| Robinia pseudoacacia L,a member of Robinia in Leguminosae was originated from North America.It possesses many important features,such as rapid growth,strong.and elastic woody fibers,anti-adversity,well-nodulated root system and so on,so that it becomes widespread in north and northwest of China for soil and water Conservation. Robinia pseudoacacia is also an indispensable species for urban avenue virescence due to its rich fragrance,easy planting,and valuable appearance for public attention.Robinia pseudoacacia ever featured in strong resistance to infection of pathogenic organisms.However,in recent years,it has been found to suffer some serious diseases, such as violet root rot and skin ulceration caused by pathogenic microorganisms,and invasion from bugs small wrinkle(leaf),pod borer,Angoumois grain moth,small bees (seeds).Though transgenic engineering has been successfully used to introduce advantageous genes into some important plant species to improve resistance to attacking from pests and diseases,it has been not available in Robinia pseudoacacia mainly due to lacking a well-established receptor system.In present study,we used the growing-well stems of Robinia pseudoacacia with axillary's buds as explants.Firstly,we studied the influence of several factors on differentiation of buds and roots,including different media,different hormones and different concentration ratio of some hormones mixtures.And then,based on availability of a high frequency regeneration system,we tested the effects of various factors on transformation efficiency of Bt CrylA gene mediated by Agrobacterium tumefaciens with histochemical assays of GUS.The transformation procedure established here was proved to be reproducible and effective,and opened up a possibility of more beneficial genes to be introduced into Robinia pseudoacacia.Detailed experiments and data were listed as the following(1) Establishment of a high frequency regeneration system,of Robinia pseudoacacia:we found that the most effective medium to induce callus is MS basic medium containing 0.3mg/L of 6-BA(6-benzyladenine),0.04mg/L of NAA (naphthalene acetic acid) and 30g/L of sucrose and 9g/L of agar,and that the mean bud differentiation rate is 80%,the mean number of buds differentiated from an explant reaches up to 7.3,and MS medium supplemented with 0.04mg/L ofNAA,0.05mg/L of KT(kinetin) and 30g/L of sucrose is very efficient to improve rooting.The highest rooting frequency is 93.3%and the mean rooting frequency is 86.7%.Our data showed that the plants cultivated in tubes should be acclimated in green house for 15～20 days before planting into outdoor soils,making the surviving rate reach higher than 83%.(2) Establishment of a high efficient transformation system of Robinia pseudoacacia:in the transformation of Bt CrylA gene mediated by Agrobacterium tumefaciens strain LBA4404,the most suitable bacteriostatic agent is 400mg/L Cef(cephalosporin) and the selecting concentration is 50mg/L of Km(kalamycin ). Based on further optimization of necessary factors to improve the transformation frequency by GUS histochemical assays,we established an efficient transformation system of Robinia pseudoacacia:including:(1) stems should be pre-cultured in-vitro for 1～2 days;(2),Agrobacterium tumefaciens was inoculated till OD600 reached 0.5～0.7;(3) they were co-cultured for 3～4 days.Our data also suggested adding 30mg/L of Acetosyringone(AS) into the co-cultured medium could dramatically improve transformation frequency;(4) the co-cultured explants were transferred to MS medium containing 400mg/L of Cef;(5) After about 15 days,they were transferred to the same medium with 50mg/L of Km to select Km-resistant shoots. |
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