Xia Guangmin

Plant regeneration from intergeneric somatic hybridization between Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel

The suspension derived protoplasts of wheat (Triticum aestivum) cv. Jinan 177 were used as a recipient to fuse with the protoplasts of the 60Co gamma-ray irradiated calli of Legmus chinensis. The wheat suspension cells and their protoplasts were not capable of differentiating to whole plants. The irradiated calli of L. chinensis were also the same. The protoplasts originated from the treated or untreated calli were both unable to divide under the conditions of this experiment. However, the fusion products grew and developed to whole plants which were identified as hybrids according to the analysis of chromosome, isozyme and morphology. The above result revealed that the lost regeneration capacity of both parents could be complementarily restored through somatic hybridization. This phenomenon also occurred with our work on Triticum aestivum (+) Haynaldia villosa, T. aestivum (+) Agropyron elongatum and T. aestivum (+) Psathyrostachys juncea.

Effect of UV dosage on somatic hybridization between common wheat (Triticum aestivum L.) and Avena sativa L.

Protoplasts of Avena sativa L. (A) irradiated with ultraviolet light (UV) at an intensity of 300 μW/cm2 for 0 (symmetric fusion), 30 s, 1 min, 2 min, 3 min, 5 min, respectively were fused by the PEG method with protoplasts of two cultivars of wheat (cv. Jinan 177 or cv. Hesheng 3). Two different sources of material were used for isolation of protoplasts of Jinan 177. Type one (T1) originated from long-term subcultured suspensions which could not be regenerated to plants, while the other (T2) derived from embryogenic calli was capable of regeneration. Protoplasts of cv. Hesheng 3 (T3) also originated from embryogenic calli possessing high regeneration capacity. Regenerated clones were produced in all fusion combinations, but only those from combinations IB and IC (irradiating A. sativa for 1 and 2 min) differentiated to albinos. The clones were recognized as somatic hybrids in nature by cytological, isozyme, RAPD and 5S rDNA spacer sequence analysis. Genomic in situ hybridization (GISH) of somatic hybrid clones at mitosis always revealed the presence of recombined and intact A. sativa chromosomes in the cells. The number of intact chromosomes of donor A. sativa decreased, while the chromosome fragments translocated or inserted to wheat chromosome increased as the dosage of UV enhanced.

Transfer of salt tolerance from Aeleuropus littorulis sinensis to wheat (Triticum aestivum L.) via asymmetric somatic hybridization

Protoplasts of wheat c.v. Jinan 177 were fused by PEG method with the UV irradiated protoplasts of A. littoralis - a salt tolerant plant intertribal to wheat. The early-formed regenerated clones were identified as hybrids by chromosome, isozyme and RAPD analysis. Their salt-tolerant ability was compared with both parents in relative growth, proline accumulation and Na(+)/K(+) ratio under salt stress, and was proved higher than wheat, indicating some corresponding genes coding salt-tolerance had been transferred into the hybrids. However, the hybrid clones could only differentiated to albino plants. Further investigations are now being conducted to solve this problem.

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd

Protolasts of Bupleurum scorzonerifolium were prepared from stem node-derived embryogenic calli with an enzyeme mixture, in which snailase was a necessary component. Follolwing cell wall regeneration protoplasts divided and directly formed somatic embryos which developed into plantlets. The conditions favorable to direct embryo formation were investigated, and the nature of the callus used for protoplast preparation was found to be a critical factor. The osmotic concentration and the composition of the culture medium including the phytohormone combinations were also important.

Asymmetric somatic hybridization between haploid common wheat and UV-irradiated Haynaldia villosa

UV irradiated protoplasts from young embryo derived calli of Haynaldia villosa were fused with protoplasts originated from anther derived calli of Triticum aestivum cv. 24-2 by the PEG method. Regenerated calli from fusion cells were obtained but none of them differentiated into plants. The cell clones that formed early were examined for their hybrid nature, chromosome counting and isozyme analysis suggested that they were nuclear hybrid. Further unequivocal confirmation was accomplished using the technique of in situ hybridization (ISH) and RAPD analysis, which facilitated the identification of the hybrid nature in fusion products. The results revealed that UV might have played an important role in breaking donor chromosomes and ensuring the introduction of alien chromatin into the recipient via asymmetric hybridization. The failure of plant regeneration of the somatic hybrids was discussed and further investigations are being conducted.