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IV. Progress in Cell and Tissue Culture Research

IV. Progress in Cell and Tissue Culture Research

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154



S. KRESOVICH E T A L .



FIG.2. Cross section of aberrant meristematicgrowth, mistakenly reported as undifferentiated callus in many sorghum studies. x61. (Courtesy of D. K. Villalon.)



Reports of callus induction from mature somatic tissue are few, and this

deficiency in the current technology precludes screening and selection of

hybrids and segregating populations through callus culture. Without a

routine method of callus induction from field-grown mature plants (Panella

et al., 1987), cell and tissue culture techniques are of limited value in early

generation screening and selection strategies.



B. ANTHER

CULTURE

Despite the potential value of anther culture as a source of homozygous

germplasm for breeding, progress toward the establishment of callus

cultures from microspores of sorghum has been slow (Jinchow Institute of

Agriculture, 1978; Liang, 1978). Possibly this void may be associated with

the great need for technical support associated with this type of investigation. An initial effort (Caulkins, 1978) directed at the induction of anther

cultures of 22 genotypes including 60M and Norghum evaluated the effects

of experimental variables such as pretreatment conditions (i.e., cold shock);

medium constituents and concentrations (i.e., minerals, auxins, cytokinins,

carbohydrates, amino acids, vitimins, and activated charcoal or other antioxidants); and incubation conditions (i.e., temperature and photoperiod

length) on culture establishment. Nonetheless, results proved futile. Recent

studies by Rose et al. (1986a,b) have demonstrated progress toward the

establishment of haploid callus cultures using the sorghum cultivar



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155



FIG. 3. Somatic embryos derived from scutellar tissue of an immature (14-day-old) embryo of IS362OC. x20. (Courtesy of J. N. Reed.)



Advance. However, despite the establishment of approximately 1200

cultures, only four yielded albino shoots. Clearly much more work will be

necessary prior to this application reaching fruition.



c.



REGENERATIONAND SOMATIC EMBRYOGENESIS



For the new gene-splicing and genetic manipulation technology to be applicable in a crop improvement program, it is necessary to have an efficient

method of plant regeneration from single cells and/or callus (Fig. 4).

Reports of plant regeneration from sorghum tissue cultures are limited,



FIG.4. Germinated somatic embryo of Regular Hegari. x 19. (Courtesy of J . N. Reed.)



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157



especially when the question of aberrant meristematic growth is considered

(Cure and Mott, 1978; Mott and Cure, 1978). Masteller and Holden (1970)

reported limited plant regeneration from callus derived from the basal node

tissue of sorghum genotypes Norghum and North Dakota 104. Plant

regeneration from callus derived from nodal tissue also was highlighted by

Thomas et al. (1977). Likewise, Bhaskaran et al. (1983) have reported

regeneration of IS3620C (a margaritiferum) from long-term (10-month-old)

callus cultures originating from excised nodal tissue. A recent report from

the USSR by El’konin et al. (1984) described plant regeneration from callus

cultures that had been maintained for a period of 45 months. One of the

first reports of plant regeneration of sweet sorghum, from the cultivars

Keller and Wray, utilized nodal tissue as the explant source (Kresovich et

al., 1986b). Mascarenhas et al. (1975b) reported plant and callus formation

from stem segments containing the growth tip; however, no distinction was

made as to the exact origin of regenerated tissue as well as the genetics of the

explant source, i.e., inbred line or hybrid.

Gamborg et al. (1977) observed morphogenesis and plant regeneration

from callus of immature embryos (12-18 days postpollination) of the

sorghum hybrid X4004A. Prolific shoot formation was reported by Thomas

et al. (1977) from immature embryos (10-30 days postpollination) of the

sorghum hybrid Oro. Brar et al. (1979a) reported embryogenesis from immature embryos (10-14 days old) of sorghum hybrids GPR-168, NK-300,

and X4004A. It must be noted that these efforts employed “immature” embryos collected 10-30 days postpollination from self-pollinated sorghum

hybrids as their explant sources. The description of 10- to 30-day-old embryos

as immature is inconsistent with the findings of Artschwager and McGuire

(1949) and Paulson (1969), which suggest that embryos of sorghum may

reach maturity in as little as 12-25 days. Furthermore, a zygotic embryo

derived from a selfed hybrid or a heterozygous line will not necessarily have

the same genetic constituent as the parent, thereby adding uncontrolled and

unwanted factors to the analysis of response.

Recent investigations (Ma et al., 1987) with the Chinese inbred C401-1

have highlighted the responsiveness of 9- to 12-day-old embryos. We have

found embryos excised 10-14 days after pollination to be a responsive explant source based on our studies of the genotypes (inbreds) RTx430,

BTx399, CS3541, MN1500, Regular Hegari, and IS3620C. In our studies,

overall embryogenesis (as calculated on a per culture basis) across genotypes

ranged from 26 to 69%. The frequency of response for embryos from individual panicles within a genotype reached values as high as 95%. These

results suggest that a high degree of phenotypic plasticity exists for in vitro

responsiveness. Therefore, potential exists for improving response through

intensive selection for this character. Furthermore, Ma et al. (1987)

reported that the ability to differentiate plants from callus was heritable,

controlled by two gene pairs, and acted as a dominant trait.



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S. KRESOVICH ET AL.



Unlike the results of Ma et af. (1987), we found no correlation between

embryo size and responsiveness. As noted in the work of Santos and Torne

(1986) with maize, we have identified factors, i.e., greenhouse

microclimatology and donor plant preconditioning, that potentially may

confound the effects of age and/or size of embryo on explant response.

Therefore, one must be sensitive to the role that explant-environment interactions play in affecting in vitro response. Ma et af. (1987) also recognized this point in the interpretation of their data.

While documenting the in vitro responsiveness of the immature embryo,

many investigators have failed to consider the potential breeding (and interpretational) problems associated with the use of sporophytic (2n) tissue

from segregating populations of germplasm, i.e., F, hybrids and selections

from early F,-F,, generations. Therefore, many of the interpretations of the

response of the immature embryo in culture may have been confounded due

to genetic recombination and segregation of the immature embryos. While

Thomas et af. (1977) also reported plant regeneration from mature embryos

excised from 2-year-old seed of the hybrid Oro, it is unclear as to whether

the tissue was derived from a segregating or nonsegregating source.

Building on the work of Thomas et al. (1977), Dunstan et al. (1978) examined the development of these embryo-like structures (Fig. 5 ) . Although

some embryos were of single-cell origin, no evidence was found that they

arose from proliferating callus cells. Instead, the shoots and embryo formed

initially from the primary explant (the zygotic embryo).

El’konin et al. (1986) recently documented somatic embryogenesis and

plant regeneration from callus obtained from mature embryos of the

genotypes Norghum 165 and Volzhskoe 2. MacKinnon et af. (1986a) reported

somatic embryogenesis and plant regeneration utilizing callus derived from

both immature and mature embryos of the sweet sorghum cultivars Keller,

Rio, and Wray. Their results demonstrated no major difference in response

with either grain or sweet sorghum cultivars; however, they concluded that

pigmentation color of the callus differed between grain and sweet types. In

our laboratory, we have found the pigmentation response to be correlated

with the phenotypic character of secondary plant color (tan, red, or purple),

rather than whether the sorghum was either a grain or sweet type.

Brettell et af. (1980) described embryogenesis from cultured immature inflorescences of several sorghum cultivars and hybrids including CK-60,

Regular Hegari, Plainsman, WAC692, G522DR, FS302, and G83F. All entries except Regular Hegari produced plants, although the hybrid G522DR

was superior in culture response. Furthermore, observations of inflorescences

from 10 to 50 mm in length indicated that those from 10 to 20 mm yielded the

greatest frequency of embryogenic cultures. Studies in our laboratory with

the genotypes (33541, MN1500, and Regular Hegari and inflorescence

lengths ranging from 16 to 105 mm have suggested a correlation between



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159



FIG. 5. Longitudinal section of a somatic embryo derived from immature (22 days

postpollination) inflorescence tissue of Regular Hegari. x LOO. (Courtesy of J. N. Reed.)



length and embryogenic response. However, the response was not consistent

across genotypes. The embryogenic responses of CS3541 and Regular Hegari

were negatively correlated with inflorescence length, while MN1500

demonstrated a positive correlation. Also, MN1500 yielded more callus per

unit length of inflorescence than CS3541. As with other tissue sources, high

variation in response between individual cultured inflorescences was observed. Again, these findings indicate that it may be possible to improve

response through selection. Average values of establishment of embryogenic

cultures across tested genotypes have ranged from 21 to 75%.

Wernicke and Brettell (1980,1982) and Wernicke et al. (1982)reported

somatic embryogenesis from immature leaves of G522DR. This group also



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160



noted the further spatially and temporally, i.e., more mature, leaf tissue

was from the apical meristem, the less responsive it became. This finding

suggests that competence in vitro, i.e., the ability to regenerate plants, is

correlated with continued meristematic activity. These reports indicate that

somatic embryogenesis from the primary explant (zygotic embryo, inflorescence, or immature leaf) is possible in a number of sorghum

genotypes, although the technique is not yet routine. While these reports are

encouraging, plant regeneration of sorghum has been generally of low frequency, limited to a few genotypes, and with great variability (as affected

by genetic segregation and donor plant preconditioning) in culture

response. Furthermore, as one attempts to integrate cell and tissue culture

techniques with plant breeding, practical considerations, i.e., amount of

technical support and availability of desired tissue types, may force a

balance between ease of explant acquisition and explant responsiveness

(Fig. 6).

Based on these concerns, complementary studies have been established in

our laboratory to identify biochemical differences (markers) discriminating

embryogenic from nonembryogenic callus. Discernible biochemical differences ultimately may be utilized as rapid screening tools for the identification of responsive cell types. Preliminary results in our laboratory suggest differences exist in the esterase isozyme patterns between embryogenic

and nonembryogenic calli of, RTx430 and Regular Hegari. Similar results

have been documented in the maize genotype B73 (Everett et al., 1985).

However, routine application of these techniques for identification and/or

screening purposes will require further resolution. Nonetheless, isozymic

analysis ultimately may prove useful in the study of the genetic regulation of

plant development.

D.



PROTOPLAST

CULTURE



Relatively little research has been directed at sorghum protoplast isolation,

culture, and regeneration. This is an important area because many genetic

transfer techniques (electroporation, microinjection, etc.) rely on an available

source of viable protoplasts (Chin and Scott, 1979). The earliest reported

establishment of sorghum protoplast cultures was by Brar et al. (1979b). A

cell suspension of the sorghum genotype GPR-168 was initiated from immature embryos, and protoplasts were isolated from 3- to 4day-old suspension cultures. Fusion was reported between sorghum protoplasts and leaf protoplasts of maize. Fusion products underwent several divisions and formed

colonies of 8-10 cells. A subsequent study by Karunaratne and Scott (1981)

isolated protoplasts from leaves of the genotypk Zulu. While reporting a

higher mitotic index than the protoplast suspensions of Brar et al. (1979b),

long-term viability of the cultures could not be maintained. Chourey and



FIG.6. Regenerated plants of Regular Hegari ready for transfer to greenhouse. x 1. (Courtesy of R. E. McGee.)



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S. KRESOVICH ETAL.



Sharpe (1 985) described cell suspension establishment and protoplast isolation from callus cultures initiated with immature embryos of a sorghum

hybrid and two cultivars, NK-300, M35-1, and SC420, respectively. Protoplasts underwent sustained cell division and callus formation. In some instances, protoplast-derived calli have been put back into suspension, giving

rise to “protoclone cell suspension cultures. ” These results indicate that

protoplast isolation and subsequent callus formation is possible in

sorghum. No observations of plant regeneration from protoplast-derived

cultures have been reported.



V.



POTENTIAL APPLICATIONS



Using plant cell and tissue culture techniques as model systems to study

and affect plant behavior has been a primary goal of biotechnology. Future

applications may include studying cellular and tissue-level mechanisms involved in stress physiology; rapid screening and selection techniques at the

cellular and tissue levels; increasing, identifying, and controlling variation;

and as adjuncts to genetic studies. A fundamental understanding and control of the in vitro system is necessary to undertake such studies. However,

our basic understanding of sorghum cell and tissue culture techniques is

limited; nonetheless, several applications have been attempted and potential

does exist in these and other areas.

A. CELL AND TISSUECULTURE! AS A MODELSYSTEM



Many processes which ultimately affect whole-plant characters occur at

the cellular level. By studying these processes using cell and tissue culture

techniques free from complicated whole-plant interactions, researchers can

gain knowledge into the physiology and biochemistry of the cell. If subsequent correlations can be drawn between cellular and higher organizational

level, e.g., whole-plant responses, then these characters may become useful

as screening or selection markers. The work of Bhaskaran et al. (1985) exemplifies this type of approach in which proline accumulation in callus

cultures of sorghum was measured in response to water stress. These studies

have been continued (Newton et al., 1986) to document changes in soluble

carbohydrates and organic acids as a function of water stress. Results indicated proline concentration to be an incidental consequence of stress;

however, soluble carbohydrate concentrations may be correlated with

osmotic adjustment.

Research in our laboratory indicating a cellular component of salinity

tolerance in sugar cane (Kresovich et al., 1986a; McGee et al., 1986) has en-



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163



couraged us to begin utilization of cell suspensions of sorghum as a model

to elucidate and assess its modes of cellular adaptation to salinity stress. By

exploiting cell and tissue cultures of sorghum in this manner, it is hoped that

a better understanding of stress physiology and biochemistry will evolve.

This model may prove useful in the study of mechanisms such as carbon

partitioning and utilization, mineral uptake and compartmentation, and

membrane stability. When integrated with an improved understanding of

the interactions between tissue types, media, preconditioning effects, and

plant regeneration, this approach offers the potential advantage of being better able to isolate and propagate variants with these particular characters.

Although there are several well-established model systems (tobacco, carrot, tomato, etc.), it is often difficult to extrapolate results across species.

Therefore, the use of sorghum cell and tissue culture techniques to verify

and study these phenomena is important.



B.



SCREENING AND SELECTION TECHNIQUES



The phenomenon of callus cultures paralleling whole plant response to

salts (Orton, 1980) and heavy metals (Meredith, 1978) in growth media has

been observed in several species. Relatively little work has been done with

sorghum regarding this application. Smith et af. (1983) tested callus from

four sorghum genotypes (BTx3197, BTx623, BTx399, and IS3620C) on

media containing 0, 100, 200, and 400 p k f aluminum-EDTA. Differences

were reported in growth among the cultivars in response to aluminum.

Plants were regenerated from IS3620C after several passages on selection

media, but no field test results are yet available.

Bhaskaran et af. (1983) reported plant regeneration from IS3620Cderived callus grown on media containing high concentrations (0.1-0.5%

w/v) of sodium chloride. Callus obtained by prolonged subculture on this

selection medium was better able to grow on a medium containing sodium

chloride than donor cells. Regenerated plants grown to maturity exhibited

low seed set, initially hindering attempts at progeny analysis. A subsequent

study (Bhaskaran et af., 1986) noted differences between selected and

nonselected progeny with respect to enhanced tolerance (reported as

vegetative growth only) to sodium chloride. However, the control population of the donor plant demonstrated a greater relative tolerance than either

tissue culture-selected population; therefore, this finding suggests that the

selection methods employed may be of little practical value for sorghum improvement. MacKinnon et af. (1986b) explored four selection methods for

the derivation of sodium chloride-tolerant plants of the cultivars Regular

Hegari, Keller, and Rio. With regard to frequency of regeneration, many

more plants were obtained from selection on solid rather than liquid



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S. KRESOVICH E T A L .



medium. However, results from field tests will be necessary to confirm the

frequency of true tolerant or resistant plants versus that of potential

escapes. From a crop improvement perspective, one needs to be concerned

more with the frequency of obtaining desired variants from a selection

technique rather than the total number of plants derived. That is, if field

evaluation suggets a great proportion of the regenerated population to be

escapes, the in vitro selection technique will be of limited value to the plant

breeder.

Smith et af. (1985) tested callus from 10 sorghum genotypes on polyethylene glycol-amended screening media (0-20070 w/v). Indications were

that preanthesis drought tolerance and performance of cultured tissues might

be related. Subsequent studies suggest that extreme variability in callus

growth makes discerning differences difficult (Panella, 1986; Reilley, 1986).

To our knowledge, no work has been directed at the screening and selection

of herbicide-resistant genotypes of sorghum via cell or tissue culture.

However, based on promising work with other cereals, e.g., maize (Shaner

and Anderson, 1985), this potential screening and selection approach warrants study.

The ontogeny of tissue cultures becomes critical when using screening and

selection techniques. Dunstan et af. (1978, 1979) and Wernicke et af. (1982),

based on their conclusions that regenerated plants arose initially via somatic

embryogenesis, albeit from the primary explant only, suggest that variation

may be more directed when selection pressure can be applied to single cells.

This hypothesis again points to the need for cultures containing synchronized, undifferentiated cells that can be readily induced to differentiate via

somatic embryogenesis into new plants. Because relatively little work has

been done with sorghum screening and selection for salt, heavy metal, and

drought tolerance, it is difficult to predict whether these potential applications of cell and tissue culture techniques can be implemented into traditional

breeding programs. In order for these new techniques to be useful, a strong

correlation between in vitro and field responses must be established. Furthermore, in vitro screening and selection must become simpler, more reliable,

and more cost-effective to be useful in a breeding program.

C. SOMACLONAL

VARIATION



Somaclonal variation, i.e., the variability expressed in plant phenotype

derived from cell and tissue culture, does occur in many, if not all, species.

This variation, depending upon how it is expressed, is similar to that caused

by a chemical mutagen and is generally detrimental. Somaclonal variation,

i.e., variation in leaf morphology and growth habit, was first reported in



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