High-efficient Plantlets Regeneration System And Genetic Transformation In Phalaenopsis, Oncidium And Cymbidium

The transgenic of Phalanopsis , Cymbidium hybridum and Oncidium were studied and the the main results are as follows:(1) Decontamiated-materials can be obtained from unsymbiotic- seed germination, or by inducing from floral stalk, and proliferation of plantlets was achieved on MS media supplemented with 6-BA7.5mg/L+NAA1.0mg/L; 6-BA and NAA can stimulate PLB formation and the effective ruslts can be obtained on MS with 6-BA10.0mg/L+NAA1.0mg/L; The optimal medias were 1/2MS and Hyponex medias for proliferation and transformation of PLBs; The relations between the concentration of sucrose and plantlets regenerations from PLBs can be formulated by conic models, and plantlets developed well on 1/2MS containing 20g/L without any growth regulators.(2) Analysis of variance and multiple range tests show that the differences of propagate ratios in different genotype Cymbidium hybridum are significance or extreme significance, but different means also tend towards cluster and scatter; The main factors that influence on bud proliferation is the concentration of cytokinins, and the relations can be formulated by conic models ; 2,4-D was a necessary factor to induce callus, and with the concentration increased, the frequency of callus formation increased, KT was the key factor to induce formation of somatic embryogenesis, and the effects was affected by 2, 4-D and low does NAA; Histological observations indicated that somatic embryogene developed into conglobulation -embryogene, then torpedo- embryogene and last hypocotyls embryogene.(3)The sixty percent of plantlets forming can be achieved on half-MS media supplemented with 6-BA2.0mg/L+NAA0.5mg/L from floral stalk of Oncidium.; Callus induction was mainly effected by [NAA]/[BA] and light intensity, and the toppest callus forming was reached 78% when light intensity was 250 lux on MS media supplemented with 6-BA0.2mg/L+NAA 1.0mg/L; Plantlets forming and commerce value can be reached on MS media supplemented with 6-BA0.4mg/L+NAA0.2mg/L; The media with all nutrient elements but medille or low concentration was benefit to the plantlets forming and plantlets from callus with length of l²cm can be transplant into MS media supplemented with AC 2.0g/L to reduce roots.Plantlet with roots can be painted into greenhouse and percent s of survivor can be reached 99%.(4)The best protocol of transformatin for genetic transformation in Phalanopsis is: 4⁵days for pre-culture time, OD)₆₀₀=0.05, 10 minutes for infection, pH5.4 and through two stages of cocultivation, then washing the explants with liquid media containing500mg/L Cef, and finally, subculture in media containing Cef 50mg/L and Km 10mg/L, if so, the percnt of Km^r PLBs canbe reached 16.2%, which is as five times as in basic media; A total of 280 KmrPLBs were obtained, from which 16 KmrPLBs were randomly selected and confirmed by PCR analyses, the results indicated that the foreign DNA was successfully integrated into the plants;(5) The present transformation method is applied into Cymbidium hybridum and Oncidium, but the low transformation can be obtained , and only got eight Cymbidium hybridum and 4 Oncidium plantlets which were positive for Km-, In order to select positive plantlets for foreign DNA early, pBin35S-GFP was constracted to establish an optium tansformation system for Cymbidium hybridum , Oncidium and some other orchids in future.