II. Plant Cell Tissue Culture

SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



41



elapsed before a mature plant was regenerated from a single cell (Braun,

1959).

The ability t o culture plant cells under defined conditions stems from the

efforts of Gautheret (1939) and White (1942). Since then many research

workers have examined and refined the nutritional and environmental requirements of plant cells in culture. Particular mention must be made of the work of

Skoog and co-workers (Murashige and Skoog, 1962; Linsmaier and Skoog, 1965)

from which most currently used tissue culture media have been derived. Plant

tissue culture media consist of inorganic salts, trace elements, vitamins, a carbon

source for energy, and plant growth regulators. Although generally not essential,

undefined organic supplements such as yeast extract, casein hydrolysate, plant

extracts, and the liquid endosperm of coconuts are often used as supplements.

The basic methodology of plant culture has been adequately covered (Kruse and

Patterson, 1973; Street, 1973; Gamborg and Wetter, 1975) and will not be

further elaborated here. A very large number of plant species representing many

families of gymnosperms, dicotyledons, and monocotyledons have been successfully cultured, and the list continues t o expand.

Virtually any part of a plant can be induced to form callus including embryos,

root or stem sections, hypocotyl, cotyledons of immature seeds, and germinating

seedlings and leaves. The use of young plants or rapidly growing plant tissue is

preferable since nonpathogenic bacteria tend to invade older plants and quiescent tissue. Cell cultures of many species have been initiated in our laboratory,

including dicotyledons, monocotyledons, and herbaceous perennials. Although

seeds are initially slower to callus than, say, stem internode segments, most

success has been achieved by initiating callus from seeds germinated directly on

the culture medium. The rigorous surface sterilization that can be applied to

seeds results in minimal contamination of the resultant callus cultures. Plant cells

can be cultured on agar media in shaken liquid suspension cultures or in

continuous culture fermentors. Cell doubling time varies from 15 hours to

several days.

Suspension cultures are particularly useful for genetic experiments because

they generally grow more rapidly than agar cultures and provide large populations of physiologically homogeneous cells. Moreover, several methods are now

available for adequate and repeatable measurement of growth rate. These include

dry cell weight, packed cell volume, direct cell counting, and turbidity measurements. The use of this latter measure of cell growth of tobacco cultures has

provided us with repeatable estimates of growth. Plant cell cultures tend to be

asynchronous during growth. This tends not to be a problem in genetic studies

but most certainly is in related studies on the cell cycle. Techniques to induce

synchrony include the use of inhibitors of DNA synthesis or of mitosis, nutrient

starvation, exclusion of kinetin (cf. Yeoman, 1974), and the partial replacement

of the aerobic gas phase with nitrogen (Constabel et al., 1974).



42



W.R. SCOWCROFT

B. FREEZE-PRESERVATION OF CELL CULTURES



A recent development is the ability to store plant cell cultures by freezepreservation. This technique is of particular value to cell culture genetics, since

any genetic program will sooner or later have a battery of cell culture mutants

which need to be stocked. Indeed such a technique is essential for callus of those

species which cannot be regenerated to produce mature plants and hence seed.

Freeze-preservation of cell cultures has also been advocated as a means of

preserving valuable genetic stocks of asexually reproducing species of agricultural

importance (Henshaw, 1975). The most extensive studies on freeze-preservation

have been done with carrot cells (Nag and Street, 1973, 1975a,b) but some

studies have been extended to include Acer cells and shoot apices of carnation

(Siebert, 1976). The procedure involves an initially controlled and slow rate of

freezing prior to transfer to liquid nitrogen at -196°C. A cryoprotectant such as

glycerol of dimethylsulfoxide is required during the freezing process. A high rate

of recovery results if the thawing process is fairly rapid and the cryoprotectant is

quickly removed by washing (Nag and Street, 1975b). Freeze-preservation is a

routine aspect of animal cell culture research. Undoubtedly this will also apply

in the plant cell tissue culture research.



C. PLANT REGENERATION FROM CELL CULTURES



From the genetic and plant improvement aspect, the ability to regenerate

fertile plants from cell cultures means that genetic manipulations at the cellular

level can be evaluated in mature plants and possibly utilized in conventional

breeding programs. The totipotency of plant cells has been invaluable in research

which seeks to understand the process of embryogenesis (Street and Withers,

1974). Eventually this might provide a set of principles so that plant regeneration from cell cultures can be achieved at will. Unfortunately, nature has so far

failed to yield her secrets. Consequently, attempts to regenerate plants from cell

cultures must be approached empirically. Regeneration has been attempted for a

very diverse number of plant species and many notable successes have been

achieved. The experimental inputs into these studies have been evaluated by

Murashige (1974) in an attempt to decipher the important variables in the

process of plant regeneration. While Murashige (1 974) was able to show that a

number of nutritional and physical parameters can affect the process of plant

regeneration, the major breakthrough of general applicability was that of Skoog

and Miller (1957). They showed that the relative concentration of the growth

regulators, auxin and cytokinin, determined the pattern of organogenesis. A

relatively high ratio of cytokinin to auxin suppressed root formation and

enhanced shoot initiation. The converse favored root formation over shoot

development. This finding has more or less been systematized to give combina-



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



43



tions of various concentrations of representatives of these two classes of growth

regulators (e.g., de Fossard et al., 1974; Kartha, 1975; Kartha et al., 1976).

An aspect of plant regeneration which has become apparent is that there is

significant genetic variability affecting the ease of plant regeneration from callus.

In a number of studies on regeneration of plants from callus of species such as

tomato (Gresshoff and Doy, 1972), Brassica oleracea (Baroncelli e t al., 1973),

maize (Green and Philipps, 1975), and alfalfa (Bingham et al., 1975) several

varieties were used. In each case the capacity to regenerate plants differed

between varieties. We have had a similar experience with barley, where only two

of 30 varieties tested were successfully regenerated into plants. More recently,

Bingham et al. (1975), by recurrent selection, have developed a line of alfalfa

which regenerates at very high frequency. After only two cycles of recurrent

selection the frequency of regenerating genotypes increased from 12 to 67%.

This indicates that regenerative capacity is highly heritable.

In attempts to develop tissue culture and regeneration for a particular species,

most workers tend to utilize a limited number of varieties (or genotypes) and

vary the culture media and growth regulator concentrations. The alternate

option is to restrict the variability in the culture media and utilize diverse

genotypes. For particularly important agricultural species, benefit in terms of

ease of regeneration would most certainly be derived from a few cycles of

recurrent selection for plant regeneration.

The number of plant species in which the potential for plant regeneration from

callus has been demonstrated is impressive. The recent compilation of species by

Murashige (1974) even now can be considerably expanded. The majority of

successes have been achieved with exotic and horticultural species but, among

the agricultural gramineae, plants can be regenerated from callus of wheat

(Shimada et al., 1969), rice (Nishi et al., 1968, 1973), sugar cane (Nickel1 and

Heinz, 1974), maize (Green and Philipps, 1975), barley (Cheng and Smith,

1975), and oats (Lorz ef al., 1976). Success has also been achieved in regenerating plants from callus of coffee (Herman and Hass, 1975), cassava

(Kartha and Gamborg, 1975), potato (Skirvin et al., 1975), and for some tree

and shrub species (Sommer et al., 1975; Pierik, 1975).

Until recently the legumes have proven recalcitrant to differentiation. Despite

extensive research with soybean cell cultures, the differentiation of mature

plants from callus has not been reported. The two legume species where plants

can be regenerated from callus with ease are alfalfa (Bingham et al., 1975) and a

tropical pasture legume, Stylosanthes hamata (Scowcroft and Adamson, 1976).

It is noteworthy that S. hamata is a perennial legume, and that the more

successfully regenerating varieties of alfalfa were creeping rooted types which

develop adventitious shoots from root cells.

Apart from the “mere” difficulties of translating the potentialities of plant

regeneration into everyday reality there are other limitations. Among these the

maintenance of long-term embryogenic potential is paramount (Murashige,



44



W. R. SCOWCROFT



1974; Thomas and Davey, 1975). Street (1975) and Sheridan (1975) have

discussed the loss of regenerative capacity as a function of an increase in

aneuploidy. Some cell lines of Haplopappus, Crepis, and Lilium do not tend

toward aneuploidy and retain long-term embryogenic potential.

The cellular cloning of plant species is used in the asexual propagation of many

species (Murashige, 1974) and in the production of “disease-free’’ plants. In the

ensuing discussion of the genetic modification of plants at the cellular level, the

ability to regenerate plants from such cells is essential for such modification to

be of value to plant improvement. First, the cellular modification must be

evaluated in the mature plant so that the plant breeder may judge their potential

contribution to the gene pool. Second, the plant breeder must be able to inject

such novel genotypes into his breeding population by conventional sexual

hybridization for which he needs mature fertile plants.

Ill. Anther Culture and Haploids



The potential value of plants with the gametic number of chromosomes

(haploids) in basic and applied genetic research, has been recognized ever since

Blakeslee et al. (1922) first described a haploid mutant in Datura. The semantic

arguments surrounding the use of the term “haploid” are discussed by de

Fossard (1974). Because of the great potential to plant improvement, advances

in haploid plant research have been closely monitored. Kimber and Riley (1963)

reviewed the then known origins of haploidy. They listed 71 species in which

haploids had occurred spontaneously, as a consequence of hybridization, from

twin embryos or by experimental induction. More recent progress was considered in depth at a conference in Canada in 1974 (Kasha, 1974). The production of haploids through parthenogensis or by chromosome elimination in barley

(Kasha and Kao, 1970) and recently in wheat (Barclay, 1975) have as much

potential importance to basic and applied genetic research as those generated by

anther culture. I will not discuss the former two methods further (cf. Kasha,

1974), but acknowledge that the practical and theoretical consequences of

producing haploids by anther or pollen culture also apply to these other

methods.



A. METHODOLOGY



The technique of producing haploid genotypes by anther culture was pioneered by Guha and Maheshwari (1964) in DQmra. This methodology was

developed by many, but most notably by J. P. and C. Nitsch and co-workers

(Nitsch and Nitsch, 1969; Nitsch, 1972). More recently Nitsch (1974, 1975) has



SOMATIC CELL GENETICS AND PLANT IMPROVEMENT



45



demonstrated that isolated microspores of tobacco can be cultured to produce

haploid plants. Extensive cytological analyses in Nicotiana and Datura have

elucidated the events which lead to the production of haploids (see Sunderland

and Dunwell, 1974; Sunderland, 1974). Under appropriate culture conditions

the normal process of pollen development is arrested between the tetrad stage

and the conclusion of the first pollen mitosis. Subsequently the generative cell

degenerates while the normally quiescent vegetative cell divides to form an

embryolike structure. Cytological events indicate that haploidy follows one of

either of two developmental pathways, each of which leads to the ultimate

degeneration of the generative partner (Sunderland, 1974). Following induction

of cell division in the pollen, embryos develop which give rise to plantlets

actually growing out of the anther, or callus may be formed which then has to

be differentiated to regenerate a plant. The former sequence is characteristic

primarily of tobacco and Datura, and the latter is the more general consequence

in other species. In cases where callus, hopefully with a haploid complement, is

formed, controlling the ploidy level in the callus may be a serious limitation to

its practical use for generating haploids. On the basis of the use of p-fluorophenylalanine (PFP) to increase frequency of haploid segregation in fungi (Day

and Jones, 1971), Gupta and Carlson (1972) claimed that PFP inhibited the

growth of diploid, but not haploid, cells of tobacco. This claim has not been

sustained (Zenk, 1974; Dix and Street, 1974) or at best is not very reproducible

(Chaleff and Carlson, 1974). An additional problem, as pointed out earlier, is the

difficulty of regenerating plants from callus. This varies from species to species,

and indeed from genotype to genotype within a species. With haploid callus I

would judge that the problem of regenerating a representative sample of haploid

genotypes might be even more difficult.

Notwithstanding, haploid plants have been successfully produced in species

other than tobacco and Datura. Catalogs of species in which haploids have been

produced are provided by Smith (1974), McComb (1974), and Sunderland

(1974). Although specific reports cannot be cited, a haploid information exchange service, edited by the Haploid Project Group, Max Planck Institute fur

Biologie, Rosenhof, Germany (see Kasha, 1974, p. 41 l), carries reports of both

successful and unsuccessful attempts to induce haploids. Apart from tobacco,

other important crop species in which haploids have been produced by anther

culture include rice (Niizeki and Oono, 1968; Oono, 1975; Wang e t al., 1974;

Laboratory of Genetics, 1975; Woo and See, 1975), wheat (Ouyang et al., 1973;

C. Wang et aZ., 1973;Picard and de Buyser, 1973), barley (Clapham, 1973; Dale,

1975), triticale (Y. Wang et al., 1973; Sun et al., 1974), tuberous Solanum

species (Irikura and Sakaguchi, 1972; Dunwell and Sunderland, 1973), and

turnip rape (Brassica campestris) (Keller e t al., 1975).

The use of haploids in plant improvement, or indeed any research where a

gametophyte is required from the haploid sporophyte, requires that the chro-