Study On Shoot Regeneration In Vitro And ISSR Analysis Of Loquat

In this paper, four cultivars of loquat(Eriobotrya japonica Lindl.) were used as test mateiral, namely, Tianzhong', 'Bingtangzhong', 'Baiyu' and 'Guanyu'. The technical scheme of inducing loquat anther callus was preliminarily selected. And then, the system of shoot regeneration in vitro from cotyledons of loquat was established. Besides, ISSR reaction system of loquat was established, and the genetic diversity of 24 genotypes of Jiangsu and Zhejiang province was analyzed using ISSR markers.1) The anthers of the four loquat cultivars were cultured in vitro. Experiment results show: The optimal date for flower buds collecting was in the metaphase of blooming. Low temperature treated for three days and dark condition is more helpful for inducing loquat anther callus which was cultured best on MS supplemented with 1.0mg/L 2,4-D,1.0mg/L IAA and 2.0mg/L BA. But activated carbon(0.5%～1%)was negative for loquat anther callus inducing.2) The cotyledons of the four loquat cultivars were cultured in vitro. Experiment results show: For 'Tianzhong' and 'Bingtangzhong', shoots regenerated best on MS supplemented with 4.0mg/L TDZ, while for 'Baiyu' and 'Guanyu', shoots regenerated best on MS supplemented with 3.0mg/L TDZ. When 'Bingtangzhong' used as test material, it was found that univalve cotyledons with embryonic axis and wounded in the surface had the best capability to regenerate shoots, and the optimal days for dark treatment was three weeks. For 'Tianzhong' and 'Bingtangzhong',shoots propagate best on MS supplemented with 0.5mg/L BA and 0.1 mg/LNAA,and for 'Baiyu' and 'Guanyu',shoots propagate best on MS supplemented with 0.75mg/L BA and 0.2 mg/L NAA. For the four cultivars, the best rooting medium was moiety MS supplemented with 0.25mg/L NAA and 0.25mg/L IBA.3) The optimal reaction of ISSR in 'Xiaobaisha' was studied with primer 5'-CCA(GTG)4-3'. The results showed that the optimum concentration of five important components i.e. Tag DNA polymerase, Mg²⁺, primer, template DNA and dNTPs in 25μL reaction system of ISSR were 2.0U, 1.0mM, 0.2μmol/L, 40ng and 0.20mmol/L, respectively. With 8 ISSR primers, 88 bands were amplified in 24 cultivated, of 50 bands (56.8%)were polymorphic. The genetic similarity coefficients of 24 cultivated accessions were ranged from 0.167 to 0.778. The culstering of 24 cultivated accessions showed that the cultivars are divided into 2 groups which is related to there origin of 0.200 at the threshold.