Plant Regeneration Medium Research Articles

Tall Fescue Doubled Haploids via Tissue Culture and Plant Regeneration1

Genetic improvement of tall fescue (Festuca arundinacea Schreb.) by conventional breeding methodology is slow, and use of haploids and doubled haploids may accelerate the process. Androgenic haploid lines were evaluated in the field to identify differences in forage quantity and quality. Chromosome doubling to obtain fertility was attempted with some of the haploid lines. Direct regeneration of shoots from expanded (“aged”) leaf midveins, as can be done with tobacco (Nicotiana tabacum L.), was not successful for tall fescue. However, calli from tall fescue were established in vitro from the lower end of rapidly elongating (“young”) peduncles. Calli developed on such tissue explants within a few weeks on modified Linsmaier and Skoog medium with 2 mg of 2,4‐D per L and no cytokinin. After 5 weeks, the calli were removed from the explants and subcultured on the same medium formulation at 24±2°C for 15 weeks to “age” the callus tissue. The calli grew rapidly at first, but had stopped detectable growth and were beginning to senesce at 15 weeks. The “aged” calli were subdivided and placed on a plant regeneration medium (the same formulation but with only 0.25 mg of 2,4‐D per L and no cytokinin). Leafy shoots began to emerge from the calli after 3 to 4 weeks of culture under continuous illumination from cool‐white fluorescent lamps at about 15 μmol m−2s−1 in a room maintained at 23±2°C. There were numerous green shoots and a few albinos. The green shoots were excised when they were about 1 cm tall and transferred to a rooting medium (devoid of both 2,4‐D and cytokinin). The shoots developed roots within a few weeks and were transferred to sterile soil at high humidity. The humidity was decreased and light intensity increased over a several day period. After the plants were established, root tips were examined for somatic chromosome numbers. Of the normal‐appearing plants, some had 21, others had 42, and a few had 81 to 84 chromosomes. Doubled haploids were developed by this method from six different haploid lines. One of the lines also produced a few quadrupled haploids. The regeneratd doubled and quadrupled haploids are being evaluated for forage characteristics and their potential usefulness to rapidly isolate desired characteristics.

Plant regeneration from tissue cultures of Pokkali rice is promoted by optimizing callus to medium volume ratio and by a medium-conditioning factor produced by embryogenic callus

The frequency of plant regeneration from seed-derived Pokkali rice callus has been substantially increased. Four conclusions were drawn from the study: (1) Non-embryogenic callus consisting of elongated, highly-vacuolated cells did not produce regenerated plants. Embryogenic callus consisting of small, non-vacuolated cells produced somatic embryos and regenerated plants. (2) The numbers of plants could be markedly increased by optimizing a medium for embryogenic callus production and a second medium for plant regeneration from embryogenic callus. (3) The optimization of callus to medium volume ratio of 6.5 mg embryogenic callus per 1.0 ml of medium significantly increase plant production on regeneration medium. (4) A further significant increase was obtained by using regeneration medium previously conditioned for one or two weeks by optimal amounts of embryogenic callus. At present, the callus derived from a single seed in six months could theoretically be used in the seventh month to produce 127500 plants.

Sodium Chloride Tolerant Callus of Sorghum bicolor (L.) Moench.

Summary Callus cultures of Sorghum bicolor (L.) Moench cv. Margaritiferum obtained by prolonged culture on NaCl-containing medium were able to grow better on NaCl-containing medium than the parent cells. After every passage on the stress selection medium, part of the cultures were transferred to hormone-free medium for plant regeneration. Plants were regenerated from calli grown on NaCl-containing media for up to five passages. Some of these plants were albinos. A few green plants have been grown to maturity in the greenhouse. There was very low seed set in these plants. Attempts are underway to regenerate more plants from stress selection media and also to get better seed set from the plants obtained.

Long-duration, high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 μm in diameter; NE callus consists of long tubular cells averaging 52 μm in width and 355 μm in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.

In vitro Growth Responses and Plant Regeneration from Cryopreserved Meristems of Cassava (Manihot esculenta Crantz)

Summary A droplet-freezing method has been developed for the cryopreservation of cassava, Manihot esculenta Crantz, meristems. The meristems treated with a 15 % DMSO + 3% sucrose solution for 15 min were frozen over an 18 μm aluminium foil in 2–3 μl droplets of the cryoprotectants in plastic petri dishes at a cooling rate of 0.5°C/min to various sub-zero temperatures (-20; -25; -30 and -40°C) and stored in liquid nitrogen (-196°C). The meristems retrieved from liquid nitrogen storage, upon thawing and return to in vitro culture on plant regeneration medium, exhibited various morphogenetic responses such as differentiation of callus and leaves and whole plantlets. The plantlets were successfully grown in pots.

Improved protoplast culture and plant regeneration from protoplast-derived callus in Arabidopsis thaliana

Summary Protoplasts of Arabidopsis thaliana race Columbia were prepared from fast growing suspension culture cells. About 50–60% of the protoplasts formed calli when plated at a density of about 3 × 105 protoplasts per ml in the medium of Gamsorg et al. (1968) modified to contain 1 mg 2,4-D, 0.15 mg BA and 1000 mg CaCl2 × 2H2O per liter and 0.4 M glucose. About 40% of these calli formed shoots when transferred onto plant regeneration medium.