Developing Cell and Tissue Culture Systems for the Improvement of Cereal and Grass Crops

The components of plant tissue culture media II. Organic additions, osmotic and pH effects, and support systems

Immature embryos as well as explants obtained from young inflorescences of Pennisetum atnericanum (pearl millet) give rise to callus tissues on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). A compact and pale-yellow callus that arises from the peripheral cells of the scutellum, and from the young inflorescences, undergoes further organized growth. When transferred to a 2,4-D-free medium, supplemented with indole-acetic acid or kinetin, or both, embryoids are formed in the organized areas of the callus. Embryoids show a bipolar organization with a shoot-coleorhiza (root) axis and have a coleoptile-like structure surrounded at the base by a cup-shaped structure that resembles the scutellum in texture and morphology. Embryoids show bilateral or radial symmetry and "germinate" in vitro to form plants that have been grown to maturity in soil. Similar embryogenic callus cultures have been produced from young inflorescence tissues of hybrid Pennisetum, a triploid sexually sterile hybrid of P. americanum x P. purpureum. Plants derived from these have also been transferred to soil. The regenerated plants showed normal chromosome numbers. REGENERATION of plants from cultured immature embryos and inflorescence tissues of several species of cereals and grasses has been described (Thomas and Wernicke, 1978; Thomas, King and Potrykus 1979; I. K. Vasil, Ahuja and Vasil, 1979). In cereals it is the scutellar tissue of the immature embryo which proliferates in vitro (LaRue, 1952; Narayanaswamy, 1959; Norstog, 1970), and has the maximum potential for organogenesis and plant regeneration (Green and Phillips, 1975; Cummings, Green and Stuthman, 1976; Gamborg et al., 1977; Thomas, King and Potrykus, 1977; Gosch-Wackerle, Avivi and Galun, 1979; Rybczynski, 1979). Plant regeneration from hypocotyledonary explants of Pennisetum typhoideum has been reported by Rangan (1976). The differentiation of shoots in tissue cultures of cereals and grasses is not too frequent, and is often sporadic and short-lived. Plants are formed from shoot primordia that arise in centers of meristematic activity located in the surface layers of callus tissue cultures (Rangan, 1974; Dunstan et al., 1979; Nakano and Maeda, 1979; Shimada and Yamada, 1979; Springer, ' Received for publication 2 June 1980; revision accepted 11 October 1980. Florida Agriculture Experiment Station Journal Series No. 2462. This research was supported by the Florida Agriculture Experiment Station, and by the Science and Education Administration of the U.S. Department of Agriculture under Grant No. 5901-0510-8-0141-0 from the Competitive Research Grants Office. Green and Kohn, 1979). The phenomenon of somatic embryogenesis in cereals and grasses is even rarer (Gamborg, Constabel and Miller, 1970; Norstog, 1970; V. Vasil and Vasil, 1980, 1981). The formation of shoots and embryoids cannot be ascribed to any known plant growth substances, although it is generally known that shoot differentiation follows the transfer of callus tissues from a medium containing 2,4dichlorophenoxyacetic acid (2,4-D) to media devoid of this synthetic auxin, or containing very low concentrations. We report rapid clonal propagation via somatic embryogenesis in Pennisetum americanum (L.) K. Schum. = P. typhoides (Burm.) Stapf & C. E. Hubb. (pearl or Indian millet), and hybrid Pennisetum (P. americanum x P. purpureum Schumach, Napier or elephant grass; Muldoon and Pearson, 1979). Both plants belong to the tribe Paniceae of the Gramineae, and are widely grown in the arid tropical and subtropical parts of Asia and Africa. Pearl millet is an important human food, and along with hybrid Pennisetum is also used extensively as a fodder crop. Our interest in the clonal propagation of these plants in vitro is related to our attempts to develop the necessary technology for the somatic hybridization and genetic manipulation of cereal crops through unconventional means. The regeneration of plants from cultured protoplasts, single cells, and callus tissue is a basic prerequisite that must be satisfied before any of these potentially useful techniques can be adopted for cereals (I. K. Vasil and Vasil, 1980). Pearl

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM TISSUE CULTURES OF PENNISETUM AMERICANUM, AND P. AMERICANUM x P. PURPUREUM HYBRID

Suspensions composed of small, richly cytoplasmic starch-containing cells (embryogenic cells) and elongated thick-walled cells with large vacuoles were established from cultured inflorescence segments of pearl millet in Murashige and Skoog's medium supplemented with 2.5 mg 2,4-D/l + 5% coconut milk. When 2- to 3-wk-old suspensions were transferred to agar with reduced 2,4-D levels, hundreds of globular and early cotyledonary embryoids were produced which developed into green plants when transferred to medium + ABA. Atypical embryoids, some containing many shoot meristems and a leafy scutellum were also observed

Characterization of an embryogenic cell suspension culture derived from cultured inflorescences of pennisetum americanum (pearl millet, gramineae)

Somatic embryogenesis was induced in proliferating leaf segments of Panicum maximum Jacq., cultured on Murashige and Skoog's nutrient medium containing 2,4-dichlorophenoxyacetic acid and coconut milk. The embryoids gave rise to plants on a medium containing gibberellic acid. The plants were successfully transplanted to soil and grown to maturity. Histological examination of proliferating leaves showed that the embryogenic callus tissue was formed by divisions in cells of the lower epidermis as well as the mesophyll tissue. The regenerated plants showed the normal chromosome number of 2n=4x=32.

Somatic embryogenesis and plant regeneration from leaf tissues of Panicum maximum Jacq.

Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple

Growth and Plantlet Regeneration in Tissue Cultures of Some Indian Millets: Paspalum scrobiculatum L., Eleusine coracanaGaertn. and Pennisetum typhoideumPers.

T.S. Rangan

Papers

Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple

Growth and Plantlet Regeneration in Tissue Cultures of Some Indian Millets: Paspalum scrobiculatum L., Eleusine coracanaGaertn. and Pennisetum typhoideumPers.

Morphogenic investigations on tissue cultures of Panicum miliaceum

Growth and regeneration of plantlets in callus cultures of pineapple

Micro-Propagation of Ananas sativus in vitro