A Study On Tissue Culture And Somaclonal Variation Of Three Major Salt-and Alkali-Tolerant Forage Grasses Indigenous To The Songnen Prairie Of China

Efficeient tissue culture protocols were developed for plant regeneration from young inflorescence-derived calli of Hordeum brevisubulatum (Trin.) Link, mature seed–derived calli of Puccinellia tenuiflora and mature seed–derived calli of Puccinellia chinampoensis, respectively. Genetic and epigenetic instabilities in the calli and regenerated plants (regenerants) from these three forage grasses were assessed by three molecular markers, AFLP, S-SAP and MSAP. The results showed that there were both genetic and epigenetic variations in calli and regenerants from the three forage grasses at the molecular level.Two pools of calli derived from young inflorescences of a single donor plant and 44 randomly chosen regenerants in Hordeum brevisubulatum (Trin.) Link were subjected to AFLP analysis. Results showed that 74 out of 793 scored bands yielded by the selected 18 primer pairs were polymorphic among the studied samples, giving rise to a genetic variation frequency of 9.3%. The number of variant bands as compared to the donor plant varied greatly among the regenerants (range: 2-55), with a small number of regenerants accumulated a large number of variant bands, while the majority of regenerants(68.2%) showed only 2-3 variant bands. A subset of regenerants together with the two pools of calli was selected for S-SAP and MSAP analysis to detect possible retrotranspositional activity of a prominent retroelement family called BARE-1 in the genomes of Hordem species, and alterations in cytosine methylation. S-SAP analysis showed that of the 768 scored bands generated from the selected 24 primer combinations, 151 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 19.7%, albeit no evidence for retrotranspositional event was obtained. From 170 to 196 methylated CCGG sites were detected among the analyzed samples by MSAP with the selected 22 primer pairs. MSAP analysis revealed that levels of hemi-methylation at external C, full-methylation at internal C and the total methylation at CCGG sites in regenerants were all apparently lower than those of the donor plant, the most patterns of changes were demethylation at internal C of CCGG sites. Sequencing of selected variant bands indicated that both protein-coding genes and transposon/retrotransposons were underlying the genetic and epigenetic variations. Correlation analysis of the genetic and epigenetic instabilities by Mentel test indicated that there exists a significant correlation between MSAP and S-SAP (r=0.8118, 1,000 permutations, p<0.01), whereas the correlation between MSAP and AFLP (r=0.1048) is not statistically significant.With the seedling as a control, three pools of calli and 8 randomly chosen regenerants derived from an individual seed of Puccinellia tenuiflora were subjected to AFLP, S-SAP and MSAP analysis. The 19 primer pairs yielded 925 fragments by AFLP, of which 16 fragments were polymorphic, and the percentage of polymorphic sites is 1.7%. Seven-hundred and eighty-nine bands were scored by S-SAP using 24 primer pairs, 36 out of which were polymorphic with a genetic variation frequency of 4.6%. From 163 to 170 methylated CCGG sites were amplified among the analyzed samples by MSAP using the selected 16 primer pairs.