Somatic Hybridization Of Triticum Aestivum With Intertribal Plants And Genetic Basis Of Wheat Somatic Hybrid

The progress of somatic hybridization between wheat (Triticum aestivum L.) and its phylogenetically remote species was hindered by the difficulty of plant regeneration. There is not any success about remote hybrid plant of wheat regeneration before this research. Up to now, only a little investigation was reported on the composition and relationship of nuclear and cytoplasmic genomes of somatic hybrids, e.g. in the hybrid between wheat and Haynaldia vilosa Schur, as well as wheat and Zea mays L. As for the genetic stability of wheat somatic hybrid, only an initial study was conducted.In this work, somatic hybridization between wheat and its intertribal cereals and grasses was studied. The aim is: to establish an efficient system for hybrid plant regeneration; to investigate UV dose effect on elimination and fragmentation of donor chromosomes, as well as growth and differentiation of hybrid; to understand the hybrid plant regeneration in relation to the genotype of parents and the genome complementary of two kinds of wheat with donor, the component of nuclear and cytoplasmic genomes of the hybrid; to study the traits and heredity of hybrid lines in the inbred progenies and the introgression of donor chromatin into wheat genome and their stability. The important role of the introgression on wheat breeding was discussed.The main process and results of this research were as follows: 1. Somatic hybridization between wheat and phylogenetically remote speciesProtoplasts of Bromus inermis Leyss and Avena sativa L. irradiated with ultraviolet light (UV) at an intensity of 380 μ W/cm~2 for 0s (symmetric fusion) , 30 s, 1 min- 2 min, 3 min, 5 min respectively were fused by PEG method with protoplasts of three cultivars of wheat (Triticum aestivum L.) (cv. 99P, (cv. Jinan 177 or cv. Hesheng 3 ). Regenerated clones were produced in all fusion combinations, but only albinos was obtained:1) Wheat(cv. 99P) (+) Bromus inermis: Three clones (No.l-No.3) were regenerated from the fusion products and differentiated into albino seedlings. The clones and the seedlings were all verified as hybrids by chromosome counting, isozyme and RAPD analysis. The isozyme and RAPD patterns contained the characteristic bands of both parents and new band(s). The chromosome numbers of albinos were in the range of 42-54 with small chromosomes and chromosome fragments of Bromus inermis. The above results confirmed that hybrid albinos were obtained.2) Wheat (cv. Jinan 177 suspension, Jinan 177 calli, Hesheng 3 calli respectively) (+) Avena sativa: 191 regenerated clones were produced in all fusion combinations. Of which, only 8 ones differentiated into albinos. The clones were recognized as somatic hybrids in nature by analysis of cytological, isozyme, RAPD and 5S rDNA spacer sequence. GISH (genomic in situ hybridization) analysis revealed that only a few intact chromosomes and chromosome fragments of A. sativa existed in the somatic hybrid. A dose effect of UV on elimination of donor chromosomes was observed in these experiments: the number of intact chromosomes of donor A. sativa decreased, while that of the chromosome fragments translocated or inserted to wheat chromosome increased as the dose of UV enhanced.2. Somatic hybridization between "mix-type"of wheat and intertribal speciesProtoplasts of two types of the same wheat (from cell suspensions (Tj) and from embryogenic calli (T2) were mixed and fused either with that of UV-treated Setaria italica (S) or Avena sativa (A) by PEG method. The hybrid plants were obtained:1) Tj (+) T2 (+) S: Twenty-three putative hybrid clones were produced in the fusion combinations, but only clone No.5 could regenerate plant. Eight clones and the regenerated plants possessed nuclear genomes of T|, T2 and S. italica, as it was revealed by analysis of cytological, isozyme, RAPD and 5S rDNA spacer sequence.2) Ti (+) T2 (+) A: Many clones and normal green plants were regenerated from all fusion combinations. They were also verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis.3. The component and the localization of A sativa chromatin in the hybrid plants between wheat and Avena sativa1) The chromosomes number of A> B, D genome were 9, 7> 8 in Tt, and 12^. l(h 12 in T2 respectively. Chromosomes 3A> 4B and 4D were easily loosed in both Tt and T2. Chromosome distribution of Ti and T2 was different in A^ B, D genome, but both had a high frequency of 4A^ 5A, 2B, 3B> 7B, 3D, 6D. The rates of chromosome mutation were more than 25% in group B and D, which were higher than that of group A. The duration time of the calli subculture had no relation to chromosome mutation.2) The genomic constitution of hybrid plants: There was basically intact wheat chromosome existed in hybrid plants including A, B and D chromosome set3) The existence and location of oat chromatin: The oat chromatin as chromosome fragments translocated and inserted to wheat chromosomes of 4AL, 3BSn 4BL> 3DS, 6DL and 7DL, and the percentage distance from the centromere to the hybridization site is 60.55 + 11.12 , 21.63 + 10.19, 29.71 + 11.25, 40.57+12.33 > 58.58 + 11.02 |P 27.29 ±11.57 respectively.4. Analysis of nuclear and cytoplasmic genomes of somatic hybridsRandom Amplified Polymorphism DNA (RAPD) and Genomic in situ hybridization (GISH) analysis were employed to identify the nuclear genomes of somatic hybrids and their parents. We applied Restriction Fragment Length Polymorphism (RFLP) and Simple Sequence Length Polymorphism (SSLP) to analyze the cytoplasm constitution.Regenerated calli and plants from the wheat (+) Setaria italica and wheat (+) Avena sativa were analyzed:4.1 Genome analysis by GISH1) GISH results of hybrids showed that the chromosome institution of the hybrid calli favored wheat. Statistical result of the chromosome number indicated that the plant regeneration of the hybrid was related to the chromosome number of recipient. The chromosome number of wheat in hybrid clone with regeneration capacity was over 42 , while that of non-regenerable one was less than 42.2) The observation and statistics of the donor chromosome in hybrid calli from the different UV dosage showed that: the intact chromosome decreased and chromatin introgression increased as the dosage of UV enhanced. So, the dosage effect of UV on donorchromosome elimination and fragmentation is evident in this experiment. There was no obvious difference on the small chromosome fragments of the donor existed in the regeneration or non-regeneration clones.4.2 Analysis of the cytoplasmic genomes of the hybrid clones by RFLP and SSR1) RFLP profiles of the mitochondrial and choroplast genomes of the hybrid clones revealed biparental DNAs co-existence with a characteristic of biased to the recipient. Integration and recombination of mitochondrial and chloroplast genomes existed in some clones without obvious effect of UV dosage.2) According to the statistical analysis, RFLP fragments of 31.4% were derived from donor and 71.0% from receptor mitochondrial genomes. These results showed that the contribution of receptor to the hybrid mitochondrial genomes was dominant as it done in nuclear genomes, which suggested the correlation between nuclear and mitochondria! genomes of somatic hybrids.3) Of seven microsatellite loci of chloroplast DNA, six were wheat-type, one had an intact wheat bands and an oat band. All hybrid plants had the same pattern.4) The occurrence of albinos and mosaic plantlets was related to the variation of DNA fragment in chloroplast genome.4.3 Analysis of the nuclear and the cytoplasmic genomes of ovary-derived plantsRAPD analysis of nuclear genomes in regenerated plants induced from ovaries showed the specific patterns from both parents. 75% of plants retained the same RAPD patterns, while 25% showed various degree of DNA recombination or exclusion. Although these plants were derived from the ovaries of same clone origin, some variations produced in the course of subcultures. SSRs for chloroplast genomes were surveyed across the ovary-derived plants and their original plants, which showed the same patterns and indicated the stability.In above research, we reported a successful technique for intertribal somatic hybridization of wheat, the results of nuclear-cytoplasmic genome constitution of somatic hybrids with the mix-type of wheat The results provide abundant information of theory and technology for the remote hybridization of wheat.