Asymbiotic Germination Of Interspecific Hybrids And Their Genetic Evaluation Via ISSR In Orchidaceae

Distant hybridization was carded out among eight orchid species and the hybrid seeds were germinated under asymbiotic condition.The progenies and their parent combination Cymbidium cyperifolim var.szechuanicum×C.floribundum were analysed by ISSR markers in molecule level.The experiment aimed to creat new germplasm by distant crossing and evaluate the behavior of the generation by molecular markers and morphological traits,and thus accumulate experience and knowledge for further orchid breeding.The main results are as follows:1.Eight species of orchid were used as parents for wide cross.Among sixteen cross combinations,twelve cross combinations produced capsule fruits,in which seeds of five cross combinations were germinated successfully under asymbiotic experimental condition.The results showed combinations,such as C.cyperifolim Wall var.szechuanicum×C. floribundum,C.floribundum×C.cyperifolim Wall var.szechuanicum,C.goeringii var. longibracteatum×C.cyperifolim Wall var.szechuanicum,C.cyperifolim Wall var.szechuanicum×C. goeringii var.longibracteatum,C.goeringii×C.cyperifolim Wall var.szechuanicum,C. floribundum×C.goeringii were easy for producing hybrid generation,while other combinations, including Luisia teres×C.nanulum,Aerides rosea×C.nanulum,Paphiopedilum micranthum×C. cyperifolim Wall var.szechuanicum,C.cyperifolim Wall var.szechuanicum×Paphiopedilum micranthum were difficult to obtain hybrid generation.2.The seeds of C.cyperifolim var.szechuanicum×C.floribundum were germinated in asymbiotic culture.Seven-month-embryo-aged-seeds were found to have the highest germination rate,when treated with 0.1 mol/L KOH for 10 min.After germination,the best medium for PLBs propagation was Z₄(KC+BA 0.4 mg/L + NAA 0.6 mg/L+KT 0.2 mg/L),the rate of PLBs proliferation could reach 12.8.Adding activated carbon to medium prevented the cut browning in the process of PLB's multiplication and differentiation.3.The process of asymbiotic germination of the offspring seeds between C. cyperifolim var.szechuanicum×C.floribundum was observed.The morphologic character of first offspring presented between their parents.Ninety days after germination the PLBs seems to be a complex of PLBs and rhizomes,combination of their parents. After differentiation they still remained some structure like rhizomes beneath the root of PLBs.Considering they originated from the combination of terrestrial orchid and epiphytic orchid,the adaptability to the envioronment of the offspring need further study.4.Orthogonal design was adorpted to optimize ISSR-PCR amplification conditions, 14 ISSR primers with rich polymorphism were screened from 80 ISSR primers.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.5.107 polymorphic stable and clear bands were produced by 14 ISSR primers,every primer had 7.64 bands in average.The length of amplified DNA fragments ranged from 90 bp to 2100 bp,there were nine kinds of bands.The polymorphism of ISSR marker in the F1 progenies was high,and the segregation loci was 43.92%.87.23 percent of segregation loci accorded with the segregation patterns of Mendelian segregation model, the segregation ratio nearly 1:1 and 3:1,12.77 percent of segregation loci deviated from Mendelian segregation model,and the reason was dimness.