Identification And Evaluation Germplasm Resources And Study On In Vitro Culture Technique Of Renanthera Spp

Renanthera spp. was a kind of rare tropical Orchidaceae orchid. The flowers was bright red, can be widely used for cutting flower, garden scenery, and the production of potted plant, and had a very high application value, and economic value of gardening. Renanthera hybridization affinity was strong, so it had high breeding value. Comprehensively identification and evaluation of Renanthera germplasm from plant characters, ornamental characters and genetic diversity was carried out. The in vitro culture system of Renanthera was optimized through the screening of explants, basic media, hormone types and concentrations, and other factors. The result in this study laid the foundation for Renanthera germplasm innovation, selection of new varieties and commercial development. The results were summarized as follow:21ornamental characters of Renanthera germplasm were evaluated using principal component analysis method. Three principal component factors had92.36%variance contribution rate of all the character information. Three principal component factors were flower number factor, flowering period factor and plant stature factor. According to the evaluation of the Renanthera ornamental characters,12Renanthera germplasms were divided into four types:long inflorescence with large number of flowers (R8), long flowering period (R14and R18), large flower (R22), different flower color (the other8types).4excellent Renanthera germplasms were selected through the calculation of principal component scores and the screening of ornamental characters.Two in vitro regeneration systems of Renanthera were developedin this study: Single-node stem culture and Prorocorm-Like bodies (PLB) culture. In vitro regeneration of Renanthera was conducted with single-node stem as the explants. The results showed the the best shoot induction medium of Renanthera was abtained on:MS+6-BA3.0mg/L+NAA0.5mg/L, Adding0.3g/l activated carbon could effectively control the browning phenomenon in shoot induction culture.; the optiamal rooting and elongation medium was1/2MS+6-BA0.2mg/L+NAA1.0mg/L+AC0.5g/L; The comparative experiment on the effects of cutting and plant growth regulator concentration was conducted with axillary bud、 single-node stem and leaf as the explants. The results showed the best medium of PLBs induction was:1/2MS+BA8.0mg/L+NAA0.8mg/L+20mg/L Ad+AC0.2g/L with cutting axillary buds longitudinally; The proliferation medium was1/3MS+BA3.0+NAA0.5+AC0.1g/L+Pepton2g/L; the optimum medium of plant regeneration was1/3MS+BA3.0mg/L+NAA1.0mg/L+AC0.2g/L+Pepton2g/L; the optimal rooting medium was1/3MS+BA1.0mg/L+NAA1.0mg/L+AC0.5g/L+Pepton2g/L, The transplanting survival rate of Renanthera plantlets were more than98%.It was the first time using SRAP molecular marker technique to investigate the genetic diversity of Renanthera germplasm. The SRAP-PCR system was constructed and optimized in this study:the total volume of20μL containing60ng genomic template DNA, Mg2+2.0mmol/L,1.0μmol/L Primer,1.2U Taq DNA polymerase,0.20mmol/L dNTP. The PCR reaction procedure were as follows:5min of denaturing at94℃,1min of degenerating at94℃,1min of annealing at35℃and1min of elongation at72℃. In the following35cycles, the annealing temperature was increased to50℃, with a final elongation step of10min at72℃.190primer combinations were screened with24Renanthera germplasm DNA, of which18primer combinations gave stable and reproducible amplification patterns. Among the total286amplified fragments,278(97.2%) were polymorphic, with an average of15fragments for each primer combination. Using NTSYS-pc2.10e to cluster analysis, the18Renanthera germplasm can be classified into6groups:the first group included Rl-. R23and R10; the second group included R4、 R5and R6; the third group included R2andR15; the forth group included R14、 R19、 R17、 R18、R22、R3、 R13、R20、 R21、 R8、 R11、 R16、 R12andR9; the fifth group was R7, the sixth group was R24.