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IV. Progress in Cell and Tissue Culture Research
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.
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
CELL AND TISSUE CULTURE TECHNIQUES
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.
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.)
CELL AND TISSUE CULTURE TECHNIQUES
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.
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
CELL AND TISSUE CULTURE TECHNIQUES
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
S. KRESOVICH ET AL.
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
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
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.)
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.
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
Research in our laboratory indicating a cellular component of salinity
tolerance in sugar cane (Kresovich et al., 1986a; McGee et al., 1986) has en-
CELL AND TISSUE CULTURE TECHNIQUES
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.
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
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
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.
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