determine which were
needed for optimum plant development. The MS medium was used in the
following studies since this was one of the media which promoted the
most root, shoot, and plant formation in sweet potato (Table 4-1). In
addition, MS contains a substantial quantity of ammonium which may be
critical for embryo to plant conversion. The ammonium form of N has
been associated with improved production and quality of soybean somatic
Media comparison study. Effect of various media (nutrient
components and concentrations) on the percentage and mean
days to root, shoot, and plant formation when embryos were
placed in hydroxyethyl cellulose gel.z
Media? Root Shoot Plant
--------------- Final percentage response" ---------------
Gamborg (B5) 68av 26a 25a
Hoagland 43b 3b lb
Skoog 59ab 29a 24a
White 48b 3b 3b
----------------- Mean days to response ------------------
Gamborg (Bs) 8.7a 15.3a 13.8a
Hoagland 9.2a 16.0a 16.0a
Skoog 9.5a 13.6a 13.8a
White 7.4a 15.Oa 15.Oa
ZData combined from two experiments.
YMedia formulations contained only inorganic salts.
XEmbryos placed in a gel treatment containing 87 mM
yielded no roots, shoots, or plants.
"Final percentage roots, shoots, or plants obtained from embryos were
determined at 21 days. Significance determined after arc sine
VMeans separated within columns using Duncan's multiple range test, 5%
Fig. 4-2. Plant production and growth as influenced by nutrient media
and media concentration of Murashige and Skoog from embryos
placed in hydroxyethyl cellulose gel.
A. Plant production and growth were greatest when MS and
Gamborg nutrient formulations were incorporated in the
gel compared with White's medium and Hoagland's
B. Half and full-strength concentrations of MS resulted
in the greatest plant production.
ASE AND SKOOG
embryos as measured by improved plantlet production (Stuart and
The number of embryos which formed roots was similar regardless of
the MS basal medium concentration (Fig. 4-3). The exception was when
the MS medium was excluded (control treatment) from the gel which
resulted in no root production. It has been suggested that the gel
material supplies nutrients for improved germination and early seedling
growth of zygotic seeded species (Schultheis et al., 1988; Taylor,
1986). This did not occur with the somatic embryos in these studies
since no root formation (germination) occurred when the gel lacked
The final percentage of plants produced in gels containing half-
strength MS basal medium were comparable to those produced in the full-
strength concentration (Fig. 4-3). However, as the concentration of the
basal medium was reduced to quarter-strength, so was the percentage of
plants produced compared with basal medium at either half or full-
strength concentrations. No plants were produced in gels without MS
This study gives further support to the idea that N is of primary
importance for optimum embryo to plant conversion. As with MS and
Gamborg media, full-strength and half-strength MS medium contained 60
and 30 mM N respectively, and led to equivalent percentage plant
production, while quarter and eighth-strength (15 and 7.5 mM N,
respectively) resulted in a reduced number of plants (Fig. 4-2). The
mean days for root and plant production were similar regardless of MS
concentration (Table 4-2).
0 0.125 0.25 0.5 1.0
MURASHIGE AND SKOOG MEDIUM CONCENTRATION
Effect of concentrations of Murashige and Skoog medium
standard error on the percentage root and plant production at
28 days. Data combined from two experiments.
S------- 0 Plant
Table 4-2. MS concentration study. Effect of Murashige and Skoog (MS)
concentrations on mean days to root and plant formation when
embryos were placed in hydroxyethyl cellulose gel.z
Mean days to Mean days to
MS concentration root formation plant formation
0.25 9.8 18.3w
0.5 10.3 19.5
1.0 9.7 15.2
zData combined from two experiments. Final percentage roots and plants
obtained from embryos were determined after 21 days. Significance
determined after arc sine transformation.
YNo roots or too few plants formed.
XLSD (0.05) for comparing mean days to root formation 3.2 days.
WLSD (0.05) for comparing mean days to plant formation 6.3 days.
A third nutrition experiment was conducted to determine which
constituents in the medium were needed for plant development. The
greatest percentage of embryos rooted when macro nutrients were added to
the gel, while root formation was less when N, P, and K were the only
elements incorporated in the gel (Table 4-3). Shoot production and thus
plant production was best with and without organic constituents, while
the elimination of micronutrients reduced the formation of plants. Most
protocols used for plant production from somatic embryos have used a
basal medium recipe which included vitamins (Lazzeri et al., 1987; Ranch
et al., 1986; Gupta and Durzan, 1986; etc). However, vitamins were not
needed to optimize plant production from somatic embryos of sweet
Gel treatments which had no nutrient amendments or contained only
N, P, and K had no shoot or plant production (Table 4-3). The inclusion
of both the macro and micro inorganic salts were needed for optimum
plant production in sweet potato. These essential elements were most
likely required since the nutritional reserves that are typically found
in seeds were probably much lower or lacking in somatic embryos as a
result of underdeveloped cotyledons and no endosperm. The lack of well-
developed cotyledons in somatic embryos compared with zygotic embryos
has been reported for sweet potato (Chee and Cantliffe, 1988). A
comparison of rapeseed somatic embryos with zygotic embryos revealed
that proportionally lower quantities of the same proteins were found in
somatic versus zygotic embryos (Crouch and Sussex, 1981).
No difference in the rate of root, shoot, or plant production
could be attributed to the treatments in the constituent study or in any
of the other nutrition studies. The rate of plant formation is probably