Enzymatic and physiological studies of low temperature response in vegetative and somaclonal pangola

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Enzymatic and physiological studies of low temperature response in vegetative and somaclonal pangola
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Ackerman, Eugene Balliet, 1956-
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Thesis (Ph. D.)--University of Florida, 1990.
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Includes bibliographical references (leaves 75-88).
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by Eugene Balliet Ackerman.
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ENZYMATIC AND PHYSIOLOGICAL STUDIES OF LOW TEMPERATURE
RESPONSE IN VEGETATIVE AND SOMACLONAL PANGOLA






By

EUGENE BALLET ACKERMAN


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







1990


ACKNOWLEDGEMENTS



I wish to convey my heartfelt appreciation to my major

professor, Dr. Sherlie H. West. His insight, guidance, and

assistance kindled a creative and exciting environment

throughout my graduate program. Combining his professional

stature with a very caring humanistic approach allows for a

meaningful multilevel relationship.

Sincere appreciation is expressed to the members of my

committee, Drs. Jerry M. Bennett, Robert H. Biggs, Dennis J.

Gray, and Rex L. Smith, for their technical guidance,

constructive criticism, and philosophical opinions.

I am also very grateful to the Institute of Food and

Agricultural Sciences, to the University of Florida, and to

the United States Department of Agriculture for support and

providing a vital professional community. I am indebted to

many of my fellows within this community. Among the scores

that have assisted me stands David Block. His detailed

support in electronics is genuinely appreciated.

My deepest gratitude is felt for the members of my

extended family. Their true commitment of heart and hand

enabled this period of study to become a reality. In the







light of such love, challenges are met and obstacles

overcome.

To my wife Amy, and our children, Jacob, Rachel, and

Taryn, I am truly grateful for their love which is tempered

with perception and patience.


iii














TABLE OF CONTENTS


Page

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

LIST OF TABLES. .................... ............ ...... vi

LIST OF FIGURES... ........ .......................... vii

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

CHAPTERS

I INTRODUCTION.... ............................ 1

II PRODUCTION OF PANGOLA SOMACLONES AND
SELECTION FOR INCREASED TOLERANCE TO FREEZING
AND CHILLING TEMPERATURE..................... 5

Materials and Methods...................... 17
Results and Discussion..................... 28
Conclusions................................ 41

III EVALUATION OF CHILLING TEMPERATURE AND
GIBBERELLIC ACID ON THE EXPRESSION OF
HYDROLYTIC ENZYMES............................ 42

Materials and Methods...................... 51
Results and Discussion..................... 57
Conclusions... .................... ........ 64

IV EVALUATION OF HYDROLYTIC ENZYMES IN FREEZING-
TEMPERATURE-TOLERANT SOMACLONES OF
PANGOLA ................................. ........ 63

Materials and Methods...................... 65
Results and Discussion..................... 66
Conclusions................................ 70

V SUMMARY AND CONCLUSIONS....................... 71







Page

REFERENCES..... ............ ........ ... .............. 75

BIOGRAPHICAL SKETCH.................................. 89













LIST OF TABLES


Table Page

1 Percentage of pangola callus surviving low
temperatures and supporting plant regeneration... 31

2 Percentage of regenerated pangola plants
with less electrolyte leakage than the
source plants .................................. 37

3 Freeze induced electrolyte leakage of selected
regenerated pangola plants and source
plants ........................................... 39

4 Percentage F, remaining in selected regenerated
pangolagrass plants and source plants after
exposure to chilling temperature................. 40

5 Reagents and gel preparation for native starch
PAGE slab gel electrophoresis..................... 53













LIST OF FIGURES


Figure Page

1 Decrease in the rate of fluorescent emission
(FR) from pangola chilled at 100C for
45 minutes. Peak fluorescence (P) is the
maximum fluorescent emission. ................... 18

2 Configuration of LaCroy transient recorder. ..... 26

3 Configuration of LaCroy transient recorder. ..... 27

4 Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 MM 2,4-D. X 7. ................................ 29

5 Pangola callus with embryogenic (e)
and nonembryogenic (n) callus. On MS media
containing 9 AM 2,4-D. X 58 ..................... 30

6 Longitudinal section of pangola callus
with nonembryogenic (n) callus, embryogenic (e)
callus, and young somatic embryo (em). On MS
media containing 2 MM 2,4-D. X 58. ............... 33

7 Longitudinal section of pangola callus
with embryogenic (e) callus supporting a
maturing somatic embryo; scutellum (sc),
coleoptile (co), and shoot apex (s). On MS
media without 2,4-D. X 75. ........................ 34

8 Regenerated pangola plant with plumule (p)
and young root (r). On MS media without 2,4-D.
X 7. ............................................. 35

9 Effect of temperature and GA3 on expression of
starch-degrading-hydrolytic enzymes in
pangola. White bands (bands 1-8) show
endoamylase activity, blue band (band 9)
indicates debranching enzyme activity. F refers
to the bromophenol blue front marker. Micrograms
protein refers to the total protein. ............. 59


vii









10 Effect of temperature and GA3 on production and
expression of starch-degrading-hydrolytic enzymes
in pangola. Zymograms (z) of hydrolytic
activity and coomassie protein stains (c) are
presented for each treatment. Micrograms protein
refers to the total protein. ..................... 61

11 Zymograms of starch-hydrolyzing enzymes isolated
from pangola plants that were exposed to
300C or 100C. White bands (bands 1-8) show
endoamylase activity, blue band (band 9)
indicates debranching enzyme activity. Lanes 1
and 3 represent the vegetatively propagated control
plants. Lanes 2 and 4 represent plants regenerated
from tissue culture. F refers to the bromophenol
blue front marker. ............................... 67

12 Zymograms of starch-hydrolyzing enzymes and total
protein profiles from pangola plants exposed
to 300C or 100C. Lanes 1 and 4 are zymograms. White
bands (bands 1-8) show endoamylase activity,
blue band (band 9) indicates debranching enzyme
activity. Coomassie blue stained proteins are
represented in lane 2 vegetativelyy propagated
control plants) and lanes 3 and 6 (plants
regenerated from tissue culture). ................ 69


viii


Figure


Page








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


ENZYMATIC AND PHYSIOLOGICAL STUDIES OF LOW TEMPERATURE
RESPONSE IN VEGETATIVE AND SOMACLONAL PANGOLA

by

Eugene Balliet Ackerman

May 1990

Chairman: Dr. S. H. West
Major Department: Agronomy

Conventional breeding programs are unable to generate

variation in 'pangola' digitgrass (Diqitaria decumbens

Stent.). Pangola is a highly sterile cultivar which resulted

from hybridization of closely related Diqitaria species.

Pangola is a triploid (2n = 27). A lack of variation causes

growth rates among individual plants to be uniformly reduced

by chilling and freezing temperatures. Shortages of forage

from pangola pastures occur throughout the winter months in

northern portion of central Florida.

The objectives of this research were to produce somatic

tissue cultures of pangola, regenerate plants, and select

variant plants exhibiting chilling and freezing-temperature

tolerance. Evaluation of freezing-temperature resistance was

based on in vitro survival and integrity of the plasmalemma

membrane. Evaluation of chilling-temperature resistance was

based on in vitro survival, integrity of the photosynthetic







electron transport chain, and the number of assimilatory

starch-hydrolyzing enzymes.

Somatic embryogenesis in pangola followed a pattern of

development similar to those described for other grasses.

Freezing or chilling temperature treatment did not

predispose plants that were regenerated from these calli to

be freeze tolerant. A range of freeze damage occurred

between regenerated plants with respect to plasma membrane

damage. A subset of plants selected for a decrease in

freeze-induced plasmalemma damage had no reduction in chill

damage to the photosynthetic electron transport chain.

Chill-induced alterations in assimilatory starch-

hydrolyzing enzymes resulted in the identification of two

chill-sensitive endoamylolytic enzymes. One debranching and

six endoamylolytic enzymes were unaffected by chilling

temperature. The number of hydrolytic enzymes was unaffected

by applying gibberellic acid (GA3) to the plant prior to

chilling temperature exposure.

All of the regenerated plants that were selected for an

increase in freezing tolerance were identical with respect

to the number of starch-hydrolyzing enzymes regardless of

temperature.

Somatic variation in freezing or chilling temperature

response of pangola was evident in vitro. Variant

regenerated plants that sustain less damage from freezing

temperature can be identified by a low-electrolyte leakage.













CHAPTER I
INTRODUCTION



Pastures that are established with 'pangola' digitgrass

(Diqitaria decumbens Stent.) produce an abundance of lush,

high-quality forage. Pangola can be used as a pasture, hay,

or silage crop (Hodges et al., 1975). During the early 1930s

pangola was introduced into the US Gulf Coast areas and

southern California (USDA, 1932). Pangola was registered by

the Crop Science Society of America (registration number 28)

in 1972 (Schank et al., 1972). The establishment of pangola

resulted in pastures that sustain abundant high-quality

yields in the hot summer months and drastically reduced

yields in the cooler months. Pangola is very freeze

susceptable as characterized by the death of grass crowns

(Hodges and Jones, 1958). Winterkilling results from a

combination of frost and freezing temperature.

The livestock industries in Florida, particularly the

beef industry, desire a pasture grass that provides a year-

round supply of abundant forage. Unfortunately, during the

cooler months, pangola production falls short of the

farmers' needs. Farmers are forced to purchase additional

feed stocks after their banked pangola pastures have been







2

depleted. Increasing the cost effectiveness of beef

production in Florida strongly depends on the year-round

production of abundant and inexpensive forage. Extending the

summertime growth characteristics of pangola into the cooler

months would substantially reduce the farm and ranch

investments in livestock feed.

Whole plant growth has been characterized by Burgess

(1985) as a natural process that is regulated by genetic,

biochemical, morphological, and physiological changes in

each cell. Multiplication and differentiation of plant cells

results from selective expression of parts of the genome.

Selective expression of the genome keeps the plant in

balance with its environment.

During the winter months, the most important

environmental factor restricting plant growth is chilling

(120C-00C) and freezing (0C and lower) temperatures

(Christiansen and St. John, 1984). Chilling and freezing

conditions are defined by these temperature ranges. A wide

range of tolerance to low-temperature stress often exists

within crop species due to the genetic diversity generated

by gene recombination during meiosis (Jones, 1985). Sterile

crops such as pangola are lacking in gene recombination and

therefore retain a high degree of genotypic and phenotypic

uniformity. Tissue culture methods have been used to

regenerate plants with increased genetic variation







3

(Scowcroft and Larkin, 1988) and increased tolerance to

unfavorable environments (Tal, 1983).

Many agronomically important plant species of tropical

origin are sensitive to chilling temperatures in the range

of 200C down to 0C. Below some critical temperature,

grasses are sharply restricted in growth (Gusta et al.,

1980). Several plant factors contribute to reduced growth

under chilling-temperature conditions. Membrane damage is a

universal manifestation of chill-temperature damage in

biological systems and is commonly regarded to be the

primary cause of injury (Lyons et al., 1979). Photosynthesis

is one of the first processes affected by chilling

temperatures (Oquist, 1987). Alterations in the light

reactions are evident at various stages in the

photosynthetic electron transport chain. Changes in the dark

reactions (i.e., carbohydrate anabolism) result from

alterations in the activity (Carter et al., 1972) and

synthesis (Jacobsen and Higgins, 1982) of amylolytic

enzymes. Despite having distinct genetical, biochemical, and

physiological factors, the action and interaction of all

these factors contribute to a common result: reduced growth.

Several of the above factors were studied and reported

herein. The objectives of the study reported in Chapter II

were a) to generate somatic cell lines of pangola; b) to

select freezing and chilling resistant cell lines in vitro;

c) to regenerate whole plants from selected cultures; and d)







4

to test regenerated plants for increased tolerance to

freezing and chilling temperature at the organelle and

cellular level.

The studies reported in Chapter III were conducted with

the objectives a) to evaluate the effect of chilling

temperature on the expression of amylolytic enzymes in

pangola, b) to identify amylolytic proteins that may assist

in starch breakdown after exposure to chilling conditions,

and c) to determine the effect GA3 has on the synthesis of

these proteins.

The objectives of the study presented in Chapter IV

were a) to evaluate low-temperature-tolerant pangola

somaclones for variation in the expression of amylolytic

enzymes and b) to select plants that retain amylolytic

proteins which may assist in starch breakdown after exposure

to chilling conditions.












CHAPTER II
PRODUCTION OF PANGOLA SOMACLONES AND SELECTION
FOR INCREASED TOLERANCE TO FREEZING AND CHILLING TEMPERATURE



This study tested the tentative assumption that

somaclonal variation may be obtained in pangola plants that

are regenerated from tissue culture. Hypothetically, somatic

variants may be selected for increased low-temperature

tolerance.

To test this hypothesis, the objectives of this study

were a) to generate somatic cell lines of pangola, b) to

select freezing and chilling resistant cell lines in

culture, c) to regenerate plants from the selected callus,

and d) to test for increased tolerance to freezing and

chilling temperature at the whole plant level.

Pangola establishes excellent summertime pastures but

is an unproductive fall and winter forage crop in Florida.

Chilling temperatures (00C-120C) encountered by pangola

during the cooler seasons reduce growth rates and eventually

stop productive growth (West et al., 1968).

The genetic diversity that permits a fertile plant

species to adapt to temperature extremes is not present in

pangola. Pangola is highly sterile due to irregularities in

both microsporogenesis and macrosporogenesis (Sheth, 1955).






6

Sheth et al. (1956) and Schank et al., (1972) reported that

pangola produced very few viable seed. This genetic crossing

barrier eliminates any genetic variation that is acquired

from independent gene segregation and gene recombination.

An increase in genetic variability, from sterile

plants, may be obtained by subjecting the tissue to somatic

cell culture (Reisch, 1983). The concept of somaclonal

variation as a new source of genetic variability was

presented by Larkin and Scowcroft (1981) and has been

reviewed a number of times (Scowcroft, 1985; Ahloowalia,

1986; Larkin, 1987, Ryan et al., 1987). Variation arising as

a consequence of tissue culture has been reported in the

following forage grasses: ryegrass (Lolium multiflorum L.)

(Ahloowalia, 1983; Torello and Symington, 1984; Skene and

Barlass, 1983), tall fescue (Festuca arundinacea L.)

(Kasperbauer and Eizenga, 1985), guineagrass (Panicum

maximum L.) (Bajaj et al., 1981), and crabgrass (Digitaria

sanquinalis L.) (Elmore et al., 1988). This suggests that

somatic variation in pangola could be used to generate

variation.

A fundamental requirement for obtaining somatic

variation is the ability to support in vitro callus growth

and plant regeneration. Urata and Long (1968) were first to

report the regeneration of grass plants from tissue culture.

Pangolagrass was one of the several tropical grasses that

they regenerated through organogenesis. Recently, using






7

pangolagrass, Marousky and West (1988) reported shoot

development from ovarian explant tissue and somatic embryo

formation from callus.

Until recently, it was thought that the only way to

obtain useful nonchimeral mutants was by regeneration of

somatic embryos (Gamborg et al., 1970). Both organogenic

shoots (Horsch et al., 1985) and somatic embryos (Williams

and Maheswaran, 1986) can initiate from single cells. This

indicates that both modes of regeneration reported in

pangola may give rise to the more useful nonchimeral

regenerated plants.

Regenerated plants expressing altered phenotypes are

not necessarily expressing altered genes but may be

expressing epigenetic changes. Epigenetic changes arising

due to stress placed on the cultured cell may result in

alterations of either transcription and/or translation from

an unaltered genome.

Epigenetic variation may be useful in an asexually

propagated crop such as pangola, provided that the

characteristic remains stable. Meins (1974) stated that this

type of variation is usually, but not necessarily

reversible. Secor and Shepard (1981) reported that potato

(Solanum tuberosum L.) somaclones, which had gone through a

number of vegetative generations in the field, still

retained selected characteristics.







8

Mitra and Steward (1961) were among the first to report

the existence of true genetic change in somatic cell

culture. They observed chromosomal abnormalities, polyploidy

and aneuploidy in carrot (Daucus carola L.) cell cultures.

These genetic changes were gross chromosomal mutations and

are not stably inherited or useful. According to Orton

(1984), stably inherited or Mendelian variation accounts for

chromosomal changes that are heritable and segregate in

crosses with individuals exhibiting a distinct phenotype.

Using this criteria, any heritable phenotypic alteration

caused by a base change, deletion, or rearrangement in the

DNA is included. The DNA may be located in the nucleus,

mitochondria, or chloroplast. The site of mutation may be at

loci that results in either major qualitative effects or

subtle quantitative effects.

Larkin et al. (1985) cited reports of 14 different

species where chromosomal modifications had been induced and

then inherited in a Mendelian manner. The DNA alterations

included point mutations, deletions, interchanges,

inversions, and amplifications. In two of the species, corn

(Zea mavs L.) and alfalfa (Medicago sativa L.), increased

transposable element activity during tissue culture has been

reported by Benzion et al. (1986) and Gross and Bingham

(1986), respectively.

The reason why tissue culture results in an increased

frequency of chromosomal aberration is yet to be explained.







9
Benzoin et al. (1986) hypothesized that the late replicating

heterochromatin may occasionally replicate so late that

bridge formation and subsequent chromosome breakage occurs

at anaphase. The resulting breakage-fusion-bridge cycle

would lead to exchanges, interchanges, or translocations.

Larkin et al. (1984) studied heritable somaclonal

variation in wheat (Triticum aestivum L.) and concluded that

two mechanisms, one operating to create a mutant gene and a

second to make the mutant homozygous, accounted for the

somaclonal variation. Their conclusions were based on the

observations that variation was evident in morphological and

biochemical traits, traits under simple genetic control

(grain color), quantitatively inherited characteristics

(heading date), and a number of traits within a single

somaclone having subsequent independent assortment.

Homozygous and heterozygous mutants were present at

different loci within a single somaclone. Chromosomal loss

or addition was not attributed to have been the primary

cause of variation.

Phenotypic expression may be altered by a variety of

structural changes in chromosomes. An understanding of how

to relate specific cultural conditions to favor one class of

mutation is beginning to emerge. The following is a partial

review of reports on the use of culture variables to affect

phenotypic changes: explant source (Demarly, 1986), media

constituents (Van Harten et al., 1981; Hughes, 1983), cell







10

cycle duration (Muller and Grafe, 1978; Bayliss, 1976), and

regeneration (Hughes, 1983; Mahfouz et al., 1983).

The length of time in culture influences the cultures

ability to regenerate. Matthews and Vasil (1975) reported

that increasing the length of time in culture favors variant

cells and results in more precocious regeneration. This

decline has been attributed, at least partly, to genetic

mutations (Smith and Street, 1974). In pangola, Marousky and

West (1988) reported that after 5 weeks in culture

embryogenesis occurred at a 90-100% frequency. Genetic

mutations in in vitro pangola cells may be favored by

extending the length of time in culture beyond 5 weeks while

not totally compromising the regenerative capacity.

Isolation of variant cells can be achieved by positive

selection, i.e., the application of a stress, followed by

the harvesting of surviving cells. The stress may be exerted

either stepwise or gradually. Larkin et al. (1985) reported

that a stepwise application of pressure is more apt to favor

novel genotypes which arise from either gene amplification

and/or gene mutation. Variant phenotypes produced by this

procedure have resulted in regenerated plants that are

resistant to disease (Foroughi-Wehr et al., 1986; Larkin and

Scowcroft, 1983), mineral toxicity (Ahloowalia, 1982; Oono,

1981), mineral deficiency (Qureshi et al., 1981), and

herbicide damage (Chaleff and Bascomb, 1987).







11

The few reported attempts to select chilling or

freezing-temperature-resistant cell lines that maintain

resistance in culture have had mixed results. The earliest

attempts to select chilling or freezing-temperature-

resistant callus was reported by Steponkus (1972). Steponkus

(1972) obtained cell lines of ivy (Hedera helix L.) which

survived the selection procedure of freezing, but none of

the lines maintained a stable enhanced resistance to the

freezing temperature (Dix, 1980). Based on respiration rates

in mitochondria, Dix and Street (1976) obtained some cell

lines of tobacco (Nicotiana sylvestris L.) and pepper

(Capsicum annum L.) that maintained chill resistance and

other lines that lost chill resistance. Templeton-Somers et

al. (1981) selected freezing-temperature resistant carrot

callus. Resubmission of the resistant callus to freezing

conditions resulted in considerable variation in the ability

of the cell lines to tolerate the selection conditions. Chen

et al. (1982) selected chilling-tolerant sugarcane

(Saccharum spp.) cell lines, most of which retained chill

tolerance in culture.

It has not been determined if the chilling or freezing-

temperature resistance of the selected callus will be

expressed in regenerated plants. Cell lines, selected for

chilling or freezing-temperature tolerance, often lose their

ability to differentiate and regenerate plants (Dix and

Street, 1976; Chen et al., 1982). Dix (1977) sexually







12

crossed plants that were regenerated from chill-sensitive

callus. Callus, obtained from progeny plants, had lost their

chill sensitivity. He concluded that the resistance was not

heritable. Malmberg (1979) showed that culturing leaves from

a tobacco plant, that was regenerated from a chilling-

temperature-sensitive callus, resulted in callus that

retained chill sensitivity. Templeton-Somers et al. (1981)

reported that chilling-temperature resistance in selected

callus is not always expressed during embryo development.

They stated that although there is no concrete evidence for

increased chill resistance in the somatic embryos, further

work is necessary to determine the expression in more mature

plants.

A visual symptom of freezing-temperature injury in

mature plant tissue is the infiltration of tissue

intercellular space with water. Chen et at. (1976) used this

soaked appearance, along with loss of turgor, as a criterion

for evaluating freezing injury. Another common result of

freezing injury is the leakage of ions from cells. Until

recently, reports such as Levitt (1972) and Sukurman and

Weiser (1972) attributed the efflux of ions from frozen and

thawed tissue to the breakdown of the semipermeable property

of the cell membrane. Palta et al. (1977a) has shown that in

onion (Allium cepa L.) bulb cells the semipermeable

properties remain intact during the process of freezing,

whereas the active transport properties of the cell






13
membranes are damaged. They reported that inactivation of

the active transport system results in a large passive

efflux of ions and sugars. During thawing, as ice melts in

the extracellular space, ions and sugars move down

concentration gradients vacuolee to extracellular solution),

as the active transport system is unable to pump them back

into the vacuole. As a result of osmotic equilibrium between

the outside solution and the cell sap, the cell is unable to

absorb water. The tissues become flaccid due to the

infiltration of the tissue with extracellular solution and

lose of turgor.

Using onion bulb cells Hansteen-Cramer (1922) first

reported that Ca is needed for the stability of natural

membranes. Palta et al. (1977b) reported a small but

increasing efflux of Ca from onion bulb cells as a result of

freezing injury. They concluded that such removal of Ca from

the membranes causes instability of the membrane structure

and finally results in breakdown of the membrane system.

Based on the leakage of ions, Dexter et al. (1932)

reported that measurement of electrical conductivity of the

ions can provide a quantitative evaluation of freezing

injury. Electrolyte leakage has been used by Patterson et

al. (1976) to measure low-temperature sensitivity in species

of passionflower (Passiflora) having a range of climactic

requirements. Wiltbank and Oswalt (1984) reported the use of







14

electrolyte leakage to determine changes in the killing-

point temperature of several citrus cultivars.

Use of electrolyte leakage to measure variation in low-

temperature stress should be applicable in assessing

variations in low-temperature sensitivities of regenerated

pangolagrass plants.

Damage to the photosystem apparatus occurs at

temperatures above those which cause breakdown of cellular

integrity (Berry and Bjorkman, 1980). This damage can be

used as an indication of the amount of low-temperature

stress in the plant. Drake and Salisbury (1972) reported

that growth inhibition in C4 species is proportional to the

severity of chilling and to the intensity of light received

during chilling. Chatterton et al. (1972) attributed reduced

growth in pangola, during chilling, to damage in the

photosynthetic apparatus.

Early indicators of reduced photosynthetic activity in

chill-sensitive plants were believed to be thylakoid

membrane phase transitions (Murata and Fork, 1975; Shneyour

et al., 1973) and increases in stomatal resistance to CO2

flux (Crookston et al., 1974; Drake and Salisbury, 1972).

However, work by Low et. al. (1984) has indicated that both

chilling-sensitive and chilling-tolerant plants exhibit

equivalent thylakoid phase transitions. Low et al. (1984)

and Martin (1986) have reported that there is insufficient

thylakoid membrane damage to account for the impairment of







15

photosynthesis. Further, Hallgren et al. (1982) reported

that stomatal closure does not necessarily inhibit CO2

uptake because intercellular CO2 pressure can remain

constant despite a decreased stomatal conductance.

Chilling corn in light decreased quantum yield of CO2

assimilation, rate of 02 evolution, and variable fluorescent

emission of photosystem II (Long, 1983). Hillard and West

(1970) reported that pangola exhibited decreased rates of

CO2 assimilation after chilling in the dark. Chill-stressed

pangola plants also had a reduction in the Hill reaction

(West, 1970). The extent of low-temperature alterations in

variable fluorescent emission of photosystem II in pangola

is unknown.

Steinback and Mall (1986) reported that the loss of

photosynthetic competence in low temperature is due to

photoinhibition of photosystem II activity. Chill-induced

damage to photosystem II has been associated with altered

energy flows in the electron transport chain (Percival et

al., 1987; Heinrich and Lasch, 1987). The active center of

photosystem II is not immediately affected by chilling

(Smillie, personal communication). Chilling-temperature

inhibition of the water-splitting side of photosystem II was

reported by Margulies (1972) and 6quist (1983), as assayed

by electron transfer and fluorescent excitation of

chlorophyll a. No chilling temperature effects on

photosystem I were observed by Oquist (1983). Hetherington







16

et al. (1983) and Yung-ling et al. (1987) reported that the

damaged water-splitting enzyme systems produce thermal

rather than chemical energy. Chilled leaves having an

inhibited water-splitting protein complex sustained a lower

yield of fluorescent emission from chlorophyll a

(Papageorgiou, 1975).

The lower yield of chlorophyll fluorescent emission due

to chilling temperature is evident in FR measurements. A FR

measurement is defined as the linear increase of chlorophyll

a fluorescent emission per unit surface area as a function

of time between I and P, where the fluorescence at time I is

the initial fluorescent emission and the fluorescence at

time P is the maximum fluorescent emission (Figure 1).

Lawlor (1987) stated that high FR values are indicative of

an intact electron transport chain. The quantity of

fluorescent emission at time I is regulated by the redox

state of the quinone protein (Q), the primary electron

acceptor of photosystem II. The more that Q and

plastoquinone (PQ) are in the reduced states the higher will

be the initial fluorescence. Munday and Govindjee (1969)

reported that the amount of variable fluorescence,

fluorescent emission from I to P, is limited by the

photoreduction of Q via the photosystem II reaction linked

to the water-splitting enzyme system.

Smillie and Hetherington (1983) used FR as a chilling

stress measurement. Hetherington et al. (1983) found that







17

chill stress reduced the FR value in corn. Their work with

corn led to the identification of chill-tolerant corn

populations based on FR values.

Machado et al. (1984) rated increased atrazine

resistance using somatic variants regenerated from tissue

culture, by testing differences in the emission of

chlorophyll fluorescence. To date, no reports on somatic

variation in chilling or freezing-temperature tolerance have

been presented using fluorescent emission measurements.



Materials and Methods

Plant Material and Growing Conditions

A 'pangola' digitgrass (Digitaria decumbens Stent.)

plant was obtained from the University of Florida's Agronomy

Seed Laboratory in September of 1986. Eleven vegetatively

propagated source plants were grown in a greenhouse with

natural lighting. The average daily temperature range was

300C to 350C. The source plants were grown in 15-cm pots

containing Metro-mix 350 growing medium. Rapid growth was

maintained by supplying six grams of Osmocote (W. R. Grace,

Cambridge, MA) a slow-releasing fertilizer (14-14-14, N-

P205-<20) to each pot. The plants received adequate water on

a daily basis and were clipped to 15 cm above the soil

surface every two weeks. Care was taken to prevent root

binding by vertically halving the entire plant and repotting

when needed.





















RELATIVE

FLUORESCENCE


FR,
!


FR
/


27C


TIME


Fig. 1. Decrease in the rate of fluorescent emission
(F,) from pangolagrass chilled at 100C for 45
minutes. Peak fluorescence (P) is the maximum
fluorescent emission.










Cell Culture Initiation

Source plants were no longer clipped after the first

of May, 1987, in order to promote floral development. In

early June of 1987 immature inflorescence 10 to 15 cm in

length were excised from culms at approximately 8:00 AM.

Entire inflorescence was surface sterilized in 0.8% (v/v)

sodium hypochlorite (15% Clorox in water). A drop of Tween-

20 (Fisher Scientific Company, Fair Lawn, NJ) was added per

100 mL of solution. Flower heads were totally immersed in

the sterilizing solution for 45 min while being shaken at 35

rpm on a New Brunswick Scientific G10 gyrotory shaker

(Edison, NJ). The remaining decontamination procedures and

transferring to culture media was done under an Edgeguard

laminar flow hood (The Baker Co., Sanford, ME). The flower

heads were rinsed four times with sterile deionized water

(dHO2) and then cut longitudinally by making an incision

with a scalpel through the leaf sheaths. The immature

florets were exposed by spreading the tissue apart on either

side of the incision with a pair of curved tip microforceps.

The spikes were excised with smooth tip microforceps and cut

into 5-mm segments. Ten to 15 segments, or explants, were

cultured in each of 100 X 15 mm petri dishes containing a

solid MS media (Murashige and Skoog, 1962). The MS media

contained premixed MS salts (Sigma Chemical Co., St. Louis,

MO), 8% T C agar (Carolina Biological Supply Co.,







20

Burlington, NC), 4% sucrose, and 9 jIM 2,4-

dichlorophenoxyacetic acid (2,4-D). The petri dishes were

wrapped in parafilm (American Can Co., Greenwich, CT) and

placed in a Percival growth chamber (Boone, IA) at 270C with

a 12 h light/dark cycle. Three weeks later, the explant

material was discarded after being separated from callus

tissue. Calli were transferred to fresh media of the same

composition at three-week intervals, for three cycles.



Temperature Treatments

After the third transfer cycle, calli were subjected to

different temperature treatments. Compact white embryogenic

callus was sectioned into 1 cm2 pieces and four of these

calli were placed in each petri dish. The petri dishes

contained the same MS media composition as used for callus

initiation. A minimum of 30 plates (containing a total of

120 calli) were placed in each temperature treatment. The

treatments were 270C, 20C for 12 d, 20C for 24 d, -120C for 4

h, -12C for 8 h, -120C for 23 h. All cultures were

maintained in the dark. The 20C treatments were placed in a

Percival growth chamber whereas the -120C treatments were

kept in a Whirlpool freezer (Benton Harbor, MI).

Callus viability was estimated by the ability to regrow

on fresh MS media containing 2 pM 2,4-D at 270C in 12 h

light/dark for 42 d.









Plant Regeneration

After exposure to temperature treatments the callus was

transferred to a MS media containing 2 jM 2,4-D and placed

at 270C with a 12 h light/dark period. Chilling or freezing

temperature damage and subsequent developmental morphology

of the callus were observed by using an Olympus SZ-Tr stero

microscope (Tokyo, Japan). Viable embryogenic calli and

developing embroids were separated from necrotic calli and

transferred to fresh MS media of the same composition every

other week, for three cycles. The number of surviving calli

were recorded.

Subsequent embryo maturation and plantlet regeneration

was advanced by transferring cultures to MS media devoid of

2,4-D. Cultures were transferred to fresh media of the same

composition every other week, for three cycles. After the

third transfer, individual regenerated plants that had a

well-defined root system consisting of several adventitious

roots and leaf lengths of 2 to 4 cm were separated and

counted. The regenerated plantlets were transferred to septic

6-cm pots. The pots contained Metro mix 350 growing media

which was kept constantly moist. The plants were withheld

from direct sunlight for seven days, after which time the

plants were exposed to full sun and fertilized with Peters

fertilizer (20-20-20, N-P205-K20) (W. R. Grace and Co.,

Foglesville, PA). The plants were watered as needed for the







22

next three weeks. The plants were then transferred to 15-cm

pots and maintained as previously described.



Scanning Electron Microscope

Micrographs of embryo development were obtained with a

Hitachi S-450 scanning electron microscope (Tokyo, Japan).

Selected tissue was fixed in FAA and dried through an

alcohol dehydration series. Some of the tissue was sectioned

with a single-edge razor blade. The tissue was dried in a

Balzer CPD 030 critical point dryer (Furstentum,

Liechtenstein) and coated with gold in a Samsputter 2a

automatic coating apparatus (Tousimis Research Corp.,

Rockville, MD).



Plant Selection: Electrolyte Leakage

A total of 263 regenerated plants and three

vegetatively propagated source plants were tested for

freezing- temperature damage using modifications in the

electrolyte leakage procedure presented by Patterson et al.

(1976) and Wiltbank and Oswalt (1984).

Tissue was obtained from plants maintained in the

greenhouse. Six young, fully expanded leaves from each plant

were cut from the plant and washed three times in deionized

water. Leaf tips and 6 cm of leaf tissue were immersed in a

55 mL test tube containing 20 mL of deionized water. Care

was taken to avoid submerging cut tissue. The temperature of






23

the leaf tissue and water was decreased by approximately 2C

per min to a final temperature of -5C for 30 min in a Lauda

refrigerated circulator (Brinkmann Instruments Co.,

Westburry, NY) containing ethylene glycol. After 30 min at -

50C the supercooled water was crystallized by seeding with a

small deionized ice crystal. Leaf tissue remained in the ice

for an additional 30 min. The test tubes were then

readjusted to 250C for 90 min. Leaf material was removed

from the water and electrical conductivity of the water was

measured with an ASA 610 electrical conductivity bridge

(Agro Sciences Inc., Ann Arbor, MI).

Mean electrolyte conductivity of the solute leakage

from individual plants, 263 regenerated plants and three

vegetatively propagated source plants, was calculated based

on two replications. The proportion of regenerated plants

with electrolyte conductivity less than the mean electrolyte

conductivity the standard deviation (S.D.) of the four

source plants are reported for each in vitro temperature

treatment. Ten regenerated plants were selected,

irrespective of in vitro temperature treatment, based on

their low quantity of electrolyte leakage relative to the

electrolyte leakage of the source plants.

Six replications of the 10 selected regenerated plants

and 4 vegetatively propagated plants were tested in a

completely randomized design for analysis of mean

electrolyte leakages. MSTAT, a PC statistical program, was







24

used for data analysis. Means were calculated and data were

analyzed by Duncan's New Multiple-range Test. The same

plants were used for chlorophyll fluorescence

determinations.



Plant Selection: Chlorophyll Fluorescence

Experiments were conducted during March, 1988. The 10

previously selected plants were removed from the greenhouse

at 8:00 AM and dark adjusted for 60 min at 270C before

obtaining initial fluorescent emission measurements. The top

third of the first fully extended leaf was excised from the

plant and placed in a 270C Hansatech LD2 leaf chamber

(King's Lynn, Norfolk, UK). The leaf was positioned above a

pad wetted with a 1 M sodium bicarbonate solution. The

sodium bicarbonate maintained an adequate supply of CO2 for

photosynthesis (Walker, 1987). A template was constructed

which limited the light exposure to 50 mm2 of leaf tissue.

The template did not interfere with gas flow within the

chamber. Excitation energy was applied two minutes after the

leaf tissue was placed in the leaf chamber. A quantum flux

density of 60 W m-2 (660 nm) was supplied by a Hansatech LS1

light source (King's Lynn, Norfolk, UK) for photosynthesis.

The LD2 unit was attached to a Lauda RM6 refrigerated

circulator (Brinkmann, Westbury, NY) and maintained at 27C.

Chlorophyll fluorescent emission was detected by a Hansatech

fluorescence detector that was fitted with a 740 nm







25
interference filter. Data points were obtained every 6.9 ms

with a LeCroy transient recorder (Figs. 2 and 3) (Spring

Valley, NY). The signals were relayed to an IBM PC for

operating the Waveform-catalyst software program version 2

(LeCroy Research Systems Corp., Spring Valley, NY), storage

of data, and retrieval of data.

Fluorescent emission from chill-stressed plants was

obtained after measuring the initial fluorescent emission.

Chilling treatment was imposed by placing the plants in a

darkened 100C Percival growth chamber for 45 min. The top

third of the first mature leaf was excised from the plant

and placed in the 270C leaf chamber for two minutes before

excitation energy was applied. Measurements were obtained as

previously described.

Measurements were expressed as FR as described by

Hetherington et al. (1983). The effect of chilling on

chlorophyll fluorescence was gauged from the chilling

induced decrease in FR (Smillie and Hetherington, 1983). FR

was measured before chilling (Reading 1) and again 45 min

after beginning chilling (Reading 2). Percentage FR

remaining after chilling was calculated as (Reading

2/Reading 1) X 100.

Data means + S.D. of individual plants (10 regenerated

plants and four source plants) were calculated on six

replicates.





















*~;'~~


b .


Fig. 2. Configuration of LeCroy transient recorder.
a 8013A crate.
b 8100 gain differential amplifier, in crate
slot #2.
c 8800A memory, in crate slot #5.
d 8210 waveform analyzer, in crate slot #6.
e 8901A GPIB interface, in crate slot #12.














































Fig. 3. Configuration of LaCroy transient recorder.
a 8100 gain differential amplifier.
control lock.
filter in.
gain 1.
multiply 1.
input to fluorescent detector.
output to 8210 waveform analyzer.
b 8210 waveform analyzer.
post trigger samples 7.
sample interval external clock.
input channels 4.
display channel 4.
input to 8100.










Results and Discussion

Somatic embryogenesis in pangola followed a pattern of

development similar to that reported by Marousky and West

(1988). Within 10 d of initiation on MS media containing 9

AM 2,4-D, soft friable callus formed from the pedicils and

ovarian tissue. Prolific growth was observed. After two

weeks of culture, two types of callus were produced (Figures

4, 5). Embryogenic callus was pale yellow to white in color,

nodular and compact in appearance, and was composed of

small, densely cytoplasmic cells. Alternatively, non-

embryogenic callus was friable and contained loose cells

which were often elongated and highly vacuolated.

Isolation of cell lines with an increased tolerance to

freezing or chilling temperature was achieved by a stepwise

exertion of low temperature and positive selection. Larkin

et al. (1985) reported that a stepwise exertion of selective

pressure tends to select the novel genotypes that arise from

either gene amplification and/or gene mutation. Freezing and

chilling-temperature stress adversely affected the viability

of embryogenic callus (Table 1). Callus viability decreased

when the temperature severity or duration of exposure

increased. An LD 50 was approximated when chilling (20C)

temperature was imposed for 24 d or when freezing (-120C)

temperature was imposed for 23 h. Damaged and necrotic

callus became apparent several days after the temperature
















































Fig. 4. Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 gM 2,4-D. X 7.





















































Fig. 5. Pangola callus with embryogenic (e) and
nonembryogenic (n) callus. On MS media containing
9 AM 2,4-D. X 58.









Table 1. Percentage of pangola callus surviving low
temperatures and supporting plant regeneration.



Treatment Pangola callus


Tempera-
ture Duration+ Total Viable Supporting regeneration

C d or h % %

27 constant 125 74 14
2 12d 101 73 12
24d 437 45 9
-12 4h 234 63 7
8h 284 65 2
23h 198 48 0


+Period of exposure to temperature treatment.

Percentage of viable callus from which plants regenerated.







32

treatments ended. The affected callus appeared dark brown in

color, softer in consistency, and discolored the surrounding

media. The surviving embryogenic calli retained their

characteristic appearance. Theoretically the surviving cells

had a higher degree of chill tolerance than the chill-

susceptible cells. Selection procedures reported by Dix and

Street (1976), Breidenbach and Waring (1977), Dix (1980),

Templeton-Somers et al. (1981), and Chen et al. (1982) have

successfully isolated cell lines that tolerated chilling or

freezing temperatures.

Somatic embryos were obtained from the surviving calli

by reducing the level of 2,4-D in the medium was reduced to

2 MM (Figure 6). Maturing embryos contained the

characteristic organs of a grass embryo: scutellum,

coleoptile, and shoot apex (Figure 7). Gross abnormalities

in embryo development were absent. Variation in the degree

of typical embryo development was not assessed. Vasil (1983)

reported embryo development ranging from atypical to typical

in somatic embryos of different grasses.

Eleven weeks after calli were initiated plant

regeneration from somatic embryos was advanced on MS media

without 2,4-D (Figure 8). Matthewes and Vasil (1975)

reported that increasing the length of time in culture

favors variant cells and results in more precocious

regeneration. Table 1 shows that after 11 weeks in culture,

















































Fig. 6. Longitudinal section of pangola callus with
nonembryogenic (n) callus, embryogenic (e) callus,
and young somatic embryo (em). On MS media
containing 2 jM 2,4-D. X 58.












































Fig. 7. Longitudinal section of pangola callus with
embryogenic (e) callus supporting a maturing somatic
embryo; scutellum (sc), coleoptile (co), and shoot
apex (s). On MS media without 2,4-D. X 75.

















































Fig. 8. Regenerated pangola plant with plumule (p) and
young root (r). On MS media without 2,4-D. X 7.







36

at 270C, 14% of the viable embryogenic calli supported plant

regeneration. Marousky and West (1988) observed

embryogenesis in pangolagrass callus at a 90-100 percent

frequency after five weeks of culture. The percentage of

viable embryogenic callus capable of supporting plant

regeneration decreased when the temperature severity or

duration of exposure increased. Callus surviving -120C for

32 h was unable to regenerate plants. Dix and Street (1976)

and Chen et al. (1982) found that tobacco cell lines which

were selected for low-temperature resistance were unable to

support plant regeneration.

An observed increase in the concentration of Ca in the

efflusate of freeze-injured leaf tissue (data not shown)

agreed with Palta et al. (1977a) and implied a loss of

plasmalemma integrity. The percentage of regenerated plants

exhibiting electrolyte conductivity values less than the

vegetatively propagated source plants is given in Table 2.

The percentage of regenerated plants ranged from a high of

approximately 50 % (270C, 20C for 12 d) to a low of 12%

(-120C for 8 h). Increasing the severity of the in vitro

temperature treatment on calli resulted in fewer regenerated

plants with less electrolyte leakage (Table 2). Steponkus

(1972) reported a total loss of chilling-temperature

tolerance in cells during tissue culturing. Other

researchers reported a partial loss of chilling or freezing-

temperature tolerance in cells during culturing (Dix and











Table 2. Percentage of regenerated pangola plants with
less electrolyte leakage than the source plants.



Callus treatment Regenerated plants


Temperature Duration+ Total Electrolyte conductivity
less than source plant

C d or h %

27 constant 89 47
2 12d 16 50
24d 65 14
-12 4h 51 29
8h 42 12


+Length of time for callus exposure to temperature
treatment.

Percentage of regenerated plants with lower electrolyte
conductivity than the mean electrolyte conductivity of three
source plants + S.D.







38

Street, 1976; Templeton-Somers et al., 1981; Chen et al.,

1982). Templeton-Somers et al. (1981) reported that

chilling-temperature resistance expressed in carrot culture

cells are not necessarily expressed in the somatic embryo

stage of development. These observations are supported by

the present study where freeze-tolerant cell lines supported

the regeneration of freeze-susceptible plants.

Electrolyte conductivity of the 10 selected freeze-

tolerant-regenerated plants was less than that for the

source plants (Table 3). Six of the regenerated plants had

significantly reduced electrolyte leakage when compared to

the vegetatively propagated source plants.

Data presented in Table 3 supported the hypothesis that

somatic variation in pangolagrass was obtainable and that

variant regenerated plants were able to have been selected

for an increase in freeze tolerance.

The percent FR remaining in regenerated plants and

source plants after chilling-temperature exposure is

presented in Table 4. No variation in the percent FR

remaining after chilling was detected between regenerated

plants or between regenerated plants and source plants.

These results do not support the hypothesis that somatic

variants may be selected for chilling tolerance using

chlorophyll fluorescence. Hetherington et al. (1983)

reported that chill stress reduced the FR value in corn, a











Table 3. Freeze-induced electrolyte
regenerated pangola plants


leakage of selected
and source plants.


Plant No. Mean conductivity

gamps

Source plant
1 78.2 a
2 65.0 ab
3 63.0 abc
4 61.2 a b c
Regenerated plant
59 48.4 a b c d
91 40.4 b c d e
40 40.0 b c d e
8 32.6 cde
103 29.6 d e
84 29.0 de
76 22.8 de
24 15.2 e
4 14.8 e
65 8.8 e



Means not followed by same letter are significantly
different at P < 0.05 (DMRT).









Table 4. Percent F, remaining in selected regenerated
pangola plants and source plants after
exposure to chilling temperature.


Regenerated plants Source plants


Plant No. F, remaining* Plant No. FR remaining*

% %

59 39 +15 2 57 + 9
91 40 + 8 3 56 + 9
40 58 + 9 4 51 +10
8 51 +11 1 51 +11
103 48 + 7
84 56 + 7
76 70 + 9
24 70 +10
4 48 + 8
65 35 +12


+Percent F, remaining = (Fg 230C / FR after 45 min at 100C)
100. Mean of six replications + S.D.






41

C4 grass. Their work with corn led to the identification of

chill-tolerant corn populations based on FR values.



Conclusions

Callus growth and subsequent somatic embryogenesis was

obtained in pangolagrass. Increasing the in vitro chilling

or freezing-temperature treatments reduced callus

survivability and competency for supporting embryogenesis.

Regenerated pangolagrass plants sustained a range of

freeze-induced damage between individuals as evidenced by

plasmalemma integrities. The ability of in vitro cells to

survive freezing or chilling temperatures did not predispose

plants, regenerated from those cells, to be freeze tolerant.

Apparently, increasing the chilling or freezing temperature

treatments in culture reduced the proportion of regenerated

plants that were freezing-temperature tolerant.

Somaclones with increased freeze tolerance relative to

vegetative control plants were obtained. Within this group

of plants, no variation in the extent of chill damage to the

photosynthetic electron transport chain within the thylakoid

membrane was observed.













CHAPTER III
EVALUATION OF CHILLING TEMPERATURE AND GIBBERELLIC ACID
ON THE EXPRESSION OF HYDROLYTIC ENZYMES



The hypothesis tested in this section was that chilling

temperatures reduce the number of debranching enzymes,

endoamylases, and exoamylases that break down starch.

Hypothetically, gibberellic acid (GA3) may nullify the

influence of low temperature on the reduction in the number

of starch-degrading enzymes.

The objectives of this study were a) to evaluate the

influence of low temperature on the expression of starch-

hydrolyzing enzymes; b) to identify hydrolytic enzymes which

may assist in starch breakdown under low- temperature

conditions; c) to evaluate the effect of GA3 on the

expression of starch-hydrolyzing enzymes during low-

temperature conditions; and d) to identify GA3 induced

isozymic forms of hydrolytic enzymes which may assist in

starch breakdown under low-temperature conditions.

The severe growth depression of pangola that results

from low nighttime temperatures has been associated with the

accumulation of starch granules in mesophyll cells and

bundle sheath cells (Hillard and West, 1970; Chatterton et

al., 1972). Hillard (1975) reported that cultivars of







43
Digitaria whose growth was least affected by chilling

temperature were those that retained the least amount of

starch in their chloroplasts after chilling nights.

The accumulation of large assimilated starch grains in

the chloroplast has been shown to reduce growth rates and

net photosynthesis via physical damage of the chloroplast

membrane (West, 1970), and disruption of photophos-

phorylation and electron transport (Pearson and Derrick,

1977).

Garrard and West (1972) determined that low leaf

temperature drastically reduces starch anabolism and

translocation, even when nearby sinks are maintained at high

temperatures. Jones (1985) concurred that translocation of

photosynthate to the growing region and utilization of

photosynthate by this region was limited by chilling

temperature. Kleinedorst and Brouwer (1972) reported that

restricting chilling to the leaves had little effect on

meristematic cell division. They observed a reduction in

cell elongation and postulated that this was due to reduced

carbohydrate availability for cell wall formation in the

region of cell elongation.

The biosynthesis of chloroplastic starch is well

characterized and has been reviewed by Preiss (1982), Preiss

and Levi (1979), and Preiss et al. (1987).

Most starches are a composite of two different types

of polysaccharides (Meyer and Gibbons, 1951). One is







44

unbranched and composed of long chains of a-1,4 linked

glucose units; the other is branched and consists of shorter

chains of a-1,4 linked glucose residues which are joined

through the a-1,6 positions to form a large molecule. Meyer

called the linear component amylose and the branched one

amylopectin. The number of glucose units in various

amylopectins ranges from 2,000 to 200,000 whereas amylose

may contain a few thousand glucose units (Salisburry and

Ross, 1985b).

Assimilatory starch granules in photosynthetic tissue

are located in the chloroplast. Viewed under a scanning

electron microscope, starch granules in pea (Pisum sativum

L.) are consistently surrounded by thylakoid membranes

(Beck, 1985). Beck (1985) reported the appearance of the

granules to be dramatically different between illuminated

and darkened pea leaves. The starch granules from an

illuminated leaf, unlike a darkened leaf, appeared pasty and

had a lack of sharp contours. The pasty starch mantle

surrounded

a crystalline starch core (Beck, 1985; Steup et al., 1983).

Beck (1985) investigated the water content of the starch

granule and reported that the mantle had a higher water

content than the core. Accompanying the higher water

content, the mantle also contained more a-1,6 branching than

the core.







45

Confusion in the literature concerning the mode of

starch degradation reflects the fact that the predominant

pathway of starch degradation within the chloroplast is

unresolved. Until recently, many researchers believed that

starch phosphorolysis by phosphorylase (Stitt and Heldt,

1981; Levi and Preiss, 1978; Stitt and Rees, 1980) was the

predominant pathway of starch degradation. Other researchers

believed that starch hydrolysis by endoamylase, exoamylase,

R-enzyme (debranching activity), and D-enzyme

(transglycosylase) (Herold et al., 1981; Peavy et al., 1977;

Chang, 1982) was the predominant pathway of starch

degradation. Assimilated starch is now believed to be

degraded by the combined action of hydrolysis and

phosphorolysis (Lin et al., 1988; Kakefuda et al., 1986;

Robinson and Preiss, 1987; Echeverria and Boyer, 1986).

Starch phosphorylase catalyses the phosphorolysis of

a-1,4 glucosyl chains yielding glucose-l-phosphate. a-

amylase attacks a-1,4 linkages of amylose or amylopectin

yielding glucose and small amounts of maltose (Salisburry

and Ross, 1985a). P-amylase attacks only the nonreducing

ends of chains, splitting off pairs of glucosyl units as

maltose (Salisburry and Ross, 1985a). R-enzyme attacks the

1,6 branching points of amylopectin making this component of

starch susceptible to further attack by hydrolysis or

phosphorolysis (Salisburry and Ross, 1985a). D-enzyme can

transfer groups of glucosyl units between short chain







46

dextrins producing a mixture of longer and shorter chain

dextrins and glucose (Salisburry and Ross, 1985a).

Dextrins were described by Meyer and Gibbons (1951) as

starch breakdown products that were composed of mixtures of

chain fragments of low but varying molecular weights.

Dextrins are routinely analyzed using a potassium-iodide-

iodine reaction (Jensen, 1962). Kakefuda and Duke (1984)

reported that electrophoretic gels containing amylopectin

stain dark violet with iodine and reveal areas of enzyme

activity as unstained or differentially stained regions

according to the type of starch-degrading enzyme present.

Unstained areas indicate complete amylopectin breakdown due

to the action of endoamylases, and pink regions reveal the

degradation of amylopectin to P-limit dextrin by

exoamylases. Debranching enzyme debranches amylopectin to

form amylose, which stains blue with iodine (Ziegler and

Beck, 1986).

Beck (1985) noted that during the nocturnal breakdown

of the starch granule, the mantle was degraded more rapidly

than the core. Endogenous hydrolytic enzymes accounted for

the degradation of the pasty mantle. Beck et al. (1981) and

Steup and Schachtele (1981) reported that assimilatory

starch is a very poor substrate for the chloroplastic

phosphorylase in spinach (Spinacia oleracea). They found

that amylolysis of assimilatory starch resulted in

degradation products that were acted on by phosphorylase






47
three to four times more rapidly than starting materials.

These results demonstrate the cooperativity and not a

competition between the amylolytic system and phosphorylase

in assimilatory starch breakdown.

Beck (1985) stated that, although amylases cooperate

with phosphorylases in the degradation of assimilatory

starch, the overall cellular location and physiological role

of these enzymes in leaves is far from clear. For example,

spinach leaf chloroplasts contain endoamylase, debranching

enzyme, phosphorylase, and D-enzyme (Okita and Preiss, 1980;

Preiss et al., 1980). In contrast, pea leaf chloroplasts

contain phosphorylase, R-enzyme, D-enzyme (Kakefuda et al.,

1986), and a small amount of P-amylase (Levi and Preiss,

1978). The a-amylase is either absent or in very low

activity in the chloroplast (Jacobsen and Beach et al.,

1985; Kakefuda et al., 1986). Beers and Duke (1988) reported

that the location of extrachloroplastic a-amylase is

primarily apoplastic.

Although amylase and phosphorylase activities

constitute the major degradative activity in the

chloroplast, only endoamylase has been demonstrated to

attack assimilated starch granules (Steup et al., 1983). The

extrachloroplastic location of a-amylase presents a logistic

problem. No extrachloroplastic homoglucans which could act

as substrates for such amylases are known and no







48

explanations have been presented to account for these

enzymes outside the chloroplast.

In corn, Echeverria and Boyer (1986) reported starch

accumulation in the chloroplasts of bundle sheath cells.

Starch was absent from the chloroplasts of mesophyll cells.

Analysis of starch-degradative enzymes showed that 30% of

the degradative enzymes were located in mesophyll cells,

where no starch accumulated. The remaining 70% of starch-

degradative enzymes were located in the bundle sheath cells.

In bundle sheath cells, 60% of the activity of these enzymes

were chloroplastic and 40% were located in the cytosol.

Starch phosphorylase was limited to bundle sheath cells with

only 70% of phosphorylase activity being chloroplastic.

In pangola, low temperature causes an excessive

accumulation of assimilated starch and a decrease in the

activity of starch-degrading enzymes (Carter et al., 1974).

Hillard (1975) reported that cultivars of weeping lovegrass

(Eragrostis curuvla L.) and digitgrass whose growth was

least affected by low temperature retained the least starch

in their chloroplasts after chilling nights and had the

highest amyolytic enzyme activities at chilling

temperatures. Karbassi et al. (1972) reported a reduction in

starch degrading activity when pangolagrass plants were

pretreated at chilling night temperatures or when the enzyme

assay was conducted under chilling conditions.







49

Carter et al. (1972) reported the localization of two

bands of amyolytic enzymes by polyacrylamide gel

electrophoresis using amylose containing gels. The relative

activity of both bands were decreased when exposed to 100C

incubation temperatures with the band nearest the origin

decreasing the least under low temperature. Carter et al.

(1973) modified the electrophoretic procedure to separate

the amyolytic enzymes into seven bands. The bands separated

into a fast migrating pair (bands 1 and 2) and a slow

migrating group (bands 3 to 7). Chilling temperature

decreased the activity of band 2 the most, while band 7 was

decreased the least. No change in the number of bands was

detected under the different temperature treatments.

Production of a-amylase in the barley (Hordeum vulgare

L.) aleurone layers has been shown to respond to GA3

(Jacobsen and Higgins, 1982). Addition of the hormone to the

de-embryonated seed or isolated aleurone layers results in a

large increase in a-amylase activity (Atzorn and Weiler,

1983) and debranching enzyme activity (Bewley and Black,

1985). GA3 acts at the transcriptional level increasing a-

amylase mRNAs(Bewley and Black, 1985). That increase in a-

amylase activity is the result of production of multiple

isozyme forms (Callis and Ho, 1983). Nolan and Ho (1988) and

Nolan et al. (1987) reported that GA3 differentially

controls the expression of two a-amylase genes or a group of







50

genes giving rise to two groups of a-amylase isozymes with

different properties.

Atkin et al. (1973) reported that chilling-temperature

exposure of corn plants reduces the amount of gibberellin in

the xylem exudate. They suggested that slower growth rates

during chilling-temperature exposure may be related to a

decrease in the gibberellin concentration in the leaf

tissue. Foliar application of GA3 to 'Tifdwarf,' a

bermudagrass (Cynodon dactylon L.) that is damaged by

chilling temperature, stimulates growth during chilling

temperatures (Dudeck and Peacock, 1985). GA3 has been shown

to improve growth rates of tropical forage grass when

applied 10 hours prior to chilling (Karbassi et al., 1971;

Whitney et al., 1973).

Foliar application of GA3 on pangola increases the

amylolytic activity in the plants at either ambient or

chilling temperatures (Carter et al., 1973; Karbassi et al.,

1972). Karnok and Beard (1986) showed that GA3 application

on chill-stressed bermudagrass reduced the number of starch

granules in the bundle sheath chloroplast.

Carter et al. (1973) reported that the application of

GA3 increased the relative activity of the seven amylolytic

enzymes that were separated by gel electrophoresis. The

activity of band 2 was increased the most while.the activity

of band 7 was least influenced. Of the 3-7 group, the enzyme

of band 3 was more responsive and those of bands 5 and 7







51

were the less enhanced by GA3. GA3 application did not cause

the production of additional amylolytic isozymes. This

response is unlike that in the aleurone layers of barley

seed and may be unique to chloroplasts.



Materials and Methods

Plant Material and Treatment Application

Experiments were conducted during the months of July

and August, 1989, at which time the photoperiod decreased

from 14 h to 13 h and the quantum flux density averaged

1,500 to 1,700 Meinsteins m-2 s1 (400-700 nm) at midday.

Average daily temperature in the greenhouse was maintained

between a range of 300C to 350C. Vegetatively propagated

source plants were grown in the greenhouse as described in

chapter II. Gibberellic acid was applied to a vegetatively

propagated source plant following the procedures outlined by

Karbassi et al. (1971) with the following modifications. A

10 uM GA3 solution, adjusted to pH 6.8 with KOH, was

sprayed to the drip point on all vegetative parts of the

plant 10 h before the 100C temperature treatment was

imposed. Temperature treatments were initiated at the end of

the photoperiod. One vegetatively propagated source plant

was placed in a darkened 300C Percival growth chamber. A

second source plant and the GA3 treated source plant were

placed in an identical growth chamber at 100C. Plants

remained in the growth chambers for 12 h until sampling.







52

Treatment application and subsequent analysis of starch-

hydrolyzing enzymes were replicated 18 times using different

plants.



Whole Leaf Extract Preparation

One gram fresh weight of the first fully extended leaf

was obtained at the end of the temperature controled 12 h

dark period. The tissue was washed with deionized water and

cut into 0.5 to 1.0 mm segments with a straight edge razor

blade. Segments were ground for 3 min in a chilled mortar

containing 3 mL of ice cold extraction buffer (Table 5). 3-

mercaptoethanol and polyvinylpolypyrolidone were added to

the extraction buffer just prior to grinding in a porcelain

mortar, Coors 522, size No. 6 having an unglazed grinding

surface. Ground glass was added to facilitate a fine grind.

The slurry was maintained at 20C during centrifugation at

20,000 g for 20 min. The supernatant was maintained at 20C

and used as samples for electrophoresis.

Protein controls were prepared for electrophoresis by

diluting a-amylase from bacillus (Sigma Chemical Company,

St. Louis, MO), E-amylase from sweet potato (Sigma Chemical

Company, St. Louis, MO), and pullanase a debranching enzyme

from Enterobacter aerogenes (Sigma Chemical Company, St.

Louis, MO) with extraction buffer.











Table 5. Reagents and gel preparation for native starch PAGE
slab gel electrophoresis.


Extraction buffer
0.5 M Tris, pH 6.8
Glycerol
B-Mercaptoethanol
polyvinylpolypyrolidone


Electrode buffer
Trizma base
(Tris[hydroxymethyl]aminomethane)
Glycine
pH 8.3

Separating gel 7.5% gel, 0.375 M Tris
dH20
Soluble starch
1.5 M Tris, pH 8.8
Acrylamide/bis (30%T, 2.67%C)
Ammonium persulfate (10%)
TEMED
N,N,N',N'-Tetramethylethylenediamine

Stacking gel 4.0% gel, 0.125 M Tris
dHO2
0.5 M Tris, pH 6.8
Acrylamide/bis (30%T, 2.67%C)
Ammonium persulfate (10%)
TEMED

Marker buffer
0.5 M Tris, pH 6.8
Glycerol
B-Mercaptoethanol
Bromophenol blue


0.5
11.0
0.1
20.0


M
%
%
mg/mg fw
plant


24.8 mM

191.8 mM



24.8 mL
100.0 mg
12.5 mL
12.5 mL
250.0 pL
50.0 gL



6.1 mL
2.5 mL
1.3 mL
50.0 gL
10.0 AL


0.1 M
11.0 %
0.1 %
0.002 %


The pH of preparations were adjusted with HC1


at 12'C.








Slab Electrophoresis

Reservoir, stacking, and separating gel buffers for SDS

PAGE gels as described by Laemmli (1970) were modified for

native PAGE gel preparations (Table 5). Native proteins are

separated based on their charge, molecular weight, and

configuration. The separating gel was amended by

substituting the water phase with a soluble starch solution.

A SE600 vertical slab gel electrophoresis unit (Hoeffer

Scientific Instruments, San Francisco, CA) was operated with

a discontinuous buffer system using two 160 mm long, 180 mm

wide, and 1 mm thick gels.

The 7.5% acrylamide separating gel (Table 5) was

prepared by boiling 100 mg of soluble starch in 24.8 mL of

dHO2 for two min and allowed to cool. Then 12.5 mL of 1.5 M

TRIS (pH 8.8) and 12.5 mL of acrylamide/bis (30%T, 2.67%C)

were added. The solution was degassed for 10 min before

adding 250 jL of ammonium persulfate (10%) and 25 pL of

TEMED. Twenty two and a half milliliters of the acrylamide

solution were immediately transferred to between the glass

sandwich with a pipette and overlayed with a thin layer of

butanol. After 15 min a sharp gel/butanol interphase was

visible. The butanol was poured out of the glass sandwich

and the remaining butanol was rinsed with dH2O before drying

the glass.

The stacking gel solution (Table 5) was prepared by

adding 6.1 mL dHO2 and 2.5 mL of 0.5 M TRIS (pH 6.8) to 1.3






55

mL of acrylamide/bis. The solution was degassed for 10 min

before adding 50 lL of ammonium persulfate (10%) and 10 pL

TEMED. The stacking gel solution was then pipetted into the

remaining space, between the glass sandwich, until 2 mm from

the top of the glass. The comb was inserted into the

stacking gel solution and removed after polymerization.

Wells, formed by the comb, were each loaded with 90 pL

of an appropriately dilute enzyme preparation. Forty

microliters, 60 ML, and 70 AL of enzyme preparation were

adjusted to 90 iL with the marker buffer (Table 5) before

loading into separate wells. After loading, the

electrophoresis was conducted at 35 volts for 2.5 hours at

120C. Each sample was replicated twice on each gel so that

after electrophoresis half the gel could be used for protein

staining and half for enzyme activity staining.



Protein and Enzyme Activity Staining

The quantity of protein loaded into each well for

electrophoresis was determined spectrophotometrically using

Bio-Rad's protein assay (Bio-Rad, Richmond, CA). A protein

standard curve was constructed ranging from 20-140 Mg

protein using bovine serum albumin (United States

Biochemical Corporation, Cleveland, OH). Absorbance at 595

nm was measured with a Hewlett Packard 8451A diode array

spectrophotometer (United States). Twenty microliters, 40

ML, and 60 AL of enzyme preparation were adjusted to 100 ML







56

with extraction buffer and subjected to the standard assay

procedure. The quantity of protein in the extracts was

determined by comparison to the protein standard

curve.

Gels were stained for protein using the method of

Schuler and Zielinski (1989). The gel was placed in 300 mL

of a fixative containing 40% methanol, 10% glacial acetic

acid, and 0.1% coomassie brilliant blue for 30 min and

gently shaken. The gel was then destined in 300 mL of 40%

methanol and 10% glacial acetic acid. The destaining

solution was changed twice over a three hour period. After

clearing the background stain, the gels were photographed as

quickly as possible and dried on a slab gel dryer (Hoeffer

Scientific Instruments SE 1160).

Staining of the gel for starch-degrading enzymes

followed the procedure outlined by Vallejos (1983). The gel

was incubated in a solution consisting of 50 mM sodium

acetate and 1 M calcium chloride adjusted to a pH of 5.6

with HC1 at 350C for 60 min. After incubation, the gel was

rinsed thoroughly with dHO2 and stained in 10 mM iodine and

14 mM potassium iodide. When the zymogram developed a dark

purple background it was rinsed in dHO2 and photographed

quickly before being dried on a slab gel dryer (Hoeffer

Scientific Instruments SE 1160).







57

Results and Discussion

Native starch PAGE gels stained differentially

according to the type of hydrolytic enzyme (data not shown).

a-Amylase activity resulted in a clear band, P-amylase

activity resulted in a red band, and debranching activity

produced a blue band. These results support previous

reports. Ziegler and Beck (1986) and Kakefuda and Duke

(1984) reported the identification of the type of hydrolytic

enzyme activity based on the color of bands after iodine

staining of starch impregnated native gels.

Following electrophoresis, incubation, and staining of

gels containing pangola leaf extracts distinct banding

colors and banding patterns were evident. Interpretation of

the type of enzyme activities were based on the band color

as related to the known a-amylase, P-amylase, and pullanase

samples and Ziegler and Beck (1986) and Kakefuda and Duke

(1984) reports. For reference purposes, zymograms will be

identified by plant treatment with regards to night

temperature and GA3 application as follows: 300C(control),

100C, 100C+GA.

Red bands resulting from exoamylolytic enzyme activity

were not detected regardless of temperature or GA3 treatment

(Figure 9). Enoamylolytic enzyme activity was evident in the

300C treatment by the appearance of eight clear bands. The

bands separated into a fast migrating group (bands 1-5), a

slower migrating pair (bands 6 and 7), and a single slowly







58

migrating band (band 8) (Figure 9). Carter et el. (1972)

previously found one fast migrating band and a slower

migrating pair of bands in pangola. By altering the

electrophoretic conditions, they further separated

endoamylolytic enzymes into a fast migrating pair of bands

and a slower migrating group of five bands (Carter et al.,

1973). The results from this study differ from the

previously cited literature in that an additional

endoamylolytic enzyme was resolved and bands on the gels did

not form similar migratory patterns.

Treatment at 100C reduced the number of visable

endoamylolytic bands. Bands 6 and 8 were not visable when

compared to the 300C treatment (Figure 9). Based on

densitometer tracings, Carter et. al. (1972) and Carter et

al. (1973) reported a reduction in the enzyme activity

within the bands and not a reduction in the number of bands.

Approximately doubling the total protein load in the 10C

treatment relative to the 300C treatment, 83 pg to 42 Ig

respectively, did not result in the appearance of band 6 or

8. This indicates the presence of two chill-sensitive-

endoamylolytic isozymes.

Data presented in Figure 9 supported the hypothesis

that chilling temperature reduced the number of starch-

degrading enzymes; specifically endoamylolytic isozymes. The

remaining endoamylolytic isozymes may not be able to

satisfactorily hydrolyze starch, particularly if the









Treatment


30C


10C


10"C+GA


5-
4-
3-
2-
1-


Protein


9g
42 83 55 73 48

Fig. 9. Effect of temperature and GA3 on expression of
starch-degrading-hydrolytic enzymes in pangola.
Areas of endoamylase activity show up as white
(bands 1-8) that of debranching enzyme activity blue
(band 9) against the dark background of the
amylose-amylopectin containing gel. F refers to
the bromophenol blue front marker. Micrograms
protein refers to the total protein.







60

remaining endoamylases have a lower activity as reported by

Carter et al. (1972) and Carter et al. (1973). The

consequence of is a starch buildup.

Coomassie stain for total protein did not reveal a

specific protein band at endoamylase enzyme position 6 or 8

respective of protein load or treatment (Figure 10). The

mechanism of sensitivity remains unclear, i.e.

continued protein synthesis with reduced enzyme activity or

termination of protein synthesis.

The number of hydrolytic enzyme bands in the 100C

treatment zymogram were not altered as a result of treatment

with GA3 (Figure 9). Approximately doubling the total

protein load in the 100C+GA treatment relative to the 10C

treatment did not increase the number of detectable bands

(Figure 9). Carter et al. (1973) and Karabassi et al. (1971)

reported that relative activities of amylolytic enzymes and

not the number of isozymes were increased by applying GA3.

In both studies 10 AM GA3 was applied to pangolagrass 10

hours prior to low-temperature treatment.

The data presented in Figure 9 did not support the

hypothesis that GA3 nullifies the effect of low temperature

on the reduction in number of starch-degrading enzymes.

A slow moving blue band (band 9), indicative of

debranching enzyme activity, was evident in the 30C

treatment. Kakefuda et al. (1986) identified a debranching








61


Treatment


10'C


7 C C


Protein


42 42 82


55 55 83
55 55 83


48 48 73


Fig. 10. Effect of temperature and GA3 on production and
expression of starch-degrading-hydrolytic enzymes
in pangola. Zymograms (z) of hydrolytic
activity and coomassie protein stains (c) are
presented for each treatment. Micrograms protein
refers to the total protein.


30"C


7. .


1 -C+GA







62

enzyme in crude pea leaf extract using electrophoresis as

described by Kakefuda and Duke (1984). The slow moving blue

band remained unchanged regardless of temperature or GA3

treatment (Figure 9).



Conclusions

Current studies indicate that anabolism of assimilated

starch in chloroplasts is accomplished by the cooperation of

hydrolytic and phosphorolytic enzymes. The cellular location

and physiological role of these enzymes remain unclear and

need to be identified for the type of plant in question.

Eight endoamylolytic enzymes and one debranching enzyme

have been identified in pangola leaf tissue by native starch

gel electrophoresis. Two temperature-sensitive-

endoamylolytic enzymes were identified. The mechanism of

sensitivity remains unclear i.e., continued protein

synthesis with reduced enzyme activity or termination of

protein synthesis.

The number of starch-degrading-hydrolytic enzymes was

unaffected by applying GA3 to the plant prior to low-

temperature exposure.












CHAPTER IV
EVALUATION OF HYDROLYTIC ENZYMES IN FREEZING-TEMPERATURE-
TOLERANT SOMACLONES OF PANGOLA



This study tested the tentative assumption that chill-

tolerant-endoamylolytic isozymes may be present in low-

temperature-tolerant somaclones of pangolagrass.

The objectives of this study were a) to evaluate low-

temperature-tolerant-pangola somaclones for variation in the

expression of hydrolytic enzymes and b) to select plants

that retain hydrolytic enzyme activity which may assist in

starch breakdown after exposure to low temperatures.

The rapidity of screening and simple inheritance make

isozymes a powerful tool in the analysis of variation.

Variation in isozyme patterns of starch-hydrolyzing enzymes

has been studied extensively during the germination process

in seeds (Bewley and Black, 1985; Jacobsen and Beach, 1985;

Atzorn and Weiler, 1983; Nolan and Ho, 1988).

Starch-hydrolyzing isozyme patterns have been used to

show induced variation within the same seed type. Henke

(1981) used a half-seed a-amylase assay capable of screening

thousands of half-seeds in a short time. After screening

100,000 mutagenized barley seeds for the ability to express

a-amylase in the absence of GA3 or in the presence of GA3







64

plus an inhibitor, he found 1% of the seeds to be variant.

Embryos of the variant half-seeds were grown to maturity and

selfed. The resulting seed provided evidence for inheritance

of a mutant trait. Ho et al.(1980) used a comparable

approach and obtained similar results.

Few studies used starch-hydrolyzing enzymes for the

identification of somatic variants. A somaclonal variant

which effects the isozyme pattern of one of the seed protein

complexes of wheat, namely a-amylase, was reported by Larkin

et al. (1984). Seeds from 68 somaclones were assayed to

identify eight somatic variants. Of the variant plants, four

were GA3-insensitive, one was GA3-supersensitive, and four

were ABA-insensitive. The characteristics were heritable

through two seed generations.

Ryan and Scowcroft (1987) obtained somatic variation

within P-amylase isozymes in wheat seeds. One variant seed

phenotype was observed from among 149 regenerated plants.

This variant was characterized by five new isozyme bands, as

well as an increased intensity of two previously existing

bands. The variant segregated in a mendelian manner, without

recombination between bands, and mitosis and meiosis were

cytogenetically normal in both the homozygous variant and in

the F1 backcross. This new variant has not been previously

observed in the 10 known p-amylase phenotypic groups and

gives the appearance of being a new gene.







65

To date no reports have been made of variation in the

starch-hydrolyzing isozyme banding patterns in

photosynthetic tissue of somatically regenerated plants.

Exposing photosynthetic tissue of regenerated plants to low-

temperature treatments may be useful in identifying variant

plants which express an increased number of hydrolytic

enzymes under low-temperature conditions.



Materials and Methods

Plant Material and Treatment Application

Sixteen vegetatively propagated plants were produced

from each of the 10 previously selected low-temperature

tolerant regenerated plants. Vegetatively propagated

regenerated plants and vegetatively propagated source plants

were maintained in the greenhouse as described in chapter

II. Experiments were conducted during the month of August,

1989. Temperature treatments were imposed at the end of the

photoperiod. One vegetatively propagated regenerate plant,

from each of the 10 selected regenerated plants, and one

vegetatively propagated source plant was placed in a

darkened 300C Percival growth chamber. An identical

combination of plants was placed in a darkened 10C Percival

growth chamber. Plants remained in the growth chambers for

12 hours until sampling.







66

The application of temperature treatment and subsequent

analysis of starch-hydrolyzing enzymes were replicated eight

times.



Whole Leaf Extract Preparation

Protein extracts were prepared for native starch PAGE

slab gel electrophoresis as described in Chapter III.



Slab Electrophoresis

Electrophoretic separation of the protein extracts was

preformed as presented in Chapter III.



Protein and Enzyme Activity Staining

The quantity of total protein, coomassie blue staining

procedure for the total protein, and I2-KI stain for starch-

degrading enzyme activity was followed as described in

Chapter III.



Results and Discussion

Zymograms of the 10 regenerated plants from Chapter II

contained the characteristic hydrolytic banding pattern

found in the vegetatively propagated control plants. Figure

11 shows the comparison of a typical zymogram from the

regenerated plants to that of a vegetatively propagated

plant. No variation in the isozyme banding pattern was







67

Treatment


30C


10"C


1 2 3 4


7-

6-



5-
4-
3-
1--













F-


Fig. 11. Zymograms of starch-hydrolyzing enzymes isolated
from pangola plants that were exposed to 300C
or 10 C. White bands (bands 1-8) show endoamylase
activity, blue band (band 9) indicates debranching
enzyme activity. Lanes 1 and 3 represent the
vegetatively propagated control plants. Lanes 2 and
4 represent plants regenerated from tissue culture.
F refers to the bromophenol blue front marker.







68

observed between the regenerated plants or between

regenerated plants and vegetatively propagated control

plants respective of temperature treatment.

Nine hydrolytic bands were evident in zymograms from

the 300C treatment (Figure 11). Exoamylolytic activity was

not detected. Endoamylolytic activity resulted in eight

clear bands which separated into a fast migrating group

(bands 1-5), a slower migrating pair (band 6 and 7), and a

single slowly migrating band (band 8). Debranching enzyme

activity resulted in a single extremely slow migrating blue

band.

The 100C treatment resulted in a banding pattern

identical to the 300C treatment except for the elimination

of bands six and eight (Figure 11). Data do not support the

hypothesis that somatic variation in low-temperature

tolerant somaclones can be used to obtain chill-tolerant

endoamylolytic isozymes. Amplifying the number of test

plants would increase the probability of detecting a desired

variation.

Coomassie staining of the protein did not show specific

banding for the endoamylolytic protein at band number six or

eight for regenerated plants or vegetatively propagated

control plants in either temperature treatment (Figure 12).

Evidently coomassie stain was not sensitive enough to detect

the quantity of protein at band six or eight position.

Silver stain which detects 10-50 ng of protein and is







69



Treatment

300C 100C

1 2 3 4 5 6

9-

8-



7- .
6-



5-
4-
3-
2-
1-












F-

Fig. 12. Zymograms of starch-hydrolyzing enzymes and total
protein profiles from pangola plants exposed
to 300C or 100C. Lanes 1 and 4 are zymograms. White
bands (bands 1-8) show endoamylase activity, blue
band (band 9) indicates debranching enzyme
activity. Coomassie blue stained proteins are
presented in lane 2 vegetativelyy propagated
control plants) and lanes 3 and 6 (plants
regenerated from tissue culture).







70

approximately 100 times more sensitive than coomassie stain

may detect protein at band six or eight position.



Conclusions

All of the regenerated plants that were selected in

chapter II for freezing-temperature tolerance were similar

in that they showed no somaclonal variation in starch-

hydrolyzing enzymes or total protein banding patterns. The

characteristic banding patterns of hydrolytic enzymes in the

30C treatment (eight endoamylolytic and one debranching

enzyme) and the 100C treatment (six endoamylolytic and one

debranching enzyme) were consistent between regenerated

plants and between regenerated plants and vegetatively

propagated control plants. The regenerated plants and the

vegetatively propagated control plant had two similar

temperature-sensitive-endoamylolytic enzymes.












CHAPTER V
SUMMARY AND CONCLUSIONS



A large number of factors contribute to the reduction

of growth of pangola under freezing and chilling-temperature

conditions. Several of the major factors are loss of

plasmalemma integrity, reduction in photosynthetic electron

transport, and decreased activity of assimilated starch-

hydrolyzing enzymes. Irrespective of being distinct factors,

the action and interaction of all these factors contribute

to a common result: reduced growth resulting from freezing

or chilling-temperature exposure.

Several experiments were conducted to induce phenotypic

variation and select variant plants for a reduction in low-

temperature damage. The studies reported in Chapter II were

conducted with the objectives: a) to generate callus lines

of pangola; b) to select freezing or chilling-temperature

resistant cell lines in vitro; c) to regenerate whole plants

from selected cultures; and d) to test regenerated plants

for increased tolerance to freezing and chilling temperature

at the organelle and cellular level. Somatic embryogenesis

in pangola followed a pattern of development similar to

those described for other grasses. Increasing the in vitro







stress reduced callus survivability and competency for

supporting embryogenesis. Regenerated pangola plants

sustained a range of freeze-induced damage to their

plasmalemma membranes. Selection of in vitro cells that

survived freezing or chilling temperature did not result in

plants, regenerated from these cells, that were freezing-

temperature tolerant. Apparently, increasing the freezing or

chilling-temperature selection pressure in culture reduced

the proportion of regenerated plants that were freezing-

temperature tolerant. Somaclones with increased freeze

tolerance relative to vegetative control plants were

obtained. Within this group of plants, no variation in the

extent of chill damage to the photosynthetic electron

transport chain within the thylakoid membrane was observed.

The objectives reported in Chapter III were a) to

evaluate the influence of chilling temperature on the

expression of starch-hydrolyzing enzymes; b) to identify

hydrolytic enzymes which may assist in starch breakdown

after chilling-temperature exposure; and c) to evaluate the

effect of GA3 on the expression of starch-hydrolyzing

enzymes during chilling-temperature conditions.

Current studies indicate that anabolism of assimilated

starch in chloroplasts is accomplished by the cooperation of

hydrolytic and phosphorolytic enzymes. The cellular location

and physiological role of these enzymes remain unclear and

need to be identified for the type of plant in question.







73

Eight endoamylolytic and one debranching enzyme have been

identified in pangola leaf tissue by native starch gel

electrophoresis. Two temperature-sensitive-endoamylolytic

enzymes were identified. The mechanism of sensitivity

remains unclear, i.e., continued protein synthesis with

reduced enzyme activity or termination of protein synthesis.

The number of starch-degrading-hydrolytic enzymes was

unaffected by applying GA3 to the plant prior to chilling-

temperature exposure.

Chapter IV reports on research conducted with the

objectives: a) to evaluate the chill-temperature-tolerant

pangola somaclones for variation in the expression of

hydrolytic enzymes and b) to select plants that retain

hydrolytic enzyme activity which may assist in starch

breakdown after exposure to chilling temperature. All of the

regenerated plants that were selected for Freezing-

temperature tolerance in Chapter II showed no somaclonal

variation with respect to the number of starch-hydrolyzing

enzymes or total protein banding patterns. The

characteristic banding patterns of hydrolytic enzymes in the

300 treatment (eight endoamylolytic enzymes and one

debranching enzyme) and the 100C treatment (six

endoamylolytic enzymes and one debranching enzyme) were

consistent between regenerated plants and between

regenerated plants and vegetatively propagated control

plants. The regenerated plants and the vegetatively







74

propagated control plant had two temperature-sensitive-

endoamylolytic enzymes.

Tissue cultured pangola circumvents sterility barriers

which prevent plant improvement via conventional breeding

programs. Somatic variants of pangola can be regenerated

from tissue culture and selected for increased tolerance to

freezing-temperature conditions. The selection factor for

freeze tolerance is plasmalemma integrity.

Direction for future research should be threefold.

First, field evaluations of the selected freeze-temperature

tolerant plants should be continued. Forage characteristics

such as plant spread, leaf area, yield, flowering date, rust

resistance, crude protein, organic matter, and percent

organic matter that is digested by rumen microorganisms are

being studied. Second, densitometer tracings of gels should

be obtained for analyzing relative activities of the

identified amylolytic enzymes. Temperature and GA3

treatments should be imposed as presented in these studies.

Third, a larger number of regenerated plants (larger than

263 regenerates) should be produced and screened for

freezing and chilling-temperature tolerance. This may lead

to the identification of individuals with increased

tolerance in all three selection factors.












REFERENCES


Ahloowalia, B. S. 1982. Plant regeneration from callus
culture in wheat. Crop Sci. 22: 405-410.

Ahloowalia, B. S. 1983. Spectrum of variation in somaclones
of triploid ryegrass. Crop Sci. 23: 1141-1147.

Ahloowalia, B. S. 1986. Limitation to the use of somaclonal
variation in crop improvement. p. 14-27. In J. Semal
(ed.) Somaclonal variations and crop improvement.
Maritnus Nijhoff Publishers, Boston.

Atkin, R. K., G. E. Barton, and D. K. Robinson. 1973. Effect
of root-growing temperature on growth substances in
xylem exudate of Zea mavs. J. Environ. Bot. 24:475-487.

Atzorn, R., and E. W. Weiler. 1983. The role of endogenous
gibberellins in the formation of a-amylase by aleurone
layers of germinating barley caryopses. Planta 159:
289-299.

Bajaj, Y. P. S., B. S. Sidhu, and V. K. Dubey. 1981.
Regeneration of genetically diverse plants from tissue
cultures of forage grass Panicum sp. Euphytica 30: 135-
140.

Bayliss, M. W. 1976. Variation of cell cycle duration within
suspension cultures of Daucus carota and its
consequence for the induction of ploidy changes with
colchicine. Protoplasma 88: 279-285.

Beck, E. 1985. The degradation of transitory starch granules
in chloroplasts. p. 27-44. In R. L. Heath, and J.
Preiss (eds.) Regulation of carbon partitioning in
photosynthetic tissue. American Soc. Plant
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BIOGRAPHICAL SKETCH


Eugene Balliet Ackerman was born on November 4, 1956,

in Bainbridge, Maryland. He completed high school at the

Hill School, Pottstown, Pennsylvania, in 1976. In May, 1981

he received his Bachelor of Science degree in agronomy from

The Pennsylvania State University in College Park,

Pennsylvania. From June 1981 to January 1983, he was granted

an assistantship at the University of Arkansas to work

toward a Master of Science degree in agronomy.

In 1983 he was granted a fellowship to study at the

Marine Biological Laboratory, Woods Hole, Massachusetts. He

conducted research on microbial ecology.

In August 1985 he joined the plant stress unit of the

United States Department of Agriculture-Agricultural

Research Service (USDA-ARS), located at Gainesville,

Florida. He is currently researching plant growth under

stress conditions.

In August 1986 he initiated work towards a Ph.D. degree

in agronomy at the University of Florida. He is currently a

Ph.D. candidate, and upon completion of his graduate work,

he will be employed by Hoffman-LaRoche as an agricultural

specialist in the business development department.







90

In 1989 he was certified as a professional crop

scientist by the American Registry for Certified

Professionals in Agronomy, Crops, and Soils.

He is a member of the American Society of Agronomy and

Crop Science Society of America.

He is married to Amy Lynn and has three children:

Jacob, Rachel, and Taryn.