IV. Mutant Isolation and Selection

SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



49



plant biology. Moreover, defined mutants greatly facilitate the recognition of

rare genetic events such as might result from genetic recombination, mutation,

somatic hybridization, and genetic transformation. Apart from these more

fundamental uses of biochemical mutants, selecting mutants which cause lesions

or alterations in biochemical pathways may be of importance in several aspects

of plant improvement. For example, biochemical mutants could be selected for

disease resistance, improvement of nutritional quality, adaptation of plants to

stress conditions such as occurs in saline soils, elimination of toxins and antimetabolites deleterious to man and animals, and to increase the biosynthesis of

plant products used for medicinal or industrial purposes.

There are only a few cases where mutants which cause a block in a particular

biosynthetic pathway have been recovered in whole plants. These include

thiamine-deficient mutants in Arubidopsis (Langridge, 1955) and tomato (Langridge and Brock, 1961), nitrate reductase deficiency in Arubidopsis (OostindierBraaksma and Feenstra, 1973), and a proline auxotroph in maize (Gavazzi et al.,

1975). Slightly more success has been achieved in isolating mutants which affect

photosynthesis primarily because they affect chloroplast development and can

be readily selected (Levine, 1969; Miles and Daniel, 1974; Miles, 1976). Such

mutants have been valuable in analyzing basic processes in photosynthesis. The

relatively depauperate collection of biochemical mutants in plants probably

results from the expense of screening large populations of whole plants for

relatively rare mutants. As pointed out by Chaleff and Carlson (1974), the

organizational complexity of plants with morphologically and biochemically

different, yet interdependent, cells and structures also hinders the isolation of

defined biochemical mutants.

The ability to manipulate large populations of homogeneous plant cells provides the opportunity to isolate biochemical mutants. Technically it is relatively

simple to screen 106-107 cells in culture; screening a similar number of whole

plants is very resource-consuming. Because plants can be regenerated from cells

of some species the effect of such mutants may be evaluated in mature plants.

Dominant and co-dominant mutants can be isolated from diploid, or indeed,

polyploid cells. It might appear axiomatic that haploid cell lines would be

required to isolate recessive biochemical mutants. However, this might not be

the case. Recessive mutants occur in diploid animal cell lines at a frequency

considerably greater than would be expected from the frequency of a double

mutation event (Terzi, 1974). Recently, Williams (1976) found in the slime

mold Dictyostelium discoideum that the frequency of spontaneous mutation to

the recessive state at a single locus was only an order of magnitude greater in

diploids relative to that in haploids.

Indeed, plant cell cultures have been used to successfully isolate biochemical

mutants. A discussion of some of these mutants can be found in Chaleff and

Carlson (1974, 1975), Widholm (1974b), and Zenk (1974). The only report



50



W.R. SCOWCROFT



dealing with the recovery of auxotrophic mutants is that of Carlson (1970). In

this study he utilized the lethality to growing cells of the incorporation of

5-bromodeoxyuridine as an enrichment procedure for nongrowing auxotrophic

mutants. By this procedure Carlson was able to isolate tobacco cell clones which

required amino acids, vitamins, or a nucleic acid for growth. These mutants were

leaky in that they continued to grow, albeit slowly, on unsupplemented

medium. This may have been due to multiple gene copies, for although Carlson

(1970) used callus cultures derived from haploids, such haploids do in fact

contain two genomes because tobacco is an amphidiploid. It is also possible that

plants have alternate biosynthetic pathways.

By far the greatest success in isolating biochemical mutants has resulted from

selecting mutants resistant to antimetabolites. When nitrate is the sole source of

nitrogen for tobacco cells, the inclusion of L-threonine in the medium inhibits

cell growth, presumably by blocking the nitrate assimilation pathway (Heimer

and Filner, 1970). Under such conditions Heimer and Filner were able to recover

a ceIl line which was resistant to the growth inhibitory effects of threonine. The

resistance was due to a mutant in the nitrate uptake pathway so that nitrate

could be assimilated in the presence of threonine.

Mutant cell lines have also been reported which are resistant to the base

analogues 5-bromodeoxyuridine (Maliga et aZ., 1973a) and 8-azaguanine

(Lescure, 1973; Bright and Northcote, 1975). The BUdR-resistant mutant is

controlled by a simple Mendelian gene (Marton and Maliga, 1975). Bright and

Northcote (1975) demonstrated that 8-azaguanine resistance resulted from a

decrease in hypoxanthine phosphoribosyl-transferase, so lessening the incorporation of the base analogue. Cell lines resistant to the drug streptomycin have been

found in Petunia (Binding et aZ., 1970) and tobacco (Maliga et aZ., 1973b). The

streptomycin resistance was maternally inherited, and since streptomycin affects

the greening of plant tissue it is likely that the mutation occurred in the

chloroplasts. It is likely that the chloroplast ribosomes were affected, since

streptomycin resistance in bacteria is associated with 70s ribosomes and chloroplast ribosomes are similar to those of bacteria.



A. AMINO ACID ANALOGUE-RESISTANT MUTANTS



Mutants resistant to amino acid analogues are the most thoroughly studied

recent biochemical mutants (Widholm, 1974b). Amino acid analogues inhibit the

growth of plant cells for several reasons, but the main thrust has been with those

that may act as false-feedback inhibitors, i.e., they mimic the natural amino acid

in inhibiting one of the enzymes in the biosynthetic pathway. Widholm (1971)

obtained circumstantial evidence that tryptophan biosynthesis in cell cultures of

several plant species was regulated by feedback inhibition of anthranilate syn-



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



51



thetase. With the tryptophan analogue, 5methyltryptophan, mutants were

selected in tobacco (Widholm, 1972a) and carrot (Widholm, 1972b) which were

resistant to growth inhibitory concentrations of the analogue. All of the mutants

had an altered anthranilate synthetase. Inhibition studies on cell extracts indicated that the enzyme was not inhibited by the analogue, nor indeed by

tryptophan, to the same extent as was the enzyme of nonmutant cell cultures.

Moreover, an expectation was fulfilled, namely that mutants which lacked

feedback regulation would overproduce the specific amino acid. The mutant

carrot and tobacco lines had free tryptophan levels 27- and 10-fold higher than

normal, respectively. Subsequently, Widholm (1974a) selected an additional

5-methyltryptophan resistant mutant carrot cell line in which the mechanism

was due to decreased uptake of the analogue. In this mutant the free tryptophan

level was also elevated but the mechanism of how this occurred is unknown.

Mutants have also been isolated in carrot and tobacco which are resistant to

the phenylalanine analogue, p-fluorophenylalanine (Palmer and Widholm, 1975).

The analogue is normally toxic because it is incorporated into protein. The basis

for the resistance in the mutants was probably due to decreased incorporation

into protein as a result of increased cellular levels of phenylalanine in the carrot

mutant, and also presumably for the tobacco mutant, where it appears that the

increased phenylalanine was converted to phenolic compounds. The enzyme

chorismate mutase from the mutant tobacco cells had reduced sensitivity to

inhibition by phenylalanine or its analogue. In the carrot mutant, chorismate

mutase was unchanged. The basis for the resistance in the carrot mutant is

unknown.

A preliminary report (Chaleff and Carlson, 1975) has also indicated that the

lysine analogue, S-(P-aminoethyl)-cysteine, which inhibits the growth of rice

cells, can be used to select resistant rice cell cultures which have elevated levels

of lysine, both in the free amino acid pool and in total amino acids. The levels of

other amino acids are also elevated in these mutants. The mechanism of resistance and the basis for the increased synthesis of lysine and other amino acids

have not been determined. It would also be of considerable value t o know the

lysine content of the grain of plants regenerated from lysine analogue-resistant

lines.

The isolation of mutants which have elevated levels of certain amino acids is of

interest and of possible value to plant improvement because the grains of most

crops are deficient in certain amino acids important to human and monogastric

nutrition. The limiting amino acid in all cereals is lysine, and maize is also

deficient in tryptophan, and wheat and rice are deficient in threonine. Legume

grains tend to be deficient in methionine. The principal mechanism which

regulates amino acid pools in plants is feedback inhibition, and indeed this has

been confirmed by some mutant cell cultures reported previously. Brock et al.

(1973) discuss the principles of feedback inhibition in relation to obtaining



52



W. R. SCOWCROFT



mutants which overproduce lysine. They suggest that the feedback receptor site,

presumably on the enzyme aspartokinase, can be inactivated by mutation so that

the enzyme is no longer sensitive to feedback inhibition. Such mutants could be

selected in the presence of the lysine analogue S-(0-aminoethy1)-cysteine which

normally inhibits growth because it mimics lysine in inhibiting the activity of

aspartokinase. The cell culture mutants of Charleff and Carlson (1975) tend to

support the expectation of Brock et al. (1973). Increasing the level of lysine in

the free amino acid pool is of course only the first step. Ideally it is the lysine in

the grain storage protein that needs to be increased. The assembly of amino acids

into proteins, both catalytically active and storage proteins, is a complex process

involving messenger ribonucleic acid (mRNA) synthesis, the coupling of amino

acids to transfer RNAs (tRNAs), polypeptide chain initiation, elongation, and

termination. As has been pointed out by Brock and Langridge (1975), genetic

alterations in the amino acid specificity of tRNAs, which has been done in

prokaryotes, could alter the amino acid composition of storage proteins.



B. DISEASE-RESISTANT MUTANTS



The susceptibility of agronomic crops to pathogenic diseases is probably still

the major constraint on maximizing yield. The battle against crop pathogens is a

continuing one, since pathogenic variants arise by genetic events which render

previously resistant crop varieties susceptible. Many bacteria are pathogenic

because they secrete toxin lethal to plant cells. The ability t o screen large

numbers of plant cells in culture provides a means whereby direct selection for

clones resistant to the bacterial toxin could yield resistant genotypes. Mutant

clones have been isolated from tobacco cultures which when regenerated into

plants have increased resistance to the pathogen Pseudomonas tabaci which

causes wildfire disease (Carlson, 1973). The resistance of these plants is not as

complete as that in naturally resistant varieties. Methionine sulfoximine was used

as the antimetabolite to select the resistant cell clones because it would elicit the

same chlorotic response in tobacco as did the bacterial toxin. The relationship

between methionine sulfoximine and wildfire toxin is not precisely clear but in

bacteria the former interferes with the activity of glutamine synthetase

(Brenchley, 1973) and the bacterial toxin is considered to inhibit glutamine

synthetase in plants (Sinden and Durbin, 1968). Glutamine synthetase has been

shown to be extremely important in cellular metabolism in bacteria (Magasanik

et al., 1974), and the same is probable for plants. It is unfortunate that Carlson

(1973) did not compare the enzymatic characteristics of the glutamine synthetase in the mutant clones with that in susceptible cells.

In 1969, 1970, and 1971 there was an epidemic of southern corn leaf blight

(Helminthosponum maydis) because a new pathogenic race arose which attacked



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



53



maize hybrids and inbreds which carried the widely used “Texas” (T) source of

male sterile cytoplasm. It has been established that susceptibility t o this pathogenic race was due to a toxin which binds to the mitochondrial membranes of

susceptible lines, leading to the uncoupling and inhibition of mitochondrid

electron transport (Peterson et al., 1975). Gegenbach and Green (1975) found

that the growth of cell cultures derived from maize with T cytoplasm was

mhibited by the toxin, whereas the growth of normal cytoplasm cultures was

not. Subsequently, they selected a cell clone from the T cytoplasm cultures

which was resistant to growth inhibitory concentrations of the toxin. The

mitochondria of this resistant clone were no longer sensitive t o the toxin.

Resistance was retained when cultures were grown in the absence of the toxin

suggesting that the basis of resistance was genetic. Since plants have not been

regenerated from this toxin-resistant clone it cannot be established whether the

genetic change was nuclear or mitochondrial or whether the cytoplasm was

similar t o the parent in respect to male sterility.

For any plant disease in which pathogenicity is associated with a toxin, it is

relatively inexpensive to treat cell cultures of susceptible, but otherwise desirable, varieties to obtain resistant clones. This would provide an assessment of

the chance of recovering resistant mutants by directly exposing the plant

population to the toxin. Moreover, if the resistant clones could be regenerated to

produce fertile plants, and provided the plant and field resistance correlated well

with resistance in cell culture, then plant breeders may have a way of hastening

the development of new disease-resistant varieties. The use of tissue culture in

this way could provide an alternative t o the expensive and time-consuming

conventional method of transferring disease resistance into susceptible but

otherwise highly regarded varieties.



C. STRESS-RESISTANT AND OTHER MUTANTS



Plant improvement depends primarily on the evaluation of a phenotype and

this of course is a function of many different genetic and biochemical components. However, there is a reductionist approach in biochemistry, plant

physiology, and genetics which attempts to provide an elemental description of

plant processes in biochemical and genetic terms. As this is achieved, plant tissue

culture can be utilized to develop genotypes which have genetic alterations

affecting a specific biochemical function.

Salinity, particularly in irrigated areas, is a major restriction on realizing yield

potential. Also there is an immense crop potential if saline water, and indeed

seawater, could be used without the expensive process of desalination. There is a

need for salt-tolerant agricultural varieties and this may be achieved by selection

since there is a genetic basis to salt tolerance (Dewey, 1960; Abel, 1969; Rush



54



W. R. SCOWCROFT



and Epstein, 1976). However, where natural genetic variability is absent, tissue

culture may provide a solution. It would appear that salt-tolerant clones can be

rapidly isolated from plant cell cultures (Nabors et al., 1975; Dix and Street,

1975). These cultures can withstand 4-5 times the salt concentration that

inhibits growth of normal cells. Again, the evidence to judge whether plants

regenerated from such tolerant clones are also tolerant to high salt concentrations has not yet been provided. Since internal ion concentration is regulated by

cellular restriction of ion uptake, or by excretion of adsorbed ion, it is likely

that there would be a high correlation between cellular and plant salt tolerance.

Crops are often subjected to flooding and it has been postulated that the

injury results from the accumulation of alcohol as a consequence of anaerobic

respiration in the roots (McManmon and Crawford, 1971). Under anaerobic

conditions alcohol dehydrogenase (ADH) catalyzes the reduction of acetaldehyde to alcohol. There are electrophoretic variants of ADH and the “fast”

varient apparently is catalytically more active than the “slow” variant (Felder

and Scandalios, 1971). Marshall et al. (1973) have shown that maize plants

which carry the presumptive catalytically less active form of ADH are more

tolerant to flooding. It can be argued that plants deficient for ADH might be

even more tolerant to flooding conditions. Indeed a selection system exists for

such ADH-deficient genotypes. M y 1 alcohol is converted to the highly toxic

acrylaldehyde by ADH (Megnet, 1967), and Schwartz and Osterman (1976) have

utilized allyl alcohol as a pollen selection system in maize. Our own research has

shown that plant cells are very sensitive to low concentrations of allyl alcohol.

We are currently attempting to select allyl alcohol-resistant clones which we

predict will be deficient for ADH. This selection system also has the added

advantage that ADH can be contraselected, namely, that ADH-mutants will be

sensitive to exogenous acetaldehyde which is toxic to plant cells unless metabolized. Using tissue culture cells this selection system may provide a precise

genetic means of increasing plant tolerance to flooding.

There is no doubt that mutants which are altered in some specific biochemical

function can be isolated from cell culture. As has been the case with microorganisms, this will greatly increase the understanding of biochemical processes

in plants. The degree to which genetic alterations at the cellular level correlate

with altered metabolism in whole plants is as yet largely unknown. The direct

value of somatic cell mutants in plant improvement will depend on the extent of

t h ~ scorrelation. However, other aspects of genetic manipulation at the cellular

level, e.g., somatic hybridization, do require biochemical mutants to provide

efficient hybrid selection systems.

Although the studies are not specifically related to plant improvement, plant

tissue culture is also being evaluated for the production of physiologically active

substances, particularly those of medical importance such as steroids and cardiac

glycosides (Misawa et al., 1974; Reinhard, 1974). Microorganisms have been



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



55



used extensively for the biosynthesis of commercially valuable metabolites and

the efficiency of such biotransformation has been increased by the use of

mutants having altered biosynthetic functions. If plant cell cultures prove to be

useful for the production of medically important compounds, then assuredly

mutants will be selected which yield greater quantities of such compounds.



V.



Plant Cell Protoplasts



A. METHODOLOGY OF ISOLATION



For the purposes of this discussion a protoplast refers t o a cell from which

the cell wall has been removed by mechanical or enzymatic methods. It has been

possible to isolate protoplasts from plants by mechanical methods, but the yield

and quality of the protoplasts is generally low. In 1960 Cocking used a crude

enzyme preparation of the fungus Myrothecium yemearia to isolate protoplasts

from tomato roots. Since that time, and particularly as a result of the commercial availability of cell wall degrading enzyme complexes (Gamborg and

Wetter, 1975), protoplast technology has developed enormously. Several recent

reviews examine various aspects of the isolation, culture, and current and

proposed uses of plant protoplasts (Cocking, 1972; Tempe, 1973; Eriksson et al.,

1974; Gamborg and Wetter, 1975; Vasil, 1976; Gamborg, 1976). Therefore it is

not intended to go into specific details or to cite from the extensive literature

which is largely covered by these reviews. Only recent and key references will be

cited.

Protoplasts can be isolated from virtually any plant structure that is not

lignified, including leaves, petals, and microsporocytes, and also from plant cell

cultures. Leaves have been used extensively for such isolation. To expose the

mesophyll cells to the enzyme preparations the epidermis can be physically

removed or injured by the use of carborundum (Beier and Bruening, 1975), or

leaves can be gently macerated. Enzymatic digestion can be by a sequential

process, where mesophyll cells are first released by the action of crude pectinase

and the cell walls then degraded by cellulase (Nagata and Takebe, 1970), or by

the more common, single-step procedure using an enzyme mixture containing

pectinase and cellulase. Since protoplasts are subject to osmotic damage and

rupture, an osmotic stabilizer such as mannitol, sorbitol, glucose, or sucrose is

required in the culture medium. Factors which affect the quality, quantity, and

osmotic stability of isolated protoplasts include the immediate environmental

and nutritional history and age of the plants (Shepard and Totten, 1975). Cell

suspension cultures are proving extremely valuable for the isolation of protoplasts because of the greater control over the physiological state of the cells and

the sterility of the starting material. Cells in early to mid-log phase of growth



56



W. R. SCOWCROFT



appear to be in the most favorable state for protoplast isolation (Uchimiya and

Murashige, 1974).

Crude enzyme preparations contain various impurities some of which are

probably toxic. While unpurified enzymes can be used with success, an improvement both in yield and in protoplast quality is obtained if the enzyme mixture is

cleansed of low molecular weight impurities by passage through a Sephadex

G-25 column (Schenk and Hildebrandt, 1969). In our laboratory we have found

a substantial improvement in protoplast viability by merely dialyzing the cell

wall degrading enzyme mixture overnight in the cold against several changes of

distilled water. This appears to remove phenolics, salts, and other low molecular

weight impurities. Protoplast preparations have varying degrees of cellular and

subcellular debris. Several methods involving repeated sedimentation and resuspension or two-phase liquid partitioning have been used with varying success. We

have confirmed a recent report by Larkin (1976) that commercial density

buffers containing sodium metrizoate and Ficoll (Lymphoprep, Nyegaard A/S

Oslo, Norway; Ficoll-Paque, Pharmacia, Uppsala, Sweden) are excellent for

removing debris.

Protoplasts as experimental systems per se have already found widespread use

in studying virus infection and multiplication (Takebe, 1 9 7 9 , cell organelles and

vacuoles (Wagner and Siegelman, 1975), photosynthesis (Nishimura and

Akazawa, 1975), the cellular response to toxins from pathogens (Pelcher et al.,

1975; Strobel, 1975), and cell wall biosynthesis and deposition (Fowke et al.,

1974; Willison and Cocking, 1975).



B. PROTOPLAST CULTURE



Protoplasts are significant to both fundamental and applied genetic research

because at least some species can be induced to form a cell wall, divide, and

undergo regeneration into plants. This means that genetic modifications that

are facilitated using protoplasts may possibly be evaluated in mature plants.

Protoplasts can be cultured by embedding in agar medium or suspending in

liquid, either as large-volume (25-50 ml) or dropsuspension (about 100 pliters)

cultures or as suspensions on agar. The nutritional requirements of cultured

protoplasts are similar to those for culturing plant cells but with the addition of osmotic stabilizers (mannitol, sorbitol) and possibly antibiotics (Watts

and King, 1973) if bacterial and fungal infection is a problem. The general

references previously cited examine the various aspects of the culture and

regeneration of protoplasts. Recently, Uchimiya and Murashige (1 976) have

systematically examined the nutritional requirements for the recovery of

dividing cells from tobacco protoplasts. They found that while growth regulators

are not essential for cell wall regeneration, an exogenous auxin is required for



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



57



cell division and a lower than normal sucrose concentration (1.5%) seems

optimal. Protoplasts have been successfully cultured in a range of media containing widely varying total salt concentrations (Gamborg, 1976). Uchimiya and

Murashige (1976) found Murashige and Skoog’s (1962) basic salts most successful, although they do point out that a systematic approach t o the nutritional

requirements for successful protoplast regeneration for a chosen species is most

desirable.

The time course of cell wall formation and cell division is variable and is

treated in detail by Vasil(1976), Willison and Cocking (1975), and Williamson et

al. (cited in Gamborg, 1976). The deposition of microfibrils on the plasmalemma

membrane begins immediately after removal of the cell wall degrading enzymes,

and wall formation can be observed macroscopically, using fluorescent brighteners (Calcofluor), usually within 48 hours. Cell division proceeds thereafter and

according to Uchimiya and Murashige (1976), 30% of the protoplasts had

r e - f m e d into dividing cells within 5-6 days. The general case is that cell wall

formation precedes cell division, but according to Meyer and Abel (1975)

division in tobacco protoplasts can occur without rigid cell wall formation. The

precise relationship between nuclear division and cytokinesis in plant cells is not

known, but at least in Chlamydomonas rheinhardtii, mutants without cell W ~

have been recovered (Hyams and Davies, 1972). A similar mutant in plant cells

would be extremely useful particularly if it were a conditional (temperaturesensitive) lesion, whlch under certain conditions could regenerate a cell wall. A

mutant clone with a genetic lesion affecting cell wall formation should be

relatively easy to select because such a clone would tend to disaggregate in

culture. Of course such a mutant might be effectively lethal if cell wall formation is an absolute prerequisite for cell division.

The number of species for which plants have been regenerated from protoplasts more or less parallels regeneration studies with normal tissue culture cells.

Vasil (1976) and Gamborg (1976) provide lists (and appropriate references) of

such species, which include tobacco (Nicotiana tabacum), rapeseed (Brassica

napus), asparagus (Asparagus officinalis), carrot (Daucus carota), petunia

(Petunia hybrida and P. parodii), tomato (Lycopersicon esculentum), bromegrass

(Bromus inermis), Datura innoxia, Ranunculus scleratus, A tropa belladona, and

orange (Citrus sinensis). The development of callus from isolated protoplasts has

been observed in an additional twenty-odd plant species including soybean

(Glycine m a ) , cowpea (Vigna unguiculata), pea (Pisum sativa), sugarcane

(Sacchaium sp.), and flax (Linum usitatissimum). Even at this level not one of

the major cereal crops is represented. While this list of species might seem

impressive, considering that the technology to isolate and culture plant protoplasts has been available for less than a decade, it is unfortunate that no success

has been achieved with the cereals and only limited success with the legumes.

However, this has not been through a lack of application of protoplast tech-



S



58



W. R. SCOWCROFT



niques to cereal and legume species. The recalcitrant nature of these species at

the protoplast level is a reflection of the difficulties experienced in normal tissue

culture studies.

Apart from the earlier mentioned uses of protoplasts (Section V, A), as

potential cell and plant regeneration systems, protoplasts could facilitate the

genetic modification of plant species. There are two broad areas where this

might apply. First, they provide a physically amenable system for the uptake of

large particles and macromolecules such as DNA, aspects of studies of which will

be considered later (Section VI, C). Second, since the rigid cell wall is removed

protoplasts can fuse.



C. PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION

This topic has been recently and extensively reviewed (Cocking, 1975;

Melchers et al., 1975; Vasil, 1976;Gamborg, 1977), and only the broad outlines

and recent developments will be presented here. Protoplast fusion does occur

spontaneously and this appears to be a consequence of the isolation procedure

rather than a result of contact between isolated protoplasts. Fusion between

isolated protoplasts can be induced using NaN03, Ca2+ at high pH, and polyethylene glycol (PEG). A comparative evaluation of these methods indicates that

PEG-induced fusion is the most effective and reproducible method (Burgess and

Fleming, 1974). At a concentration of 20-30% PEG, immediate and extensive

protoplast aggregation occurs whch is enhanced by Ca2+enrichment. Fusion is a

consequence of the removal of PEG. The relative importance of Ca” in protoplast fusion is also a feature of animal cell fusion, where it has been found that

agents which increase the cytoplasmic concentration of Ca2+, e.g., cation ionophores, may enhance fusion (Ahkong et al., 1975).

Following protoplast fusion the heterokaryon may form a cell wall and

proceed to divide to form callus. Gamborg and co-workers (Gamborg, 1977)

have observed division of heterokaryocytes resulting from the fusion of protoplasts obtained from the leaf mesophyll of several species, on the one hand, with

protoplasts from cell cultures, primarily soybean, on the other. Nuclear fusion

has also been observed in heterokaryocytes of pea and soybean (Constabel et al.,

1975a) and carrot and barley (Dudits et al., 1976).

For plant improvement, the real value of somatic hybridization lies in the

capacity t o transfer genetic information from one species to another. There are

now a number of instances where hybrid plants have been recovered following

somatic hybridization by protoplast fusion. In each case the recovery of hybrid

cell clones depended on the use of a selection system which favored the growth

of the hybrid cell. Carlson et al. (1972) recovered a somatic hybrid between two

species of tobacco, Nicotiana glauca and N langsdorfii. These two species will



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



59



hybridize sexually, and cells of the tumorous hybrid grow on a media devoid of

growth regulators; neither of the two parents grows on such media. T h s

provided the basis of Carlson’s selection system and, following the induction of

fusion, a number of presumptive somatic hybrid calluses were recovered of

which three were analyzed in detail. On morphological, electrophoretic, and

chromosome number grounds the somatic hybrid was similar to the sexual

hybrid. Fraction I protein analysis of the somatic hybrid revealed that the

nuclear-coded small subunits of both parents were present but only the chloroplast-coded large subunit polypeptides of N. glauca (Kung et d., 1975).

Carlson’s results have recently been confirmed (Smith et d , 1976) and 23

mature hybrid plants, representing 19 independent fusion events, have been

regenerated following PEG-induced protoplast fusion and selection on growthregulator-free medium. Plants from at least 14 of these 19 events were fertile,

and corolla, leaf, and plant habit were characteristic of, but somewhat different

from, the sexual hybrid. Cytological examination of the 23 hybrid plants

revealed a somatic chromosome number of 56-64, which differs from that of

Carlson et QZ. (1972) who found a somatic number of 42, which is the chromosome number of the sexual amphiploid representing the 24 from Nicotiana

glauca plus 18 from N . lungsdorffi.Smith et QZ. (1 976) explained their results by

assuming that successful hybrids resulted from triple fusions with subsequent

chromosome loss, which indeed they observed in hybrid plants regenerated from

a hybrid callus at different times.

Melchers and Labib (1974) recovered somatic hybrids following fusion of

protoplasts from two mutant lines of Nicotiana tQbQCUm.These mutant lines

carry nonallelic nuclear mutations which affect chlorophyll formation and grow

very slowly in strong light. The F1 hybrid between them is normal. Twenty

independent hybrids were obtained, and genetic segregation of the two nonallelic mutations in the F2 of the somatic hybrid was similar to that of the

sexual hybrid. A similar, although less elegant system, has been used by Gleba et

d. (1975) to recover presumptive somatic hybrids also in N. tabacum.

Somatic hybrids have also been obtained following fusion of protoplasts from

two closely related, sexually compatible species of Petunia (Power et aL, 1976).

Hybrid callus was isolated by a selection procedure based on naturally occurring

differences between the two species, P. parodii and P. hybrids. On a particular

medium P. parodii protoplasts, at best, only produced small (50-cell) colonies

and then ceased to grow, whle P. hybrids protoplasts produced viable callus.

The complementary part of the selection system was based on the greater

sensitivity of P. hybrids protoplasts to actinomycin D. Plants regenerated from

selected “hybrid” callus had the expected chromosome number range of 24-28,

and flower color and morphology were identical to the sexual hybrid which was

distinguishable from either parent. Peroxidase isoenzyme banding patterns differed between the two species. The isoenzyme patterns of the sexual and