Studies On The Technique Of The Isolation, Purification And Culture Of Protoplast In Apple

Apple is one of the most important fruit species in China, but the apple industry is interrupted by both by low unit yield and by poor quality. One of the best practical practices for solving the problem is to develop new desired cultivars and rootstocks. Apple is highly hybrid in hybridity, which has caused much difficulty in routine breeding prospects. The technique of protoplast isolation, culture and fusion, which can enrich the germplasm resources and transferable distant characteristics, have shown great potential in breeding. Certain progress of the technology of protoplast culture has been obtained in recent years, however, only a few reports on regenerated plants from apple protoplast has been released in limited genotypes and unfortunately, the test can not be repeated. Those problems made the researcher puzzled. Therefore it is significant to further study protoplast culture and fusion in order to create new germplasm and improve genetic characteristics of cultivars and rootstocks in apple.l.In the recent study callus from leaves and young ovule were induced and there was marked difference in induction if different combination and ratio of hormone were applied. The induced callus were sub-cultured on MS medium for several times and the grainulated callus can be obtained. Adventitious buds formed at the same time when the callus was induced. In this test, we obtained the callus from the three kind of material of 77-34,Gala and Zhumei.2.Culture condition of cell suspension was optimized, the growth of suspension cell was affected by Gin content added and interval of subculture. The optimal interval of subculture ranged from 6d to 7d.Adding 300mg/L Gin into MS medium can increase the specific weight of suspension cells.3.Protoplasts were isolated from leaves, young ovule and friable callus. The36result indicated that the variation of physiological condition resulted in different yields and viability of protoplast. The highest yields and viability of the protoplast isolated from newly expending leaves has been observed. The yields and viability of protoplast isolated from young ovule culture in dark for 2~7d are higher than those cultured in light for the same period. Higher yields and viability of protoplasts isolated from callus sub-cultured for 2~4months have been figured out.4.An appropriate culture medium for apple protoplast was developed. The component of the medium is as follows:KM8p+ 0.6rnol/LGIucose+300mg/L CH+20mg/LArg+300mg/LGIn +500mg/LME +0.5mg/L2,4-D+0.5mg/LBA.The protoplasts were cultured on shallow liquid medium by a density of 0.5 X 105/mL.The first division of protoplast can be observed at 4lh or 5th day after culture and the third division after 10lh day and multiple division after 15lh day.