Plant Tissue Culture

Nancy A. Reichert

The science of plant tissue culture is based on the concept of totipotency, which is the ability of individual plant cells to grow into complete adult plants. The plant cells do this by responding to "cues" in the tissue culture media, most important of which are the plant growth regulators (PGR's). Other media components include all the nutrients (organic and inorganic) necessary for growth in culture (in vitro).

Explant choice (tissue or organ placed in culture) also aids in determination of outcome. If true­to­type plants are desired, shoot tips and axillary buds are usually employed for direct growth and regeneration. If the goal is plant improvement, plants of altered type are desired. Therefore, explants, such as leaf and internodal stem sections, would be utilized in adventitious (indirect) regeneration protocols.

In plant improvement strategies, tissues in culture can be manipulated in various ways, depending on the desired outcome. Regardless of desired end­product, the first goal is to develop reliable adventitious regeneration protocols for the plant of interest.

With kenaf, our research group at Mississippi State University was the first to regenerate intact kenaf plants in vitro (McLean et al., 1992). Internodal stem explants of Tainung 1 were tested on media containing different combinations and concentrations auxins and cytokinins (PGR's).

Within 5 days, callus (growth of undifferentiated cells) formed around the periphery of the explants. Within 30 days, adventitious shoots developed from the callus on various media. The shoots were excised and placed on a different medium for root formation. Intact plants were then transferred to soil for continued growth.

Since the initial research, we have optimized adventitious regeneration protocols for kenaf, starting with internodal stem and leaf sections. We can reliably regenerate three new varieties: Everglades 41 (E41), Guatemala 45 (G45), and G48 (Reichert and Liu, 1994: manuscript in preparation).

In 1993, we field­tested 28 E41 tissue culture regenerants (R0), and currently are in the process of analyzing their progeny (R1). In 1994, we will be field testing hundreds (and perhaps, thousands) of R0 regenerants from each of these varieties.

Another explant type used in adventitious regeneration protocols are protoplasts, which are plant cells without cell walls. Hydrolytic enzymes (commercially available) are used to digest away the plant cell walls. Typical enzymes used for this purpose are a cellulase (digests cellulose) plus a pectinase (digests away pectins). Generally, millions of protoplasts can be harvested from each gram of leaf tissue (approximately 1/30 oz).

Once cell walls are removed, various manipulations can be performed on these "naked" cells. Manipulations include genetic engineering and cell fusion strategies (discussed below).

We have optimized protoplast isolation and culture protocols for seven kenaf varieties (Cubano 2032, E41, E71, G4, G45, G51, and Tainung 1) and are currently modifying our adventitious regeneration protocols to fit into our protoplast protocol (Reichert and Liu, 1994; manuscript in preparation).

With the development of adventitious regeneration protocols, plant tissue culture can be used as a tool for use in crop improvement strategies. Three projects interrelated with kenaf tissue culture, plant breeding, and genetic engineering are briefly described below.

(1) Screening for improved traits resulting from somaclonal variation. Plant cells in tissue culture have mutation rates much higher than the rate that normally occurs in nature. Because of this, plants regenerated from these cells display a higher frequency of new or altered traits. These altered plants

arising from culture are called somaclonal variants.

Many researchers have used the somaclonal variation phenomenon in the past to improve other plants. Some altered traits that have been observed include variations in pathogen/disease resistance, leaf shape, growth habit, maturity

date and yield (Larkin and Scowcroft, 1981; Evans, 1989). Examples of plants improved in this manner include ornamental, vegetable and agronomic crops. New carrot, celery, geranium, pepper, and tomato varieties have been developed in this manner.

We have, and will continue to screen our regenerants (R0 and R1) for new or altered traits. Superior plants will then be incorporated into the kenaf breeding project at Mississippi State University.

(2) Develop tetraploid kenaf via protoplast fusions (electrofusion) for trait assessment and breeding to related species. Normal kenaf has 36 chromosomes (diploid two complete sets of chromosomes) in each cell. Protoplast fusions (combining two plant cells into one) are used to increase the total numbers of chromosomes in each cell. Two kenaf cells fused together would create a cell containing 72 chromosomes (tetraploid: four complete sets of chromosomes). Plants regenerated from this cell would contain 72 chromosomes in all cells. Tetraploid plants, in general, display more vigorous growth habits than their diploid counterparts.

We would like to perform electrofusions with kenaf protoplasts for two distinct reasons. Since kenaf is harvested for its fiber, we want to determine if tetraploid kenaf can generate greater amounts of fiber per plant. We also

would determine any effects on fiber quality.

Tetraploid kenaf would also be incorporated into a breeding program to introduce resistance/tolerance to a devastating plant pathogen. Kenaf is extremely susceptible to root­knot nematode (Meloidogyne incognita) damage, which can greatly affect yield. Hibiscus sabdariffa, (roselle; tetraploid; 72 chromosomes) a species related to kenaf, displays nematode tolerance (no yield

reductions). Because differences in chromosome numbers, sexual crosses between the two species are nearly impossible. With generation of tetraploid kenaf, sexual crosses to roselle should be possible for incorporation of nematode tolerance into kenaf.

We have developed reliable electrofusion protocols for kenaf protoplasts designed to be incorporated into our protoplast isolation and culture protocols for the eventual generation of tetraploid kenaf.

(3) Improve kenaf via genetic engineering. Previous research by others (Banks et al., 1993) proved that kenaf can be genetically engineered. Unfortunately, because of their experimental design, they were unable to regenerate transgenic (engineered) plants. Because of our regeneration protocols, we should be able to genetically engineer kenaf tissues and regenerate transgenic kenaf for field growth and analysis. In fact, plant transformation protocols are currently being developed to coincide with our defined regeneration protocols for immediate use once genes are identified for transfer into kenaf.

References

Banks, S.W., D.R. Gossett, M.C. Lucas, E.P. Millhollon, and M.G. LaCelle. 1993. Agrobacterium­mediated transformation of kenaf (Hibiscus cannabinus L.) with the B­glucurondidase (GUS) gene. Plant Mol. Biol. Rep. 11:101­104.

Evans, D.A. 1989. Somaclonal variation ­ genetic basis and breeding applications. Trends Genet. 5:46­50.

Larkin, P.J., and W.R. Scowcroft. 1981. Somaclonal variation ­ a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197­214.

McLean, K.S., G.W. Lawrence, and N.A. Reichert. 1992. Callus induction and adventitious organogenesis of kenaf (Hibiscus cannabinus L.). Plant Cell Rep. 11:532­534.

Reichert, N.A. and D. Liu. 1994. Manipulations and regeneration of kenaf (Hibiscus cannabinus L.) in vitro. (manuscript in preparation)

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Nancy A. Reichert is an Assistant Professor of Horticulture, Department of Plant and Soil Sciences, Mississippi State University.