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Biochemical and molecular analysis of regenerants derived from somatic embryos of Pennisetum purpureum K. Schum

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Biochemical and molecular analysis of regenerants derived from somatic embryos of Pennisetum purpureum K. Schum
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Shenoy, Vivek Bhaskar, 1961-
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vii, 170 leaves : ill., photos. ; 29 cm.

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Callus ( jstor )
Chloroplasts ( jstor )
Corn ( jstor )
DNA ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Genomes ( jstor )
Mitochondrial DNA ( jstor )
Plastids ( jstor )
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Pennisetum purpureum -- Analysis ( lcsh )
Pennisetum purpureum -- Genetics ( lcsh )
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non-fiction ( marcgt )

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Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 150-169).
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Typescript.
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Vita.
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by Vivek Bhaskar Shenoy.

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BIOCHEMICAL AND MOLECULAR ANALYSIS OF REGENERANTS
DERIVED FROM SOMATIC EMBRYOS OF PENNISETUM PURPUREUM K.
SCHUM.















By
VIVEK BHASKAR SHENOY















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


1991





























Dedicated to my family for their support and understanding and to my nieces,
Renuka, Sai, and nephew, Shirish, all of whom I have yet to see.















ACKNOWLEDGEMENTS


I would like to acknowledge the valuable guidance and encouragement of my

committee chairman, Dr. Indra K. Vasil. I am forever grateful for his constant push

towards perfection. I am indebted to Dr. Daryl R. Pring for all his technical

supervision, the use of his laboratory and the various DNA probes he provided. I

thank Dr. S. C. Schank for providing the plant material and initial field space. Drs.

Robert J. Ferl and Henry C. Aldrich, I thank for their time, help and guidance

whenever I needed it. I am particularly grateful to Dr. William B. Gurley for

consenting to attend my final examination.

I also thank Dr. M. K. U. Chowdhury, Mr. Mark G. Taylor and Mr. Luis F.

Pedrosa for all the useful discussions, suggestions and their invaluable help. I am

grateful to all other colleagues for their friendship and support.

For technical help and guidance, I thank Drs. Rex L. Smith and C. E.

Vallejos.

Finally, I would like to thank the Dav6, Gokhale, Gor6 and Navath6 families

for their help in making my stay in Florida both enjoyable and pleasant.













TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ..................................................................................... iii

ABSTRA CT ................................................................................................................ .....vi

CHAPTERS

1 INTRODUCTION......................................................................... 1

2 LITERATURE REVIEW.......................... .............. ............... 3

Variation in tissue culture............................................................. 3
Isozym es ................................................................................................... 4
Mitochondrial DNA .................................................... ............... 9
Chloroplast DNA .......................................................................... 16
Nuclear DNA ................................................................................. 25

3 REGENERATION OF PLANTS FROM SOMATIC EMBRYOS OF
PENNISETUM PURPUREUM K. SCHUM. BY TISSUE
CULTURE OF IMMATURE LEAF SEGMENTS ............... 29

Introduction.................................................... ................................ 29
Materials and methods ..................................................................... 31
R esults............................................................................................... 32
D discussion ......................................................................................... 33

4 BIOCHEMICAL ANALYSIS OF A POPULATION OF
PENNISETUM PURPUREUM K. SCHUM. DERIVED
FROM TISSUE CULTURE USING, ISOZYMES AS
M AR KER S................................................................................... 36

Introduction...................................................................................... 36
Materials and methods................................... ......................... 38
R esults............................................................................................... 40
D discussion ......................................................................................... 44

5 RESTRICTION AND HYBRIDIZATION ANALYSIS OF
MITOCHONDRIAL DNA FROM A POPULATION OF
PENNISETUM PURPUREUM K. SCHUM. REGENERANTS
DERIVED FROM SOMATIC EMBRYOS.......................... 77

Introduction...................................................................................... 74
Materials and methods ................................... ......................... 75
R results ............................................................................................... 80
D iscussion.............................................................................................. 119


iv *








6 CHLOROPLAST DNA ANALYSIS OF PENNISETUM
PURPUREUM K. SCHUM. REGENERANTS DERIVED
FROM TISSUE CULTURE OF YOUNG LEAF
SEGMENTS .................................................................................. 121

Introduction ............................................................................................ 121
Materials and methods ...................................................................... 122
R esults............................. .......................................................... 125
D discussion ........................................................................................ 126

7 DNA HYBRIDIZATION ANALYSIS OF NUCLEAR DNA
FROM TISSUE CULTURE DERIVED REGENERANTS OF
PENNISETUM PURPUREUM K. SCHUM. ........................ 134

Introduction..................................................................................... 134
Materials and methods ...................................................................... 135
R esults.................................................................................................... 138
D discussion ........................................................................................ 138

8 CONCLUSIONS .................................................................................. 148

REFEREN CES .......................................................................................................... 150

BIOGRAPHICAL SKETCH ...................................................................................... 170













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

BIOCHEMICAL AND MOLECULAR ANALYSIS OF REGENERANTS
DERIVED FROM SOMATIC EMBRYOS OF PENNISETUM PURPUREUM K.
SCHUM.

By
Vivek Bhaskar Shenoy

June 1991

Chairman: Indra K. Vasil
Cochairman Robert J. Ferl
Major Department: Botany

A population of Pennisetum purpureum plants regenerated from embryogenic

callus cultures was analyzed for the occurrence of variation at the biochemical and

molecular levels. Fifty-seven P. purpureum regenerants obtained from the tissue

culture of young leaf segments of a single clone were used for the analysis. The

biochemical analysis consisted of screening the entire population for the activity of

several isozymes using electrophoretic techniques. Molecular analyses were carried

out to identify any aberrations at the DNA level.

For the biochemical studies, both polyacrylamide (native) as well as starch

gel techniques were tested. The starch gel technique was used for a mass analysis of

the regenerants for isozyme activity. A total of fourteen enzyme systems yielded

good zymograms and were used to screen the entire population. The isozymes

successfully stained for activity included Acid phosphatases (a and P), Alcohol

dehydrogenase, Aryl esterases (a and P), Aspartate aminotransferase,

Endopeptidase, Glutamate dehydrogenase, Hexokinase, Malate dehydrogenase,

Malic enzyme, 6-Phosphogluconate dehydrogenase, Phosphohexose isomerase and









Shikimic acid dehydrogenase. In all the isozyme systems that showed activity, no

variation was observed in banding patterns between tissue culture regenerants and

control plants.

Twenty-three regenerants were selected randomly, in addition to the parental

clone, for the extraction of mtDNA. These DNA samples were primarily analyzed

by comparing their restriction patterns on agarose gels. The four restriction enzymes

used individually for this analysis were BamHI, HindIII PstI and Sall. The

comparative analysis of restriction patterns from all the extracted samples did not

yield any unique fragments, suggesting that there was no variation at the mtDNA

level. These gels were blotted to nylon membranes which were used for

hybridization analysis of the restricted DNA. The membrane blots corresponding to

each restriction enzyme were probed using six different mitochondrial genes ie.

atpA, atp6, atp9, coxl, coxlI and the 18S ribosomal gene. In addition to this, the blots

were probed using random probes from the wheat mitochondrial genome and

cosmids cloned from the maize mitochondrial genome, each cosmid had an insert
averaging 35 kb. The hybridization analyses of all the samples mentioned above also

showed no unique patterns.

Analysis of cpDNA and nuclear DNA was carried out using total DNA

extracted from twenty-two randomly selected regenerants in addition to the parent.

Total DNA from each sample was restricted and blotted from gels for hybridization

analysis. The enzymes used to restrict the total DNA were EcoRI, HindIII and PstI.

The blots corresponding to each restriction enzyme were probed using two cosmid

clones, which together represented more than 75 kb or 60% of the maize chloroplast

genome. The blots were also probed using three different random P. purpureum

nuclear probes and the Nor locus gene from wheat. All the blots thus probed showed

no variation within the individuals.













CHAPTER 1
INTRODUCTION

For the past few decades, tissue culture has been studied very closely and has

opened up innumerable possibilities in its use as a technique to obtain clonal

populations. Since the process of in vitro culture does not involve the zygotic

process, tissue culture was expected to provide progeny with clonal fidelity.

However, it has been shown that tissue cultures and plants derived from them

undergo many changes at the cytogenetic and morphological levels (Murashige and
Nakano 1966, 1967; Heinz and Mee 1971). These anomalies were claimed to be

beneficial to the plant breeder, as a novel method of introducing new varieties

(Larkin and Scowcroft 1981). Most of the aberrations identified have been found to

occur at low frequencies in normal sexual crosses. Other variations have been

epigenetic and hence not heritable through a sexual cycle. Variation in culture also

depends on the nature and source of the explant tissue. While a large number of

reports concerning variation in tissue culture have focused on the use of immature

embryos from inbred lines to provide clonal populations, Breiman et al. (1989) have

observed the occurrence of variations at a very low level between individuals of an

inbred line. The anomalies observed were similar to the ones they reported from a

tissue culture-derived population, in an earlier publication (Breiman et al. 1987a).

In contrast to the reports of variation, there have been others accentuating

the stability of plants from tissue culture. It has been documented that

embryogeniccallus cultures are largely euploid and plants derived from such cultures

are both euploid and genetically stable (Swedlund and Vasil 1985; Rajasekaran et al.

1986; Gmitter et al. 1991). The stability of callus and tissue culture derived plants is








important for genetic manipulations in biotechnology, to be able to predict the

outcome of such manipulations, barring spontaneous mutations.

Although plants derived from embryogenic cultures are known to be

cytogenetically and morphologically stable, it is important to screen the regenerants

for changes at the biochemical and molecular levels to ascertain their fidelity to the

explant source. Biochemical analyses have generally involved isozymes and total

proteins, while molecular analyses involve the scrutiny of the nuclear and

cytoplasmic genomes for restriction fragment length polymorphisms (RFLPs).

This study involved biochemical and molecular analyses of a population

derived from somatic embryos obtained from a single field grown clone of

Pennisetum purpureum (napiergrass). The study differs from other studies in that

the parental clone is used as a control for comparative analysis of the regenerant

population. Biochemical analyses consisted of screening the population by staining

for the activity of several isozymes on starch gels. Molecular analyses involved the

study of the nuclear and cytoplasmic genomes. Restriction profiles of the

mitochondrial genome were visualized on agarose gels using four restriction

enzymes. The DNA from these gels was blotted onto membranes for use in DNA

hybridization analyses using known gene probes from the maize mitochondrial

genome, and random clones from the maize and wheat mitochondrial genomes.

Chloroplast and nuclear DNA analyses were carried out using random cosmid

clones from the maize chloroplast genome and random nuclear probes from the

napiergrass genome to probe total DNA blots.













CHAPTER 2
LITERATURE REVIEW

Variation In Tissue Culture

Totipotency and Plant Regeneration

The concept of single cell autonomy and totipotency is contained in the

independent works of Schleiden and Schwann during the earlier part of the
nineteenth century (Gautheret 1985). Totipotency refers to the ability of a single

cell to give rise to an entire individual, and implies that all the genes present in the

zygote are conserved in each subsequent cell. Many researchers have attempted to

establish long term totipotent cell and callus cultures in a variety of plant species.

All such attempts proved unsuccessful until the mid-1930s, when continuously

growing callus cultures were independently obtained by Gautheret (1934, 1935),
Nob6court (1939) and White (1939), which formed the basis for further studies on

the possibility of regenerating plants from such cultures. These efforts culminated

with the demonstration of totipotency by Vasil and Hildebrandt (1965a) who

cultured isolated single cells of Nicotiana glutinosa x N. tabacum in microchambers

and documented their development into entire plants.

Plants regenerated from tissue culture should normally result in clones that

are phenotypically and genotypically identical to the explant from which they have

been originally derived. However, plant cell and callus cultures accumulate

chromosomal variability and lose their regenerative capacity over time (Murashige

and Nakano 1966, 1967; Orton 1980). Therefore, embryogenic cultures, in which

plants are derived from somatic embryos of single cell origin, are considered more

useful because there is a selection away from chromosomal








variants in the formation of somatic embryos (Hanna et al. 1984; Karp and Maddock

1984; Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986; Cavallini et al.

1987; Kobayashi 1987; Feher et al. 1989; Gmitter et al. 1991). Plants derived from

embryogenic calli are mostly euploid and devoid of any discernible morphological or

cytological variability.

The Concept of Variability

Although tissue culture has been used extensively for the clonal propagation

of plants, there are numerous reports of cytological variation in tissue culture

(D'Amato 1978). Larkin and Scowcroft (1981) proposed the term "somaclonal

variation" for variability arising in culture, and suggested that somaclonal variants

recovered from tissue culture could be utilized as novel breeding lines for plant

improvement.

While there have been several reports of morphological and cytogenetic

variation in tissue culture, this review will be limited to the literature concerning

variation at the biochemical and molecular variation.

Isozymes

Multiple Forms of Enzymes

The technique of staining electrophoretic gels to test for the activity of

enzymes was first described by Hunter and Markert (1957) and the stained gels were

called zymograms. The term isozymes (isoenzymes) was coined later by Markert

and Moller (1959) to designate multiple forms of enzymes occurring in organisms

belonging to the same species. For over three decades, isozymes have proved useful

as biochemical tools in plant breeding, chromosome mapping, developmental

biology, tissue and organ specific studies and in a variety of tissue culture

experiments. Isozymes were initially used to classify the different molecular forms

of enzymes belonging to major biochemical pathways in plants as well as animals

(Markert and Moller 1959).








Isozymes as Markers in Plant Breeding
The use of isozymes in plants is obviously not limited to the recognition of

variations in banding patterns in different tissues and at distinct developmental

stages, but they are an invaluable tool in plant breeding studies and evolutionary
analyses (Chiang and Kiang 1987; Doong and Kiang 1987; Quiros et al. 1987; Whitt

1987; Nevo 1990). The diversity of isozymes in nature is not only well structured in

populations, species and higher taxa, but also partly correlated with and predictable

by ecological heterogeneity (Nevo 1990). This claim may be corroborated by the
process of natural selection, which exerts a major differentiating and orienting force

at the evolutionary level (Nevo 1990). In restriction fragment length polymorphism

(RFLP) studies of wild emmer wheat (Triticum diccoccoides), a significant

correlation between RFLPs and certain isozymes was observed (Nevo 1990).

Isozymes have also been used for the "fingerprinting" of various plant species and

useful crop cultivars to help identify individual genotypes and hybrids between two
different accessions (Smith 1984, 1986; Smith and Wych 1986; Tsaftaris 1987).
Plant breeders have classified breeding techniques into three different

categories: (i) self-pollinated plants, (ii) cross-pollinated plants, and (iii) clonally

propagated plants. As mentioned earlier, the plant breeding industry has greatly
benefited from the use of isozymes as markers for the development of newer and

better varieties of cultivated plants (Tsaftaris 1987). Outcrossing rates and mating

systems have been determined for various plant species (Tsaftaris 1987), by using

isozymes. Other reports have used isozymes for quantitative estimates of mating

systems in corn (Tsaftaris 1987) and for self and cross pollination analyses (Smith

1984, 1986; Smith and Wych 1986). In the production of hybrids, the F1 generation

can be screened for the occurrence of self pollination, by using specific isozyme

markers in the parental generation (Smith 1984, 1986; Smith and Wych 1986;

Tsaftaris 1987).








Many of the important cultivated plants are polyploid (Tsaftaris 1987).

While most of these are allopolyploids (wheat, tobacco, cotton, sugarcane,

napiergrass, etc.), some are autopolyploids (potato, alfalfa, etc.). Polyploids have

also been artificially created by breeders, such as ryegrass and red clover which are

autopolyploids, and triticale and raphanobrassica which are allopolyploids (Tsaftaris

1987). While the ancestry of artificial polyploids is well known, isozymes are

extremely useful in combination with RFLP analyses in phylogenetic studies of

naturally occurring polyploids (Suiter 1988; Glaszmann et al. 1989; Dahleen and

Eizenga 1990). The use of isozymes also extends to the study of a wide array of

plants, since plants can be biochemically "fingerprinted" with the help of isozymes

(Smith 1984, 1986; Smith and Wych 1986; de Kochko 1987; Tostain et al. 1987;

Tsaftaris 1987; Vorsa et al. 1988; Gaur and Slinkard 1990). Isozymes have also been

widely used in genetic studies for the mapping of enzyme gene loci (Knapp and

Tagliani 1989; Vaquero 1990).

Isozymes in Tissue Culture Studies

Tissue culture studies have benefited from the use of isozymes as a tool for

various applications (Tsaftaris 1987). Zymograms of ADH from callus cultures of

wheat, rye and triticale were similar to those of their roots grown under anaerobic

conditions, and varied from those of their stems (Suseelan et al. 1982), suggesting

that the enzymes were developmentally regulated. Regenerants obtained from

tissue cultured ovules of seedless grape have been used for isozyme analysis, to

predict the polyembryonic origin of sexual crosses of the parents (Durham et al.

1989).

As documented in plants at various stages of development, so also, in tissue

culture studies it has been observed that certain isozymes show definite and

predictable changes at different stages of development in vitro. During the

differentiation of Vigna unquiculata callus tissue from an undifferentiated state into









tracheids and xylem vessels, De and Roy (1984) observed the presence of a new

band at the anionic end of the acid phosphatase zymogram. Peroxidase patterns

have been shown to differ appreciably between selected and non-selected lines of

rice, wherein the selected line was capable of differentiating into root and shoot

primordia, while the nonselected line was incapable of such differentiation (Abe

and Futsuhara 1989). There have been reports showing the appearance of specific

banding patterns on peroxidase and esterase zymograms that can be used to predict

the advent of embryogenesis, or to discern between embryogenic and non-

embryogenic callus cultures. Such studies have been conducted on maize (Everett et

al. 1985; Rao et al. 1990) and barley (Coppens and DeWitte 1990).

Tissue Culture Variation

The use of biochemical markers in the identification of variants from in vitro

culture has included the analysis of isozymes and specific proteins. Although

morphological and cytogenetic variations have been shown to occur in plant tissue

culture, one of the earliest studies that reported the comparative use of isozymes

was on callus derived regenerants of sugarcane (Heinz and Mee 1971). Two

cultivated lines were used in this study, one of which was a chromosomal mosaic.

The plants derived from this mosaic line revealed distinct differences in the isozyme

banding patterns of four isozyme systems. The regenerants derived from the stable

line exhibited no variation, suggesting that the variation observed may have been

caused by the instability of the explant genome. Selby and Collin (1976) analyzed

callus tissues from Allium cepa for alliinase activity and showed similar levels of

activity between the calli and normal plants, but the precursor levels in the callus

tissue were only 2-10 percent of that in the plant.

Isozymes have been especially useful in studies attempting to document

tissue culture-induced variation in a variety of plants. In potato plants regenerated








from tissue culture, the ADH and aspartate aminotransferase (AAT) zymograms

showed the loss of one band when compared to the parental cultivar. This variation

was believed to be caused by rearrangement of DNA sequences in tissue culture

(Allichio et al. 1987). Dahleen and Eizenga (1990) reported a variant

phosphoglucoisomerase pattern in four plants derived from a monosomic line of

Festuca arundinacea. In addition to morphological and cytogenetic variation in

tissue culture derived plants of Botriochloa sp., Taliaferro et al. (1989) observed

changes in the banding patterns of esterase and peroxidase isozymes. The frequency

of variation at the morphological level and in the electrophoretic banding patterns

of certain seed proteins in wheat plants derived from tissue culture, was very low

(about 1%) (Maddock et a. 1985). Other reports in wheat have shown higher

frequencies of somaclonal variation at different isozyme loci (Davies et al. 1986;

Ryan and Scowcroft 1987). These changes were shown to be heritable through a

sexual cross and hence believed to be at the DNA level as opposed to epigenetic

changes.

In contrast, other studies using isozymes or proteins as markers to detect

tissue culture derived variation have shown a high degree of stability with very little

or no variation. In a population of 645 maize plants derived from tissue cultures of

immature maize embryos, Brettell et al. (1986a) reported a single plant showing an

altered pattern of alcohol dehydrogenase (ADH). In the analysis of over 550

immature embryo derived plants of wheat, Davies et al. (1986) detected only 4

euploid plants with an altered ADH pattern. Thirteen other plants with similar

irregularities were aneuploid. Ryan and Scowcroft (1987) recovered one plant, out

of a population of 149 regenerants from tissue cultured immature embryos of wheat,

that exhibited a variation in the p-amylase isozyme pattern. It was, however,

unresolved whether the variation was inheritable by the progeny. In a population of

plants regenerated from the tissue culture of immature embryos of triticale, Jordan









and Larter (1985) were unable to detect any variation between parental clones and
their progeny. No variation was observed in pearl millet regenerants from cultured

immature inflorescences that were analyzed for total protein content and ADH

activity (Swedlund and Vasil 1985). In 25 protoplast derived plants of orange,

Kobayashi (1987) found no significant variations in four isozyme systems that were
tested. A population of 42 barley plants failed to show any variations in esterase
and aspartate aminotransferase banding patterns (Karp et al. 1987). The same

population had one plant with abnormal meiosis, which also produced one seed with

a variant hordein protein. In a large number of plants of red clover analyzed for the

occurrence of tissue culture variation, Wang and Holl (1988) observed stable

banding patterns for 5 different isozyme systems. Taliaferro et al. (1989) observed

identical peroxidase and esterase banding patterns in the progeny of Botriochloa sp.
derived from the in vitro culture of two lines used as explants. However, the banding

patterns of the progeny differed from that of the original explants. Bebeli et al.

(1990) reported the presence of a single individual containing a variant pattern of
40K r-secalins, from a population of over 350 regenerants derived from the culture

of immature embryos of a selfed line of rye. Zymograms of 95 tissue culture derived

regenerants of Festuca arundinacea assayed for seven isozyme systems did not show

any variation from the parental genotype (Eizenga and Dahleen 1990).

Mitochondrial DNA

Organellar DNA

All eukaryotic cells have organelles which compartmentalize the cell. Plant

cells differ from other forms of life in that they have both mitochondria and plastids

which are believed to have an endosymbiotic origin, and possess their own DNA

(Penny and O'Kelly 1991). Both of these genomes are distinct from the nuclear

genome, but efficient interaction between these three systems is absolutely

necessary for normal development of plants (Palmer 1985a; Lonsdale 1989).









Modern molecular research in plants therefore involves studies on mitochondrial,
plastid, and nuclear genomes.

Plant Mitochondrial DNA

The study of plant mtDNA gained impetus with the discovery that the

expression of cytoplasmic male sterility (CMS) in maize was associated with mtDNA

(Pring and Levings 1978; Laughnan and Gabay-Laughnan 1983). This discovery has

led to a better understanding of the structure, evolution and coding properties of the
mtDNAs of angiosperms (Palmer 1985a). The present study involved the use of

mtDNA as a parameter to study variation induced by in vitro culture.

MtDNA is larger and more complex than its chloroplast counterpart (Stern

and Palmer 1984b; Palmer 1985b; Lonsdale 1989). There have been various

attempts made to determine the physical size of mtDNA from many different plant

species (Leaver and Gray 1982; Lonsdale 1989).

Bailey-Serres et al. (1987) used electron microscopy to estimate the sizes of

mtDNA molecules obtained from seven species of plants belonging to diverse

families and observed a range of molecules varying in size from 1 kb to 126 kb.

Another method exemplified by Ward et al. (1981) for estimating the size of the

mitochondrial genome was to study the renaturation kinetics of mtDNA. The

authors used this technique to determine the size of mitochondrial genomes from

pea, maize and four species from the Cucurbitaceae.

Complexity of The Mitochondrial Genome.

The complexity of the mitochondrial genome has been studied using

hybridization studies and in vitro protein synthesis on isolated intact mitochondria

(Lonsdale 1989). Restriction endonuclease digests of mtDNA from a variety of

plants, when probed with radioactively labelled fragments from other genomes, have

exhibited a set of conserved sequences (Stern et al. 1983; Stern and Newton 1985;

Lonsdale 1989). Experiments using cloned DNA fragments from the mitochondrial









genome of Brassica campestris to probe RNA detected 24 transcripts totalling
approximately 60 kb. These results are consistent with those reported by Makaroff

and Palmer (1987). Results from RNA excess hybridization studies in cucurbits

suggest that the proportion of the mitochondrial genome transcribed varies from

20% in muskmelon to 70% in watermelon (Bendich 1985). The translational

expression and polypeptide processing of mitochondria extracted from tissues at

different stages of development show quantitative as well as qualitative differences

(Boutry et a. 1984; Newton and Walbot 1985), suggesting that these changes may be

developmentally regulated. Mitochondrial genomes also contain open reading

frames (ORFs) which are DNA sequences that may be transcribed but one cannot

assume that these are pre-mRNAs, and unassigned reading frames (URFs) which

are DNA sequences that may be transcribed and translated but the function of the

coded polypeptide is unknown (Lonsdale 1989). In addition, mitochondrial

genomes may also contain gene chimeras, nonfunctional genes and nonfunctional

transcribed sequences (Lonsdale 1989).

Repeat Elements

MtDNA has been shown to possess repeated sequences that range in size

from 0.5 kb to 14 kb in maize (Lonsdale 1989), and may exist as direct repeats or

inverted repeats. The presence of repeated sequences in an inverted orientation,

may lead to homologous recombinational events which cause sequence inversions in

the genome (Lonsdale et al. 1983, 1984; Palmer and Shields 1984; Stern and Palmer

1984a, 1986). On the other hand, if the repeated sequences are present in the same

orientation, a recombination between them would lead to the formation of smaller

circular molecules from a larger molecule.

The mitochondrial genome of plants is believed to exist as a single master

circle of DNA (Lonsdale et al. 1984; Palmer 1985a; Lonsdale 1989) and many

smaller circles that have arisen by recombinational events between direct repeats on









the master chromosome (Lonsdale et al 1983, 1984; Stern and Palmer 1984a;
Falconet et al. 1984; Lonsdale 1989). The entire 218 kb mitochondrial genome of

turnip, for example, consists of three distinct circular chromosomes (Palmer and

Shields 1984). The large master circle possesses two copies of a 2 kb element as a

direct repeat, separated by 135 and 83 kb and the two smaller circles are 135 and 83

kb in size. These three circles are believed to interconvert from one form to the

other (Palmer and Shields 1984). Such repeat elements have also been reported in

normal maize mtDNA (Lonsdale et al. 1983, 1984; Lonsdale 1984; Palmer 1985a;

Lonsdale 1989), although the maize mitochondrial genome (570 kb) is much larger

than that of turnip (218 kb). The maize genome also differs from the turnip genome

in that it possesses six pairs of large repeated sequences, five of which are present as

direct repeats and hence may be recombinationally active (Lonsdale et al. 1984).

Two of these five sites are considered to be preferred sites and the majority of the

mtDNA exists as four smaller circles of 503, 253, 250 and 67 kb in addition to the

master circle of 570 kb (Lonsdale et al. 1984). Such recombinational events

between the different repeat elements could be a source of heterogeneity in the

restriction profiles of mtDNA of a single plant species (Spruill et al. 1980; Lonsdale

et al. 1981; Borck and Walbot 1982). While McNay et al. (1984) have found distinct

differences in the relative stoichiometry of mtDNA bands in the restriction profile

of tissue cultured cells of maize.

MtDNA in Tissue Culture

MtDNA has been widely used in the field of tissue culture for the analysis of

restriction profiles from somatic hybrids (Belliard et al. 1979; Nagy et al 1981;

Galun et al. 1982; Boeshore et al. 1983, 1985; Chetrit et al. 1985; Vedel et al. 1986;

Ozias-Akins et al. 1987; Rothenberg and Hanson 1987; Tabaeizadeh et al 1987;

Kemble et al. 1988a,b; Jourdan et al. 1989), the effect of tissue culture on the

stoichiometry of minicircular mtDNAs (Negruk et a. 1986; Shirzadegan et al. 1989),









supercoiled mtDNAs (Dale et al. 1981), restriction analysis (McNay et al. 1984) and

filter hybridization studies of tissue culture progeny for the detection of variation in

tissue culture (Gengenbach et al. 1981; Boeshore et al. 1985; Oro et al. 1985;
Chowdhury et a. 1988; Aubry et al. 1989; Brears et al. 1989; Shirzadegan et al. 1989;
Saleh et al. 1990). Tissue culture cells have also been studied for the presence of
unique populations or changes in the stoichiometry of the plasmid-like DNAs (Kool

et al. 1985; Negruk et al 1986; Meints et a. 1989). Negruk et aL (1986) observed an

increase in the percentage of minicircles in suspension cultures of Vicia faba.

MtDNA from two different culture lines of a single cultivar of tobacco showed

differences in the size classes of supercoiled molecules but their restriction profiles

were almost identical (Dale et al. 1981).
The recombinational ability of the mitochondrial genome is clearly
elucidated in fusion of protoplasts of two varieties or species when the somatic

hybrids exhibit restriction profiles that differ from either fusion parent (Belliard et

al. 1979; Nagy et al. 1981; Galun et al. 1982; Boeshore et al. 1983; Boeshore et al.

1985; Chetrit et al. 1985; Vedel et al. 1986; Ozias-Akins et al. 1987; Rothenberg and

Hanson 1987; Tabaeizadeh et al. 1987; Kemble et al. 1988a,b; Jourdan et al. 1989).

It is interesting to note that the restriction patterns of individual plants regenerated
from the same fusion experiment are not identical. Such variations are not observed

in fusion products regenerated from protoplast lines with identical restriction

profiles (Nagy et al. 1981; Boeshore et al. 1983). Boeshore et al. (1983) suggested

two possible explanations for the mode of recombinations that they observed: (1)

The parental molecules of mtDNA may undergo intermolecular recombination

following protoplast fusion or (2) Separate parental molecules may assort

independently following protoplast fusion. Later work has shown that the
mitochondrial genomes of the parental clones do recombine to give unique

restriction profiles (Boeshore et al. 1985).









Tissue Culture Variation

From cell cultures of the Texas type cytoplasmic male sterile maize,

Gengenbach and Green (1975) recovered callus cultures resistant to the pathotoxin

of Helminthosporium maydis and regenerated disease resistant plants that stably

transmitted the resistant trait to their sexual progeny (Gengenbach et al. 1977).

These resistant plants were also revertants to male fertility. Cultures that gave

fertile revertants from callus cultures in the absence of pathotoxin were later

reported by Brettell et al. (1980). Upon closer scrutiny of this reversion from male

sterile and disease susceptible to male fertile and disease resistant, it was discovered

that the change involved a rearrangement in the mtDNA of the male sterile cell

cultures to cause the change in phenotype (Gengenbach et al. 1981). This variation

was exclusively associated with the reversion of the CMS-T strain to fertility

(Gengenbach et al. 1981; Lonsdale et al. 1981; Umbeck and Gengenbach 1983;

Fauron et al. 1987; Wise et al. 1987: review Pring and Lonsdale 1989; Levings 1990).

It has now been documented that a partial or complete loss of the T-urfl3

mitochondrial gene or its disruption caused by a frame shift causes a reversal to

male-fertile phenotype in the CMS-T type cytoplasm of maize (Rottmann et al.

1987; Wise et al 1987). Such a rearrangement has been observed only in tissue

cultured cells, providing direct evidence to the ability of in vitro cultures to give rise

to variation. It is believed that the T-urfl3 gene produces a polypeptide that acts as

a receptor for the pathotoxin molecules (Dewey et al. 1987, 1988).

Recent reports also show the presence of variation derived in vitro in sugar

beet, wheat and Brassica campestris. The restriction profile of B. campestris showed

variations caused by rearrangements which were at least two inversions and a large

duplication. The native plant tissue, however, shows the presence of the rearranged

molecules at a very low level, hence they appear to be sorted out and amplified in

tissue culture (Shirzadegan et al. 1989). The restriction profile of mtDNA from









maize tissue cultures showed changes in the relative stoichiometry of bands in the

restriction profile, although no differences were observed in the restriction profiles
(McNay et al. 1984). Wilson et al. (1984) and Chourey et a. (1986) have reported a
high degree of variation in specific regions of the mitochondrial genome of sorghum
and maize respectively. Tissue cultures of CMS varieties of sugar beet showed a
single regenerant with a rearranged mtDNA pattern, detected by hybridization with

cosmid clones (Brears et al. 1989). Callus cultures of wheat were shown to exhibit a

different mtDNA pattern in non-embryogenic cultures when compared to

embryogenic cultures (Hartmann et al. 1987). In callus cultures obtained from
immature embryos of wheat, Rode et al. (1987) reported extensive changes in
mtDNA corresponding with the loss of a fraction of the mitochondrial genome.

Hartmann et al. (1989) have reported the occurence of unique organization of the

mitochondrial genome in plants regenerated from the callus cultures of wheat. The
mtDNA profile in all plants regenerated from short-term cultures of wheat except

one appeared to resemble either that of the parent plant or that of the embryogenic

cultures. However, all plants except for one regenerated from long-term cultures

exhibited a mitochondrial genome organization similar to that of the long-term non-

embryogenic cultures (Hartmann et al. 1989). Similar variations have also been

reported in the mtDNA from albino cultures and plants regenerated from anther

cultures of wheat (Aubry et al. 1989). Chowdhury et al. (1988) reported variation in

the mtDNA organization of long term cell cultures of rice when hybridized with

mitochondrial gene clones. In another case involving the use of mtDNA, Kemble

and Shepard (1984) reported the appearance of low molecular weight DNA in
addition to a sequence alteration in the mitochondrial genome of potato plants

regenerated from protoplasts.
In tobacco, Dale et al. (1981) observed differences in the stoichiometry of

different supercoiled molecules but practically identical restriction patterns of









mtDNA from two culture lines of a single cultivar. Breiman et al. (1987a) observed

a complete absence of variation in DNA-DNA hybridization patterns of total DNA

blots of barley probed with mitochondrial genes from maize and wheat. In a ten

year old cell suspension culture of carrot cells, Matthews and DeBonte (1985)

reported the complete lack of variation in the restriction patterns of mtDNA. A

population of Brassica napus derived from protoplasts was shown to harbor no

variations in either mtDNA or cpDNA (Kemble et al. 1988a). In a recent study, 3

month old callus cultures, 2 month old suspension cultures, a totipotent suspension

and 19 month suspension cultures of rice, had identical mtDNA restriction profiles.

The same study reported, however, that a 30 month old suspension showed a

different restriction profile (Saleh et al. 1990).

Chloroplast DNA

Plants and algae are known to possess a unique class of organelles which are

collectively or individually called plastids. These include amyloplasts, chloroplasts,

chromoplasts, elaioplasts, etioplasts and proplastids. Proplastids are believed to be

the precursors for most of the plastid types. Chloroplasts are responsible for the all

important process of photosynthesis. They impart a green color and an autotrophic

mode of life to the organisms that possess them. The fact that chloroplasts are

pigmented and larger than mitochondria probably aroused the curiosity of the early

plant scientists, leading to the elucidation of their role in photosynthesis. This

discovery generated obvious interest among scientists, and hence it is logical that, in

plants, chloroplasts have been studied as organelles for a longer time when

compared to mitochondria (Palmer 1985a). A large volume of the research on

chloroplasts has been conducted on green algae, however, this review is limited to

the study of plastids in higher plants.









Endosymbiotic Origin of Chloroplasts
Chloroplasts of algae and higher plants with one known exception are all

known to contain DNA, usually in multiple copies (Possingham and Lawerence

1983). The single exception is the green alga Acetabularia; chloroplasts in many of

its species do not contain any detectable DNA (Coleman 1979; Luttke and Bonnoto

1982). There seems to be little doubt if any that, like mitochondria, chloroplasts

have an endosymbiotic origin from a prokaryotic precursor (Palmer 1985a,b, 1987;

Palmer et al. 1988; Penny and O'Kelly 1991). This assumption is based on the fact

that rRNA genes in the plastid genomes of most plants and algae have a striking

resemblance to those of the eubacterium Escherischia coli (Gray 1983; Spencer et al.

1984; Dale et al. 1984; Palmer 1985a). In the light of this information, plastid and
eubacterial genomes almost certainly had a more recent common ancestry than

plastid and nuclear genomes. Chloroplast DNA (cpDNA) sequences from both

algae as well as flowering plants share a lot of homology with cyanobacteria. This

provides almost irrefutable evidence that plastids evolved by the endosymbiotic

association of an autotrophic prokaryote with a primitive eukaryote (Gray and

Doolittle 1982; Gray 1983; Palmer 1985a,b, 1987; Palmer et al. 1988).

Consequently, the present day occurrence of plastid and nuclear genomes in a single

cell appears to be the result of horizontal evolution, i.e. endosymbiosis (Palmer

1985a). The genome of all the different plastid types within a single plant, according

to all available data, appears to be identical (Palmer 1987).

Interaction Between Organelles

The genomic size of the endosymbiont was probably reduced by the transfer

of most of its genes to the host nucleus while retaining only those genes that were

vital for the proper functioning of the organelle. This may be endorsed by the fact

that a large number of structural polypeptides for both mitochondria as well as

plastids are encoded by the nucleus (Lonsdale 1989). Such a transfer of genetic








material thus guarantees interaction between the nucleus and plastids, whereby

polypeptides coded for by the nucleus are synthesized in the cytoplasm and
dispatched to the plastids with an attached target polypeptide (Lonsdale 1989).

Promiscuous transfer of DNA from the plastid genome to the nuclear genome has

been observed in spinach on a very large scale, where each haploid nuclear genome

has been shown to possess the equivalent of up to five plastid genomes (Scott and

Timmis 1984). Such a shift of genetic information from the organelle to the nucleus

is not without mishap. It is quite probable that a plastidd gene" could acquire a

mitochondrial targeting sequence as observed in the mitochondria of the alga

Ochromonas danica. In this alga, the small subunit of RuBPCase is found in the

mitochondria, although it is possible that the mitochondria possess an entire or

partially active copy of the small subunit gene (Lonsdale 1989). Stern and Palmer

(1984b) have documented several homologies between the chloroplast and

mitochondrial genomes of several plant species at the inter- and intraspecific levels.

Inheritance of Plastids

Most angiosperms show a maternal inheritance of organelles, while few show

paternal inheritance and yet others show a biparental inheritance. Amongst

gymnosperms, conifers almost exclusively exhibit a uniparental-paternal pattern of

plastid inheritance as documented by microscopic (Whatley 1982) and molecular

studies (Neale and Sederoff 1988). The paternal plastids are observed to enter the

egg cell while the maternal plastids degenerate (Whatley 1982). Paternal

inheritance of chloroplasts has also been confirmed by restriction fragment length

polymorphisms (RFLPs) on the cpDNA of the parents and their sexual progeny in

many conifers (Neale et al. 1986; Szmidt et al 1987,1988; Wagner et al. 1987; Neale

and Sederoff 1989). In some sexual hybrids between two larch species, biparental as

well as maternal inheritance of plastids was observed (Szmidt et al. 1987) while in

crosses between Pinus rigida and P. taeda the inheritance of plastids is paternal but









mitochondria are maternally inherited (Neale and Sederoff 1989). In redwood

(Sequoia semipervirens), the inheritance of mitochondria as well as plastids is
paternal (Neale et al. 1989).
The pattern of plastid transmission in angiosperms is, as mentioned earlier,
largely maternal (Palmer 1987; Palmer et al. 1988). However, biparental

inheritance has been implicated in many angiosperm species (Corriveau and

Coleman 1988). Four types of plastid inheritance are believed to exist in

angiosperms : (1) In the Lycopersicon type, the plastids in the microspore selectively

segregate to the vegetative cell. Nevertheless, in Nicotiana, paternal inheritance of
plastids has been shown to occur at very low frequencies (Medgyesy et al 1986), (2)
In the Solanum type, plastids are equally divided between the generative cell and

the vegetative cell of the microspore but the plastids in the generative cell are

selectively lost (or eliminated), hence the sperm cells are devoid of plastids, (3) In

the Triticum type, which is found in most grasses, plastids are found in the
generative cell as well as the vegetative cell. In spite of that, when the sperm enters
the egg cell, enucleated cytoplasmic bodies containing plastids and mitochondria are

left outside (Mogensen and Rusche 1985; Mogensen 1988), and (4) In the

Pelargonium type, plastid inheritance is biparental, although, in alfalfa (Medicago

sativa) the paternally derived plastids predominate and in Oenothera the maternally

derived plastids are prevalent. This may suggest the existence of additional

mechanisms of influencing plastid inheritance (Lee et al. 1988, 1989; Smith

1988,1989). In the genus Brassica, the relationships between different species and

the ancestry of certain amphidiploids have been determined by identifying the

cytoplasmic type of the maternal parent (Erickson et al. 1983; Palmer et al. 1983,
1988; Palmer 1987). The chloroplast genome of B. napus, however, is believed to

have evolved by introgression from some unidentified species (Palmer et al. 1983,

1988; Palmer 1987).









CpDNA in Interspecific Hybrids
In interspecific hybrids, or in cases of biparental inheritance, it is seen that

the two chloroplast types neither fuse nor do their genomes recombine (Scowcroft

and Larkin 1981; Kemble and Shepard 1984; Palmer 1987; Palmer et aL 1988). As

discussed earlier, in sexual inter- or intraspecific hybrids, one of the parental plastid

types is usually selected against. Somatic hybrids created by the fusion of

protoplasts from two different plant species also exhibit an independent assortment

of chloroplasts from the two parental species (Morgan and Maliga 1987), wherein

the chloroplasts do not fuse or produce any recombination between the two

genomes. In interspecific somatic hybrids between two species of Daucus

(Matthews and Widholm 1985), Petunia (Clark et al. 1986), Medicago (D'Hont et al.

1987) and Brassica (Kemble et al. 1988a), the hybrids showed inheritance of the

plastid genome from only one of the parental species. Thanh et al.. (1988) have

reported the intergeneric transfer of chloroplasts from Salpiglossis sinuata to the

cytoplasm of Nicotiana tabacum. The donor cytoplasm was irradiated before fusion

and appropriate streptomycin-resistant donor or light-sensitive recipient mutants

were used.

The Chloroplast Genome

The chloroplast genome of all land plants is relatively uniform in size (120-

217 kb) when compared to the mitochondrial genome (Palmer et al. 1988). Its

complexity varies between 110-150 kb, because most of the variation in size is

observed to arise from a few major expansions or contractions in the large inverted

repeat (Palmer 1985a; Palmer et al. 1988). The total size variation of angiosperm

cpDNAs may be misleading; the lower extreme of this size variation occurs in a

single group of legumes which have lost one copy of the large two copy inverted

repeat (Palmer 1985a), while the variation in genome size of the upper extreme

range of 55 kb is observed only in two species so far, i.e. Spirodela oligorrhiza (Van









Ee et al. 1980) and Pelargonium hortorum (Palmer 1985a). The variation in size of

cpDNAs among most of the angiosperms observed falls within the relatively narrow

range between 135-160 kb, when compared to the mitochondrial genome, with most

plants having only a 20-30 kb inverted repeat. The increase in the size of the
Pelargonium cpDNA is attributed to an enlarged inverted repeat which is over 75 kb
in size (Palmer 1985a).

Evolution in the chloroplast genome has been shown to occur at a very

conservative rate of about 1.5 X 10-9 substitutions per site per year (Zurawski and

Clegg 1987). In comparison, the rate of silent substitutions in cpDNA may be as
much as a hundred times lower than that observed in animal mtDNA and two to

three times lower than nuclear DNA, but it is three to four times higher than in
plant mitochondrial genes (Zurawski et al. 1984; Palmer 1987; Palmer et al. 1988).
One striking difference between cpDNA and plant mtDNA is that, plastid DNA

completely lacks any minicircular or plasmid DNAs that are characteristic of plant

mtDNAs (Palmer 1985a, 1987).

Any change in the complexity of a genome is wrought by the addition of new

sequences or the deletion of existing ones (Palmer 1987). It seems highly unlikely

that such changes in complexity occur by the gradual drift of repeated elements until

they effectively become single copy (Palmer 1987). The infiltration of cpDNA
sequences into mitochondria has been exhibited in extremely diverse species like

maize (Zea mays), cauliflower (Brassica oleracea), mung bean (Phaseolus aureus),

spinach (Spinacia oleracea) and evening primrose (Oenothera berteriana) (Carlson et

al. 1986a,b; Marechal et a. 1987; Schuster and Brennicke 1987, 1988; Nugent and

Palmer 1988; review Lonsdale 1989). On the other hand, very rarely has the

chloroplast genome been shown to possess genes from any extraneous sources
(Palmer 1987; Schuster and Brennicke 1988). In cpDNAs compared from hundreds

of plant species, there are only two significantly large sized mutations that have been









observed, i.e. the addition of a 7-9 kb sequence in Nicotiana acuminata (Shen et al.
1982) and the addition or deletion of a 13 kb sequence in a collection of Linum

species (Coates and Cullis 1987). There are other mutations that have been

reported which are significantly smaller in size and less frequent in occurrence.

These involve changes ranging from 50 to 1200 bp (Gordon et al. 1982; Bowman et

al. 1983; Salts et al. 1984; Palmer et al. 1985; Palmer 1987). The maximum number

of mutations occurring in cpDNA usually take place either as additions or deletions
involving 1 to 10 bp, probably according to the "slippage-mispairing" model

(Takaiwa and Sugiura 1982; Zurawski et al. 1984; Palmer 1987). One interesting

fact is that cpDNAs from algae as well as higher plants completely lack any

modified bases such as 5-methylcytosine (Bohnert et al. 1982; Loiseau and Dalmon

1983; Palmer 1985a).

The Inverted Repeat of the Plastid Genome

The plastid genome of a majority of land plants has an extremely similar

arrangement of genes (Palmer 1987). The gene order in the cpDNA of spinach is

believed to be similar to that of the ancestral vascular plant (Palmer and Stein 1986)

and is also representative of many of the angiosperm species studied (Fluhr and

Edelman 1981; Palmer and Thompson 1982; Palmer et al. 1983). In all the

angiosperm families except in one section of subfamily Papilionoideae of the legume

family Fabaceae, the plastid genome has a characteristic inverted repeat (Chu and

Tewari 1982). This inverted repeat is always positioned asymmetrically and divides

the entire genome into a large single copy part and a small single copy part (Chu

and Tewari 1982; Palmer 1985b, 1987). As mentioned earlier, this repeat is also

responsible for the variation in size of the chloroplast genome amongst various plant

species. Although the inverted repeat can vary up to six times in size, it always

contains an entire set of ribosomal RNA genes (Chu and Tewari 1982). The two









arms of the inverted repeat are identical in an individual, and all mutations in the
repeat elements are symmetrical (Palmer 1985a, 1987).

Considering the static nature of the plastid genomes and their arrangement

of genes, Palmer (1987) has elucidated six generalizations regarding internal

rearrangements: (1) all well characterized rearrangements are inversions, (2) the

cases of rearrangement are usually simple and involve only one or two discrete
inversions, (3) in cases where the inverted repeat is greatly altered, e.g. Pelargonium

and in legumes that lack the inverted repeat, the rearrangements are extreme, (4)

the flanking regions of the best known inversions are located within largely

noncoding regions, (5) some of the highly rearranged genomes have families of

somewhat large dispersed repeats of several hundred bp, and (6) rearrangements

are not known to disrupt the functions of groups of genes that are transcriptionally

linked. The lack of disruption observed may be a direct consequence of the fact that

plastid genomes have a high density of genetic information. Any disruption of these

genes by the insertion of foreign sequences would probably cause lethal mutations

which would obviously be selected against (Lonsdale 1989). This contrasts sharply

with nuclear and mtDNA where the functional genes are widely dispersed and

inserted sequences stand a better chance of being retained (Lonsdale 1989).

Variation in the Plastid Genome

The inherent nature of cpDNA, whereby both the structural and sequence

fidelity are maintained, strongly limit its use as a marker for variability in studies

involving large populations (Palmer 1985a, 1987; Palmer et al. 1988). However,

minor variations have been detected at specific and intraspecific levels as in Lupinus

texensis (Banks and Birky 1985), Brassica nigra (Palmer et al. 1983) and Lycopersicon

peruvianum (Palmer and Zamir 1982). The lack of widespread variation, or its

presence at very low levels, is an excellent tool for phylogenetic studies and has been

used in several studies using RFLP techniques (Palmer and Zamir 1982; Palmer et









al. 1983, 1985a; Banks and Birky 1985; Sytsma and Gottlieb 1986a,b). There have

been numerous other studies involving a wide range of plants using cpDNA for
cladistic analysis and the subsequent construction of phylogenetic trees (Palmer

1985a, 1987; Palmer et al. 1988).

Although the cpDNA evolves very slowly, there are several reported

examples of base substitutions and changes in genome structure. Zurawski et a.

(1984) and Zurawski et al. (1984) conclude that most nucleotide substitutions occur

as silent changes in the third position of codons and missense substitutions are

clustered at the ends of genes. As mentioned earlier, all changes in the inverted

repeat occur symmetrically (Palmer 1987). The absence of the inverted repeat in

the chloroplast genome is believed to have a profound effect, causing the genome to

be prone to more frequent rearrangements as seen in Pisum and Trifolium (Palmer

and Thompson 1982; Palmer 1985a,b; Palmer et al. 1987).

Use of the Plastid Genome as a Marker

The plastid genome has been used in comparative analyses as a marker for

genetic variation using at least three different methods, as outlined by Palmer

(1987). Purified samples of cpDNA, from individuals to be compared, may be

subjected to a restriction analysis. In cases with complex restriction patterns,

restriction maps of the genome using several enzymes may be used for a

comparative analysis. A certain part of the genome may also be used for sequencing

studies to study a defined segment of the genome.

Plastid DNA in Tissue Culture

Recombination between the genomes of two different chloroplast types has

been observed in a somatic hybrid of Nicotiana tabacum and N. plumbaginifolia

(Medgyesey et al. 1985). The two chloroplast types were selectable on either
streptomycin or lincomycin, while the somatic hybrid progeny showed recombinant









cpDNA patterns. The plastid genome of the hybrid was believed to contain at least

six recombination sites (Medgyesey et al. 1985).

There are fewer reports concerning the use of the chloroplast genome for the

purpose of identifying variation in tissue culture. This may be due to the conserved

nature of the plastid genome (Lonsdale 1984, 1989; Chowdhury et al. 1988). Day

and Ellis (1984, 1985) reported that plants regenerated from anther culture of wheat

lacked pigmentation and linked this to deletions in the cpDNA of the regenerants.

A study on a population of alfalfa regenerants from protoplasts revealed the

occurrence of a chloroplast genome that varied from the parental type (Rose et al.

1986). There was an apparent selection towards two types of banding patterns, in

regenerated protoclones of Medicago sativa L., that were different from the parental

type. Twenty-two of the twenty-three clones observed had either one or the other of

the variant banding patterns observed. Kemble and Shepard (1984) reported the

absence of any variation in a population of potato plants regenerated from leaf

mesophyll protoplasts. Matthews and DeBonte (1985) also reported a complete

lack of variation in the cpDNA restriction profiles of a 10 year old carrot cell

suspension.

Nuclear DNA

Restriction Fragment Length Polymorphisms

It is well known that the genetic complement of all species has evolved by

selection, and in the process the DNA from related species and different individuals

from the same species have accumulated minor aberrations (mutations) that have

become part of the genome. Variations like single base substitutions have, in recent

times, been found to be extremely useful as genetic markers present in close

association with certain genes of interest when they cause unique restriction profiles

among different individuals of a single species. Recently there have been many

reports involving the use of RFLPs as markers in plant breeding (Clarke et a. 1989;









Smith et al. 1989), fingerprinting of genotypes (Appels and Dvorak 1982; May and

Appels 1987; Smith et al. 1989; Riedel et al. 1990; Sano and Sano 1990),

phylogenetic analysis (Appels and Dvorak 1982; Hintz et al. 1989), chromosome
linkage analysis (Landry et al. 1987; Sharp et al. 1989) and analysis of regenerants

derived from tissue cultures (Landsmann and Uhrig 1985; Brettell et al. 1986a,b;

Breiman et al. 1987a,b, 1989; Karp et al. 1987; Rode et al. 1987; Zheng et al. 1987;

Benslimane et al. 1988; Miller et al. 1990).

Ribosomal DNA in Plant Breeding and Tissue Culture

Examination of nuclear ribosomal DNA (rDNA) is another aspect of

molecular analysis for the detection of tissue culture derived variation. Unlike

mtDNA or cpDNA, restricted nuclear DNA does not yield a profile that can be used

for comparative purposes. Therefore, Southern blots of nuclear DNA cut with the

restriction enzyme of choice are probed with cloned DNA fragments to provide

autoradiograms in order to accurately estimate changes (Southern 1975).

Landsmann and Uhrig (1985) reported two plants from a population of twelve to

possess a variant Southern-hybridization pattern of nuclear DNA when probed with

a ribosomal DNA (rDNA) clone. Zheng et al. (1987) observed an amplification of

some highly repeated nuclear DNA sequences in rice suspension cultures. rDNA

has also been used as a probe to detect variation at the nuclear DNA level in

dihaploid plants of wheat, derived from tissue culture (Rode et al. 1987a,b;

Benslimane et al. 1988). The nucleolar organizer region (Nor) consisting of rDNA

genes has been used as a marker in several cereal crop plants. In triticale, an

analysis of the Nor loci located on chromosomes 1B, 6B and 1R revealed that one

out of six phenotypes tested had a marked reduction in the number of rDNA units

present at the locus (Brettell et al. 1986b). Such a discrepancy was also detected in a

study involving plants regenerated from wheat callus. One out of three genotypes

tested in this study, showed a similar reduction at the Nor loci (Breiman et al.









1987a). In an independent report, wild barley (Hordeum spontaneum) plants

derived from immature embryo-derived callus were also observed to possess such a

reduction in the intergenic spacers of the rDNA (Breiman et al. 1987b). In a later

publication, however, Breiman et al. (1989) expressed serious doubts about the

ability of tissue culture to cause such variations at the Nor loci. These doubts were

expressed when the parental lines were observed to possess similar variations at the

Nor loci at a very low frequency. Karp et al (1987) reported no variation at the Nor

loci in a population of forty-two barley plants regenerated from cultured immature

embryos.

DNA Methylation in Tissue Culture

Methylation of DNA bases is believed to play an important role in the

expression of genes vital to plant development. The methylation of cytidine, and on

occasion adenine bases, is believed to regulate the expression of genes during plant

and animal development (Jones and Taylor 1980; Theiss and Follmann 1980; Theiss

et al 1987). In a study involving cultured cells of soybean, a restriction analysis of

5S RNA genes revealed that the DNA from the explant material and long-term

cultures was highly resistant to digestion by the enzyme HpallI which recognizes

methylated cytosine bases in the sequence CCGG. DNA extracted from freshly

cultured tissues, however, was easily restricted by HpaII and its isoschizomer MspI,

which is sensitive to methylation of the cytosine bases in the same sequence. Brown

(1989) attempted to use a 5-methylcytosine analog 5-Azacytidine to study its effect

on the methylation and possible promotion of protoplast division in maize and

tobacco cell cultures, but failed to detect any correlation. An analysis of

phenotypically variant regenerants of maize from cultured immature embryos

revealed that housekeeping as well as structural genes had significantly altered

levels of methylation (Brown 1989). The author suggested that such changes may

play a role in the variation of plants derived from tissue culture. Miller et al. (1990)





28


found a close correlation between tissue culture-derived regenerants of rice that

showed rearrangements in their DNA and methylation of the genome.













CHAPTER 3
REGENERATION OF PLANTS FROM SOMATIC EMBRYOS OF
PENNISETUM PURPUREUM K. SCHUM. BY TISSUE CULTURE OF
IMMATURE LEAF SEGMENTS

Introduction

Tissue culture is an established procedure for obtaining clonal plant

populations. Many diverse plant species have been successfully initiated into culture

using different tissues as explants (Vasil 1986). However, the most important group

of plant species induced into culture is without doubt the cereals and grasses (Vasil

and Vasil 1986). While plants regenerated from in vitro culture are expected to be

identical clones of the explant, it is also known that a certain amount of variation

arises in cell cultures and plants obtained from in vitro culture (Heinz and Mee

1971; Edallo et al. 1981; McCoy et al. 1982; Swedlund and Vasil 1985). The term

"somaclonal variation" was introduced by Larkin and Scowcroft (1981) to

characterize variation observed in tissue culture and included all types of

morphological, biochemical, cytogenetic and molecular variation.

Variation obtained from tissue culture derived plants has been considered

potentially beneficial to plant breeders, in the hope of recovering unique and

commercially profitable cultivars (Larkin and Scowcroft 1981). There have been

many conflicting reports, however, on the ability of the process of tissue culture to

cause such widespread useful variation. It is generally argued that at least a part of

the variation observed in cell cultures and populations derived from them is a result

of preexisting variation in the differentiated cells of the explant which may be









amplified or selected for in vitro (D'Amato 1985; Swedlund and Vasil 1985; Vasil

1988; Morrish et al. 1990). A majority of the variation obtained in vitro is not novel

and is very similar in range to the variation resulting from mutations in sexual

crosses. Furthermore, a great deal of the variation obtained in vitro is epigenetic in

nature and is not transmitted to sexual progeny. It is thus of no interest to the plant

breeder. It is, therefore, not surprising that there is not a single example of any

important variety of a major crop species developed as a variant from tissue culture,

which is grown commercially anywhere in the world (Vasil 1990).

There are reports of the genetic stability of long and short term cell and

callus cultures as well as plants regenerated from them (Edallo et al. 1981; Hanna et

a. 1984; Karp and Maddock 1984; Swedlund and Vasil 1985; Maddock and Semple

1986; Binarovi and Dolezel 1988). The ability of the tissue culture process to

perpetuate and amplify preexisting variations in the explant has been amply

demonstrated in Pennisetum glaucum (Morrish et al. 1990). Although many studies

have shown the occurrence of chromosomal aberrations in cell and callus cultures,

there appears to be a definite exclusion of such variants in the formation of somatic

embryos and the plants regenerated from them (Vasil 1988; Hanna et al. 1984; Karp

and Maddock 1984; Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986;

Cavallini et al. 1987; Gould 1986; Kobayashi 1987; Feher et al. 1989; Gmitter et al.

1991). Such genetic and chromosomal fidelity in somatic embryos is useful,

especially in light of the fact that tissue culture is an invaluable tool in modern

biotechnology to provide uniform and stable transformed plants.

This study was undertaken to examine a population derived from somatic

embryos of a single clone of Pennisetum purpureum (L) K. Schum., by the in vitro

culture of young leaves (Haydu and Vasil 1981). Since morphological variations can

be epigenetic in nature, the only avenues used to examine the regenerants were

biochemical analyses at the isozyme level and molecular analyses at the cytoplasmic








and nuclear genome levels. The decision to use somatic tissue from a single clone

as explant material was made to minimize the introduction of any variation inherent
to the tissue.
Materials and Methods

Callus Initiation and Maintenance

Actively growing shoots of P. purpureum were collected from field grown

plants (field accession number PP10). Approximately 75 mm. of the proximal,

tightly coiled innermost 5-6 leaves were used for initiation of callus, after surface
sterilization by wiping with 95% ethanol. Leaf segments 1-2 mm long, were placed

on MS medium (Murashige and Skoog 1962) supplemented with 0.5 mg/1 2,4-

dichlorophenoxyacetic acid (2,4-D), 0.5 mg/1 6-benzylaminopurine (BAP), 1.0 mg/1

a-naphthalene acetic acid (NAA) and 50 ml/1 coconut milk (Haydu and Vasil 1981),

solidified using 2 g/1 Gelrite (Scott Laboratories Inc., Fiskeville, RI). Cultures were

maintained at 270C in the absence of light. After approximately 3 weeks, the

explants yielded embryogenic and non-embryogenic callus. The compact

embryogenic callus was carefully selected and subcultured onto similar medium and

maintained by routine subculture at 3 week intervals.

Embryogenic callus was also initiated from approximately 1 cm long

immature inflorescences of P. purpureum. The culture conditions and medium used

were identical to those used for the culture of immature leaf segments.

Plant Regeneration

For the regeneration of plants, embryogenic calli from immature leaf

explants as well as immature inflorescence explants were placed on MS medium
supplemented by 0.5 mg/1 NAA and 1.0 mg/1 BAP. These cultures were transferred

to an illuminated growth chamber at 270C with a 16 hour light cycle. After 4 weeks,

the plants were transferred to the same medium in tubes ( 150 mm L. X 25 mm

dia.) and thus maintained for 3 weeks to allow for root and shoot elongation.








Plantlets were then transferred to soil (4 parts Metromix 300 [Grace Horticultural

Products, Cambridge, MA] to 1 part Perlite [Chemrock Industries]) in Conetainers

(Ray Leach Conetainer Nursery, Canby, OR) and maintained in a closed

environment with a humidifier, in a 16 hour light cycle, for 4 days before being

transferred to the greenhouse. Plantlets were transplanted into successively larger

pots from the Conetainers, before transfer to the field. Regeneration procedures

from embryogenic calli were initiated 3, 6, 12, 18 and 24 weeks after the initiation of

cultures. The plants regenerated from leaf explants were identified by their callus

pedigree (e.g. C1, C2, C3, ...etc.). Individual regenerant plants within each pedigree

were assigned ascending numbers (e.g. R1, R2, R3, ...etc.). A total of 57 plants

were obtained from 11 callus pedigrees. A single pedigree established from the

inflorescence explant was prefixed Inf. and the regenerants were assigned similar

numbers to those obtained from leaf callus. Six plants were obtained from the

pedigree established from embryogenic callus of immature inflorescence segments.

A single clump of the parental clone PP10 was also transferred to the same

plot along with the 62 regenerants from both the pedigrees to expose all the plants

to uniform field conditions.

Results

Embryogenic calli obtained were white and compact (Fig. 3.1). Roots and

shoots were formed in the regeneration medium (Fig. 3.2). The plants were

transferred to the field after a well established root and shoot system were achieved

in pots (Fig. 3.3). Growth of all plants in the field was uniform (Fig. 3.4). Three

plantlets from the C1 line, and one plantlet each from the C5 and Inf3 lines failed to

survive the transition from the regeneration medium to soil. Some callus pedigrees

yielded only a single regenerant while others provided as many as ten regenerants.

The plants that made a successful transition from the greenhouse to the field

showed uniform growth and did not show any obvious phenotypic differences.








Rajasekaran et al. (1986) have shown the morphological, cytological and
physiological uniformity of a similar population, hence, no specific measurements

were made at these levels. The entire population was subjected to a biochemical

analysis described in Chapter 2, and individuals selected at random were analyzed

using molecular techniques detailed in Chapters 3, 4 and 5.

Discussion

Regeneration of plants from in vitro cultures is essentially a mitotic process,

hence eliminating variation caused by meiotic recombination. The use of different

clones, or immature embryos as explants can introduce existing variation between

individuals into culture and the population thus obtained may lack uniformity. Such

variation can be minimized by the use of inbred lines for explant tissue. The most

productive variety from such an analysis may be used for the establishment of a

population. Another simple method of obtaining a uniform population of

regenerants is, as described here, the use of a single clone for the establishment of

embryogenic callus cultures. The use of a single clone for the production of a tissue-

culture derived population of regenerants also helps to maintain and identify the

lineage of the regenerants.

Somatic embryos have been shown to arise from single cells (Vasil and Vasil

1982). The absence of variation in plants obtained from embryogenic cultures is

considered to be due to the selection of chromosomally stable cells in the formation

of somatic embryos (Vasil 1988; Hanna et al. 1984; Karp and Maddock 1984;

Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986; Gould 1986; Feher et

al. 1989; Gmitter et al. 1991). Adventitious meristems in organogenic calli, on the

other hand, are multicellular in origin and give rise to chimeric plants (D'Amato

1978), which are undesirable.



























Fig. 3.1 Compact embryogenic callus
obtained from the culture of young leaves
of P. purpureum.



Fig. 3.3 P. purpureum plants regenerated
from embryogenic calli, ready for transfer
to the field.


Fig. 3.2 Differentiation of plantlets
from embryogenic calli of P.
purpureum, placed on regeneration
medium.


Fig. 3.4 Tissue culture-derived P.
purpureum plants in the field.




















































































--LC~_I*~C I.CrYLtL.E"W -~r














CHAPTER 4
BIOCHEMICAL ANALYSIS OF A POPULATION OF PENNISETUM
PURPUREUM K. SCHUM. DERIVED FROM TISSUE CULTURE USING,
ISOZYMES AS MARKERS.

Introduction

Isozymes are defined as multiple forms of an enzyme with similar or identical

substrate specificity occurring within the same organism (Markert and Moller 1959).

Most organisms may exhibit two principal alterations in metabolic activity within

their cells. These changes can be classified as quantitative and qualitative changes

in protein (enzymatic) activity (Scandalios 1974). Increases in enzyme activity may

be due to de novo synthesis of the enzyme molecules or the activation of an

existing enzyme precursor, while qualitative variations in enzyme activity may be the

result of changes in the immediate environment of the cell or tissue.

Three different classes of isozymes have been recognized: a) those that are

distinctly different molecules and presumed to arise from different genetic loci, b)

those that evolve from secondary alterations in the structure of a single polypeptide

species which may also be in vitro artifacts or the binding of different co-factors to a

single polypeptide (Scandalios 1974), and c) those arising as a result of a gene

mutation or recombinational events in the gene that codes for the enzyme molecule.

A zymogram is the stained representation of enzyme activity usually visualized on a

starch or polyacrylamide gel. Any alteration in the molecular structure of the

enzyme can easily be detected on a zymogram as a change in migration of the

molecule which is represented as a single band. Isozymes can, therefore, provide an

accurate picture of any biochemical differences that may exist between separate
individuals.









Isozymes are ideal for use as markers in tissue culture because of : i) their

ease of detection, ii) the abundance of naturally occurring variant molecules of

enzymes in most populations, iii) their applicability to small amounts of tissue and

minimum sample preparation due to the use of crude extracts, and iv) the fact that

in most cases the marker is expressed in the undifferentiated state of cell culture.

Native gel electrophoresis is used for the activity staining of isozymes. In this

technique, the proteins are separated on the basis of size and charge. One
limitation of this technique is that it does not discriminate between molecules which

may have the same net charge due to a single substitution but similar catalytic

activities.

Zymograms have been used to illustrate unique banding patterns of various

isozyme systems and elucidate differences in embryogenic and non-embryogenic

tissues and callus cultures (Abe and Futsuhara 1989; Coppens and Gillis 1987; Rao

et al. 1990; Everett et al. 1985; Coppens and Dewitte 1990). Isozyme analyses have

been carried out to study different types of callus tissue (Suseelan and Bhatia 1982),

differentiation in callus tissue (De and Roy 1984) and to study exo-isozymes in the

nutrient medium of suspension cultures (Berger et al. 1988).

The most common use for the isozyme technique in tissue culture studies is

the identification of variation that may occur in vitro. In the belief that isozymes are

excellent indicators of biochemical variation, many investigators have used

zymograms to evaluate such variation (Maddock et al. 1985; Taliaferro et al. 1989;

Ryan and Scowcroft 1987; Allicchio et al. 1987).

This study involves the biochemical analysis of 57 leaf tissue derived

regenerants of Pennisetum purpureum K. Schum. (Napiergrass). These regenerants

were analyzed for the activity of 13 different isozymes. Isozymes were selected

based on the involvement of each enzyme in diverse and major metabolic pathways

and the availability of staining techniques.









Materials and Methods

Starch and Polyacrylamide

Polyacrylamide as well as starch gel techniques were used in this study for the

analysis of isozymes. The drawback of the polyacrylamide technique was the

amount of sample processing that was required. Part of the activity of the enzymes

was lost during this period. The advantage of this technique was the excellent

resolution of the isozyme bands upon staining for activity. Starch gels needed a

minimal amount of sample processing and the extracts could be stored in the form

of wicks at -800C. The resolution of isozyme patterns on starch gels was good.

Sample Processing

Collection of Plant Material.

Leaf material from field grown plants was used for isozyme analysis. Tissue

samples were collected on dry ice to prevent the loss of enzyme activity during

transportation from the field to the laboratory. Each sample was collected and

tagged as a cylindrical segment approximately 200 mm long, which included the

shoot tip.

Preparation of Plant Material.

Storage

The young leaf tissue was cut into approximately 6 mm long segments after

peeling off the older leaves from the outside and wrapped in small pieces of

aluminum foil numbered 1 to 7, beginning from the last internode near the apical

meristem. All segments from one sample were included in a large piece of

aluminum foil marked with the corresponding accession number, frozen in liquid

nitrogen and later transferred to -800C for long term storage.

Grinding

Samples were removed from the freezer prior to grinding and maintained on

dry ice. The tissue was ground in a home-made multiple well plexiglass grinding








unit, designed to handle small quantities of tissue and 20 samples. Care was taken

to use the same numbered segments) from each sample. The tissue was weighed,

and ground in 0.5 v/w grinding buffer (0.2 M Tris-HCI pH 7.8, 60 % Glycerol and

0.2 % 2-Mercaptoethanol added just before use). The tissue was maintained on ice

at all times except while weighing and grinding.

Wicks

Wicks were placed in the crude extract obtained by grinding the samples and

allowed to saturate. Each wick was made of gel blot paper GB003 (Schleicher &

Schuell) cut approximately 1.5 mm wide and 12 mm long. After saturation (about 2

min.), the wicks were transferred to a multi-well dish maintained on dry ice, with

separate wells marked for each sample. The sample wicks were then frozen in an
ultra-low freezer at -80C for use at a later date.

Gel Processing

Preparation of the Gel.

Each gel was prepared for the various systems described in Table 2.1 (Stuber

et al. 1988), using 300 ml of gel buffer and 13% w/v starch (Sigma catalog # S-

4501). A cold slurry of starch and buffer was rapidly mixed with boiling buffer and

degassed under vacuum. After the hot mixture appeared homogeneous and

translucent, the vacuum was broken gently and the gel was poured into a home-

made mould with gel dimensions 184 mm X 158 mm X 6 mm and covered with a

larger glass plate to prevent desiccation. The gel was allowed to solidify at room

temperature for at least 4 hours before incubating for 1 hour at 4C.

Running Conditions.

The sample wicks were transferred 30 min prior to loading from the -800C freezer to

a -20C freezer and allowed to thaw on ice just before loading onto the gel. The gel

was removed from 4C and the glass plate on the top was carefully removed. The

samples were loaded on the gel as described by Stuber et al. (1988). The buffer was









kept in contact with the gel by using a large piece of spongecloth at each electrode,

one end of which was placed in contact with the gel and the other was allowed to

soak in the electrode buffer reservoir. To cool the gel during the run, the whole
setup was transferred to a refrigerator at 4C, with the power supplied from a source

placed outside the refrigerator. The gels were run at constant current of 35 mA for

4 hours. All the power values for the running of the gels were determined after

using several combinations to yield the most favorable results.

Staining of Gels.

Isozyme patterns were visualized by staining the gel for activity ofspecific

enzymes upon completion of the run. The gel was weighed down lightly with the

help of an 11 mm thick acrylic plate and sliced to the appropriate thickness by

running the steel wire alongtwo smooth strips of the desired thickness along the two

sides of the gel. Each gel yielded two 3 mm thick slices which could be used for the

staining of separate isozymes. For best results, the freshly cut gel surface was placed

face up in the staining tray. The stains were mixed from stocks according to recipes
in Table 4.3. All the isozymes studied were anodal in migration. Chemicals and

stains used were from recommended sources and in recommended quantities

(Stuber et al. 1988).

Results
Selection of Stains

Isozyme analysis was carried out on the basis of the availability of recipes for

the stains. Attempts were made to stain at least twenty-three enzymes (Table 2.4)

using several of the buffer systems described in Table 2.1. Fourteen enzymes

stained well (Figs. 2.1 to 2.14), but the other nine either did not yield a

distinguishable pattern or did not stain at all.

All of the isozymes chosen for staining were from prominent metabolic pathways.








TABE 14 Electrode and Gel nffer Formulae


System Electrode Buffer Gel Buffer
A 0.05 M L-Histidine (7.75 g/L) 0.004 M L-Histidine
pH 5.0 0.024 M Citric acid.HzO (ca. 5 g/L; 0.002 M Citric acid.H20 (13-fold
pH adjusted with Citric acid) dilution of electrode buffer)
B 0.065 M L-Histidine (10.88 g/L) 0.009 M L-Histidine
pH 5.7 0.02 M Citric acid.H20 (ca. 4.125 g/L 0.003 M Citric acid H20 (7-fold
pH adjusted with Citric acid) dilution of electrode buffer)
C 0.19 M Boric acid (11.875 g/L) 9 parts Tris-citric acid
pH 8.3 0.04 M Lithium hydroxide (ca. 1.6 g/L buffer [0.05 M Tris base (6.2
pH adjusted with Lithium hydroxide) g/L), 0.007 M Citric acid.H20
(1.5 g/L) pH 8.3] : 1 part
electrode buffer
CT 0.04 M Citric acid.H20 (8.41 g/L) 0.002 M Citric acid.H20
pH 6.1 0.068 M N-(3-Aminopropyl) 0.0034 M N-(3-Aminopropyl)
Morpholine (9.8 g/L) Morpholine (20-fold dilution
of electrode buffer)
D 0.065 M L-Histidine (10.088 g/L) 0.016 M L-Histidine
pH 6.5 0.007 M Citric acid.HO2 (ca. 1.5 g/L) 0.002 M Citric acid.H20 (4-fold
(pH adjusted with citric acid) dilution of electrode buffer)
F 0.135 M Tris base (16.35 g/L) 0.009 M Tris base, 0.003 M
pH 7.0 0.04 M Citric acid.H20 (ca. 9 g/L) Citric acid.H20 (15-fold
pH adjusted with citric acid) dilution of electrode buffer)
Formulae from Stuber et a._(1988)
TABLE 4.2 Recipes for Activity Staining of Isozymes

Enzyme Stains Amount Incubation
a-Acid phosphatase 0.1 M Sodium Acetate- 100 ml 60 minutes in dark at
(a-ACP) acetic acid pH 5.0 room temperature
Fast Garnet GBC 50 mg
MgCl2 50 mg
a-Naphthyl acid 50 mg
phosphate (Na)
p-Acid phosphatase 0.1 M Sodium Acetate- 100 ml 60 minutes in dark at
(p-ACP) acetic acid pH 5.0 room temperature
Fast Garnet GBC 50 mg
MgCl2 50 mg
p-Naphthyl acid 50 mg
phosphate (Na)


TABLE 41


Electrode and Gel Rnff~t Formulae








TABLE 4.2 (continued)

Enzyme Stains Amount Incubation


Alcohol dehydrogenase
(ADH)



a-Aryl esterase
(a-EST)



p-Aryl esterase
(P-EST)


0.05 M Tris-HCl pH 8.0
95% Ethanol
NAD
MTT
PMS

0.2 M Phosphate
buffer (Na) pH 6.0
N-Propanol
a-Naphthyl acetate
Fast garnet GBC

0.2 M Phosphate
buffer (Na) pH 6.0
p-Naphthyl acetate
N-Propanol
Fast garnet GBC


50ml
2 ml
20 mg
20 mg
5 mg


30 minutes in dark at
room temperature


50 ml 45 minutes in dark at
room temperature
2.5 ml
20 mg
25 mg

50 ml 45 minutes in dark at
room temperature
20 mg
2.5 ml
25 mg


Aspartate aminotransferase A 0.1 M Tris-HCI pH 8.5
a-ketoglutarate
(AAT) Aspartic acid
B Pyridoxal-5-P
Fast Blue BB salt


100 ml
100 mg
200 mg
10 mg
150 mg


2 hours in dark at
room temperature
after mixing
A and B


Endopeptidase
(ENP)





Glutamate dehydrogenase
(GDH)


0.2 M Tris-Maleate
pH 5.6
a-N-Benzoyl-DL-
arginine-p-Naphth-
ylamide-HCL
MgCl2
Black K salt

0.1 M Tris-HC1 pH 8.5
L-Glutamic acid
CaC12
NAD
NBT
PMS


50 ml 60 minutes in dark at
room temperature

25 mg

50 mg
25 mg


50 ml
150 mg
50 mg
20 mg
15 mg
5 mg


60 minutes in dark at
room temperature


Hexokinase
(HEX)


0.05 M Tris-HCl pH 8.0 50 ml
P-D(+)-Glucose 125 mg
ATP 125 mg
MgC12 50 mg
NAD 10 mg
MTT 5 mg
PMS 1.25 mg
NAD dependent glucose- 56.25 u
6-phosphate dehydrogenase


2 hours in dark at
room temperature







TABLE 4.2 (continued)

Enzyme Stains Amount Incubation


Malate dehydrogenase
(MDH)



Malic enzyme
(ME)




6-Phosphogluconate
dehydrogenase
(6-PGD)



Phosphohexose isomerase
(PHI)






Shikimic acid
dehydrogenase
(SAD)


0.1 M Tris-HC1 pH 9.1
DL-Malic acid
NAD
NBT
PMS

0.1 M Tris-HC1 pH 8.5
DL-Malic acid
MgC12
NADP
NBT
PMS


50 ml
100 mg
20 mg
10 mg
1.25 mg

50ml
100 mg
50 mg
15 mg
10 mg
2 mg


0.05 M Tris-HCl pH 8.0 50 ml
6-Phosphogluconic acid (Na3)20 mg
MgCl2 50 mg
NADP 5 mg
MTT 5 mg
PMS 1.5 mg

0.05 M Tris-HCl pH 8.0 50 ml
D-Fructose-6-phosphate 50 mg
MgCI2 50 mg
NADP 5 mg
MTT 5 mg
PMS 1.5 mg
NADP-dependent Glucose- 10 u
6-phosphate dehydrogenase


0.1 M Tris-HCl pH 9.1
Shikimic acid
NADP
MTT
PMS


60 ml
60 mg
10 mg
5 mg
1.33 mg


60 minutes in dark at
room temperature



overnight at room
temperature after
30 minutes at 36C



60 minutes in dark at
room temperature




60 minutes in dark at
room temperature






2 hours in dark at
room temperature


Recipes from Stuber et al.


(1988) and Vallejos (1983)


Lack of Variation.


Among all the isozymes stained, alcohol dehydrogenase (Fig.


4.3), aryl


esterase (Fig. 4.6 and Fig. 4.7), endopeptidase (Fig. 4.5), glutamate dehydrogenase

(Fig. 4.8), malate dehydrogenase (Fig. 4.10) and phosphohexose isomerase (Fig.

4.13) showed very distinct and crisp banding patterns. The other variation in all the

regenerants, at the scrutinized loci. isozymes provided a good resolution of the









banding pattern although not exceptional. Acid phosphatase and aryl esterase were

each assayed using two forms of their respective substrates and were distinguished

by using prefixes a- and F-.

All the gels had one lane dedicated to each of the regenerants and a parental

clone as the control. None of the regenerants showed any variation in the banding

patterns of the isozymes. In other words, no regenerant showed any unique isozyme

banding pattern in comparison to the parent. Hence, there was a complete lack of

any quantitative variability was due to the inherent inability in starch gel systems to

quantitate the amount of protein on sample wicks.

Discussion

The results of this work show no variation in isozyme patterns among the

regenerants derived from tissue culture of leaf segments of napiergrass. Each clone

produces scores of tillers and is hence ideal for induction into culture for obtaining a

population from a single clone. Isozymes show distinct patterns at different stages
of development. The fidelity of the population derived from somatic embryos of a

single clone was, therefore, tested by using plant tissue at the same stage of

development. This was done to minimize any variation in the tissue, inherent to the

developmental phase. Several reports on somaclonal variation (Larkin and

Scowcroft 1981; Larkin et al. 1984; Maddock et al. 1985; Breiman et al. 1987a; Ryan

et al. 1987; Ryan and Scowcroft 1987; Taliaferro et al. 1989) have focused on

regeneration using immature embryos as the explant. This study differs from the

above mentioned ones in its use of leaf tissue as the only explant material used.

Biochemical analyses of the somatically derived regenerants were carried out

to detect any variation that may exist at the tissue level, which may not be expressed

morphologically. Changes in isozyme banding patterns may be developmentally

regulated or the result of altered protein structure due to DNA rearrangement in

the genome. Isozyme analysis was, therefore, supplemented with molecular analysis































Fig. 4.1 Gels stained for enzyme a-Acid phosphatase (a-ACP) after run using
buffer system B.























noonnonooooo000
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0
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Fig. 4.2 Gels stained for enzyme p-Acid phosphatase (p-ACP) after run using
buffer system B.




















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Fig. 4.3 Buffer system C gels showing Alcohol dehydrogenase (ADH) activity.




















dpsiw


00000000
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1-> F-A wS w -j w w A(b


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Fig. 4.4 Aspartate aminotransferase (AAT) activity observed on buffer
system C gels.






























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ha, ha ha ha ha hta ih a iha ha h 0% i.t- 0% ha 0. 0- .- t -n I- L-J J -JI- I iJ .4






























Fig. 4.5 Activity of enzyme Endophosphatase (ENP) observed on buffer
system C gels.



















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A lp


% r t)Hf<^ a


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Fig. 4.6 a-Aryl esterase (a-EST) activity observed on buffer system C gels.


















It,4,t).*41


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(00000000 0 no a 60 0 rt '' ''' 0
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Fig. 4.7 p-Aryl esterase (p-EST) activity observed on buffer system C gels.




















__, _~~ _---,----~I *-




rq pst Cr I~L~~ M~lu~ I ~q


H H ) UH p .) Ln 0 -4 0w H 0 H- ") w H w) w P. 01


U------ ---




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Fig. 4.8 Buffer system C gels showing the activity of enzyme Glutamate
dehydrogenase (GDH).













+

















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2Q22S2;88t 1,'
;555558555535&585 Beffl~ rotllK^


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Fig. 4.9 Hexokinase (HEX) activity seen after staining gels prepared using buffer
system C.
















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OOOOOOOO~0OO0~or0or~O


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W39 9 39 U3 903 0 9 9


.fir~r






























Fig. 4.10 Activity of enzyme Malate dehydrogenase (MDH) observed on buffer
system B gels.























* T I -. .4*

041


* *z .* z r p w









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00


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Fig. 4.11 Malic enzyme (ME) activity observed on buffer system B gels.













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Fig. 4.12 Gels prepared using buffer system D exhibiting activity of enzyme
6-Phosphogluconate dehydrogenase (6-PGD).
















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C


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Fig. 4.13 Banding pattern of enzyme Phosphohexose isomerase (PHI) obtained on
buffer system B gels.
4





























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Fig. 4.14 Activity of enzyme Shikimic acid dehydrogenase observed on gels
prepared using buffer system D.










































00000 0 00000000000000()00.00
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l- h' l' t' -' l' -' l' -' l- -' l- O -' h- -' l- -' t- l' t- l' l- l' l' -
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39?!0 !0 9 ?a ?50 iio0
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TABLE 4- 3 Summary of En.....mes and Buffers Anal-zed-


Activity
# ENZYME Present* Absent*
1 Acid phosphatase (-a-) a-ACP) B, D
2 Acid phosphatase (-P-) P-ACP) B
3 Acomtase ACO) C, F
4 Alcohol dehydrogenase ADH) C
5 Aldolase ALD) C, D
6 Aryl esterase (-a-) a-EST) C
7 Aryl esterase (-16-) p-EST) C
8 Aspartate aminotransferase AAT) C
9 Catalase CAT) C, D
10 Diaphorase DIA) C, F
11 Endopeptidase ENP) C
12 Glucosidase (-3-) (p-GLU) CT, B
13 Glutamate dehydrogenase (GDH) C
14 Hexokinase (HEX) C C, F
15 Isocitric dehydrogenase IDH) CT, D
16 Malate dehydrogenase MDH) B
17 Malic enzyme ME) B
18 Phosphoglyceraldehyde dehydrogenase PGALDH) C, D
19 Phosphoglucomutase PGM) CT, D
20 Phosphogluconate dehydrogenase (6-) 6-PGD) D C
21 Phosphohexose isomerase PHI) B, D C, F
22 Shikimic acid dehydrogenase SAD) D
23 Triose phosphate isomerase TPI) C, F

* buffer systems described in Table 4.1

at the nuclear and cytoplasmic levels. The uniformity of banding patterns in all the

isozyme systems tested is conclusive proof of the absence of any aberrations at the


loci tested.


TART ~F, 4.3 Summary of Enzymes and


Buffers Analyzed














CHAPTER 5
RESTRICTION AND HYBRIDIZATION ANALYSIS OF
MITOCHONDRIAL DNA FROM A POPULATION OF PENNISETUM
PURPUREUM K. Schum. REGENERANTS DERIVED FROM SOMATIC
EMBRYOS

Introduction

Mitochondrial DNA (mtDNA) analysis has been employed in a variety of

tissue culture studies. The products of protoplast fusion may be scrutinized for the

presence of recombinational events in DNA at the extranuclear level using

restriction and hybridization analyses of mtDNA (Belliard et al. 1979; Boeshore et

al. 1985; Chetrit et al. 1985; Vedel et al. 1986; Rothenberg and Hanson 1987; Ozias-

Akins et al. 1987; Tabaeizadeh et al. 1987). The use of mtDNA in tissue culture

analyses does not limit itself exclusively to somatic hybrids. MtDNA analyses have

been applied for the identification of cultivars most suitable for induction into

culture (Rode et al. 1988). However, one of the most common applications of

mtDNA for in vitro studies is its use in the identification of variation that may arise

during the process of tissue culture.

The mitochondrial genome in the Texas type male sterile cytoplasm of maize

is directly involved in the reversion to fertility (Gengenbach et al. 1981). This

reversion is caused by the deletion or loss of activity by disruption, of the Turfl3

mitochondrial gene (Pring and Lonsdale 1989). Hartmann et al. (1987) and Rode et

al. (1987b) have reported the presence of a "hypervariable" region on the

mitochondrial genome of wheat tissue cultures. The induction of wheat into culture

has been termed responsible for the aberrations caused in this region. MtDNA

samples from long-term cultures of rice have been shown to possess altered

restriction patterns (Chowdhury et al. 1988; Abdullah et al. 1990). However, these








long-term cultures have also been reported to have lost their embryogenic and

regenerative capacity. Hartmann et al. (1989) have shown a correlation between a

specific restriction pattern of the mitochondrial genome and embryogenic capacity
of wheat tissue cultures, although the organization of the mitochondrial genome
may not be the cause of the embryogenic capacity. In maize cell cultures, McNay et

al. (1984) reported changes in the stoichiometry of bands in the restriction pattern

of mtDNA from maize tissue cultures, although the restriction profile remained

unchanged. Variant restriction profiles of mtDNA from Brassica campestris, caused

by DNA rearrangements, have also been found in the native plant tissue at very low

levels (Shirzadegan et al. 1989). These variations are, hence, believed to be the

result of in vitro amplification of existing variation.
The purpose of this study was the comparative evaluation of mtDNA from a

population of Pennisetum purpureum regenerants obtained from in vitro culture.

The establishment of callus cultures and regeneration of the population is described

in Chapter One.

Materials and Methods

Extraction of mtDNA

The procedure used for the extraction of mtDNA was described by Smith et

al. (1987). The soft basal regions(including young leaves and stem) of tillers

obtained from field grown plants were used for the extraction of mtDNA. The

tissue was ground in 10 volumes / FW cold buffer. The homogenate was filtered

through 4 layers of cheesecloth and 1 layer of Miracloth (Calbiochem). The filtrate

was centrifuged for 10 minutes at 1000 x g (4C) to pellet the nuclei and

chloroplasts. The supernatant was transferred to fresh bottles and centrifuged for
10 minutes at 17,000 x g (4C) to pellet the mitochondria. After discarding the
supernatant, the pellet was carefully resuspended in 5 volumes of saline buffer. The

resuspended mixture was transferred to a 30 ml tube and centrifuged for 10 minutes








at 18,000 x g at 4C. The pellet was resuspended in Saline buffer (20 ml / 50 g FW)
with 1 M MgC12 (100 pl / 10 ml). DNase (Sigma Chemical Co.) was added to
obtain a final concentration of 0.02 mg / ml and mixed well. The mixture was
incubated for 60 minutes at room temperature after which it was underlayered with
20-25 ml Shelf buffer and centrifuged for 20 minutes at 16,000 x g at 40C. The pellet
was resuspended in 30 ml Saline wash and centrifuged for 20 minutes at 16,000 x g
at 4C. This pellet was resuspended in 5 ml NN buffer with 250 p of Proteinase K

(2 mg/ml) and 250 l 10% SDS and incubated for 1 hour at 37C. An equal volume
of 2X Extraction buffer was added and further incubated for 15 minutes at 650C.
Potassium acetate (5 M) was added to a final concentration of 1.25 M). This
mixture was maintained on ice for 30 minutes with frequent mixing. After
centrifuging for 10 minutes at 16,000 x g at 4C, the supernatant was filtered through

Miracloth into a mixture of isopropanol and ammonium acetate (0.5 volume

isopropanol : 0.05 volume 5 M ammonium acetate) and incubated at -20C for 1
hour, after which it was centrifuged for 20 minutes at 16,000 x g. The supernatant
was discarded and the pellet was washed in 70% ethanol before dissolving it in 700
il TE buffer. This was extracted once with an equal volume of phenol followed by
one extraction each with equal volumes of phenol : chloroform (1:1) and

chloroform. Centrifugation at each step was for 5 minutes in a microfuge at full

speed and the aqueous phase was retrieved. After extraction with chloroform, 0.11

volume of 3 M sodium acetate and 0.7 volume of isopropanol were added to the

sample. This mixture was incubated for 1 hour at -200C to precipitate the DNA.
The tubes were centrifuged to pellet the DNA after which it was washed in 80%
ethanol and vacuum dried. The pellet was resuspended in about 100 pl of DNA
buffer.

Plants from each of the callus lines described in Chapter Three were used to

obtain mtDNA. Plants were selected at random from groups with more than two








individuals. A total of twenty one regenerants were used for the extraction of
mtDNA. Two of the six plants obtained from immature inflorescence derived calli
were also selected at random for the isolation of mtDNA, in addition to the parental

clone. The total number of mtDNA samples thus obtained, including the parent,

was twenty-four.
Restriction Analyses
Restriction endonuclease fragment analyses were conducted on the 24
samples using two restriction enzymes (HindIII and PstI). Restriction analyses using
enzyme BamHI were conducted on 22 samples representing all the callus types. All
representatives of the callus types were also included in the analysis of 21 samples
using enzyme SalI. Samples were digested using 10-15 units of enzyme for each
reaction, at 37C for 90 minutes. The reaction was stopped using 6X loading dye

(0.25% bromophenol blue, 40% sucrose). Digested samples were run on a gel unit
(gel dimensions 260 mm X 210 mm) at approximately 2 volts/cm for 16 hours using
TPE buffer (0.9 M Tris-Phosphate pH 8.0, 0.002 M EDTA). One of the lanes on
each gel contained DNA from bacteriophage lambda digested with HindIII as a
molecular size marker. The banding patterns were visualized on an ultraviolet

transilluminator (Fotodyne, model 3-3500) after staining them in a 0.5 A/g/ml

solution of ethidium bromide for 45 minutes and destaining in distilled water for 20

minutes. The gel was photographed using a UV filter, a red filter and Polaroid film

(Type 55).








TABLE 5.1 Buffers Used for Bidirectional Blotting of Mitochondrial DNA


Buffer Ingredients Molarity

Depurinating HC1 0.25 M
Denaturing NaCI 0.6 M
NaOH 0.2 M

Neutralize NaCI 3.0 M
pH 7.5 Tris Base 1.0 M
20 X SSC NaCI 3.0 M
pH 7.0 Citrate (Na3) 0.3 M


Bidirectional Southern Blotting (Sandwich Blotting)

The photographed gel was transferred to a large tray on a table top rotary

shaker and covered with 0.25 M HCI (Table 5.1) for 10 minutes, to allow for

depurination. The increased mobility of the high molecular DNA facilitated a good

transfer onto the nylon membrane. After 10 minutes the HCI was poured off and

the gel rinsed twice with deionized water. The gel was then covered with

Denaturing buffer (Table 5.1) and maintained on a shaker for 30 minutes. To

neutralize, the gel was rinsed two times with deionized water, the Neutralizing

buffer (Table 5.1 was poured on the gel and shaken for 30 minutes. Concurrent to

the pretreatment of the gel, two pieces of the nylon membrane were cut to the size

of the gel and equilibrated in 20 X SSC (Table 5.1) for 15 minutes. A blot block

approximately 1 inch thick was placed on the counter top on which three dry sheets

of 3MM paper were placed. Three sheets of 3MM paper, previously equilibrated in

20 X SSC, were placed on top of the dry 3MM sheets and all bubbles rolled out with

the help of a brayer. One of the two equilibrated nylon membranes was placed on

the wet 3MM sheets after flooding these with 20 X SSC. The gel was then carefully

placed on the membrane after rolling out all the air bubbles from under the

membrane and flooding the membrane with 20 X SSC. The gel was flooded with 20

X SSC and the second sheet of nylon membrane was placed on top, while carefully








excluding all air bubbles. Three sheets of wet 3MM paper were placed on the
membrane and the bubbles rolled out. The three dry sheets of 3MM paper were

placed on top of the wet 3MM and topped off with a 1 inch thick blot block. A glass
plate was placed on top of the stack and weighed down. After three hours, the
membranes were carefully taken apart and rinsed in 3 X SSC, wrapped in plastic

wrap and the DNA was crosslinked to the membrane by a 5 minute exposure to UV
light on a transilluminator (Fotodyne 3-3500).

DNA Hybridization
The sandwich blots obtained from the gels described above were probed
using the Southern hybridization technique (Southern 1975). The restriction
profiles produced by each enzyme were probed using at least six different

mitochondrial genes cloned from maize; viz. F1-FO ATPase subunit a (atpA, 4.2 kb)

(Braun and Levings 1985), F1-FO ATPase subunit 6 (atp6, 0.9 kb) (Dewey et al.

1985a), F1-FO ATPase subunit 9 (atp9, 2.2 kb) (Dewey et at 1985b), cytochrome c

oxidase subunit I (coxl, approximately 10 kb) (Isaac et al. 1985), cytochrome c
oxidase subunit II (coxll, 2.4 kb) (Fox and Leaver 1981) and 18S-5S ribosomal DNA
(18S, 6.0 kb) (Chao et al. 1984). Probes were provided by Dr. C. S. Levings, III, of
North Carolina State University, Raleigh, USA. Some of the blots were also probed

using random clones from the pearl millet mitochondrial genome (obtained from

Dr. R. L. Smith, University of Florida, Gainesville. FL). Each of the two

membranes corresponding to a single restriction enzyme was scrutinized using a

different probe.
The nylon blots corresponding to each gel were probed separately with
individual cloned fragments of DNA named above. The blots were prehybridized
with 30 ml of 0.5 M Sodium phosphate buffer pH 7.2, 1% BSA and 7% SDS

supplemented with approximately 2.5 mg of denatured Herring sperm DNA

(obtained by boiling the DNA solution for 5 minutes and immediately transferring









to ice for 5 minutes), in 25 cm X 30 cm plastic bag. Care was taken to exclude all air

bubbles. The sealed pouches were incubated at 650C for a minimum of 4 hours
before injecting the labelled probe into the pouch. Procedures for hybridizing
specifically with the membrane were initiated by radioactively labelling a cloned
fragment of DNA which was to be used as the probe.

The random priming reaction was carried out, according to procedures

described by Feinberg and Vogelstein (1983), in a total volume of 50 p1, which

consisted of 10 Ml OLB (Table 5.2), 6 pl BSA (1 mg/ml), 2 Al 32P-dCTP (20 iCi), 2

Al DNA polymerase (2 units), approximately 100 ng denatured DNA and the
volume was brought to 50 Ml with sterile double deionized water. The reactants

were added to the tube individually, and the mixture was incubated at 370C for 45
min. To stop the reaction, 150 p1 of OLB Stop mix (Table 5.2) was added after 30-

45 minutes of incubation. The volume was brought to 600 p with TE (10 mM Tris,

1 mM EDTA) after denaturing and injected into the pouch using a 1 ml tuberculin

syringe while taking measures not to introduce any air bubbles. The pouch was

incubated in a 650C water bath for 16-24 hours. After incubation, the membrane

was removed after draining the pouch and subjected to two washes in 3 X SSC at

65C for 15 minutes each. Following the second wash, the membrane was drained of

any excess buffer and wrapped in plastic wrap. The membrane was then monitored

for radioactivity with the help of a Geiger counter and exposed to X-ray film (Kodak

X-Omatic AR 5) with a Cronex Lightning (Du Pont) intensifying screen. The

exposure of the film depended on the amount of radioactivity detected on the

membrane. Autoradiograph exposure times were typically 24-48 hours.
Results

Restriction Analyses

Digestion patterns of mtDNA with the four enzymes were complex, yielding

between 30 and 50 fragments with the enzymes BamHI HindIII, PstI and Sall.








TABLE 5.2 Buffers Used for DNA-DNA Hybridization Procedures


Buffer

OLB TE
pH 7.0

Tris-MgC12
pH 8.0

dATP
dGTP
dTTP

Solution A




Solution B
pH 6.6

Solution C

OLB


OLB Stop Mix
pH 7.5


Formulae from Feinberg and Vogelstein (1983)

Although the gels obtained from restriction analyses were used for making nylon

blots for DNA-DNA hybridization analyses, they were not subjected to a

densitometric analysis to expose differences in stoichiometry between different

bands.

BamHI Digests

Each sample of DNA yielded at least 40 bands after being digested with

BamHI. The restriction profiles showed no differences in banding patterns between

lanes (Figure 5.1). Any discrepancies in intensity of the bands were correlated to


---


Volume

100 ml

100 ml

170 ll
169 1l
172 Al

1.033 ml




50 ml

0.555 ml

0.25 ml


100 ml


Ingredients

Tris
EDTA

Tris
MgCl2.6H20
dATP
dGTP
dTTP

Tris-MgCl2 solution
p3-Mercaptoethanol
dATP
dGTP
dTTP

Hepes

Hexamers

Solution A
Solution B
Solution C

Tris Base
Sodium chloride
EDTA
SDS


Molarity

0.003 M
0.0002 M

1.25 M
0.125 M

0.1 M
0.1 M
0.1 M






2M







0.02 M
0.02 M
0.002 M
0.0025%


Amount

0.36 gm
0.075 gm

15.14 gms
2.54 gms

0.01 gm
0.01 gm
0.01 gm

1.0 ml
0.018 ml
0.005 ml
0.005 ml
0.005 ml

23.8 gms

50 units

0.05 ml
0.125 ml
0.075 ml

0.24 gm
0.12 gm
0.075 gm
0.0025 gm









differences in amounts of DNA used in the restriction reactions. Blots from these

restrictions were probed using several DNA fragments as probes (Figs. 5.2 to 5.9).

HindIII Digests

The restriction profile of mtDNA using enzyme HindIII yielded

approximately 50 bands from every sample of mtDNA that was tested (Figure 5.10).

One of the lanes showed a single band which appeared stronger in intensity than in

the rest of the samples. The accentuated intensity of the band was, however, not

apparent when the same sample was digested with an increased amount of

restriction enzyme. Blots from these restrictions were probed using several DNA

fragments as probes (Figs. 5.11 to 5.18).

PstI Digests

Digestion of mtDNA using the enzyme Pstl yielded about 40 bands in the

restriction profile of every sample (Figure 5.19). In this case, there was no

difference detected whatsoever in the restriction patterns of any of the samples.

Differences in relative intensities between bands from distinct samples were few,

and not subjected to stoichiometric analysis. Blots from these restrictions were

probed using several DNA fragments as probes (Figs. 5.20 to 5.27).

Sail Digests

The Sall restriction profile yielded about 40 bands, and there was no

variation observed between individual samples of the population (Figure 5.28).

Hybridization Analyses

Blots were hybridized to assess them for qualitative characters. After

probing the blots with the maize mitochondrial clones atpA, atp6, atp9, coxl, coxll

and 18S rDNA, no difference was observed in any of the hybridization patterns.

Blots from the Sall digests were probed with the K', K3 and X2 clones (Figure 5.29)

from the "hypervariable region" of the wheat mitochondrial genome. These probes






























Fig. 5.1 Restriction profile of mtDNA from P. purpureum after restriction
with enzyme BamHI. Lane 1 contains DNA from bacteriophage x
digested with enzyme HindIII, used as molecular weight
markers.







So 0o
] H r^ c 0; H; Hi H; B0 C- C C-- CO CO '4
,-i a s s ,,0 a s o s o o Hi i p co cow
. U U U U U U U U U 0 U U U 0U U U U U U


ols


11j


-i




























Fig. 5.2 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene for 18S rRNA.
Fig. 5.3 BamHI digested mtDNA from P. purpureum probed with the
mitochondrial gene coding for the a subunit of ATPase (atpA).













0 1 O rH H M(i n nl
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aI U U U U U U U U


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Fig. 5.4 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene atp6.

Fig. 5.5 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene atp9.












0 4D
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000 000 00 00 O U U UU H


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Fig. 5.6 MtDNA from P. purpureum probed with the mitochondrial gene for
cytochrome oxidase subunit I (coxl), after digesting with restriction
enzyme BamHI.

Fig. 5.7 MtDNA from P. purpureum probed with the mitochondrial gene for
cytochrome oxidase subunit II (coxll), after digesting with restriction
enzyme BamHI.












0 0W

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9.0 4 me so 4040 40 40 40


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Fig. 5.8 BamHI digested mtDNA from P. purpureum probed with a random
mitochondrial probe 4D5.

Fig. 5.9 BamHI digested mtDNA from P. purpureum probed with a random
mitochondrial probe 4D12.

















0 %0
o H m a H- t-4 m~C m f














2.8 @~- ~m *

















r4 r-I N 0 V M uI 0 -4 N
0 rI ('1 '3 vr -4 M M IA 0: Ix CZ 9 9 M A m
0.~~~ ~~ 0- H Ml V4 wA wA 0 r -I r -I w- -






mo~
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Fig. 5.10 Restriction profile of mtDNA from P. purpureum after restriction
with enzyme HindIIl. Lane 1 contains DNA from bacteriophage A
digested with enzyme HindIII, used as molecular weight
markers.




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FILES



BIOCHEMICAL AND MOLECULAR ANALYSIS OF REGENERANTS
DERIVED FROM SOMATIC EMBRYOS OF PENNISETUM PURPUREUM K.
SCHUM.
By
VIVEK BHASKAR SHENOY
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
1991

Dedicated to my family for their support and understanding and to my nieces,
Renuka, Sai, and nephew, Shirish, all of whom I have yet to see.

ACKNOWLEDGEMENTS
I would like to acknowledge the valuable guidance and encouragement of my
committee chairman, Dr. Indra K. Vasil. I am forever grateful for his constant push
towards perfection. I am indebted to Dr. Daryl R. Pring for all his technical
supervision, the use of his laboratory and the various DNA probes he provided. I
thank Dr. S. C. Schank for providing the plant material and initial field space. Drs.
Robert J. Ferl and Elenry C. Aldrich, I thank for their time, help and guidance
whenever I needed it. I am particularly grateful to Dr. William B. Gurley for
consenting to attend my final examination.
I also thank Dr. M. K. U. Chowdhury, Mr. Mark G. Taylor and Mr. Luis F.
Pedrosa for all the useful discussions, suggestions and their invaluable help. I am
grateful to all other colleagues for their friendship and support.
For technical help and guidance, I thank Drs. Rex L. Smith and C. E.
Vallejos.
Finally, I would like to thank the Davé, Gokhalé, Goré and Navathé families
for their help in making my stay in Florida both enjoyable and pleasant.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE REVIEW 3
Variation in tissue culture 3
Isozymes 4
Mitochondrial DNA 9
Chloroplast DNA 16
Nuclear DNA 25
3 REGENERATION OF PLANTS FROM SOMATIC EMBRYOS OF
PENNISETUM PURPUREUM K. SCHUM. BY TISSUE
CULTURE OF IMMATURE LEAF SEGMENTS 29
Introduction 29
Materials and methods 31
Results 32
Discussion 33
4 BIOCHEMICAL ANALYSIS OF A POPULATION OF
PENNISETUM PURPUREUM K. SCHUM. DERIVED
FROM TISSUE CULTURE USING, ISOZYMES AS
MARKERS 36
Introduction 36
Materials and methods 38
Results 40
Discussion 44
5 RESTRICTION AND HYBRIDIZATION ANALYSIS OF
MITOCHONDRIAL DNA FROM A POPULATION OF
PENNISETUM PURPUREUM K. SCHUM. REGENERANTS
DERIVED FROM SOMATIC EMBRYOS 77
Introduction 74
Materials and methods 75
Results 80
Discussion 119
IV

6 CHLOROPLAST DNA ANALYSIS OF PENNISETUM
PURPUREUM K. SCHUM. REGENERANTS DERIVED
FROM TISSUE CULTURE OF YOUNG LEAF
SEGMENTS 121
Introduction 121
Materials and methods 122
Results 125
Discussion 126
7 DNA HYBRIDIZATION ANALYSIS OF NUCLEAR DNA
FROM TISSUE CULTURE DERIVED REGENERANTS OF
PENNISETUM PURPUREUM K. SCHUM 134
Introduction 134
Materials and methods 135
Results 138
Discussion 138
8 CONCLUSIONS 148
REFERENCES 150
BIOGRAPHICAL SKETCH 170
v

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
BIOCHEMICAL AND MOLECULAR ANALYSIS OF REGENERANTS
DERIVED FROM SOMATIC EMBRYOS OF PENNISETUM PURPUREUM K.
SCHUM.
By
Vivek Bhaskar Shenoy
June 1991
Chairman: Indra K. Vasil
Cochairman Robert J. Ferl
Major Department: Botany
A population of Pennisetum purpureum plants regenerated from embryogenic
callus cultures was analyzed for the occurrence of variation at the biochemical and
molecular levels. Fifty-seven P. purpureum regenerants obtained from the tissue
culture of young leaf segments of a single clone were used for the analysis. The
biochemical analysis consisted of screening the entire population for the activity of
several isozymes using electrophoretic techniques. Molecular analyses were carried
out to identify any aberrations at the DNA level.
For the biochemical studies, both polyacrylamide (native) as well as starch
gel techniques were tested. The starch gel technique was used for a mass analysis of
the regenerants for isozyme activity. A total of fourteen enzyme systems yielded
good zymograms and were used to screen the entire population. The isozymes
successfully stained for activity included Acid phosphatases (a and p), Alcohol
dehydrogenase, Aryl esterases (a and p), Aspartate aminotransferase,
Endopeptidase, Glutamate dehydrogenase, Hexokinase, Malate dehydrogenase,
Malic enzyme, 6-Phosphogluconate dehydrogenase, Phosphohexose isomerase and
vi

Shikimic acid dehydrogenase. In all the isozyme systems that showed activity, no
variation was observed in banding patterns between tissue culture regenerants and
control plants.
Twenty-three regenerants were selected randomly, in addition to the parental
clone, for the extraction of mtDNA. These DNA samples were primarily analyzed
by comparing their restriction patterns on agarose gels. The four restriction enzymes
used individually for this analysis were BamHl, Hindlll Pstl and Sail. The
comparative analysis of restriction patterns from all the extracted samples did not
yield any unique fragments, suggesting that there was no variation at the mtDNA
level. These gels were blotted to nylon membranes which were used for
hybridization analysis of the restricted DNA. The membrane blots corresponding to
each restriction enzyme were probed using six different mitochondrial genes i.e.
atpA, atpó, atp9, coxl, coxll and the 18S ribosomal gene. In addition to this, the blots
were probed using random probes from the wheat mitochondrial genome and
cosmids cloned from the maize mitochondrial genome, each cosmid had an insert
averaging 35 kb. The hybridization analyses of all the samples mentioned above also
showed no unique patterns.
Analysis of cpDNA and nuclear DNA was carried out using total DNA
extracted from twenty-two randomly selected regenerants in addition to the parent.
Total DNA from each sample was restricted and blotted from gels for hybridization
analysis. The enzymes used to restrict the total DNA were EcoRI, Hindlll and Pstl.
The blots corresponding to each restriction enzyme were probed using two cosmid
clones, which together represented more than 75 kb or 60% of the maize chloroplast
genome. The blots were also probed using three different random P. purpureum
nuclear probes and the Nor locus gene from wheat. All the blots thus probed showed
no variation within the individuals.
vn

CHAPTER 1
INTRODUCTION
For the past few decades, tissue culture has been studied very closely and has
opened up innumerable possibilities in its use as a technique to obtain clonal
populations. Since the process of in vitro culture does not involve the zygotic
process, tissue culture was expected to provide progeny with clonal fidelity.
However, it has been shown that tissue cultures and plants derived from them
undergo many changes at the cytogenetic and morphological levels (Murashige and
Nakano 1966, 1967; Heinz and Mee 1971). These anomalies were claimed to be
beneficial to the plant breeder, as a novel method of introducing new varieties
(Larkin and Scowcroft 1981). Most of the aberrations identified have been found to
occur at low frequencies in normal sexual crosses. Other variations have been
epigenetic and hence not heritable through a sexual cycle. Variation in culture also
depends on the nature and source of the explant tissue. While a large number of
reports concerning variation in tissue culture have focussed on the use of immature
embryos from inbred lines to provide clonal populations, Breiman et al. (1989) have
observed the occurrence of variations at a very low level between individuals of an
inbred line. The anomalies observed were similar to the ones they reported from a
tissue culture-derived population, in an earlier publication (Breiman et al. 1987a).
In contrast to the reports of variation, there have been others accentuating
the stability of plants from tissue culture. It has been documented that
embryogeniccallus cultures are largely euploid and plants derived from such cultures
are both euploid and genetically stable (Swedlund and Vasil 1985; Rajasekaran et al.
1986; Gmitter et al. 1991). The stability of callus and tissue culture derived plants is
1

2
important for genetic manipulations in biotechnology, to be able to predict the
outcome of such manipulations, barring spontaneous mutations.
Although plants derived from embryogenic cultures are known to be
cytogenetically and morphologically stable, it is important to screen the regenerants
for changes at the biochemical and molecular levels to ascertain their fidelity to the
explant source. Biochemical analyses have generally involved isozymes and total
proteins, while molecular analyses involve the scrutiny of the nuclear and
cytoplasmic genomes for restriction fragment length polymorphisms (RFLPs).
This study involved biochemical and molecular analyses of a population
derived from somatic embryos obtained from a single field grown clone of
Pennisetum purpureum (napiergrass). The study differs from other studies in that
the parental clone is used as a control for comparative analysis of the regenerant
population. Biochemical analyses consisted of screening the population by staining
for the activity of several isozymes on starch gels. Molecular analyses involved the
study of the nuclear and cytoplasmic genomes. Restriction profiles of the
mitochondrial genome were visualized on agarose gels using four restriction
enzymes. The DNA from these gels was blotted onto membranes for use in DNA
hybridization analyses using known gene probes from the maize mitochondrial
genome, and random clones from the maize and wheat mitochondrial genomes.
Chloroplast and nuclear DNA analyses were carried out using random cosmid
clones from the maize chloroplast genome and random nuclear probes from the
napiergrass genome to probe total DNA blots.

CHAPTER 2
LITERATURE REVIEW
Variation In Tissue Culture
Totipotencv and Plant Regeneration
The concept of single cell autonomy and totipotency is contained in the
independent works of Schleiden and Schwann during the earlier part of the
nineteenth century (Gautheret 1985). Totipotency refers to the ability of a single
cell to give rise to an entire individual, and implies that all the genes present in the
zygote are conserved in each subsequent cell. Many researchers have attempted to
establish long term totipotent cell and callus cultures in a variety of plant species.
All such attempts proved unsuccessful until the mid-1930s, when continuously
growing callus cultures were independently obtained by Gautheret (1934, 1935),
Nobécourt (1939) and White (1939), which formed the basis for further studies on
the possibility of regenerating plants from such cultures. These efforts culminated
with the demonstration of totipotency by Vasil and Hildebrandt (1965a) who
cultured isolated single cells of Nicotiana glutinosa x N. tabacum in microchambers
and documented their development into entire plants.
Plants regenerated from tissue culture should normally result in clones that
are phenotypically and genotypically identical to the explant from which they have
been originally derived. However, plant cell and callus cultures accumulate
chromosomal variability and lose their regenerative capacity over time (Murashige
and Nakano 1966, 1967; Orton 1980). Therefore, embryogenic cultures, in which
plants are derived from somatic embryos of single cell origin, are considered more
useful because there is a selection away from chromosomal
3

4
variants in the formation of somatic embryos (Hanna et al. 1984; Karp and Maddock
1984; Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986; Cavallini et al.
1987; Kobayashi 1987; Feher et al. 1989; Gmitter et al. 1991). Plants derived from
embryogenic calli are mostly euploid and devoid of any discernible morphological or
cytological variability.
The Concept of Variability
Although tissue culture has been used extensively for the clonal propagation
of plants, there are numerous reports of cytological variation in tissue culture
(D’Amato 1978). Larkin and Scowcroft (1981) proposed the term "somaclonal
variation" for variability arising in culture, and suggested that somaclonal variants
recovered from tissue culture could be utilized as novel breeding lines for plant
improvement.
While there have been several reports of morphological and cytogenetic
variation in tissue culture, this review will be limited to the literature concerning
variation at the biochemical and molecular variation.
Isozymes
Multiple Forms of Enzymes
The technique of staining electrophoretic gels to test for the activity of
enzymes was first described by Hunter and Markert (1957) and the stained gels were
called zymograms. The term isozymes (isoenzymes) was coined later by Markert
and Moller (1959) to designate multiple forms of enzymes occurring in organisms
belonging to the same species. For over three decades, isozymes have proved useful
as biochemical tools in plant breeding, chromosome mapping, developmental
biology, tissue and organ specific studies and in a variety of tissue culture
experiments. Isozymes were initially used to classify the different molecular forms
of enzymes belonging to major biochemical pathways in plants as well as animals
(Markert and Moller 1959).

5
Isozymes as Markers in Plant Breeding
The use of isozymes in plants is obviously not limited to the recognition of
variations in banding patterns in different tissues and at distinct developmental
stages, but they are an invaluable tool in plant breeding studies and evolutionary
analyses (Chiang and Kiang 1987; Doong and Kiang 1987; Quiros et al. 1987; Whitt
1987; Nevo 1990). The diversity of isozymes in nature is not only well structured in
populations, species and higher taxa, but also partly correlated with and predictable
by ecological heterogeneity (Nevo 1990). This claim may be corroborated by the
process of natural selection, which exerts a major differentiating and orienting force
at the evolutionary level (Nevo 1990). In restriction fragment length polymorphism
(RFLP) studies of wild emmer wheat (Triticum diccoccoides), a significant
correlation between RFLPs and certain isozymes was observed (Nevo 1990).
Isozymes have also been used for the "fingerprinting" of various plant species and
useful crop cultivars to help identify individual genotypes and hybrids between two
different accessions (Smith 1984, 1986; Smith and Wych 1986; Tsaftaris 1987).
Plant breeders have classified breeding techniques into three different
categories: (i) self-pollinated plants, (ii) cross-pollinated plants, and (iii) clonally
propagated plants. As mentioned earlier, the plant breeding industry has greatly
benefited from the use of isozymes as markers for the development of newer and
better varieties of cultivated plants (Tsaftaris 1987). Outcrossing rates and mating
systems have been determined for various plant species (Tsaftaris 1987), by using
isozymes. Other reports have used isozymes for quantitative estimates of mating
systems in corn (Tsaftaris 1987) and for self and cross pollination analyses (Smith
1984, 1986; Smith and Wych 1986). In the production of hybrids, the Fy generation
can be screened for the occurrence of self pollination, by using specific isozyme
markers in the parental generation (Smith 1984, 1986; Smith and Wych 1986;
Tsaftaris 1987).

6
Many of the important cultivated plants are polyploid (Tsaftaris 1987).
While most of these are allopolyploids (wheat, tobacco, cotton, sugarcane,
napiergrass, etc.), some are autopolyploids (potato, alfalfa, etc.). Polyploids have
also been artificially created by breeders, such as ryegrass and red clover which are
autopolyploids, and triticale and raphanobrassica which are allopolyploids (Tsaftaris
1987). While the ancestry of artificial polyploids is well known, isozymes are
extremely useful in combination with RFLP analyses in phylogenetic studies of
naturally occurring polyploids (Suiter 1988; Glaszmann et al. 1989; Dahleen and
Eizenga 1990). The use of isozymes also extends to the study of a wide array of
plants, since plants can be biochemically "fingerprinted" with the help of isozymes
(Smith 1984, 1986; Smith and Wych 1986; de Kochko 1987; Tostain et al. 1987;
Tsaftaris 1987; Vorsa et al. 1988; Gaur and Slinkard 1990). Isozymes have also been
widely used in genetic studies for the mapping of enzyme gene loci (Knapp and
Tagliani 1989; Vaquero 1990).
Isozymes in Tissue Culture Studies
Tissue culture studies have benefited from the use of isozymes as a tool for
various applications (Tsaftaris 1987). Zymograms of ADH from callus cultures of
wheat, rye and triticale were similar to those of their roots grown under anaerobic
conditions, and varied from those of their stems (Suseelan et al. 1982), suggesting
that the enzymes were developmentally regulated. Regenerants obtained from
tissue cultured ovules of seedless grape have been used for isozyme analysis, to
predict the polyembryonic origin of sexual crosses of the parents (Durham et al.
1989).
As documented in plants at various stages of development, so also, in tissue
culture studies it has been observed that certain isozymes show definite and
predictable changes at different stages of development in vitro. During the
differentiation of Vigna unquiculata callus tissue from an undifferentiated state into

7
tracheids and xylem vessels, De and Roy (1984) observed the presence of a new
band at the anionic end of the acid phosphatase zymogram. Peroxidase patterns
have been shown to differ appreciably between selected and non-selected lines of
rice, wherein the selected line was capable of differentiating into root and shoot
primordia, while the nonselected line was incapable of such differentiation (Abe
and Futsuhara 1989). There have been reports showing the appearance of specific
banding patterns on peroxidase and esterase zymograms that can be used to predict
the advent of embryogenesis, or to discern between embryogenic and non-
embryogenic callus cultures. Such studies have been conducted on maize (Everett et
al. 1985; Rao et al. 1990) and barley (Coppens and DeWitte 1990).
Tissue Culture Variation
The use of biochemical markers in the identification of variants from in vitro
culture has included the analysis of isozymes and specific proteins. Although
morphological and cytogenetic variations have been shown to occur in plant tissue
culture, one of the earliest studies that reported the comparative use of isozymes
was on callus derived regenerants of sugarcane (Heinz and Mee 1971). Two
cultivated lines were used in this study, one of which was a chromosomal mosaic.
The plants derived from this mosaic line revealed distinct differences in the isozyme
banding patterns of four isozyme systems. The regenerants derived from the stable
line exhibited no variation, suggesting that the variation observed may have been
caused by the instability of the explant genome. Selby and Collin (1976) analyzed
callus tissues from Allium cepa for alliinase activity and showed similar levels of
activity between the calli and normal plants, but the precursor levels in the callus
tissue were only 2-10 percent of that in the plant.
Isozymes have been especially useful in studies attempting to document
tissue culture-induced variation in a variety of plants. In potato plants regenerated

8
from tissue culture, the ADH and aspartate aminotransferase (AAT) zymograms
showed the loss of one band when compared to the parental cultivar. This variation
was believed to be caused by rearrangement of DNA sequences in tissue culture
(Allichio et al. 1987). Dahleen and Eizenga (1990) reported a variant
phosphoglucoisomerase pattern in four plants derived from a monosomic line of
Festuca anmdinacea. In addition to morphological and cytogenetic variation in
tissue culture derived plants of Botriochloa sp., Taliaferro et al. (1989) observed
changes in the banding patterns of esterase and peroxidase isozymes. The frequency
of variation at the morphological level and in the electrophoretic banding patterns
of certain seed proteins in wheat plants derived from tissue culture, was very low
(about 1%) (Maddock et al. 1985). Other reports in wheat have shown higher
frequencies of somaclonal variation at different isozyme loci (Davies et al. 1986;
Ryan and Scowcroft 1987). These changes were shown to be heritable through a
sexual cross and hence believed to be at the DNA level as opposed to epigenetic
changes.
In contrast, other studies using isozymes or proteins as markers to detect
tissue culture derived variation have shown a high degree of stability with very little
or no variation. In a population of 645 maize plants derived from tissue cultures of
immature maize embryos, Brettell et al. (1986a) reported a single plant showing an
altered pattern of alcohol dehydrogenase (ADH). In the analysis of over 550
immature embryo derived plants of wheat, Davies et al. (1986) detected only 4
euploid plants with an altered ADH pattern. Thirteen other plants with similar
irregularities were aneuploid. Ryan and Scowcroft (1987) recovered one plant, out
of a population of 149 regenerants from tissue cultured immature embryos of wheat,
that exhibited a variation in the /9-amylase isozyme pattern. It was, however,
unresolved whether the variation was inheritable by the progeny. In a population of
plants regenerated from the tissue culture of immature embryos of triticale, Jordan

9
and Larter (1985) were unable to detect any variation between parental clones and
their progeny. No variation was observed in pearl millet regenerants from cultured
immature inflorescences that were analyzed for total protein content and ADH
activity (Swedlund and Vasil 1985). In 25 protoplast derived plants of orange,
Kobayashi (1987) found no significant variations in four isozyme systems that were
tested. A population of 42 barley plants failed to show any variations in esterase
and aspartate aminotransferase banding patterns (Karp et al. 1987). The same
population had one plant with abnormal meiosis, which also produced one seed with
a variant hordein protein. In a large number of plants of red clover analyzed for the
occurrence of tissue culture variation, Wang and Holl (1988) observed stable
banding patterns for 5 different isozyme systems. Taliaferro et al. (1989) observed
identical peroxidase and esterase banding patterns in the progeny of Botriochloa sp.
derived from the in vitro culture of two lines used as explants. However, the banding
patterns of the progeny differed from that of the original explants. Bebeli et al.
(1990) reported the presence of a single individual containing a variant pattern of
40K T-secalins, from a population of over 350 regenerants derived from the culture
of immature embryos of a selfed line of rye. Zymograms of 95 tissue culture derived
regenerants of Festuca arundinacea assayed for seven isozyme systems did not show
any variation from the parental genotype (Eizenga and Dahleen 1990).
Mitochondrial DNA
Organellar DNA
All eukaryotic cells have organelles which compartmentalize the cell. Plant
cells differ from other forms of life in that they have both mitochondria and plastids
which are believed to have an endosymbiotic origin, and possess their own DNA
(Penny and O’Kelly 1991). Both of these genomes are distinct from the nuclear
genome, but efficient interaction between these three systems is absolutely
necessary for normal development of plants (Palmer 1985a; Lonsdale 1989).

10
Modern molecular research in plants therefore involves studies on mitochondrial,
plastid, and nuclear genomes.
Plant Mitochondrial DNA
The study of plant mtDNA gained impetus with the discovery that the
expression of cytoplasmic male sterility (CMS) in maize was associated with mtDNA
(Pring and Levings 1978; Laughnan and Gabay-Laughnan 1983). This discovery has
led to a better understanding of the structure, evolution and coding properties of the
mtDNAs of angiosperms (Palmer 1985a). The present study involved the use of
mtDNA as a parameter to study variation induced by in vitro culture.
MtDNA is larger and more complex than its chloroplast counterpart (Stern
and Palmer 1984b; Palmer 1985b; Lonsdale 1989). There have been various
attempts made to determine the physical size of mtDNA from many different plant
species (Leaver and Gray 1982; Lonsdale 1989).
Bailey-Serres et al. (1987) used electron microscopy to estimate the sizes of
mtDNA molecules obtained from seven species of plants belonging to diverse
families and observed a range of molecules varying in size from 1 kb to 126 kb.
Another method exemplified by Ward et al. (1981) for estimating the size of the
mitochondrial genome was to study the renaturation kinetics of mtDNA. The
authors used this technique to determine the size of mitochondrial genomes from
pea, maize and four species from the Cucurbitaceae.
Complexity of The Mitochondrial Genome.
The complexity of the mitochondrial genome has been studied using
hybridization studies and in vitro protein synthesis on isolated intact mitochondria
(Lonsdale 1989). Restriction endonuclease digests of mtDNA from a variety of
plants, when probed with radioactively labelled fragments from other genomes, have
exhibited a set of conserved sequences (Stern et al. 1983; Stern and Newton 1985;
Lonsdale 1989). Experiments using cloned DNA fragments from the mitochondrial

11
genome of Brassica campestris to probe RNA detected 24 transcripts totalling
approximately 60 kb. These results are consistent with those reported by Makaroff
and Palmer (1987). Results from RNA excess hybridization studies in cucurbits
suggest that the proportion of the mitochondrial genome transcribed varies from
20% in muskmelon to 70% in watermelon (Bendich 1985). The translational
expression and polypeptide processing of mitochondria extracted from tissues at
different stages of development show quantitative as well as qualitative differences
(Boutry et al. 1984; Newton and Walbot 1985), suggesting that these changes may be
developmentally regulated. Mitochondrial genomes also contain open reading
frames (ORFs) which are DNA sequences that may be transcribed but one cannot
assume that these are pre-mRNAs, and unassigned reading frames (URFs) which
are DNA sequences that may be transcribed and translated but the function of the
coded polypeptide is unknown (Lonsdale 1989). In addition, mitochondrial
genomes may also contain gene chimeras, nonfunctional genes and nonfunctional
transcribed sequences (Lonsdale 1989).
Repeat Elements
MtDNA has been shown to possess repeated sequences that range in size
from 0.5 kb to 14 kb in maize (Lonsdale 1989), and may exist as direct repeats or
inverted repeats. The presence of repeated sequences in an inverted orientation,
may lead to homologous recombinational events which cause sequence inversions in
the genome (Lonsdale et al. 1983, 1984; Palmer and Shields 1984; Stern and Palmer
1984a, 1986). On the other hand, if the repeated sequences are present in the same
orientation, a recombination between them would lead to the formation of smaller
circular molecules from a larger molecule.
The mitochondrial genome of plants is believed to exist as a single master
circle of DNA (Lonsdale et al. 1984; Palmer 1985a; Lonsdale 1989) and many
smaller circles that have arisen by recombinational events between direct repeats on

12
the master chromosome (Lonsdale et al. 1983, 1984; Stern and Palmer 1984a;
Falconet et al. 1984; Lonsdale 1989). The entire 218 kb mitochondrial genome of
turnip, for example, consists of three distinct circular chromosomes (Palmer and
Shields 1984). The large master circle possesses two copies of a 2 kb element as a
direct repeat, separated by 135 and 83 kb and the two smaller circles are 135 and 83
kb in size. These three circles are believed to interconvert from one form to the
other (Palmer and Shields 1984). Such repeat elements have also been reported in
normal maize mtDNA (Lonsdale et al. 1983, 1984; Lonsdale 1984; Palmer 1985a;
Lonsdale 1989), although the maize mitochondrial genome (570 kb) is much larger
than that of turnip (218 kb). The maize genome also differs from the turnip genome
in that it possesses six pairs of large repeated sequences, five of which are present as
direct repeats and hence may be recombinationally active (Lonsdale et al. 1984).
Two of these five sites are considered to be preferred sites and the majority of the
mtDNA exists as four smaller circles of 503, 253, 250 and 67 kb in addition to the
master circle of 570 kb (Lonsdale et al. 1984). Such recombinational events
between the different repeat elements could be a source of heterogeneity in the
restriction profiles of mtDNA of a single plant species (Spruill et al. 1980; Lonsdale
et al. 1981; Borck and Walbot 1982). While McNay et al. (1984) have found distinct
differences in the relative stoichiometry of mtDNA bands in the restriction profile
of tissue cultured cells of maize.
MtDNA in Tissue Culture
MtDNA has been widely used in the field of tissue culture for the analysis of
restriction profiles from somatic hybrids (Belliard et al. 1979; Nagy et al. 1981;
Galun et al. 1982; Boeshore et al. 1983, 1985; Chetrit et al. 1985; Vedel et al. 1986;
Ozias-Akins et al. 1987; Rothenberg and Hanson 1987; Tabaeizadeh et al. 1987;
Kemble et al. 1988a,b; Jourdan et al. 1989), the effect of tissue culture on the
stoichiometry of minicircular mtDNAs (Negruk et al. 1986; Shirzadegan et al. 1989),

13
supercoiled mtDNAs (Dale et al. 1981), restriction analysis (McNay et al. 1984) and
filter hybridization studies of tissue culture progeny for the detection of variation in
tissue culture (Gengenbach et al. 1981; Boeshore et al. 1985; Oro et al. 1985;
Chowdhury et al. 1988; Aubry et al. 1989; Brears et al. 1989; Shirzadegan et al. 1989;
Saleh et al. 1990). Tissue culture cells have also been studied for the presence of
unique populations or changes in the stoichiometry of the plasmid-like DNAs (Kool
et al. 1985; Negruk et al. 1986; Meints et al. 1989). Negruk et al. (1986) observed an
increase in the percentage of minicircles in suspension cultures of Vicia faba.
MtDNA from two different culture lines of a single cultivar of tobacco showed
differences in the size classes of supercoiled molecules but their restriction profiles
were almost identical (Dale et al. 1981).
The recombinational ability of the mitochondrial genome is clearly
elucidated in fusion of protoplasts of two varieties or species when the somatic
hybrids exhibit restriction profiles that differ from either fusion parent (Belliard et
al. 1979; Nagy et al. 1981; Galun et al. 1982; Boeshore et al. 1983; Boeshore et al.
1985; Chetrit et al. 1985; Vedel et al. 1986; Ozias-Akins et al. 1987; Rothenberg and
Hanson 1987; Tabaeizadeh et al. 1987; Kemble et al. 1988a,b; Jourdan et al. 1989).
It is interesting to note that the restriction patterns of individual plants regenerated
from the same fusion experiment are not identical. Such variations are not observed
in fusion products regenerated from protoplast lines with identical restriction
profiles (Nagy et al. 1981; Boeshore et al. 1983). Boeshore et al. (1983) suggested
two possible explanations for the mode of recombinations that they observed: (1)
The parental molecules of mtDNA may undergo intermolecular recombination
following protoplast fusion or (2) Separate parental molecules may assort
independently following protoplast fusion. Later work has shown that the
mitochondrial genomes of the parental clones do recombine to give unique
restriction profiles (Boeshore et al. 1985).

14
Tissue Culture Variation
From cell cultures of the Texas type cytoplasmic male sterile maize,
Gengenbach and Green (1975) recovered callus cultures resistant to the pathotoxin
of Helminthosporium mayáis and regenerated disease resistant plants that stably
transmitted the resistant trait to their sexual progeny (Gengenbach et al. 1977).
These resistant plants were also revertants to male fertility. Cultures that gave
fertile revertants from callus cultures in the absence of pathotoxin were later
reported by Brettell et al. (1980). Upon closer scrutiny of this reversion from male
sterile and disease susceptible to male fertile and disease resistant, it was discovered
that the change involved a rearrangement in the mtDNA of the male sterile cell
cultures to cause the change in phenotype (Gengenbach et al. 1981). This variation
was exclusively associated with the reversion of the CMS-T strain to fertility
(Gengenbach et al. 1981; Lonsdale et al. 1981; Umbeck and Gengenbach 1983;
Fauron et al. 1987; Wise et al. 1987: review Pring and Lonsdale 1989; Levings 1990).
It has now been documented that a partial or complete loss of the T-urf\3
mitochondrial gene or its disruption caused by a frame shift causes a reversal to
male-fertile phenotype in the CMS-T type cytoplasm of maize (Rottmann et al.
1987; Wise et al. 1987). Such a rearrangement has been observed only in tissue
cultured cells, providing direct evidence to the ability of in vitro cultures to give rise
to variation. It is believed that the T-urfl3 gene produces a polypeptide that acts as
a receptor for the pathotoxin molecules (Dewey et al. 1987, 1988).
Recent reports also show the presence of variation derived in vitro in sugar
beet, wheat and Brassica campestris. The restriction profile of B. campestris showed
variations caused by rearrangements which were at least two inversions and a large
duplication. The native plant tissue, however, shows the presence of the rearranged
molecules at a very low level, hence they appear to be sorted out and amplified in
tissue culture (Shirzadegan et al. 1989). The restriction profile of mtDNA from

15
maize tissue cultures showed changes in the relative stoichiometry of bands in the
restriction profile, although no differences were observed in the restriction profiles
(McNay et al. 1984). Wilson et al (1984) and Chourey et al. (1986) have reported a
high degree of variation in specific regions of the mitochondrial genome of sorghum
and maize respectively. Tissue cultures of CMS varieties of sugar beet showed a
single regenerant with a rearranged mtDNA pattern, detected by hybridization with
cosmid clones (Brears et al. 1989). Callus cultures of wheat were shown to exhibit a
different mtDNA pattern in non-embryogenic cultures when compared to
embryogenic cultures (Hartmann et al. 1987). In callus cultures obtained from
immature embryos of wheat, Rode et al. (1987) reported extensive changes in
mtDNA corresponding with the loss of a fraction of the mitochondrial genome.
Hartmann et al. (1989) have reported the occurence of unique organization of the
mitochondrial genome in plants regenerated from the callus cultures of wheat. The
mtDNA profile in all plants regenerated from short-term cultures of wheat except
one appeared to resemble either that of the parent plant or that of the embryogenic
cultures. However, all plants except for one regenerated from long-term cultures
exhibited a mitochondrial genome organization similar to that of the long-term non-
embryogenic cultures (Hartmann et al. 1989). Similar variations have also been
reported in the mtDNA from albino cultures and plants regenerated from anther
cultures of wheat (Aubry et al. 1989). Chowdhury et al. (1988) reported variation in
the mtDNA organization of long term cell cultures of rice when hybridized with
mitochondrial gene clones. In another case involving the use of mtDNA, Kemble
and Shepard (1984) reported the appearance of low molecular weight DNA in
addition to a sequence alteration in the mitochondrial genome of potato plants
regenerated from protoplasts.
In tobacco, Dale et al. (1981) observed differences in the stoichiometry of
different supercoiled molecules but practically identical restriction patterns of

16
mtDNA from two culture lines of a single cultivar. Breiman et al. (1987a) observed
a complete absence of variation in DNA-DNA hybridization patterns of total DNA
blots of barley probed with mitochondrial genes from maize and wheat. In a ten
year old cell suspension culture of carrot cells, Matthews and DeBonte (1985)
reported the complete lack of variation in the restriction patterns of mtDNA. A
population of Brassica napus derived from protoplasts was shown to harbor no
variations in either mtDNA or cpDNA (Kemble et al. 1988a). In a recent study, 3
month old callus cultures, 2 month old suspension cultures, a totipotent suspension
and 19 month suspension cultures of rice, had identical mtDNA restriction profiles.
The same study reported, however, that a 30 month old suspension showed a
different restriction profile (Saleh et al. 1990).
Chloroplast DNA
Plants and algae are known to possess a unique class of organelles which are
collectively or individually called plastids. These include amyloplasts, chloroplasts,
chromoplasts, elaioplasts, etioplasts and proplastids. Proplastids are believed to be
the precursors for most of the plastid types. Chloroplasts are responsible for the all
important process of photosynthesis. They impart a green color and an autotrophic
mode of life to the organisms that possess them. The fact that chloroplasts are
pigmented and larger than mitochondria probably aroused the curiosity of the early
plant scientists, leading to the elucidation of their role in photosynthesis. This
discovery generated obvious interest among scientists, and hence it is logical that, in
plants, chloroplasts have been studied as organelles for a longer time when
compared to mitochondria (Palmer 1985a). A large volume of the research on
chloroplasts has been conducted on green algae, however, this review is limited to
the study of plastids in higher plants.

17
Endosvmbiotic Origin of Chloroplasts
Chloroplasts of algae and higher plants with one known exception are all
known to contain DNA, usually in multiple copies (Possingham and Lawerence
1983). The single exception is the green alga Acetabularia\ chloroplasts in many of
its species do not contain any detectable DNA (Coleman 1979; Luttke and Bonnoto
1982). There seems to be little doubt if any that, like mitochondria, chloroplasts
have an endosymbiotic origin from a prokaryotic precursor (Palmer 1985a,b, 1987;
Palmer et al. 1988; Penny and O’Kelly 1991). This assumption is based on the fact
that rRNA genes in the plastid genomes of most plants and algae have a striking
resemblance to those of the eubacterium Escherischia coli (Gray 1983; Spencer et al.
1984; Dale et al. 1984; Palmer 1985a). In the light of this information, plastid and
eubacterial genomes almost certainly had a more recent common ancestry than
plastid and nuclear genomes. Chloroplast DNA (cpDNA) sequences from both
algae as well as flowering plants share a lot of homology with cyanobacteria. This
provides almost irrefutable evidence that plastids evolved by the endosymbiotic
association of an autotrophic prokaryote with a primitive eukaryote (Gray and
Doolittle 1982; Gray 1983; Palmer 1985a,b, 1987; Palmer et al. 1988).
Consequently, the present day occurrence of plastid and nuclear genomes in a single
cell appears to be the result of horizontal evolution, i.e. endosymbiosis (Palmer
1985a). The genome of all the different plastid types within a single plant, according
to all available data, appears to be identical (Palmer 1987).
Interaction Between Organelles
The genomic size of the endosymbiont was probably reduced by the transfer
of most of its genes to the host nucleus while retaining only those genes that were
vital for the proper functioning of the organelle. This may be endorsed by the fact
that a large number of structural polypeptides for both mitochondria as well as
plastids are encoded by the nucleus (Lonsdale 1989). Such a transfer of genetic

18
material thus guarantees interaction between the nucleus and plastids, whereby
polypeptides coded for by the nucleus are synthesized in the cytoplasm and
dispatched to the plastids with an attached target polypeptide (Lonsdale 1989).
Promiscuous transfer of DNA from the plastid genome to the nuclear genome has
been observed in spinach on a very large scale, where each haploid nuclear genome
has been shown to possess the equivalent of up to five plastid genomes (Scott and
Timmis 1984). Such a shift of genetic information from the organelle to the nucleus
is not without mishap. It is quite probable that a "plastid gene" could acquire a
mitochondrial targeting sequence as observed in the mitochondria of the alga
Ochromonas danica. In this alga, the small subunit of RuBPCase is found in the
mitochondria, although it is possible that the mitochondria possess an entire or
partially active copy of the small subunit gene (Lonsdale 1989). Stern and Palmer
(1984b) have documented several homologies between the chloroplast and
mitochondrial genomes of several plant species at the inter- and intraspecific levels.
Inheritance of Plastids
Most angiosperms show a maternal inheritance of organelles, while few show
paternal inheritance and yet others show a biparental inheritance. Amongst
gymnosperms, conifers almost exclusively exhibit a uniparental-paternal pattern of
plastid inheritance as documented by microscopic (Whatley 1982) and molecular
studies (Neale and Sederoff 1988). The paternal plastids are observed to enter the
egg cell while the maternal plastids degenerate (Whatley 1982). Paternal
inheritance of chloroplasts has also been confirmed by restriction fragment length
polymorphisms (RFLPs) on the cpDNA of the parents and their sexual progeny in
many conifers (Neale et al. 1986; Szmidt et al. 1987,1988; Wagner et al. 1987; Neale
and Sederoff 1989). In some sexual hybrids between two larch species, biparental as
well as maternal inheritance of plastids was observed (Szmidt et al. 1987), while in
crosses between Pinus rígida and P. taeda the inheritance of plastids is paternal but

19
mitochondria are maternally inherited (Neale and Sederoff 1989). In redwood
{Sequoia semipervirens), the inheritance of mitochondria as well as plastids is
paternal (Neale et al. 1989).
The pattern of plastid transmission in angiosperms is, as mentioned earlier,
largely maternal (Palmer 1987; Palmer et al. 1988). However, biparental
inheritance has been implicated in many angiosperm species (Corriveau and
Coleman 1988). Four types of plastid inheritance are believed to exist in
angiosperms : (1) In the Lycopersicon type, the plastids in the microspore selectively
segregate to the vegetative cell. Nevertheless, in Nicotiana, paternal inheritance of
plastids has been shown to occur at very low frequencies (Medgyesy et al. 1986), (2)
In the Solarium type, plastids are equally divided between the generative cell and
the vegetative cell of the microspore but the plastids in the generative cell are
selectively lost (or eliminated), hence the sperm cells are devoid of plastids, (3) In
the Triticum type, which is found in most grasses, plastids are found in the
generative cell as well as the vegetative cell. In spite of that, when the sperm enters
the egg cell, enucleated cytoplasmic bodies containing plastids and mitochondria are
left outside (Mogensen and Rusche 1985; Mogensen 1988), and (4) In the
Pelargonium type, plastid inheritance is biparental, although, in alfalfa (Medicago
sativa) the paternally derived plastids predominate and in Oenothera the maternally
derived plastids are prevalent. This may suggest the existence of additional
mechanisms of influencing plastid inheritance (Lee et al. 1988, 1989; Smith
1988,1989). In the genus Brassica, the relationships between different species and
the ancestry of certain amphidiploids have been determined by identifying the
cytoplasmic type of the maternal parent (Erickson et al. 1983; Palmer et al. 1983,
1988; Palmer 1987). The chloroplast genome of B. napus, however, is believed to
have evolved by introgression from some unidentified species (Palmer et al. 1983,
1988; Palmer 1987).

20
CpDNA in Interspecific Hybrids
In interspecific hybrids, or in cases of biparental inheritance, it is seen that
the two chloroplast types neither fuse nor do their genomes recombine (Scowcroft
and Larkin 1981; Kemble and Shepard 1984; Palmer 1987; Palmer et al. 1988). As
discussed earlier, in sexual inter- or intraspecific hybrids, one of the parental plastid
types is usually selected against. Somatic hybrids created by the fusion of
protoplasts from two different plant species also exhibit an independent assortment
of chloroplasts from the two parental species (Morgan and Maliga 1987), wherein
the chloroplasts do not fuse or produce any recombination between the two
genomes. In interspecific somatic hybrids between two species of Daucus
(Matthews and Widholm 1985), Petunia (Clark et al. 1986), Medicago (D’Hont et al.
1987) and Brassica (Kemble et al. 1988a), the hybrids showed inheritance of the
plastid genome from only one of the parental species. Thanh et al.. (1988) have
reported the intergeneric transfer of chloroplasts from Salpiglossis sinuata to the
cytoplasm of Nicotiana tabacum. The donor cytoplasm was irradiated before fusion
and appropriate streptomycin-resistant donor or light-sensitive recipient mutants
were used.
The Chloroplast Genome
The chloroplast genome of all land plants is relatively uniform in size (120-
217 kb) when compared to the mitochondrial genome (Palmer et al. 1988). Its
complexity varies between 110-150 kb, because most of the variation in size is
observed to arise from a few major expansions or contractions in the large inverted
repeat (Palmer 1985a; Palmer et al. 1988). The total size variation of angiosperm
cpDNAs may be misleading; the lower extreme of this size variation occurs in a
single group of legumes which have lost one copy of the large two copy inverted
repeat (Palmer 1985a), while the variation in genome size of the upper extreme
range of 55 kb is observed only in two species so far, i.e. Spirodela oligorrhiza (Van

21
Ee et al. 1980) and Pelargonium hortorum (Palmer 1985a). The variation in size of
cpDNAs among most of the angiosperms observed falls within the relatively narrow
range between 135-160 kb, when compared to the mitochondrial genome, with most
plants having only a 20-30 kb inverted repeat. The increase in the size of the
Pelargonium cpDNA is attributed to an enlarged inverted repeat which is over 75 kb
in size (Palmer 1985a).
Evolution in the chloroplast genome has been shown to occur at a very
conservative rate of about 1.5 X 10'^ substitutions per site per year (Zurawski and
Clegg 1987). In comparison, the rate of silent substitutions in cpDNA may be as
much as a hundred times lower than that observed in animal mtDNA and two to
three times lower than nuclear DNA, but it is three to four times higher than in
plant mitochondrial genes (Zurawski et al. 1984; Palmer 1987; Palmer et al. 1988).
One striking difference between cpDNA and plant mtDNA is that, plastid DNA
completely lacks any minicircular or plasmid DNAs that are characteristic of plant
mtDNAs (Palmer 1985a, 1987).
Any change in the complexity of a genome is wrought by the addition of new
sequences or the deletion of existing ones (Palmer 1987). It seems highly unlikely
that such changes in complexity occur by the gradual drift of repeated elements until
they effectively become single copy (Palmer 1987). The infiltration of cpDNA
sequences into mitochondria has been exhibited in extremely diverse species like
maize (Zea mays), cauliflower (Brassica olerácea), mung bean (Phaseolus aureus),
spinach (Spinacia olerácea) and evening primrose (Oenothera berteriana) (Carlson et
al. 1986a,b; Marechal et al. 1987; Schuster and Brennicke 1987, 1988; Nugent and
Palmer 1988; review Lonsdale 1989). On the other hand, very rarely has the
chloroplast genome been shown to possess genes from any extraneous sources
(Palmer 1987; Schuster and Brennicke 1988). In cpDNAs compared from hundreds
of plant species, there are only two significantly large sized mutations that have been

22
observed, i.e. the addition of a 7-9 kb sequence in Nicotiana acuminata (Shen et al.
1982) and the addition or deletion of a 13 kb sequence in a collection of Linum
species (Coates and Cullis 1987). There are other mutations that have been
reported which are significantly smaller in size and less frequent in occurrence.
These involve changes ranging from 50 to 1200 bp (Gordon et al. 1982; Bowman et
al. 1983; Salts et al. 1984; Palmer et al. 1985; Palmer 1987). The maximum number
of mutations occurring in cpDNA usually take place either as additions or deletions
involving 1 to 10 bp, probably according to the "slippage-mispairing" model
(Takaiwa and Sugiura 1982; Zurawski et al. 1984; Palmer 1987). One interesting
fact is that cpDNAs from algae as well as higher plants completely lack any
modified bases such as 5-methylcytosine (Bohnert et al. 1982; Loiseau and Dalmon
1983; Palmer 1985a).
The Inverted Repeat of the Plastid Genome
The plastid genome of a majority of land plants has an extremely similar
arrangement of genes (Palmer 1987). The gene order in the cpDNA of spinach is
believed to be similar to that of the ancestral vascular plant (Palmer and Stein 1986)
and is also representative of many of the angiosperm species studied (Fluhr and
Edelman 1981; Palmer and Thompson 1982; Palmer et al. 1983). In all the
angiosperm families except in one section of subfamily Papilionoideae of the legume
family Fabaceae, the plastid genome has a characteristic inverted repeat (Chu and
Tewari 1982). This inverted repeat is always positioned asymmetrically and divides
the entire genome into a large single copy part and a small single copy part (Chu
and Tewari 1982; Palmer 1985b, 1987). As mentioned earlier, this repeat is also
responsible for the variation in size of the chloroplast genome amongst various plant
species. Although the inverted repeat can vary up to six times in size, it always
contains an entire set of ribosomal RNA genes (Chu and Tewari 1982). The two

23
arms of the inverted repeat are identical in an individual, and all mutations in the
repeat elements are symmetrical (Palmer 1985a, 1987).
Considering the static nature of the plastid genomes and their arrangement
of genes, Palmer (1987) has elucidated six generalizations regarding internal
rearrangements: (1) all well characterized rearrangements are inversions, (2) the
cases of rearrangement are usually simple and involve only one or two discrete
inversions, (3) in cases where the inverted repeat is greatly altered, e.g. Pelargonium
and in legumes that lack the inverted repeat, the rearrangements are extreme, (4)
the flanking regions of the best known inversions are located within largely
noncoding regions, (5) some of the highly rearranged genomes have families of
somewhat large dispersed repeats of several hundred bp, and (6) rearrangements
are not known to disrupt the functions of groups of genes that are transcriptionally
linked. The lack of disruption observed may be a direct consequence of the fact that
plastid genomes have a high density of genetic information. Any disruption of these
genes by the insertion of foreign sequences would probably cause lethal mutations
which would obviously be selected against (Lonsdale 1989). This contrasts sharply
with nuclear and mtDNA where the functional genes are widely dispersed and
inserted sequences stand a better chance of being retained (Lonsdale 1989).
Variation in the Plastid Genome
The inherent nature of cpDNA, whereby both the structural and sequence
fidelity are maintained, strongly limit its use as a marker for variability in studies
involving large populations (Palmer 1985a, 1987; Palmer et al. 1988). However,
minor variations have been detected at specific and intraspecific levels as in Lupinas
texensis (Banks and Birky 1985), Brassica nigra (Palmer et al. 1983) and Lycopersicon
peruvianum (Palmer and Zamir 1982). The lack of widespread variation, or its
presence at very low levels, is an excellent tool for phylogenetic studies and has been
used in several studies using RFLP techniques (Palmer and Zamir 1982; Palmer et

24
al. 1983, 1985a; Banks and Birky 1985; Sytsma and Gottlieb 1986a,b). There have
been numerous other studies involving a wide range of plants using cpDNA for
cladistic analysis and the subsequent construction of phylogenetic trees (Palmer
1985a, 1987; Palmer et al. 1988).
Although the cpDNA evolves very slowly, there are several reported
examples of base substitutions and changes in genome structure. Zurawski et al.
(1984) and Zurawski et al. (1984) conclude that most nucleotide substitutions occur
as silent changes in the third position of codons and missense substitutions are
clustered at the ends of genes. As mentioned earlier, all changes in the inverted
repeat occur symmetrically (Palmer 1987). The absence of the inverted repeat in
the chloroplast genome is believed to have a profound effect, causing the genome to
be prone to more frequent rearrangements as seen in Pisum and Trifolium (Palmer
and Thompson 1982; Palmer 1985a,b; Palmer et al. 1987).
Use of the Plastid Genome as a Marker
The plastid genome has been used in comparative analyses as a marker for
genetic variation using at least three different methods, as outlined by Palmer
(1987). Purified samples of cpDNA, from individuals to be compared, may be
subjected to a restriction analysis. In cases with complex restriction patterns,
restriction maps of the genome using several enzymes may be used for a
comparative analysis. A certain part of the genome may also be used for sequencing
studies to study a defined segment of the genome.
Plastid DNA in Tissue Culture
Recombination between the genomes of two different chloroplast types has
been observed in a somatic hybrid of Nicotiana tabacum and N. plumbaginifolia
(Medgyesey et al. 1985). The two chloroplast types were selectable on either
streptomycin or lincomycin, while the somatic hybrid progeny showed recombinant

25
cpDNA patterns. The plastid genome of the hybrid was believed to contain at least
six recombination sites (Medgyesey et al. 1985).
There are fewer reports concerning the use of the chloroplast genome for the
purpose of identifying variation in tissue culture. This may be due to the conserved
nature of the plastid genome (Lonsdale 1984, 1989; Chowdhury et al. 1988). Day
and Ellis (1984, 1985) reported that plants regenerated from anther culture of wheat
lacked pigmentation and linked this to deletions in the cpDNA of the regenerants.
A study on a population of alfalfa regenerants from protoplasts revealed the
occurrence of a chloroplast genome that varied from the parental type (Rose et al.
1986). There was an apparent selection towards two types of banding patterns, in
regenerated protoclones of Medicago sativa L., that were different from the parental
type. Twenty-two of the twenty-three clones observed had either one or the other of
the variant banding patterns observed. Kemble and Shepard (1984) reported the
absence of any variation in a population of potato plants regenerated from leaf
mesophyll protoplasts. Matthews and DeBonte (1985) also reported a complete
lack of variation in the cpDNA restriction profiles of a 10 year old carrot cell
suspension.
Nuclear DNA
Restriction Fragment Length Polymorphisms
It is well known that the genetic complement of all species has evolved by
selection, and in the process the DNA from related species and different individuals
from the same species have accumulated minor aberrations (mutations) that have
become part of the genome. Variations like single base substitutions have, in recent
times, been found to be extremely useful as genetic markers present in close
association with certain genes of interest when they cause unique restriction profiles
among different individuals of a single species. Recently there have been many
reports involving the use of RFLPs as markers in plant breeding (Clarke et al. 1989;

26
Smith et al. 1989), fingerprinting of genotypes (Appels and Dvorák 1982; May and
Appels 1987; Smith et al 1989; Riedel et al. 1990; Sano and Sano 1990),
phylogenetic analysis (Appels and Dvorák 1982; Hintz et al. 1989), chromosome
linkage analysis (Landry et al. 1987; Sharp et al. 1989) and analysis of regenerants
derived from tissue cultures (Landsmann and Uhrig 1985; Brettell et al. 1986a,b;
Breiman et al. 1987a,b, 1989; Karp et al. 1987; Rode et al. 1987; Zheng et al. 1987;
Benslimane et al. 1988; Müller et al. 1990).
Ribosomal DNA in Plant Breeding and Tissue Culture
Examination of nuclear ribosomal DNA (rDNA) is another aspect of
molecular analysis for the detection of tissue culture derived variation. Unlike
mtDNA or cpDNA, restricted nuclear DNA does not yield a profile that can be used
for comparative purposes. Therefore, Southern blots of nuclear DNA cut with the
restriction enzyme of choice are probed with cloned DNA fragments to provide
autoradiograms in order to accurately estimate changes (Southern 1975).
Landsmann and Uhrig (1985) reported two plants from a population of twelve to
possess a variant Southern-hybridization pattern of nuclear DNA when probed with
a ribosomal DNA (rDNA) clone. Zheng et al. (1987) observed an amplification of
some highly repeated nuclear DNA sequences in rice suspension cultures. rDNA
has also been used as a probe to detect variation at the nuclear DNA level in
dihaploid plants of wheat, derived from tissue culture (Rode et al. 1987a,b;
Benslimane et al. 1988). The nucleolar organizer region (Nor) consisting of rDNA
genes has been used as a marker in several cereal crop plants. In triticale, an
analysis of the Nor loci located on chromosomes IB, 6B and 1R revealed that one
out of six phenotypes tested had a marked reduction in the number of rDNA units
present at the locus (Brettell et al. 1986b). Such a discrepancy was also detected in a
study involving plants regenerated from wheat callus. One out of three genotypes
tested in this study, showed a similar reduction at the Nor loci (Breiman et al.

27
1987a). In an independent report, wild barley (Hordeum spontaneum) plants
derived from immature embryo-derived callus were also observed to possess such a
reduction in the intergenic spacers of the rDNA (Breiman et al. 1987b). In a later
publication, however, Breiman et al. (1989) expressed serious doubts about the
ability of tissue culture to cause such variations at the Nor loci. These doubts were
expressed when the parental lines were observed to possess similar variations at the
Nor loci at a very low frequency. Karp et al. (1987) reported no variation at the Nor
loci in a population of forty-two barley plants regenerated from cultured immature
embryos.
DNA Methvlation in Tissue Culture
Methylation of DNA bases is believed to play an important role in the
expression of genes vital to plant development. The methylation of cytidine, and on
occasion adenine bases, is believed to regulate the expression of genes during plant
and animal development (Jones and Taylor 1980; Theiss and Follmann 1980; Theiss
et al. 1987). In a study involving cultured cells of soybean, a restriction analysis of
5S RNA genes revealed that the DNA from the explant material and long-term
cultures was highly resistant to digestion by the enzyme Hpall which recognizes
methylated cytosine bases in the sequence CCGG. DNA extracted from freshly
cultured tissues, however, was easily restricted by Hpall and its isoschizomer Mspl,
which is sensitive to methylation of the cytosine bases in the same sequence. Brown
(1989) attempted to use a 5-methylcytosine analog 5-Azacytidine to study its effect
on the methylation and possible promotion of protoplast division in maize and
tobacco cell cultures, but failed to detect any correlation. An analysis of
phenotypically variant regenerants of maize from cultured immature embryos
revealed that housekeeping as well as structural genes had significantly altered
levels of methylation (Brown 1989). The author suggested that such changes may
play a role in the variation of plants derived from tissue culture. Müller et al. (1990)

28
found a close correlation between tissue culture-derived regenerants of rice that
showed rearrangements in their DNA and methylation of the genome.

CHAPTER 3
REGENERATION OF PLANTS FROM SOMATIC EMBRYOS OF
PENNISETUM PURPUREUM K. SCHUM. BY TISSUE CULTURE OF
IMMATURE LEAF SEGMENTS
Introduction
Tissue culture is an established procedure for obtaining clonal plant
populations. Many diverse plant species have been successfully initiated into culture
using different tissues as explants (Vasil 1986). However, the most important group
of plant species induced into culture is without doubt the cereals and grasses (Vasil
and Vasil 1986). While plants regenerated from in vitro culture are expected to be
identical clones of the explant, it is also known that a certain amount of variation
arises in cell cultures and plants obtained from in vitro culture (Heinz and Mee
1971; Edallo et al. 1981; McCoy et al. 1982; Swedlund and Vasil 1985). The term
"somaclonal variation" was introduced by Larkin and Scowcroft (1981) to
characterize variation observed in tissue culture and included ah types of
morphological, biochemical, cytogenetic and molecular variation.
Variation obtained from tissue culture derived plants has been considered
potentially beneficial to plant breeders, in the hope of recovering unique and
commercially profitable cultivars (Larkin and Scowcroft 1981). There have been
many conflicting reports, however, on the ability of the process of tissue culture to
cause such widespread useful variation. It is generally argued that at least a part of
the variation observed in cell cultures and populations derived from them is a result
of preexisting variation in the differentiated cells of the explant which may be
29

30
amplified or selected for in vitro (D’Amato 1985; Swedlund and Vasil 1985; Vasil
1988; Morrish et al. 1990). A majority of the variation obtained in vitro is not novel
and is very similar in range to the variation resulting from mutations in sexual
crosses. Furthermore, a great deal of the variation obtained in vitro is epigenetic in
nature and is not transmitted to sexual progeny. It is thus of no interest to the plant
breeder. It is, therefore, not surprising that there is not a single example of any
important variety of a major crop species developed as a variant from tissue culture,
which is grown commercially anywhere in the world (Vasil 1990).
There are reports of the genetic stability of long and short term cell and
callus cultures as well as plants regenerated from them (Edallo et al. 1981; Hanna et
al. 1984; Karp and Maddock 1984; Swedlund and Vasil 1985; Maddock and Semple
1986; Binarová and Dolezel 1988). The ability of the tissue culture process to
perpetuate and amplify preexisting variations in the explant has been amply
demonstrated in Pennisetum glaucwn (Morrish et al. 1990). Although many studies
have shown the occurrence of chromosomal aberrations in cell and callus cultures,
there appears to be a definite exclusion of such variants in the formation of somatic
embryos and the plants regenerated from them (Vasil 1988; Hanna et al. 1984; Karp
and Maddock 1984; Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986;
Cavallini et al. 1987; Gould 1986; Kobayashi 1987; Feher et al. 1989; Gmitter et al.
1991). Such genetic and chromosomal fidelity in somatic embryos is useful,
especially in light of the fact that tissue culture is an invaluable tool in modern
biotechnology to provide uniform and stable transformed plants.
This study was undertaken to examine a population derived from somatic
embryos of a single clone of Pennisetum purpureum (L) K. Schum., by the in vitro
culture of young leaves (Haydu and Vasil 1981). Since morphological variations can
be epigenetic in nature, the only avenues used to examine the regenerants were
biochemical analyses at the isozyme level and molecular analyses at the cytoplasmic

31
and nuclear genome levels. The decision to use somatic tissue from a single clone
as explant material was made to minimize the introduction of any variation inherent
to the tissue.
Materials and Methods
Callus Initiation and Maintenance
Actively growing shoots of P. purpureum were collected from field grown
plants (field accession number PP10). Approximately 75 mm. of the proximal,
tightly coiled innermost 5-6 leaves were used for initiation of callus, after surface
sterilization by wiping with 95% ethanol. Leaf segments 1-2 mm long, were placed
on MS medium (Murashige and Skoog 1962) supplemented with 0.5 mg/1 2,4-
dichlorophenoxyacetic acid (2,4-D), 0.5 mg/1 6-benzylaminopurine (BAP), 1.0 mg/1
a-naphthalene acetic acid (NAA) and 50 ml/1 coconut milk (Haydu and Vasil 1981),
solidified using 2 g/1 Gelrite (Scott Laboratories Inc., Fiskeville, RI). Cultures were
maintained at 27°C in the absence of light. After approximately 3 weeks, the
explants yielded embryogenic and non-embryogenic callus. The compact
embryogenic callus was carefully selected and subcultured onto similar medium and
maintained by routine subculture at 3 week intervals.
Embryogenic callus was also initiated from approximately 1 cm long
immature inflorescences of P. purpureum. The culture conditions and medium used
were identical to those used for the culture of immature leaf segments.
Plant Regeneration
For the regeneration of plants, embryogenic calli from immature leaf
explants as well as immature inflorescence explants were placed on MS medium
supplemented by 0.5 mg/1 NAA and 1.0 mg/1 BAP. These cultures were transferred
to an illuminated growth chamber at 27°C with a 16 hour light cycle. After 4 weeks,
the plants were transferred to the same medium in tubes ( 150 mm L. X 25 mm
dia.) and thus maintained for 3 weeks to allow for root and shoot elongation.

32
Plantlets were then transferred to soil (4 parts Metromix 300 [Grace Horticultural
Products, Cambridge, MA] to 1 part Perlite [Chemrock Industries]) in Conetainers
(Ray Leach Conetainer Nursery, Canby, OR) and maintained in a closed
environment with a humidifier, in a 16 hour light cycle, for 4 days before being
transferred to the greenhouse. Plantlets were transplanted into successively larger
pots from the Conetainers, before transfer to the field. Regeneration procedures
from embryogenic calli were initiated 3, 6, 12, 18 and 24 weeks after the initiation of
cultures. The plants regenerated from leaf explants were identified by their callus
pedigree (e.g. Cp C2, C3, ...etc.). Individual regenerant plants within each pedigree
were assigned ascending numbers (e.g. Rp R2, R3, ...etc.). A total of 57 plants
were obtained from 11 callus pedigrees. A single pedigree established from the
inflorescence explant was prefixed Inf. and the regenerants were assigned similar
numbers to those obtained from leaf callus. Six plants were obtained from the
pedigree established from embryogenic callus of immature inflorescence segments.
A single clump of the parental clone PP10 was also transferred to the same
plot along with the 62 regenerants from both the pedigrees to expose all the plants
to uniform field conditions.
Results
Embryogenic calli obtained were white and compact (Fig. 3.1). Roots and
shoots were formed in the regeneration medium (Fig. 3.2). The plants were
transferred to the field after a well established root and shoot system were achieved
in pots (Fig. 3.3). Growth of all plants in the field was uniform (Fig. 3.4). Three
plantlets from the C] line, and one plantlet each from the C5 and Inf3 lines failed to
survive the transition from the regeneration medium to soil. Some callus pedigrees
yielded only a single regenerant while others provided as many as ten regenerants.
The plants that made a successful transition from the greenhouse to the field
showed uniform growth and did not show any obvious phenotypic differences.

33
Rajasekaran et al. (1986) have shown the morphological, cytological and
physiological uniformity of a similar population, hence, no specific measurements
were made at these levels. The entire population was subjected to a biochemical
analysis described in Chapter 2, and individuals selected at random were analyzed
using molecular techniques detailed in Chapters 3, 4 and 5.
Discussion
Regeneration of plants from in vitro cultures is essentially a mitotic process,
hence eliminating variation caused by meiotic recombination. The use of different
clones, or immature embryos as explants can introduce existing variation between
individuals into culture and the population thus obtained may lack uniformity. Such
variation can be minimized by the use of inbred lines for explant tissue. The most
productive variety from such an analysis may be used for the establishment of a
population. Another simple method of obtaining a uniform population of
regenerants is, as described here, the use of a single clone for the establishment of
embryogenic callus cultures. The use of a single clone for the production of a tissue-
culture derived population of regenerants also helps to maintain and identify the
lineage of the regenerants.
Somatic embryos have been shown to arise from single cells (Vasil and Vasil
1982). The absence of variation in plants obtained from embryogenic cultures is
considered to be due to the selection of chromosomally stable cells in the formation
of somatic embryos (Vasil 1988; Hanna et al. 1984; Karp and Maddock 1984;
Swedlund and Vasil 1985; Singh 1986; Rajasekaran et al. 1986; Gould 1986; Feher et
al. 1989; Gmitter et al. 1991). Adventitious meristems in organogenic calli, on the
other hand, are multicellular in origin and give rise to chimeric plants (D’Amato
1978), which are undesirable.

Fig. 3.1 Compact embryogenic callus
obtained from the culture of young leaves
of P. purpureum.
Fig. 3.2 Differentiation of plantlets
from embryogenic calli of P.
purpureum, placed on regeneration
medium.
Fig. 3.3 P. purpureum plants regenerated
from embryogenic calli, ready for transfer
to the field.
Fig. 3.4 Tissue culture-derived P.
purpureum plants in the field.

35

CHAPTER 4
BIOCHEMICAL ANALYSIS OF A POPULATION OF PENNISETUM
PURPUREUM K. SCHUM. DERIVED FROM TISSUE CULTURE USING,
ISOZYMES AS MARKERS.
Introduction
Isozymes are defined as multiple forms of an enzyme with similar or identical
substrate specificity occurring within the same organism (Markert and Moller 1959).
Most organisms may exhibit two principal alterations in metabolic activity within
their cells. These changes can be classified as quantitative and qualitative changes
in protein (enzymatic) activity (Scandalios 1974). Increases in enzyme activity may
be due to de novo synthesis of the enzyme molecules or the activation of an
existing enzyme precursor, while qualitative variations in enzyme activity may be the
result of changes in the immediate environment of the cell or tissue.
Three different classes of isozymes have been recognized: a) those that are
distinctly different molecules and presumed to arise from different genetic loci, b)
those that evolve from secondary alterations in the structure of a single polypeptide
species which may also be in vitro artifacts or the binding of different co-factors to a
single polypeptide (Scandalios 1974), and c) those arising as a result of a gene
mutation or recombinational events in the gene that codes for the enzyme molecule.
A zymogram is the stained representation of enzyme activity usually visualized on a
starch or polyacrylamide gel. Any alteration in the molecular structure of the
enzyme can easily be detected on a zymogram as a change in migration of the
molecule which is represented as a single band. Isozymes can, therefore, provide an
accurate picture of any biochemical differences that may exist between separate
individuals.
36

37
Isozymes are ideal for use as markers in tissue culture because of : i) their
ease of detection, ii) the abundance of naturally occurring variant molecules of
enzymes in most populations, iii) their applicability to small amounts of tissue and
minimum sample preparation due to the use of crude extracts, and iv) the fact that
in most cases the marker is expressed in the undifferentiated state of cell culture.
Native gel electrophoresis is used for the activity staining of isozymes. In this
technique, the proteins are separated on the basis of size and charge. One
limitation of this technique is that it does not discriminate between molecules which
may have the same net charge due to a single substitution but similar catalytic
activities.
Zymograms have been used to illustrate unique banding patterns of various
isozyme systems and elucidate differences in embryogenic and non-embryogenic
tissues and callus cultures (Abe and Futsuhara 1989; Coppens and Gillis 1987; Rao
et al. 1990; Everett et al. 1985; Coppens and Dewitte 1990). Isozyme analyses have
been carried out to study different types of callus tissue (Suseelan and Bhatia 1982),
differentiation in callus tissue (De and Roy 1984) and to study exo-isozymes in the
nutrient medium of suspension cultures (Berger et al. 1988).
The most common use for the isozyme technique in tissue culture studies is
the identification of variation that may occur in vitro. In the belief that isozymes are
excellent indicators of biochemical variation, many investigators have used
zymograms to evaluate such variation (Maddock et al. 1985; Taliaferro et al. 1989;
Ryan and Scowcroft 1987; Allicchio et al. 1987).
This study involves the biochemical analysis of 57 leaf tissue derived
regenerants of Pennisetum purpurewn K. Schum. (Napiergrass). These regenerants
were analyzed for the activity of 13 different isozymes. Isozymes were selected
based on the involvement of each enzyme in diverse and major metabolic pathways
and the availability of staining techniques.

38
Materials and Methods
Starch and Polyacrylamide
Polyacrylamide as well as starch gel techniques were used in this study for the
analysis of isozymes. The drawback of the polyacrylamide technique was the
amount of sample processing that was required. Part of the activity of the enzymes
was lost during this period. The advantage of this technique was the excellent
resolution of the isozyme bands upon staining for activity. Starch gels needed a
minimal amount of sample processing and the extracts could be stored in the form
of wicks at -80°C. The resolution of isozyme patterns on starch gels was good.
Sample Processing
Collection of Plant Material.
Leaf material from field grown plants was used for isozyme analysis. Tissue
samples were collected on dry ice to prevent the loss of enzyme activity during
transportation from the field to the laboratory. Each sample was collected and
tagged as a cylindrical segment approximately 200 mm long, which included the
shoot tip.
Preparation of Plant Material.
Storage
The young leaf tissue was cut into approximately 6 mm long segments after
peeling off the older leaves from the outside and wrapped in small pieces of
aluminum foil numbered 1 to 7, beginning from the last internode near the apical
meristem. All segments from one sample were included in a large piece of
aluminum foil marked with the corresponding accession number, frozen in liquid
nitrogen and later transferred to -80°C for long term storage.
Grinding
Samples were removed from the freezer prior to grinding and maintained on
dry ice. The tissue was ground in a home-made multiple well plexiglass grinding

39
unit, designed to handle small quantities of tissue and 20 samples. Care was taken
to use the same numbered segment(s) from each sample. The tissue was weighed,
and ground in 0.5 v/w grinding buffer (0.2 M Tris-HCl pH 7.8, 60 % Glycerol and
0.2 % 2-Mercaptoethanol added just before use). The tissue was maintained on ice
at all times except while weighing and grinding.
Wicks
Wicks were placed in the crude extract obtained by grinding the samples and
allowed to saturate. Each wick was made of gel blot paper GB003 (Schleicher &
Schuell) cut approximately 1.5 mm wide and 12 mm long. After saturation (about 2
min.), the wicks were transferred to a multi-well dish maintained on dry ice, with
separate wells marked for each sample. The sample wicks were then frozen in an
ultra-low freezer at -80°C for use at a later date.
Gel Processing
Preparation of the Gel.
Each gel was prepared for the various systems described in Table 2.1 (Stuber
et al. 1988), using 300 ml of gel buffer and 13% w/v starch (Sigma catalog # S-
4501). A cold slurry of starch and buffer was rapidly mixed with boiling buffer and
degassed under vacuum. After the hot mixture appeared homogeneous and
translucent, the vacuum was broken gently and the gel was poured into a home¬
made mould with gel dimensions 184 mm X 158 mm X 6 mm and covered with a
larger glass plate to prevent desiccation. The gel was allowed to solidify at room
temperature for at least 4 hours before incubating for 1 hour at 4°C.
Running Conditions.
The sample wicks were transferred 30 min prior to loading from the -80°C freezer to
a -20°C freezer and allowed to thaw on ice just before loading onto the gel. The gel
was removed from 4°C and the glass plate on the top was carefully removed. The
samples were loaded on the gel as described by Stuber et al. (1988). The buffer was

40
kept in contact with the gel by using a large piece of spongecloth at each electrode,
one end of which was placed in contact with the gel and the other was allowed to
soak in the electrode buffer reservoir. To cool the gel during the run, the whole
setup was transferred to a refrigerator at 4°C, with the power supplied from a source
placed outside the refrigerator. The gels were run at constant current of 35 mA for
4 hours. All the power values for the running of the gels were determined after
using several combinations to yield the most favorable results.
Staining of Gels.
Isozyme patterns were visualized by staining the gel for activity ofspecific
enzymes upon completion of the run. The gel was weighed down lightly with the
help of an 11 mm thick acrylic plate and sliced to the appropriate thickness by
running the steel wire alongtwo smooth strips of the desired thickness along the two
sides of the gel. Each gel yielded two 3 mm thick slices which could be used for the
staining of separate isozymes. For best results, the freshly cut gel surface was placed
face up in the staining tray. The stains were mixed from stocks according to recipes
in Table 4.3. All the isozymes studied were anodal in migration. Chemicals and
stains used were from recommended sources and in recommended quantities
(Stuber et al. 1988).
Results
Selection of Stains
Isozyme analysis was carried out on the basis of the availability of recipes for
the stains. Attempts were made to stain at least twenty-three enzymes (Table 2.4)
using several of the buffer systems described in Table 2.1. Fourteen enzymes
stained well (Figs. 2.1 to 2.14), but the other nine either did not yield a
distinguishable pattern or did not stain at all.
All of the isozymes chosen for staining were from prominent metabolic pathways.

41
TABLE 4,1 Electrode and Gel Buffer Formulae
Svstem
Electrode Buffer
Gel Buffer
A
pH 5.0
0.05 M L-Histidine (7.75 g/L)
0.024 M Citric acid.H^O (ca. 5 g/L;
pH adjusted with Citric acid)
0.004 M L-Histidine
0.002 M Citric acid.H20 (13-fold
dilution of electrode buffer)
B
pH 5.7
0.065 M L-Histidine (10.88 g/L)
0.02 M Citric acid.H20 (ca. 4.125 g/L
pH adjusted with Citric acid)
0.009 M L-Histidine
0.003 M Citric acid H20 (7-fold
dilution of electrode buffer)
C
pH 8.3
0.19 M Boric acid (11.875 g/L)
0.04 M Lithium hydroxide (ca. 1.6 g/L
pH adjusted with Lithium hydroxide)
9 parts Tris-citric acid
buffer [0.05 M Tris base (6.2
g/L), 0.007 M Citric acid.H20
(1.5 g/L) pH 8.3]: 1 part
electrode buffer
CT
pH 6.1
0.04 M Citric acid.H20 (8.41 g/L)
0.068 M N-(3-Aminopropyl)
Morpholine (9.8 g/L)
0.002 M Citric acid.H20
0.0034 M N-(3-Aminopropyl)
Morpholine (20-fold dilution
of electrode buffer)
D
pH 6.5
0.065 M L-Histidine (10.088 g/L)
0.007 M Citric acid.H^O (ca. 1.5 g/L)
(pH adjusted with citric acid)
0.016 M L-Histidine
0.002 M Citric acid.H20 (4-fold
dilution of electrode buffer)
F
pH 7.0
0.135 M Tris base (16.35 g/L)
0.04 M Citric acid.H20 (ca. 9 g/L)
pH adjusted with citric acid)
0.009 M Tris base, 0.003 M
Citric acid.H20 (15-fold
dilution of electrode buffer)
Formulae from Stuber et a/._(1988)
TABLE 4,2 Recipes for Activity Staining of Isozymes
Enzyme
Stains
Amount
Incubation
a-Acid phosphatase
(a-ACP)
0.1 M Sodium Acetate-
acetic acid pH 5.0
Fast Garnet GBC
MgCl2
a-Naphthyl acid
phosphate (Na)
100 ml
50 mg
50 mg
50 mg
60 minutes in dark at
room temperature
/3-Acid phosphatase
(/3-ACP)
0.1 M Sodium Acetate-
acetic acid pH 5.0
Fast Garnet GBC
MgCb
/3-Naphthyl acid
phosphate (Na)
100 ml
50 mg
50 mg
50 mg
60 minutes in dark at
room temperature

42
TABLE 4.2 (continued)
Enzyme
Stains
Amount
Incubation
Alcohol dehydrogenase
0.05 M Tris-HCl pH 8.0
50 ml
30 minutes in dark at
(ADH)
95% Ethanol
2 ml
room temperature
NAD
20 mg
MTT
20 mg
PMS
5 mg
a-Aryl esterase
0.2 M Phosphate
50 ml
45 minutes in dark at
(a-EST)
buffer (Na) pH 6.0
N-Propanol
2.5 ml
room temperature
a-Naphthyl acetate
20 mg
Fast garnet GBC
25 mg
/3-Aryl esterase
0.2 M Phosphate
50 ml
45 minutes in dark at
(jS-EST)
buffer (Na) pH 6.0
/3-Naphthyl acetate
20 mg
room temperature
N-Propanol
2.5 ml
Fast garnet GBC
25 mg
Aspartate aminotransferase A 0.1 M Tris-HCl pH 8.5
100 ml
2 hours in dark at
a-ketoglutarate
100 mg
room temperature
(AAT)
Aspartic acid
200 mg
after mixing
B Pyridoxal-5-P
10 mg
A and B
Fast Blue BB salt
150 mg
Endopeptidase
0.2 M Tris-Maleate
50 ml
60 minutes in dark at
(ENP)
pH 5.6
room temperature
a-N-Benzoyl-DL-
arginine-/3-Naphth-
25 mg
ylamide-HCL
MgCl2
50 mg
Black K salt
25 mg
Glutamate dehydrogenase
0.1 M Tris-HCl pH 8.5
50 ml
60 minutes in dark at
(GDH)
L-Glutamic acid
150 mg
room temperature
CaCl2
50 mg
NAD
20 mg
NBT
15 mg
PMS
5 mg
Hexokinase
0.05 M Tris-HCl pH 8.0
50 ml
2 hours in dark at
(HEX)
/3-D( + )-Glucose
125 mg
room temperature
ATP
125 mg
MgCl2
50 mg
NAD
10 mg
MTT
5 mg
PMS
1.25 mg
NAD dependent glucose-
56.25 u
6-phosphate dehydrogenase

43
TABLE 4,2 (continued)
Enzyme
Stains
Amount
Incubation
Malate dehydrogenase
0.1 MTris-HCl pH 9.1
50 ml
60 minutes in dark at
(MDH)
DL-Malic acid
100 mg
room temperature
NAD
20 mg
NBT
10 mg
PMS
1.25 mg
Malic enzyme
0.1 M Tris-HCl pH 8.5
50 ml
overnight at room
(ME)
DL-Malic acid
100 mg
temperature after
MgCl2
50 mg
30 minutes at 36°C
NADP
15 mg
NBT
10 mg
PMS
2 mg
6-Phosphogluconate
0.05 M Tris-HCl pH 8.0
50 ml
60 minutes in dark at
dehydrogenase
6-Phosphogluconic acid (Na2)20 mg
room temperature
(6-PGD)
MgCl2
50 mg
NADP
5 mg
MTT
5 mg
PMS
1.5 mg
Phosphohexose isomerase
0.05 M Tris-HCl pH 8.0
50 ml
60 minutes in dark at
(PHI)
D-Fructose-6-phosphate
50 mg
room temperature
MgCl2
50 mg
NADP
5 mg
MTT
5 mg
PMS
1.5 mg
NADP-dependent Glucose- 10 u
6-phosphate dehydrogenase
Shikimic acid
0.1 MTris-HCl pH 9.1
60 ml
2 hours in dark at
dehydrogenase
Shikimic acid
60 mg
room temperature
(SAD)
NADP
10 mg
MTT
5 mg
PMS
1.33 mg
Recipes from Stuber et al. (1988) and Vallejos (1983)
Lack of Variation.
Among all the isozymes stained, alcohol dehydrogenase (Fig. 4.3), aryl
esterase (Fig. 4.6 and Fig. 4.7), endopeptidase (Fig. 4.5), glutamate dehydrogenase
(Fig. 4.8), malate dehydrogenase (Fig. 4.10) and phosphohexose isomerase (Fig.
4.13) showed very distinct and crisp banding patterns. The other variation in all the
regenerants, at the scrutinized loci, isozymes provided a good resolution of the

44
banding pattern although not exceptional. Acid phosphatase and aryl esterase were
each assayed using two forms of their respective substrates and were distinguished
by using prefixes a- and /?-.
All the gels had one lane dedicated to each of the regenerants and a parental
clone as the control. None of the regenerants showed any variation in the banding
patterns of the isozymes. In other words, no regenerant showed any unique isozyme
banding pattern in comparison to the parent. Hence, there was a complete lack of
any quantitative variability was due to the inherent inability in starch gel systems to
quantitate the amount of protein on sample wicks.
Discussion
The results of this work show no variation in isozyme patterns among the
regenerants derived from tissue culture of leaf segments of napiergrass. Each clone
produces scores of tillers and is hence ideal for induction into culture for obtaining a
population from a single clone. Isozymes show distinct patterns at different stages
of development. The fidelity of the population derived from somatic embryos of a
single clone was, therefore, tested by using plant tissue at the same stage of
development. This was done to minimize any variation in the tissue, inherent to the
developmental phase. Several reports on somaclonal variation (Larkin and
Scowcroft 1981; Larkin et al. 1984; Maddock et al. 1985; Breiman et al. 1987a; Ryan
et al. 1987; Ryan and Scowcroft 1987; Taliaferro et al. 1989) have focussed on
regeneration using immature embryos as the explant. This study differs from the
above mentioned ones in its use of leaf tissue as the only explant material used.
Biochemical analyses of the somatically derived regenerants were carried out
to detect any variation that may exist at the tissue level, which may not be expressed
morphologically. Changes in isozyme banding patterns may be developmentally
regulated or the result of altered protein structure due to DNA rearrangement in
the genome. Isozyme analysis was, therefore, supplemented with molecular analysis

Fig. 4.1 Gels stained for enzyme a-Acid phosphatase (a-ACP) after run using
buffer system B.

o
â– rt
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.2 Gels stained for enzyme 0-Acid phosphatase (0-ACP) after run using
buffer system B.

00
•f
o
< C18R2
, C18R1
. C10R6
i C10R5
» ( C10R4
m 4 C10R3
, C10R2
C10R1
. < C8R5
, C8R4
C8R3
_«* , C8R2
. C8R1
« i
. C5R3
^ ¡ C5R2
4 C5R1
m 4| PP10
< C4R1
C3R8
* C3R7
4 C3R6
- C3R5
* C3R4
â– i C3R3
< C3R2
4 C3R1
C2R3
^ C2R2
* C2R1
^ C1R1
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
C11R10
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.3 Buffer system C gels showing Alcohol dehydrogenase (ADH) activity.

o
o
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
ÍC11R8
C11R7
,C11R6
1C11R5
>C11R4
JC11R3
lcilR2
nlcilRl

Fig. 4.4 Aspartate aminotransferase (AAT) activity observed on buffer
system C gels.

o
^cohvoin^nNHo^coh'Cin ^nMHH^cohvoiíi’tnMH
lstsrstsh*lsrs*[sh*VO'í)VÍ)^)VOH\£)^'í)^)HHHrlHHHHHH
HrlHHHrlHHHrlHrlHHPíHrlHHHHHrlHrlHrlHH
OOOUOOOOCJOOOUOCUOOOOOCJUOOOOOCJCJ
+
INrlKlTI'fnMH
KKCtíOÍOíOíOÍKin'tnfNHníNiHOHcor'voin^'nfMHnrMHH
¡ocDOOOOOOKKKKKKKfilHCUKKKíiKKKKKKííK
HriHrlHHHHCOOOWWCOiniílinflt^nnf'líinnPlnMMNH
OOCJOCJOOOOOüOUUOOa<ÜOOOCJOUUUUOOO
O
ltllBHl>uiimin ll«mt -i
zs
+

Fig. 4.5 Activity of enzyme Endophosphatase (ENP) observed on buffer
system C gels.

o
Tt
in
C18R2
C18R1
C10R6
C10R5
C10R4
IC10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
I C3R5
¡ C3R4
I C3R3
C3R2
C3R1
C2R3
C2R2
. . C2R1
C1R1
o
+
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PPIO
C16R4
C16R3
C16R2
C16R1
C11R10
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.6 a-Aryl esterase (a-EST) activity observed on buffer system C gels.

o
.o
«o
j C18R2
1 C18R1
i C10R6
’ C10R5
j C10R4
4 C10R3
4 C10R2
4 C10R1
« C8R5
( C8R4
| C8R3
4 C8R2
« C8R1
í C5R3
| C5R2
C5R1
‘ PPIO
' C4R1
‘ C3R8
C3R7
- C3R6
C3R5
C3R4
C3R3
C3R2
4 C3R1
n C2R3
t C2R2
C2R1
^ C1R1
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
J C16R8
C16R7
1C16R6
4ÉC16R5
^PPIO
4 C16R4
4C16R3
4 C16R1
4 CURIO
4C11R9
^C11R8
4C11R7
^C11R6
C11R5
jdlR4
JC11R3
iC11R2
'C11R1

Fig. 4.7 /3-Aryl esterase (/3-EST) activity observed on buffer system C gels.

o
oo
0
C18R2
C18R1
•
C10R6
•
C10R5
v'*
x
C10R4
*
♦
#
C10R3
A
C10R2
$
w
a
C10R1
•
w
C8R5
.*
•
C8R4
0
C8R3
'9-
â– m
C8R2
#
C8R1
#•
#
C5R3
*
C5R2
C5R1
«
PPIO
'4
*
C4R1
#
•
C3R8
C3R7
•
*
C3R6
*
C3R5
•
C3R4
«
C3R3
•
C3R2
4
• *
C3R1
•
C2R3
•
C2R2
•
C2R1
C1R1
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.8 Buffer system C gels showing the activity of enzyme Glutamate
dehydrogenase (GDH).

o
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
O
}
C18R2
C18R1
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.9 Hexokinase (HEX) activity seen after staining gels prepared using buffer
system C.

o
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PPIO
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.10 Activity of enzyme Malate dehydrogenase (MDH) observed on buffer
system B gels.

vO
•
^ C18R2
KT.
£
-,' 3C18R1
C10R6
*
] C10R5
*
C10R4
*
C10R3
4
C10R2
»
C10R1
4
C8R5
#
C8R4
•
C8R3
â– *
C8R2
*
C8R1
C5R3
4
C5R2
•
C5R1
4
PP10
4
4
C4R1
•
C3R8
C3R7
•
C3R6
4>
C3R5
C3R4
4
C3R3
•
C3R2
4b
C3R1
*
C2R3
.'*
C2R2
%
£
C2R1
C1R1
O
C17R9
1 '
C17R8
J «
C17R7
é *
C17R6
4 w
C17R5
$ *
C17R4
f
b
C17R3
t •
C17R2
f
C17R1
C16R9
* #
C16R8
f
C16R7
« 4
C16R6
C16R5
I
PP10
« 1
C16R4
I
C16R3
w 1
C16R2
* ♦
C16R1
- 4
C11R10
A
C11R9
C11R8
A
C11R7
w
C11R6
g
C11R5
C11R4
«
A
C11R3
A
C11R2
C11R1

Fig. 4.11 Malic enzyme (ME) activity observed on buffer system B gels.

o
'O
*
!
i
C18R2
C18R1
C10R6
• C10R5
C10R4
C10R3
C10R2
C10R1
4 C8R5
4 C8R4
i C8R3
4 C8R2
( C8R1
4 C5R3
4 C5R2
{ C5R1
i PP10
i C4R1
4 C3R8
\ C3R7
I C3R6
( C3R5
, C3R4
f C3R3
C3R2
( C3R1
4 C2R3
, C2R2
C2R1
4 C1R1
O
*
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.12 Gels prepared using buffer system D exhibiting activity of enzyme
6-Phosphogluconate dehydrogenase (6-PGD).

o
oo
'O
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PP10
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
4-
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.13 Banding pattern of enzyme Phosphohexose isomerase (PHI) obtained on
buffer system B gels.
4

o
o
r-~
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
4-
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PPIO
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

Fig. 4.14 Activity of enzyme Shikimic acid dehydrogenase observed on gels
prepared using buffer system D.

o
(N
r~-
C18R2
C18R1
C10R6
C10R5
C10R4
C10R3
C10R2
C10R1
C8R5
C8R4
C8R3
C8R2
C8R1
C5R3
C5R2
C5R1
PPIO
C4R1
C3R8
C3R7
C3R6
C3R5
C3R4
C3R3
C3R2
C3R1
C2R3
C2R2
C2R1
C1R1
-f-
O
C17R9
C17R8
C17R7
C17R6
C17R5
C17R4
C17R3
C17R2
C17R1
C16R9
C16R8
C16R7
C16R6
C16R5
PP10
C16R4
C16R3
C16R2
C16R1
CURIO
C11R9
C11R8
C11R7
C11R6
C11R5
C11R4
C11R3
C11R2
C11R1

73
TABLE 4,3 Summary of Enzymes and Buffers Analyzed
#
ENZYME
Activity
Present* Absent*
1
Acid phosphatase (-a-)
(a-ACP)
B, D
2
Acid phosphatase (-/3-)
(0-ACP)
B
3
Aconitase
(ACO)
C, F
4
Alcohol dehydrogenase
(ADH)
C
5
Aldolase
(ALD)
C, D
6
Aryl esterase (-a-)
(a-EST)
C
7
Aryl esterase (-/3-)
(/TEST)
C
8
Aspartate aminotransferase
(AAT)
C
9
Catalase
(CAT)
C, D
10
Diaphorase
(DIA)
C, F
11
Endopeptidase
(ENP)
C
12
Glucosidase (-/?-)
(/3-GLU)
CT, B
13
Glutamate dehydrogenase
(GDH)
C
14
Hexokinase
(HEX)
C
C, F
15
Isocitric dehydrogenase
(IDH)
CT, D
16
Malate dehydrogenase
(MDH)
B
17
Malic enzyme
(ME)
B
18
Phosphoglyceraldehyde dehydrogenase
(PGALDH)
C, D
19
Phosphoglucomutase
(PGM)
CT, D
20
Phosphogluconate dehydrogenase (6-)
(6-PGD)
D
C
21
Phosphohexose isomerase
(PHI)
B, D
C, F
22
Shikimic acid dehydrogenase
(SAD)
D
23
Trióse phosphate isomerase
(TPI)
C, F
* buffer systems described in Table 4.1
at the nuclear and cytoplasmic levels. The uniformity of banding patterns in all the
isozyme systems tested is conclusive proof of the absence of any aberrations at the
loci tested.

CHAPTER 5
RESTRICTION AND HYBRIDIZATION ANALYSIS OF
MITOCHONDRIAL DNA FROM A POPULATION OF PENNISETUM
PURPUREUM K. Schum. REGENERANTS DERIVED FROM SOMATIC
EMBRYOS
Introduction
Mitochondrial DNA (mtDNA) analysis has been employed in a variety of
tissue culture studies. The products of protoplast fusion may be scrutinized for the
presence of recombinational events in DNA at the extranuclear level using
restriction and hybridization analyses of mtDNA (Belliard et al. 1979; Boeshore et
al. 1985; Chetrit et al 1985; Vedel et al 1986; Rothenberg and Hanson 1987; Ozias-
Akins et al. 1987; Tabaeizadeh et al. 1987). The use of mtDNA in tissue culture
analyses does not limit itself exclusively to somatic hybrids. MtDNA analyses have
been applied for the identification of cultivars most suitable for induction into
culture (Rode et al. 1988). However, one of the most common applications of
mtDNA for in vitro studies is its use in the identification of variation that may arise
during the process of tissue culture.
The mitochondrial genome in the Texas type male sterile cytoplasm of maize
is directly involved in the reversion to fertility (Gengenbach et al. 1981). This
reversion is caused by the deletion or loss of activity by disruption, of the Tutfl3
mitochondrial gene (Pring and Lonsdale 1989). Hartmann et al. (1987) and Rode et
al. (1987b) have reported the presence of a "hypervariable" region on the
mitochondrial genome of wheat tissue cultures. The induction of wheat into culture
has been termed responsible for the aberrations caused in this region. MtDNA
samples from long-term cultures of rice have been shown to possess altered
restriction patterns (Chowdhury et al. 1988; Abdullah et al. 1990). However, these
74

75
long-term cultures have also been reported to have lost their embryogenic and
regenerative capacity. Hartmann et al. (1989) have shown a correlation between a
specific restriction pattern of the mitochondrial genome and embryogenic capacity
of wheat tissue cultures, although the organization of the mitochondrial genome
may not be the cause of the embryogenic capacity. In maize cell cultures, McNay et
al. (1984) reported changes in the stoichiometry of bands in the restriction pattern
of mtDNA from maize tissue cultures, although the restriction profile remained
unchanged. Variant restriction profiles of mtDNA from Brassica campestris, caused
by DNA rearrangements, have also been found in the native plant tissue at very low
levels (Shirzadegan et al. 1989). These variations are, hence, believed to be the
result of in vitro amplification of existing variation.
The purpose of this study was the comparative evaluation of mtDNA from a
population of Pennisetum purpureum regenerants obtained from in vitro culture.
The establishment of callus cultures and regeneration of the population is described
in Chapter One.
Materials and Methods
Extraction of mtDNA
The procedure used for the extraction of mtDNA was described by Smith et
al. (1987). The soft basal regions(including young leaves and stem) of tillers
obtained from field grown plants were used for the extraction of mtDNA. The
tissue was ground in 10 volumes / FW cold buffer. The homogenate was filtered
through 4 layers of cheesecloth and 1 layer of Miracloth (Calbiochem). The filtrate
was centrifuged for 10 minutes at 1000 x g (4°C) to pellet the nuclei and
chloroplasts. The supernatant was transferred to fresh bottles and centrifuged for
10 minutes at 17,000 x g (4°C) to pellet the mitochondria. After discarding the
supernatant, the pellet was carefully resuspended in 5 volumes of saline buffer. The
resuspended mixture was transferred to a 30 ml tube and centrifuged for 10 minutes

76
at 18,000 x g at 4°C. The pellet was resuspended in Saline buffer (20 ml / 50 g FW)
with 1 M MgCl2 (100 /¿I / 10 ml). DNase (Sigma Chemical Co.) was added to
obtain a final concentration of 0.02 mg / ml and mixed well. The mixture was
incubated for 60 minutes at room temperature after which it was underlayered with
20-25 ml Shelf buffer and centrifuged for 20 minutes at 16,000 x g at 4°C. The pellet
was resuspended in 30 ml Saline wash and centrifuged for 20 minutes at 16,000 x g
at 4°C. This pellet was resuspended in 5 ml NN buffer with 250 ill of Proteinase K
(2 mg/ml) and 250 ¡il 10% SDS and incubated for 1 hour at 37°C. An equal volume
of 2X Extraction buffer was added and further incubated for 15 minutes at 65°C.
Potassium acetate (5 M) was added to a final concentration of 1.25 M). This
mixture was maintained on ice for 30 minutes with frequent mixing. After
centrifuging for 10 minutes at 16,000 x g at 4°C, the supernatant was filtered through
Miracloth into a mixture of isopropanol and ammonium acetate (0.5 volume
isopropanol : 0.05 volume 5 M ammonium acetate) and incubated at -20°C for 1
hour, after which it was centrifuged for 20 minutes at 16,000 x g. The supernatant
was discarded and the pellet was washed in 70% ethanol before dissolving it in 700
li\ TE buffer. This was extracted once with an equal volume of phenol followed by
one extraction each with equal volumes of phenol : chloroform (1:1) and
chloroform. Centrifugation at each step was for 5 minutes in a microfuge at full
speed and the aqueous phase was retrieved. After extraction with chloroform, 0.11
volume of 3 M sodium acetate and 0.7 volume of isopropanol were added to the
sample. This mixture was incubated for 1 hour at -20°C to precipitate the DNA.
The tubes were centrifuged to pellet the DNA after which it was washed in 80%
ethanol and vacuum dried. The pellet was resuspended in about 100 ¡i\ of DNA
buffer.
Plants from each of the callus lines described in Chapter Three were used to
obtain mtDNA. Plants were selected at random from groups with more than two

77
individuals. A total of twenty one regenerants were used for the extraction of
mtDNA. Two of the six plants obtained from immature inflorescence derived calli
were also selected at random for the isolation of mtDNA, in addition to the parental
clone. The total number of mtDNA samples thus obtained, including the parent,
was twenty-four.
Restriction Analyses
Restriction endonuclease fragment analyses were conducted on the 24
samples using two restriction enzymes (Hindlll and PstY). Restriction analyses using
enzyme BamHl were conducted on 22 samples representing all the callus types. All
representatives of the callus types were also included in the analysis of 21 samples
using enzyme Sail. Samples were digested using 10-15 units of enzyme for each
reaction, at 37°C for 90 minutes. The reaction was stopped using 6X loading dye
(0.25% bromophenol blue, 40% sucrose). Digested samples were run on a gel unit
(gel dimensions 260 mm X 210 mm) at approximately 2 volts/cm for 16 hours using
TPE buffer (0.9 M Tris-Phosphate pH 8.0, 0.002 M EDTA). One of the lanes on
each gel contained DNA from bacteriophage lambda digested with Hindlll as a
molecular size marker. The banding patterns were visualized on an ultraviolet
transilluminator (Fotodyne, model 3-3500) after staining them in a 0.5 ¿ug/ml
solution of ethidium bromide for 45 minutes and destaining in distilled water for 20
minutes. The gel was photographed using a UV filter, a red filter and Polaroid film
(Type 55).

78
TABLE 5.1 Buffers Used for Bidirectional Blotting of Mitochondrial DNA
Buffer
Ingredients
Molaritv
Depurinating
HC1
0.25 M
Denaturing
NaCl
0.6 M
NaOH
0.2 M
Neutralize
NaCl
3.0 M
pH 7.5
Tris Base *
1.0 M
20 X SSC
NaCl
3.0 M
pH 7.0
Citrate (Na3)
0.3 M
Bidirectional Southern Blotting (Sandwich Blotting1)
The photographed gel was transferred to a large tray on a table top rotary
shaker and covered with 0.25 M HC1 (Table 5.1) for 10 minutes, to allow for
depurination. The increased mobility of the high molecular DNA facilitated a good
transfer onto the nylon membrane. After 10 minutes the HC1 was poured off and
the gel rinsed twice with deionized water. The gel was then covered with
Denaturing buffer (Table 5.1) and maintained on a shaker for 30 minutes. To
neutralize, the gel was rinsed two times with deionized water, the Neutralizing
buffer (Table 5.1 was poured on the gel and shaken for 30 minutes. Concurrent to
the pretreatment of the gel, two pieces of the nylon membrane were cut to the size
of the gel and equilibrated in 20 X SSC (Table 5.1) for 15 minutes. A blot block
approximately 1 inch thick was placed on the counter top on which three dry sheets
of 3MM paper were placed. Three sheets of 3MM paper, previously equilibrated in
20 X SSC, were placed on top of the dry 3MM sheets and all bubbles rolled out with
the help of a brayer. One of the two equilibrated nylon membranes was placed on
the wet 3MM sheets after flooding these with 20 X SSC. The gel was then carefully
placed on the membrane after rolling out all the air bubbles from under the
membrane and flooding the membrane with 20 X SSC. The gel was flooded with 20
X SSC and the second sheet of nylon membrane was placed on top, while carefully

79
excluding all air bubbles. Three sheets of wet 3MM paper were placed on the
membrane and the bubbles rolled out. The three dry sheets of 3MM paper were
placed on top of the wet 3MM and topped off with a 1 inch thick blot block. A glass
plate was placed on top of the stack and weighed down. After three hours, the
membranes were carefully taken apart and rinsed in 3 X SSC, wrapped in plastic
wrap and the DNA was crosslinked to the membrane by a 5 minute exposure to UV
light on a transilluminator (Fotodyne 3-3500).
DNA Hybridization
The sandwich blots obtained from the gels described above were probed
using the Southern hybridization technique (Southern 1975). The restriction
profiles produced by each enzyme were probed using at least she different
mitochondrial genes cloned from maize; viz. F1-F0 ATPase subunit a (a/pA, 4.2 kb)
(Braun and Levings 1985), F1-F0 ATPase subunit 6 (a/p6, 0.9 kb) (Dewey et al.
1985a), F1-F0 ATPase subunit 9 (a/p9, 2.2 kb) (Dewey et al. 1985b), cytochrome c
oxidase subunit I (coxl, approximately 10 kb) (Isaac et al. 1985), cytochrome c
oxidase subunit II (coxII, 2.4 kb) (Fox and Leaver 1981) and 18S-5S ribosomal DNA
(18S, 6.0 kb) (Chao et al. 1984). Probes were provided by Dr. C. S. Levings, III, of
North Carolina State University, Raleigh, USA. Some of the blots were also probed
using random clones from the pearl millet mitochondrial genome (obtained from
Dr. R. L. Smith, University of Florida, Gainesville. FL). Each of the two
membranes corresponding to a single restriction enzyme was scrutinized using a
different probe.
The nylon blots corresponding to each gel were probed separately with
individual cloned fragments of DNA named above. The blots were prehybridized
with 30 ml of 0.5 M Sodium phosphate buffer pH 7.2, 1% BSA and 7% SDS
supplemented with approximately 2.5 mg of denatured Herring sperm DNA
(obtained by boiling the DNA solution for 5 minutes and immediately transferring

80
to ice for 5 minutes), in 25 cm X 30 cm plastic bag. Care was taken to exclude all air
bubbles. The sealed pouches were incubated at 65°C for a minimum of 4 hours
before injecting the labelled probe into the pouch. Procedures for hybridizing
specifically with the membrane were initiated by radioactively labelling a cloned
fragment of DNA which was to be used as the probe.
The random priming reaction was carried out, according to procedures
described by Feinberg and Vogelstein (1983), in a total volume of 50 /¿l, which
consisted of 10 jul OLB (Table 5.2), 6 jul BSA (1 mg/ml), 2 /¿I ^“P-dCTP (20 /iCi), 2
Ml DNA polymerase (2 units), approximately 100 ng denatured DNA and the
volume was brought to 50 ¿¿1 with sterile double deionized water. The reactants
were added to the tube individually, and the mixture was incubated at 37°C for 45
min. To stop the reaction, 150 ¿d of OLB Stop mix (Table 5.2) was added after 30-
45 minutes of incubation. The volume was brought to 600 ¿d with TE (10 mM Tris,
1 mM EDTA) after denaturing and injected into the pouch using a 1 ml tuberculin
syringe while taking measures not to introduce any air bubbles. The pouch was
incubated in a 65°C water bath for 16-24 hours. After incubation, the membrane
was removed after draining the pouch and subjected to two washes in 3 X SSC at
65°C for 15 minutes each. Following the second wash, the membrane was drained of
any excess buffer and wrapped in plastic wrap. The membrane was then monitored
for radioactivity with the help of a Geiger counter and exposed to X-ray film (Kodak
X-Omatic AR 5) with a Cronex Lightning (Du Pont) intensifying screen. The
exposure of the film depended on the amount of radioactivity detected on the
membrane. Autoradiograph exposure times were typically 24-48 hours.
Results
Restriction Analyses
Digestion patterns of mtDNA with the four enzymes were complex, yielding
between 30 and 50 fragments with the enzymes Bam HI Hindlll, Pst\ and Sail.

81
TABLE 5.2 Buffers Used for DNA-DNA Hybridization Procedures
Buffer
Volume
Ingredients
Molaritv
Amount
OLB TE
100 ml
Tris
0.003 M
0.36 gm
pH 7.0
EDTA
0.0002 M
0.075 gm
Tris-MgCl?
100 ml
Tris
1.25 M
15.14 gms
pH 8.0
MgCl2.6H20
0.125 M
2.54 gms
dATP
170 Ml
dATP
0.1 M
0.01 gm
dGTP
169 Ml
dGTP
0.1 M
0.01 gm
dTTP
172 Ml
dTTP
0.1 M
0.01 gm
Solution A
1.033 ml
Tris-MgCl? solution
1.0 ml
/1-Mercaptoethanol
0.018 ml
dATP
0.005 ml
dGTP
0.005 ml
dTTP
0.005 ml
Solution B
50 ml
Hepes
2 M
23.8 gms
pH 6.6
Solution C
0.555 ml
Hexamers
50 units
OLB
0.25 ml
Solution A
0.05 ml
Solution B
0.125 ml
Solution C
0.075 ml
OLB Stop Mix
100 ml
Tris Base
0.02 M
0.24 gm
pH 7.5
Sodium chloride
0.02 M
0.12 gm
EDTA
0.002 M
0.075 gm
SDS
0.0025%
0.0025 gm
Formulae from Feinberg and Vogelstein (1983)
Although the gels obtained from restriction analyses were used for making nylon
blots for DNA-DNA hybridization analyses, they were not subjected to a
densitometric analysis to expose differences in stoichiometry between different
bands.
BamHI Digests
Each sample of DNA yielded at least 40 bands after being digested with
BamHI. The restriction profiles showed no differences in banding patterns between
lanes (Figure 5.1). Any discrepancies in intensity of the bands were correlated to

82
differences in amounts of DNA used in the restriction reactions. Blots from these
restrictions were probed using several DNA fragments as probes (Figs. 5.2 to 5.9).
Hindlll Digests
The restriction profile of mtDNA using enzyme Hinúlll yielded
approximately 50 bands from every sample of mtDNA that was tested (Figure 5.10).
One of the lanes showed a single band which appeared stronger in intensity than in
the rest of the samples. The accentuated intensity of the band was, however, not
apparent when the same sample was digested with an increased amount of
restriction enzyme. Blots from these restrictions were probed using several DNA
fragments as probes (Figs. 5.11 to 5.18).
PstI Digests
Digestion of mtDNA using the enzyme Pstl yielded about 40 bands in the
restriction profile of every sample (Figure 5.19). In this case, there was no
difference detected whatsoever in the restriction patterns of any of the samples.
Differences in relative intensities between bands from distinct samples were few,
and not subjected to stoichiometric analysis. Blots from these restrictions were
probed using several DNA fragments as probes (Figs. 5.20 to 5.27).
Sail Digests
The Sail restriction profile yielded about 40 bands, and there was no
variation observed between individual samples of the population (Figure 5.28).
Hybridization Analyses
Blots were hybridized to assess them for qualitative characters. After
probing the blots with the maize mitochondrial clones atpA, atp6, atp9, cox I, coxll
and 18S rDNA, no difference was observed in any of the hybridization patterns.
Blots from the Sail digests were probed with the K’, K3 and X2 clones (Figure 5.29)
from the "hypervariable region" of the wheat mitochondrial genome. These probes

Fig. 5.1 Restriction profile of mtDNA from P. purpureum after restriction
with enzyme BamHI. Lane 1 contains DNA from bacteriophage A
digested with enzyme Hindlll, used as molecular weight
markers.

kb
A-HindlII
PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
00
4^

Fig. 5.2 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene for 18S rRNA.
Fig. 5.3 BamHI digested mtDNA from P. purpureum probed with the
mitochondrial gene coding for the a subunit of ATPase (atpA).

o
i».
i
»
kb
PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
kb
PPIO
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6

Fig. 5.4 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene atp6.
Fig. 5.5 Hybridization pattern of BamHI digested mtDNA from P. purpureum
after probing with the mitochondrial gene atp9.

PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
kb
PPIO
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
00
00

Fig. 5.6 MtDNA from P. purpureum probed with the mitochondrial gene for
cytochrome oxidase subunit I (coxl), after digesting with restriction
enzyme BamHI.
Fig. 5.7 MtDNA from P. purpureum probed with the mitochondrial gene for
cytochrome oxidase subunit II (coxll), after digesting with restriction
enzyme BamHI.

CO
ó
kb
PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
A Ol
• • •
—4 "*>J CD
• f •
• 9 I
I t
• f I
• I I
t 9 f
• t •
• t 9
CD
b
ft
:
t
t
i
v
I
I
I
I
I
9
t
i
t
t
ft
•
kb
PPIO
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R10
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
vo
o

Fig. 5.8 BamHI digested mtDNA from P. purpureum probed with a random
mitochondrial probe 4D5.
Fig. 5.9 BamHI digested mtDNA from P. purpureum probed with a random
mitochondrial probe 4D12.

kb
PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R6
kb
PPIO
C1R1
C2R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
VO
K>
Inf3R6

Fig. 5.10 Restriction profile of mtDNA from P. purpureum after restriction
with enzyme Hindlll. Lane 1 contains DNA from bacteriophage
digested with enzyme Hindlll, used as molecular weight
markers.

0.5
N> fO
b b
O)
CO

ro
“ kb
X-Hinc
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R1C
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3Rf
Inf3R(
4*.

Fig. 5.11 HindIII digested mtDNA from P. purpureum probed using the
mitochondrial gene for the a subunit of ATPase (atpA).
Fig. 5.12 Hindi 11 digested mtDNA from P. purpureum probed using the
mitochondrial gene atp6.

rvj
A
kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R10
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
0.9
kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
vO
ON

Fig. 5.13 Hindi 11 digested mtDN A from P. purpureum probed using the
mitochondrial gene atp9.
Fig. 5.14 Hindlll digested mtDNA from P. purpureum probed using the
mitochondrial gene for cytochrome oxidase subunit II (coxll).

00
kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
6.2
00
ro
r t
i •
• t
I I
I I
I I
I 1
I I
I
I
I
I
I
I
I
•
<
:
I
f
kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R10
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
vo
oc

Fig. 5.15 HindiII digested mtDNA from P. purpureum probed using the
mitochondrial gene for cytochrome oxidase subunit I (coxl).

0.5
I CD i
kb
1 CO -
PP10
C1R1
C2R3
• 1
C3R3
11
C3R6
11
C4R1
1 1
C5R1
C5R3
1 1
C8R3
' 1 t
C8R5
II«
C10R1
«1
C10R5
I 1
CURIO
C11R2
11 (
C11R8
11
C16R4
C16R9
1 1
C17R1
1 1
C17R5
C17R8
C18R1
• •
C18R2
•# I
Inf3R5
« « 1
Inf3R6
o
o

Fig. 5.16 MtDNA from P. purpureum probed with cosmid 2A8 containing a
random mtDNA insert, after digestion with enzyme Hindlll.

kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R1C
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
o
K>
Inf3R6

Fig. 5.17 Hybridization pattern of Hindi 11 digested mtDN A from P.
purpureum after probing with a random mitochondrial probe 4E11.
Fig. 5.18 Hybridization pattern of Hindi 11 digested total DNA from P.
purpureum after probing with a random mitochondrial probe 5H2.

kb
I
(
c
f
I
I
f
l •
C
I
c
<
c
f
<
c
i
I
4
€
PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
t
kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R]
C18R2
Inf3R5
Inf3R6
o
-p*.

Fig. 5.19 Restriction profile of mtDNA from P. purpureum after restriction
with enzyme Pstl. Lane 1 contains DNA from bacteriophage A
digested with enzyme Hindlll, used as molecular weight
markers.

o
Ln
ro
O
O
<>
( f
ti tititt
:: tuts
SUBI
tt t tus
t; ;uzs
#» Mff ••
tt • •*
O
ON

Fig. 5.20 MtDNA from P. purpureum probed with the a subunit of the
mitochondrial ATPase gene (atpA), after digesting with the
restriction enzyme Pst/.
Fig. 5.21 Banding pattern of PstI digested mtDNA from P. purpureum,
after hybridization with the mitochondrial gene for 1SS rRNA.

PP10
I
I
I
t
I
I
9
t
f
I
I
I
9
I
I
•
I
I
I
I
I
I
I
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R10
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
kb
PPIO
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
C11R10
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
o
CO

Fig. 5.22 PstI digested mtDNA from P. purpureum probed using the
mitochondrial gene atp6.
Fig. 5.23 PstI digested mtDNA from P. purpureum probed using the
mitochondrial gene atp9.

kb
PP10
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
3.3
kb
PPIO
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6

Fig. 5.24 MtDNA from P. purpureum probed with the mitochondrial
gene for cytochrome oxidase subunit II (coxll), after digesting
with enzyme Pstl.
Fig. 5.25 Hybridization pattern of Pstl digested total DNA from P.
purpureum, after probing with a random mitochondrial probe 2A8.

kb
PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
kb
PPIO
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
to

Fig. 5.26 Hybridization pattern of PstI digested mtDNA from P. purpureum,
after probing with a random mitochondrial probe 4A9.
Fig. 5.27 Banding pattern of PstI digested mtDNA from P. purpureum,
after hybridization with a random mitochondrial probe 4D1.

PP10
PPIO
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6
I
9
I
I
t
I
C1R1
C2R3
C3R3
C3R6
C4R1
C5R1
C5R3
C8R3
C8R5
C10R1
C10R5
CURIO
C11R2
C11R8
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
C18R2
Inf3R5
Inf3R6

Fig. 5.28 Mitochondrial DNA digested with enzyme SalI.

A-HindiII
mo
cuu
cm
C3R6
CMl
CSR1
C8R3
C8R5
C10R1
C10R5
C11R2
C11R8
C11R10
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
Inf3R6

Fig. 5.29 Sail digested mitochondrial DNA digested with a random
mitochondrial probe K3

.
41 #
PP10
C1R1
C2R3
C3R6
C4R1
C5R1
C8R3
C8R5
C10R1
C10R5
C11I
C1U
C11K
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1
Inf3R6

119
also failed to detect variability. Samples that did show unique patterns were
analyzed again after digesting them in the presence of excess restriction enzyme.
The blots thus obtained did not exhibit anomalies between the control and the
putative aberrant lanes. Differences in the intensity of bands correlated with
differences in the loading amounts of the sample DNA.
Discussion
After restriction analyses of the mtDNA and hybridization with probes, it is
clear that no variation was introduced during the tissue culture process at the
mitochondrial genome level, at least at the sites that were analyzed. Although all
the regenerants were not tested individually, the analysis included at least one
representative from each of the eleven calli used for regeneration.
There have been several reports on the absence of variation among
mitochondrial genomes in tissue culture. McNay et al. (1984) reported no variation
in the restriction profile of mtDNA from long term cell suspension cultures of
maize, although changes in relative stoichiometry existed between different bands.
Other reports on this agree that although there may be no changes in the restriction
patterns, stoichiometric differences do occur in restricted fragments causing changes
in intensity of the respective bands (Kool et al. 1985; Matthews and DeBonte 1985;
Hause et al. 1986; Grayburn and Bendich 1987; Ozias-Akins et al. 1987; Chowdhury
et al. 1988).
Variation in the mitochondrial genome has been reported in tissue cultures
of maize (Brettell et al. 1980) and wheat (Hartmann et al. 1987, 1989; Rode et al.
1987b), where long term cultures have exhibited reorganization of the mitochondrial
genome. Wilson et al. (1984) reported the loss of a large EcoRl fragment from the
restriction and hybridization profiles of two protoclones of sorghum. This region was
termed as a hypervariable region and this was confirmed by Chourey et al. (1986) in

120
protoclones of maize. Hartmann et al. (1987) and Rode et al. (1987b) have also
reported the presence of such a hypervariable region in wheat.
Hartmann et al. (1989) reported that the mtDNA from embryogenic calli of
wheat possessed a unique restriction profile that was different from that of the
parental variety. Embryogenic calli were essentially identified as short-term
cultures, while non-embryogenic callus cultures were called long-term cultures. The
mtDNA restriction profiles of both types of cultures were distinct from each other
and could easily be correlated to either one type of callus or the other (Hartmann et
al. 1989).
Regenerants from maize cultures that showed a rearranged mitochondrial
genome also showed a reversion from cytoplasmic male sterility to a male fertile
phenotype in cultures of the CMS-T type maize line (Gengenbach et al. 1981;
Kemble et al. 1982). Closer scrutiny of these fertile revertants has revealed that
there is a loss of a fragment or a frameshift mutation that disrupts the activity of the
Turf-13 mitochondrial gene (Fauron et al. 1987; Rottmann et al. 1987; Wise et al.
1987).
No qualitative variation was observed in this study at the specific sites that
were tested, as supported by the restriction and hybridization analyses. The fact
that all the regenerants were obtained from embryogenic callus cultures may
contribute to the stability observed in the regenerated population (McCoy and
Phillips 1982; Maddock et al. 1985; Swedlund and Vasil 1985; Rajasekaran et al.
1986; Karp et al. 1987). The stability of the mitochondrial genome described in this
study can be useful in those instances where transgenic plants with a recombined
mitochondrial genome are desired.

CHAPTER 6
CHLOROPLAST DNA ANALYSIS OF PENNISETUM PURPUREUM K.
Schum. REGENERANTS DERIVED FROM TISSUE CULTURE OF
YOUNG LEAF SEGMENTS
Introduction
Chloroplasts, like mitochondria, are organelles that possess their own DNA
(Neale and Sederoff 1989). The chloroplast genome is believed to be much smaller
than the mitochondrial genome in size and complexity (Stern and Palmer 1984b;
Palmer 1985b; Lonsdale 1989). All the multiple copies of DNA in the chloroplasts
of a single cell are believed to be identical. Although the chloroplast genome is
extremely stable, there have been a few reports of chloroplast DNA (cpDNA)
recombination in higher plants as well as algae (Kung et al. 1982; Shen et al. 1982;
Tassopulu and Kung 1984; Epp et al. 1987). Like the mitochondrial genome,
cpDNA has been used widely for analyses involving phylogenetic studies (Coates
and Cullis 1982; Ishii et al. 1986; Palmer and Stein 1986; Jansen and Palmer 1988),
somatic hybrids (Scowcroft and Larkin 1981; Fluhr et al. 1984; Clark et al. 1985;
Muller-Gensert and Schieder 1985; Kushnir et al. 1987; Matsuda et al. 1988),
introgression (Szmidt et al. 1988), recombination (Tassopulu and Kung 1984; Epp et
al. 1987; Lemieux et al. 1988; Pichersky and Tanksley 1988) and tissue culture
variation (Day and Ellis 1984; Kemble and Shepard 1984). Chloroplast genomes
have been studied by the comparison of restriction profiles (Thomas et al. 1984;
Rose et al. 1986).
Qualitative differences with respect to recombination, deletions or insertions
have been identified using cloned DNA fragments from a cpDNA library, using the
121

122
Southern hybridization technique. The chloroplast genome is also believed to be
highly conserved (Palmer 1985a,b, 1989) and hence may not be the tool of choice
for recombinational analyses. Nevertheless, Day and Ellis (1984, 1985) observed
deletions in the cpDNA of plants regenerated from anther cultures of wheat. The
restriction profiles of cpDNA from protoclones of alfalfa were different from the
parental cpDNA. The regenerated plants appeared have either one of two profiles
that were dominant in the progeny (Rose et al. 1986). CpDNA analyses may also be
carried out by using cloned fragments from the chloroplast genome to probe nylon
blots containing restricted total DNA, to detect variation (Brears et al. 1989).
In this study, a population of fifty-seven regenerants obtained from tissue
cultured leaf segments from a single clone of Pennisetum purpureum was analyzed
for cpDNA variation. The analysis was carried out using cosmid clones from the
maize chloroplast genome to probe total DNA, extracted from twenty-two plants
selected randomly from each group, by the Southern hybridization technique. Each
cosmid clone was approximately 37 kb in length and the two clones used accounted
for at least 75 kb or 55% of the maize chloroplast genome.
Materials and Methods
cpDNA Isolation
The extraction of cpDNA was attempted using the published methods and
several variations thereof: Procedures described by Kolodner and Tewari (1975a,b),
Charbonnier et al. (1987), and Kut and Flick (1986), were used and most of the
variations that were attempted were modified specifically for use with napiergrass.
None of the these procedures yielded good quality cpDNA that could be used for
any sort of analyses. This was possibly due to the fact that cpDNA from napiergrass
was refractory to isolation.
One procedure described by Kolodner and Tewari (1975), did yield DNA
that was relatively free from nuclear DNA contamination, but this procedure was

123
not consistent for obtaining clean cpDNA and hence not pursued. Therefore the
analysis of cpDNA was carried out using total DNA extracted according to the
following procedure.
DNA Isolation
Extraction of total DNA from leaf tissue was accomplished using the method
described by Dellaporta et al. (1983). Approximately 1 gram of leaf tissue was quick
frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle, and
transferred to an ultra-low freezer (-80°C). To isolate DNA, 15 ml of extraction
buffer was added to the contents of each tube before the powdered tissue thawed.
After adding 1 ml of 20% SDS solution, the tubes were incubated at 65°C for 10
minutes, 5 ml of 5 M potassium acetate was added and the prep was vigorously
shaken, the resulting mixture was incubated on ice for 20 minutes, to precipitate
most proteins and polysaccharides as a complex with the insoluble potassium
dodecyl sulfate. After incubation on ice, the tubes were spun at 25,000 X g for 20
minutes and the supernatant was poured through Miracloth (Calbiochem) into a 50
ml tube containing 10 ml Isopropanol. The tubes were then incubated for 30
minutes at -20°C. The tubes were spun at 20,000 X g for 15 minutes to pellet the
DNA. After drying, the pellets were redissolved in 700 ql of fo Ijo buffer and
transferred to a microfuge tube. The microfuge tubes were then spun for 10
minutes to remove any insoluble debris. The DNA was reprecipitated using 75 ql of
3 M sodium acetate and 500 q 1 of isopropanol. The clot of DNA was precipitated
by spinning in a microfuge for 30 seconds. The pellet was washed with cold 80%
ethanol and dried in a vacuum desiccator for 30 minutes. The dried pellet of DNA
was dissolved in an appropriate amount of TiqEj buffer and stored at -20°C for
later use.

124
Restriction of DNA
DNA obtained from the above method was restricted using three different
enzymes i.e. EcoRI, HináWl and PstI. The reactions were carried out using 10 tig of
each total DNA sample,e restricted using an excess of enzyme (at least 50
units/sample). The reaction mixture was incubated at 37°C for a minimum of 3
hours. Reaction volume was limited to a maximum of 23 til, since the loading
capacity of each well was 28 til. After incubation, the reaction was stopped using 6X
loading dye (see Chapter Three). Samples were electrophoresed in a 25 cm. X 20
cm. gel unit (see Chapter Three) fitted with two 32 well combs, to accommodate
samples restricted with two different restriction enzymes. Electrophoresis was
carried out for 16 hours at 25 volts. The gel was prepared, stained and
photographed as described in Chapter Five.
Unidirectional Southern blotting
The gel was blotted onto a nylon membrane after it was visualized and
photographed on the UV transilluminator. The gel was then transferred to a tray on
a shaker and covered with 0.25 M HC1 (Table 6.1) for 10 minutes, to allow for
depurination. The DNA was denatured using Denaturing solution for 1 hour, after
rinsing twice with deionized water. The gel was covered with Neutralizing solution
following a rinse in deionized water and returned to the shaker for 1 hour. The
nylon membrane and 3MM blotting paper were equilibrated in 10 X SSC. Care was
taken to exclude all air bubbles from under the membrane used for blotting.
The transfer was allowed to take place over a 24 hour period, after which the
membrane was removed and rinsed in 3 X SSC. The membrane was wrapped in
clear plastic film and the DNA was UV crosslinked to the membrane on a
transilluminator (Fotodyne 3-3500) for 5 minutes.

125
Southern Hybridization
Since the restriction analysis did not include a comparison of the restriction
profiles of the cpDNA using different restriction enzymes, the analysis
TABLE 6.1 Formulae of Buffers for Southern blotting.
Buffer Ingredients Molarity
Depurinating
HC1
0.25 M
Denaturing
NaCl
1.5 M
NaOH
0.5 M
Neutralize
NaCl
1.5 M
pH 8.0
Tris Base
1.0 M
20 X SSC
NaCl
3.0 M
pH 7.0
Citrate (Na3)
0.3 M
Formulae from Maniatis et al. 1983.
of cpDNA was based on the Southern analysis of total DNA restricted with different
enzymes. The blots corresponding to the different restriction enzymes were each
probed using cosmid clones from the maize chloroplast genome, according to
procedures in Chapter Five. The two cosmid clones ctA5 and ctB9 (Lonsdale 1985)
accounted for more than 75 kb or more than 55% of the entire maize chloroplast
genome.
Results
Hybridization Analysis
All the restriction analyses were carried using total DNA to yield the cpDNA
profile by hybridizing with cosmid clones from the maize plastid genome. For
hybridization analysis, total DNA was restricted using three different restriction
enzymes. Restriction profiles of total DNAu produced by each of the three enzymes
showed up as a smear instead of distinct bands (Fig. 6.1). Hence, the only logical
cpDNA analysis of the total DNA was by way of radiolabelled DNA hybridization
using cpDNA probes.

126
Hybridization With cpDNA Cosmids
The nylon membrane blots of the total DNA corresponding to each of the
restriction enzymes were probed using the two cosmid clones described above.
Although there appeared to be a slight shift in the bands between the lanes, the
relative position of the bands in each lane remained unchanged. The hybridization
patterns for all three of the enzymes did not show any variation in banding patterns
for either of the two cosmid clones CtA5 and CtB9. Both the clones provided
numerous bands with DNA digested with enzyme £coRI (Fig. 6.2 and Fig. 6.3) as
well as enzyme HinúlW (Fig. 6.4 and Fig. 6.5). The size of the probes accounted
for the numerous bands displayed by each blot when hybridized with each probe.
Hence, the hybridization patterns were the closest available alternative to the
restriction profiles, and these did not show variation.
Discussion
The molecular analysis of cpDNA from tissue culture derived regenerants showed
no variation in hybridizing fragments. After scrutinizing the restriction profiles of
the chloroplast genome using different restriction enzymes, it is clear from the
results that there have been no major recombinational events at or within the sites
tested. The fact that the cpDNA clones that were used for this analysis consisted of
a majority of the maize chloroplast genome, supports the conclusion that the
passage of the explant through tissue culture does not cause any noticeable changes
in cpDNA at the molecular level due to recombination or rearrangements.
The chloroplast genome is very conserved in its organization, since cpDNA
examined from all land plants contains an inverted repeat consisting of a complete
set of chloroplast rRNA genes and that the inverted repeats are always positioned
asymmetrically (Palmer 1985a,b, 1989; Palmer and Stein 1986). It is also believed

Fig. 6.1 Total DNA from P. purpureum restricted with enzyme EcoRl.

A -Hiñe:
PP10
C1P1
C2P3
C3R6
C4R1
C5R1
C8R3
C8R5
Hi
C10R1 oo
C10R5
C11R2
C11R8
CURIO
C16R4
C16R9
C17R1
C17R5
C17R8
C18R1

Fig. 6.2 EcoRI restricted total DNA extracted from P. purpureum, probed with
maize chloroplast DNA cosmid CtB9.
Fig. 6.3 Hybridization pattern of P. purpureum total DNA when probed using
a chloroplast DNA cosmid QA5, after restriction with enzyme EcoRI.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
5=8*
PPIO
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
OJ
o

Fig. 6.4 Hybridization pattern of Hindlll digested total DNA from P. purpureum
after probing with a maize chloroplast DNA cosmid CtB9.
Fig. 6.5 Total DNA from P. purpureum restricted with Hindlll and probed
using a chloroplast DNA cosmid QA5.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
PPIO
C1R1
C2R1
C2R3
C3R2
C3R4
C4R]/
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
OJ
N>

133
that chloroplast DNA of all land plants and green algae have a common origin while
the mitochondrial genomes of plants, animals and fungi are each believed to have
separate avenues of origin (Palmer 1985a). The plastid genome, therefore, may not
easily undergo recombination or rearrangement. The fact remains, however, that
the chloroplast genome has been used as a tool in recombinational studies and has
been shown to undergo recombination in several plant species. Alfalfa plants
regenerated from protoplasts have been shown to possess a rearrangement in the
cpDNA restriction profile (Rose et al. 1986). Palmer (1983) reported the
occurrence of recombination in the Phaseolus plastid genome and its existence in
two orientations. In plants derived from anther tissue culture, cpDNA is known to
undergo deletions (Day and Ellis 1984, 1985).
Reports pertaining to somaclonal variation have traditionally focussed on
tissue culture derived regenerants obtained by culturing various tissues from
individuals belonging to a single cultivar. In cereal crops, most of the populations
regenerated from in vitro culture have been from immature embryo derived calli
(Zong-xiu et al. 1983; Ahloowalia and Sherington 1985; Maddock and Semple 1986;
Ryan and Scowcroft 1987; Ryan et al. 1987; Bhaskaran et al. 1987; Dahleen and
Eizenga 1990; Eizenga and Dahleen 1990). Each sexual embryo represents the
fusion between two unique gametes and hence, for all practical purposes, is an
unique individual. In this respect, this study differs from other reports of somaclonal
variation because all the regenerants were derived from different tillers of a single
clone that was propagated vegetatively, thereby reducing the chances of introducing
any preexisting variation from the explant material into culture. All the regenerants
were also derived from embryogenic calli. The results provide further support to
the view that plants regenerated from embryogenic cultures are normal and devoid
of any variation (Swedlund and Vasil 1985; Gmitter et al. 1991).

CHAPTER 7
DNA HYBRIDIZATION ANALYSIS OF NUCLEAR DNA FROM TISSUE
CULTURE DERIVED REGENERANTS OF PENNISETUM PURPUREUM K.
SCHUM.
Introduction
Compartmentalization of cells is first known to have occurred in eukaryotic
cells, which literally means that cells possess a "true nucleus" where the DNA is
complexed with specific proteins to form chromosomes. The chromosomes are
contained in a special membrane to form the nucleus which is the most important
organelle in a living eukaryotic cell (Palmer 1985a; Lonsdale 1989). The nuclear
genome is extremely varied in size and organization and has been studied in many
forms over the years. Yet the complexities and mysteries involved are far from
being solved.
Nuclear DNA is extremely large and complex in comparison to either
mtDNA or cpDNA, which necessitates the use of specific techniques for its study.
Most methods for the study of nuclear DNA for a comparative analysis in tissue
culture studies involve the analysis of the chromosome as a whole unit e.g.
chromosome number (Heinz and Mee 1971; McCoy et al. 1982; Karp and Maddock
1984; Swedlund and Vasil 1985; Rajasekaran et al. 1986; Cavallini et al. 1988;
Taliaferro et al. 1989; Dahleen and Eizenga 1990; Eizenga and Dahleen 1990),
chromosome banding patterns (Davies et al. 1986), and morphological studies of
chromosomes (Ahloowalia and Sherington 1985; Larkin et al. 1989; Bebeli et al.
1990; Dahleen and Eizenga 1990; Eizenga and Dahleen 1990). The nuclear genome
is therefore an important avenue for in vitro studies.
134

135
In the past few years, however, there have been a lot more reports that have
focussed attention on the molecular aspects of nuclear DNA analysis in tissue
culture. These include RFLP studies (Landsmann and Uhrig 1985; Brettell et al.
1986a,b; Breiman et al. 1987a,b, 1989; Karp et al. 1987; Rode et al. 1987a; Zheng et
al. 1987; Benslimane et al. 1988; Müller et al. 1990), studies on DNA methylation and
Southern analysis of chromosomal DNA in situ (Mouras et al. 1987; Zheng et al.
1987; Huang et al. 1988; Matthews and Kricka 1988) or after restriction of the DNA
(Zheng et al. 1987).
Tissue culture-derived plants as well as embryogenic and non-embryogenic
calli have been analyzed for the occurrence of methylation. In several plant species
that have been studied, the occurrence of methylation is believed to produce
"recombinational hotspots" which are believed to cause variation in tissue culture
(Brown 1989; Brown et al. 1989; Müller et al. 1990). Although the levels of
methylation between explant and callus were slightly different, Morrish and Vasil
(1989) failed to detect any variation in methylation levels between embryogenic and
nonembryogenic calli.
The advantage of scrutinizing nuclear DNA at the molecular level over
morphological analysis of chromosomes is the accuracy of detecting any
rearrangements or changes in the nucleotide sequence at as much as a single base
pair level, which would be impossible to detect in morphological analyses. This
study was therefore, focussed on the comparative molecular analysis of total DNA
from the parental clone of P. purpureum K. Schum. and its tissue culture derived
regenerants with random nuclear DNA probes from P. purpureum using the
Southern hybridization technique.

136
Materials and Methods
Extraction of total DNA
The total DNA from frozen samples was obtained using the extraction
procedure described by Dellaporta et al. (1984) and outlined in Chapter Four. In
addition, DNA was also extracted using a modified Appels and Dvorak (1982)
procedure described below. Modifications were by Brears et al. (1989).
Approximately 2 gms of tissue was ground to a fine powder after freezing it
in liquid nitrogen. The excess liquid nitrogen was allowed to evaporate and the
frozen powder transferred to a 14 ml polypropylene tube for long term storage at -
80°C. For extraction of total DNA, the ground tissue was removed from the freezer
and 5 ml. of extraction buffer (Table 7.1) was added and the mixture incubated for
1 hour at 37°C with occasional swirling.
After 1 hour, the contents were extracted twice with equal volumes of phenol and
once each with phenol : chloroform and chloroform : isoamyl alcohol mixture. The
mixture was centrifuged at 7500-8000 rpm in a table top centrifuge for 10 minutes,
after mixing well. DNA was precipitated from the aqueous phase with two volumes
of ethanol or one volume of isopropanol in the presence of 0.1 volume of 7.5 M
ammonium acetate. The DNA was pelleted at 3600 X g after incubating at -20°C for
one hour. Following a wash with 80% ethanol, the pellet was dried and dissolved in
400 m1 of NTE (Table 7.1) and transferred to a 1.5 ml microfuge tube. This solution
of DNA was incubated with 100 m1 of RNase mixture (Table 7.1) at 37°C for 2 hours,
to digest any RNA that may be present. The contents of the tube were extracted
once each with equal volumes of phenol and chloroform : isoamyl alcohol mixture.
The total DNA was precipitated from the aqueous fraction using two volumes of
ethanol in the presence of 0.1 volume of 7.5 M ammonium acetate. After one hour
of incubation at -20°C, the DNA was pelleted in a microfuge at maximum speed for
1-2 minutes. This pellet was washed with 80% ethanol and dried in a vacuum

137
desiccator for 30 minutes before it was dissolved in TjoEj buffer (Table 7.1) for
storage at -20°C until further use.
TABLE 7.1: Formulae of Buffers for Extraction of Total DNA.
Buffer
Ingredients
Molaritv
Ouantitv
Extraction buffer
Tris base
0.1 M
pH 8.0
Na2EDTA
0.05 M
NaCl
0.1 M
SDS
2%
Proteinase K
0.01%
NTE
Tris base
0.01 M
pH 8.0
Na2EDTA
0.001 M
NaCl
0.01 M
RNase mixture
dd H20
4.25 ml
(5 ml)
RNase Tj
100,000 units/ml
0.25 ml
RNase A
5.0 mg/ml
0.5 ml
TioEl
Tris base
0.01 M
pH 8.0
Na2EDTA
0.001 M
Formulae from Brears et al. (1989)
Restriction of DNA
Restriction of total DNA was carried out as described in Chapter Six.
Southern Blotting
The techniques used for Southern blotting of the total DNA onto the nylon
membrane were identical to those described in Chapter Six. The blotted
membranes were wrapped in plastic wrap and stored in plastic bags at 4°C.
Membranes that had been used once were "stripped", before being reused at least
three times as described in chapter 4.
DNA Hybridization
Hybridization of the probe to the membrane was carried out in exactly the
same manner as mentioned in Chapter Four. Random nuclear probes 1140 ( kb),
1181 ( kb) and 1186 ( kb) cloned from the nuclear genome of P. purpureum
(provided by Dr. M. K. U. Chowdhury) were used to probe the total DNA blots.

138
The blots were also probed using the clone of a Nor locus gene from wheat
(provided by Dr B. S. Gill, Kansas State University).
Results
As depicted in figures 7.1 to 7.8, none of the probes revealed any variation in
hybridization patterns. There were shifts in the banding patterns between lanes, but
the relative position of all the bands was unchanged. The shift in the bands was
attributed to the slight differences in loading amount of the DNA.
The Nor locus gene from the nuclear genome has been used as a molecular
probe to detect variation in other studies (Breiman et al. 1987b). This gene used as
a probe, was unable to detect variation at the corresponding loci in the regenerants
tested from the analyzed population of napiergrass. The wild type population of
napiergrass has been shown to possess RFLPs in the nuclear genome. However, the
probes 1140, 1181 and 1186, used to identify such variations, failed to reveal the
presence of RFLPs in the regenerated population.
Discussion
This study clearly demonstrates that in Pennisetum purpureum plants derived
from embryogenic cultures, no rearrangements occurred at the nuclear DNA level
at any of the sites that were examined using the random probes. Of particular
interest were the results of the hybridization with the Nor locus clone, since there
have been many reports published on the variability of this region in tissue culture
(Brettell et al. 1986; Breiman et al 1987a,b). There have also been other studies
that have disputed or raised genuine doubts about the ability of tissue culture to
cause variability in this region. The reports of variation at the Nor locus in tissue
culture-derived plants were based on the occurrence of unique banding patterns in
DNA hybridization studies on restriction digests of total DNA. Although these
variations were initially observed only in tissue culture-derived plants, later scrutiny
revealed similar patterns in individuals of the same variety at a very low frequency,

Fig. 7.1 EcoRI digested total DNA from P. purpureum, probed with a random
P. purpureum nuclear probe 1186.
Fig. 7.2 EcoRI digested total DNA from P. purpureum, probed with a random
P. purpureum nuclear probe 1181.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
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pno
4
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C16R8
C17R1
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o

Fig. 7.3 Total DNA from P. purpureum digested with EcoRI and probed using
the Nor locus gene from wheat.
Fig. 7.4 Hindlll digested total DNA from P. purpureum probed using the
Nor locus gene from wheat.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
Pf 10
Clll
C2R1
C2*>
C1I2
OM
C4R1
COR 3
C10R1
C10RS
Cl 1R1
C1 IR 1
C1O01
ClfcRS
C1 *>R0
C17R1
C17R2
C17R0
C18R2
Inf3RJ
Int 3R».
N)

Fig. 7.5 HindIII digested total DNA from P. purpureum, probed with a random
P. purpureum nuclear probe 1140.
Fig. 7.6 HindIII digested total DNA from P. purpureum, probed with a random
P. purpureum nuclear probe 1181.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
PIO
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
CIORS
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
4^

Fig. 7.8 PstI digested total DNA from P. purpureum probed using the Nor
locus gene from wheat.
Fig. 7.9 Total DNA from P. purpureum probed using a random P. purpureum
nuclear gene 1181 after digesting with enzyme PstI.

PP10
C1R1
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
C10R5
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
Inf3R6
!
PPIO
cuu
C2R1
C2R3
C3R2
C3R4
C4R1
C5R3
C8R2
C10R1
CIORS
C11R1
C11R3
C16R1
C16R3
C16R8
C17R1
C17R2
C17R8
C18R2
Inf3R2
45-
ON
Inf3R6

147
thus raising doubts of their induction from in vitro culture (Karp et al. 1987; Breiman
et al. 1989).
Napiergrass is a vegetatively cultivated plant, and the fact that different
accessions of this plant species exhibit variation amongst themselves has been amply
demonstrated by Smith et al. (1989) with the help of random RFLP probes from the
genome of napiergrass itself. The absence of any variation in the individuals tested,
using the very same probes used by Smith et al. (1989), supports the earlier studies,
on the absence of variation in the population of P. purpureum plants derived from
tissue culture.
Stability of the nuclear genome helps to maintain the genetic fidelity of the
plant and its proper functioning at the cellular level. Genetic as well as biochemical
stability of a population derived from tissue culture is especially important for plant
transformation studies involving the insertion of foreign genes into plant cells, since
an unstable nuclear genome could disrupt the proper functioning of inserted foreign
genes. The lack of variation reported in the population of tissue culture plants is
therefore significant for biotechnological manipulation.

CHAPTER 8
CONCLUSIONS
This study was conducted to investigate the incidence of variation in tissue
culture derived plants of Pennisetum purpureum. There have been many reports of
variation in the progeny of plants derived from tissue culture (Brettell et al. 1980;
Larkin and Scowcroft 1981; Breiman et al. 1987a; Rode et al. 1987b). The process of
tissue culture has been therefore claimed to be a source of obtaining variants for
their use in plant breeding.
The parameters used for analysis in this study were biochemical and
molecular analysis of the regenerants in comparison to the parental clone.
The entire population was obtained from somatic embryos, since plants
derived from somatic embryos are known to be stable. Embryogenic cultures have
been shown to deteriorate cytologically over time, although the somatic embryos
formed in these cultures are normal (Swedlund and Vasil 1985). This is attributed to
the strong in vitro selection of normal cells in the formation of somatic embryos
(Gmitter et al. 1991). Another highlight of this study was the fact that it used young
leaf tissue from the parental clone as a control for all the analyses, and this parental
clone was exposed to the same conditions as the regenerants in the field. Many of
the reports on variants derived from tissue culture have relied on the use of
immature embryos as the primary explant, which left them without an absolute
control for the explant source (Brettell et al. 1980; Larkin and Scowcroft 1981;
Breiman et al. 1987a; Rode et al. 1987b).
Biochemical variations have been reported in the tissue culture derived
progeny of several plant species (Davies et al. 1986; Ryan and Scowcroft 1987;
148

149
Taliaferro et al. 1989; Dahleen and Eizenga 1990). At the same time, biochemical
stability has also been reported in plants derived from in vitro cultures and
somaticembryos (Swedlund and Vasil 1985; Brettell et al. 1986; Karp et al. 1987;
Kobayashi 1987). The absolute lack of qualitative variation at the loci tested, in the
entire population, infers that the regenerants are biochemically stable.
In vitro cultures have been shown to undergo mutations, recombinations or
deletions in the mitochondrial genome (Brettell et al. 1980; Gengenbach et al. 1981;
Rode et al 1987b; Hartmann et al. 1989). These variations have been reported with
or without selection pressures on the in vitro cultures. The lack of molecular
variation at the tested sites in the mitochondrial genome, seen in this study, clearly
suggests the stability of the regenerants at the mtDNA level.
The chloroplast genome is conserved and not known to undergo much
change, hence none was expected and observed. The nuclear genome, however, has
been reported to undergo several changes in culture (Breiman et al. 1987a; Brown
1989; Goebel et al. 1990). However, these reports have been on cultures derived
from immature embryos. Breiman et al. (1989) reported the occurrence of similar
variations in the parent material, and raised serious doubts to the validity of
comparisons between inbred parental cultivars and plants derived from in vitro
cultures of immature embryos.
In conclusion, the results of this study reinforced the hypothesis: i) that plants
derived from somatic embryos are genetically stable, ii) Barring spontaneous
mutations, the process of regeneration from somatic embryos can be exploited in
biotechnology for the production of genetically stable and uniform plants, which is
imperative for genetic manipulation and other studies in biotechnology.

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BIOGRAPHICAL SKETCH
Vivek Bhaskar Shenoy was born on July 17, 1961, in Tirthahalli, Karnataka
State, India. He attended St. Xavier’s High School, Ville Parle, Bombay, until tenth
grade, and later attended St. Anthony’s High School. He graduated in First Class
with a bachelor’s degree (BSc.) in Botany from Chauhan Institute of Science,
University of Bombay, India, in 1982. He enrolled for a master’s degree (MSc.) in
Plant Physiology at R. Jhunjhunwala College, University of Bombay, and graduated
in First Class, in 1984. He later taught at C. H. M. College, University of Bombay,
for one year before enrolling in the Botany Department at the University of Florida,
for his Ph.D. degree.
170

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
\ ^
U-J.
Indra K. Vasil, Chairman
Graduate Research
Professor of Botany
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of PJúfpsophy. ^
Darvl R. Pring
Professor of
Plant Pathology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
1 4-Hv^ LKJm !,AjS
Henry C. Aldrich
Professor of Microbiology
and Cell Science
I certify that 1 have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
mj
Robert J. Ferl
Professor of Botany

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Stanley C. Schank
Professor of Agronomy
This dissertation was submitted to the Graduate Faculty of the Department
of Botany in the College of Liberal Arts and Sciences and to the Graduate School
and was accepted as partial fulfillment of the requirements for the degree of Doctor
of Philosophy.
December 1991
Dean, Graduate School

UNIVERSITY OF FLORIDA
1262 08285 441 4

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