| Longan(Dimocarpus longan Lour.)is an important southern subtropical fruit which has a large-scale cultivation in China. In this experiment, The long-term conserved embryogenic calli (EC) of longan were used as the materials to study the following aspects: the optimization of the restricting conservation conditions for the longan EC of three types; plant regeneration via somatic embryogenesis in 17 cultivars of longan; shoosing and optimizing the transgenic receptor system of longan; optimizing the transformation system of antisense ACS gene into longan;screening of the resistant longan EC and observation of the growth status; detection of the resistant longan EC. The main results were as follows:1. The optimization of the conditions for minimal growth conservation of longan EC of three typesThe effects of the concentrations of mannitol, sorbitol, PEG, inositol, maltose and the lights quality on the growth of embryogenic callus (EC) were compared in three types of longan EC, and the optimization of the minimal growth conservation conditions was validated in three types of longan EC. The obtained results showed that the suitable medium for CⅢEC was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.1 g·L-1 inositol and 5g·L-1 maltose; the suitable medium for CⅡa was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.2g·L-1 inositol and 5g·L-1 maltose; the suitable medium for CⅡb was the MS medium supplemented with 1mg·L-1 2,4-D, 1.5% sucrose, 0.6% agar, 20g·L-1 mannitol, 0.1g·L-1 inositol; the subculture cycle could be further prolonged from 20 d to more than 60 d for CⅢI and CⅡa on the suitable medium. For CⅡb, it could be prolonged to more than 50 d. At the same time, the EC could maintain strong growth and regeneration ability.2. Plant regeneration via somatic embryogenesis from longan EC of 17 longan cultivarsIn order to checkout the capacity of somatic embryogenesis of the minimal growth conservation of longan EC, in this experiment, longan EC of 17 cultivars were used as the materials for the studies on the induction, maturation and regeneration. And at the same time, when it was finished, it meaned the regeneration of all the conserved EC. The results showed that the somatic embryogenesis and the regeneration were affected by different varieties of longan. During the induction of somatic embryogenesis, different cultivars, concentrations of maltose, mannitol, PEG, affected the time, quantity and quality of somatic embryogenesis. And the somatic embryogenesis occurred from 15 d to 35 d, the frequency of somatic embryogenesis was 100%, the colors of the embryoids were white, the number of differentiated embryoids was 1080-14380 per gram callus. During the stage of embryoid maturation, different cultivars, the ratio of sucrose and maltose, concentrations of mannitol, PEG, and different time of desiccation effected the size, browning and the shape of matured embryoids.The embryoids were from 5 mm to 15 mm in size, white and larger. During the stage of plantlet regeneration, the results showed that the germination rate of somatic embryos and plantlet growth were affected by different cultivars, different types of somatic embryogenesis. The germination rate was 35.67% to 90.00% with a great difference. The time of normal matured somatic embryos germinated is 13-15 d.3. Choosing and optimization the transgenic receptor systems for Dimocarpus longan Lour.Because the receptor system of calli was provided with strong ability of differentiation, regeneration and favorable genetic stability. In the experiment, the EC of Honghezi was used as material for genetic transformation. Calli pre-cultured for about 15 d were proved to the appropriate genetic transformation materials for their rapid-growth and strong vitality stage. Through measuring the fresh weight increase of EC, it was suitable for transgenic. Through measuring the fresh weight increase combined with the qualitative obserbation, 100mg·L-1 kanamycin concentration was suitable for transgenic operations on EC, both the growth and regeneration were normal, while the cephamycin concentration range was 0-400 mg·L-1.4. The introduction of antisense ACS gene into longan mediated by Agrobacterium tumefaciens.Based on the above results, new transgenic strategies about transformation of longan were performed. The highly efficient and stable parameters of transgenic operations on longan showed that the receptor material of longan was pretreated with 0.15 mol·L-1 mannitol in liquid for 6 h , taking 1/2 MS liquid culture medium as re-suspension liquid, containing 80.0 g·L-1 glucose, and with 0.9 OD600 value of Agrobacterium suspension, the receptor material was infected for 30 min, then being co-cultured onto the MS medium (pH 5.6) supplemented with 1 mg·L-1 2,4-D for 6 days at 22℃in dark. The transient expression of GUS could reach 90% of the resistant EC, Through the latter part of screening in this study, 55 accessions of resistant EC were chalked up.5. The selection and screening of longan resistant EC, and the molecular detection of the GUS gene.In this experiment, the transient expression of GUS at the prior period and the detection by PCR assays of GUS for the resistant EC at the later period were examined. The gradual enhancement of concentration for kanamycin was used. At last, the resistant EC were selected on MS medium supplemented with 100 mg﹒L-1 kanamycin, 1 mg·L-1 2,4-D and 0.5 mg·L-1 NAA. 55 accessions of longan germplasm were maintained for further studies. The rapid method for trace genomic DNA extraction for identification of transgenic EC of longan with PCR, which proved that the GUS gene was integrated into the genome of these resistant EC. The resistant EC grew slowly, at the same time the capacity of somatic embryogenesis was weak. |
