Protoplasts were isolated from Agrobucterium rhizogenes A-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106per g fresh weight) was obtained from 12-d-old calluses after being subcultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2mgl-1 (9.05μM)2, 4-dichlorophenoxyacetic acid. 0.2mgl-1(0.93μM)kinetin, 0.3M mannitol, 2%(w/v)sucrose, and 500mgl-1 casein hydrolyzate at a plating density of 1.0×106perml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was ablut 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without grouwth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.

The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stein growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that TDNA had becn integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalusudsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 rag/1 2,4-D, 0.5 mg/I BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8_+0.5% and 6.5_+0.7%. respectively. Upon transfer to Murashige and Skoog (1962) medium (MS) with 1-2 mg/I BA, alone or in combination with NAA or 2,4-D at 0.1 rag/l, the protoplast-derived ealli produced complete plantlets through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5

Protoplast fusion was induced between sainfoin and alfalfa by and improved polyethylenegylcol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerated. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG sclution was previously placed. Chromosome number of regenerated hybrid buds varied from 30to 60. The genome of hybrids included the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybridity of obtained hybrid calluses was confirmed by their isozyme banding patterns and their nopaline synthetase acivity.

Callus were induced from the segments of basal part of leaf blade of rye (Secale cereal L) on MS medium supplemented with 3 mg/L 2, 4-D. Embryogenic calli were obtained when the calli derived from leaf tissues were subcultured on N6 medium plus 500 mg/L yeast cxtract, 1-2 mg/L 2, 4-D and 1-2 mg/L kinetin. Somatic embryos frequently were produced after transfering the cultures onto MS or N6 medium with 5% sucrose without growth regulators. The embryos germinated and developed into normal plantlets on hormone-free medium. The regenerated plants were potted. Histological investigation on callus initiation from leaf tissues was carried out.