| Zenia insignis Chun,belongs to the Sugaraceae family,is the second class protected tree species in China.Z.insignis is a multipurpose tree species,which is excellent in the wood material,developed in root system,strong in resistance and adaptability to the desert region of the rock,rich in protein and perishable.However,molecular biology researchs of Z.insignis like genetic assays of progeny and genetic engineering has not yet been reported.An efficient regeneration system and genetic transformation system of Z.insignis is one of the main reasons for limiting its molecular research.In this study,the seed of Z.insignis from Guangxi was used as the material,to compare the callus induction ability of different parts of anaseptic seedling,and then leaves were used as explant material,discussing the best hormone formula in the process of callus induction,adventitious bud regeneration and elongation,rooting induction.The process of callus formation and adventitious bud regeneration was observed by paraffin section in order to study the morphological changes and structural characteristics of callus induction and differentiation in Z.insignis leaves.For the first time,a complete and efficient regeneration system was established systematically.In addition,the genetic transformation system of Z.insignis was preliminarily studied on the basis of the regeneration system,which provided a technical platform for genetic engineering breeding of Z.insignis in the future.The main results are as follows:1?Seeds was treated with 0.1%mercuric chloride for 10 min was the best sterilizing scheme,and the contamination rate was 10.67%and the seed germination rate was 89.33%.2?The epicotyls,hypocotyls and leaves of aseptic seedlings with 20 days of germination were used as explants respectively.The results showed that the callus induction ability of leaves was the highest in MS medium with a same concentration of 6-BA,IBA and 2,4-D.3?MS+2.4 mg·L-16-BA+0.05 mg·L-12,4-D+0.1 mg·L-1IBA was the best medium for callus induction in leaves of Z.insignis.The rate of callus induction was 62.22%.4?The leaf induction rate was 63.33%when the leaf was placed on the medium facing upward.Callus induction in different parts of leaves is also different,The induction rate of the basal part was 14.45%higher than that of the tip part,and the induction rate was the fastest.In addition,there was no obvious gradient change of regeneration ability in different parts as the whole leaf callus induction.5?Light can inhibit the induction of callus,and the induction rate is the highest in the dark condition,which is 62.22%.6?In the process of inducing adventitious buds from callus,the optimum medium for adventitious bud differentiation was MS+0.25 mg·L-11 IBA+1.8 mg·L-11 6-BA,the differentiation rate was 51.11%,the number of buds was 7.67.It was found that the average number of effective buds was 7.00 and 6.33 when GA3 was 0.8 mg·L-11 and 1.20 mg·L-1,respectively.And the maximum elongation length was 3.1 cm and 4.2 cm,respectively,but the adventitious buds were easy to vitrification at GA3 concentration of 1.20 mg·L-1.7?The differentiation rate of adventitious buds was different under different light.The differentiation rate of adventitious buds in blue and red light sources was 56.67%and52.22%,respectively,and the shoot elongation was fastest and the growth efficiency was significant under red light.8?In the course of rooting induction,the optimum rooting medium was obtained by using different MS basic medium.The best rooting efficiency?94.44%?was achieved as adding 0.1 mg·L-11 NAA and activated carbon 1.0 g·L-11 to the 1/2 MS medium.Survival rate of regenerated plants reached 56.67%.9?The results of paraffin section showed that most of callus originated from the cut of leaves,including embryogenic callus and non-embryogenic callus.The process of callus development was divided into four stages,The initiation stage of callus began on the 9th day,and the mitotic stage began on the 21th day.The obvious meristem nodule formed at36d and the bud point appeared 45th day,and then the primordium of the callus continued to divide and differentiate into adventitious buds.10?EHA105 Agrobacterium tumefaciens was used as the mediator in the genetic transformation system.The results showed that callus,as a receptor,was infected at a OD600of 0.6for 8 min,and then co-cultured for 2 days.The explantscultured in a screening medium containing 300 mg·L-11 of cephalosporin and 12 mg·L-11 hygromycin.,the resistant adventitious buds were obtained finally. |
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