Studies On Morphogenetic Capacity And Genetic Stability Of Pear Micro-cuttings In The Process Of Long-term Subculturing In Vitro

Subculture is an important link in the process of the tissue culture and the long-term subculture is the essential method for conservation in vitro of germplasm.The propagating characteristics of Pyrus brestchneideri Rehd.cv.Jinhua and P.pyrifolia cv.Jinshui micro-cuttings repeatedly subcultured in vitro for one to fifty-five times(0～4a) was examined by analysis of the propagating efficiency,rooting,and adventitious bud regeneration rate from lesves in this experiment.The genetic relationships and fidelity among the two cultivars and their micropropagated materials were assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat(ISSR) markers.The mian results were:(1) It is lower than the propagation ability of the plantlets subcultured in vitro within sixteen times at the beginning of inoculation in term of its propagating efficiency.With the increasing of the subcultured times,the propagation ability enhanced and remained at a high level.Then the proliferation rate began to decrease.Two cultivars had different numbers of subculture.Pear tissues subcultured for over 55 times had gradually lost the propagating capability.(2) Within fifty-five times of the subculture,the lowest rooting rates were gained from shoots of 'Jinhua' and 'Jinshui' at the early time,which counted for 41.67%and 15.56%.The rooting rates had an increasing tend with increasing of subculture number and incresed to as high as 58.33%and 34.92%after shoots were subcultureed 55 times.The tissue cultures of the two cultivars studied have the capability to form roots regardless of the length of time in culture.The rooting ability of 'Jinhua'is better than that of'Jinshui'.(3) The capability to differentiate shoots were low at the 8^th subculture.The regeneration rate and the number of buds remarkably increasd after the 16^th subculture.Continued subculturing at 40^th reached the highest level.But with the procession of fifty five month's subculture,the differentiating ability of the callus,coming from pear leaf and having differentiating ability,was foud to reduced gradually.(4)In this research,by using 'Jinhua' micro-cuttings subcultured 8,16,29,40,50 times and 'Jinshui' micro-cuttings subcultured 6,13,34,40,50 times as experimental matrials and cultivars of 'Jinhua' and 'Jinshui' as CK,a total of 16 RAPD and 8 ISSR primers gave 1096, 569 distinct bands in 'Jinhua' and 1413,582 in 'Jinshui'.The genetic difference from the original line was no more than 1.65%～4.19%(RAPD) and 1.05%～5.24%(ISSR) in 'Jinhua' and 1.07%～3.66%(RAPD) and 0～4.72%(ISSR) in sterile regerants of the cultivar 'Jinshui'. Somaclonal variation were detected among micropopagation plants in pear.Most somaclonal variation took place during early culturing.But the somaclonal variation did not increase stintlessly with time in the long-term culture.The somaclonal variation began to decrease after reaching the highest level and remained at the level.The genetic variation of regnerants maintained for four years did not exeed that of plants obtained from a culture maintained for 1 year.