| Longan is an important tropical or subtropical fruit tree in south China. In this experiment the in vitro conserved embryogenic calli (EC) of longan were used as materials for the studies on in-vitro conversation of Dimocarpus longan Lour. germplasm, including the optimization of the conditions for minimal growth conservation, plant regeneration via somatic embryogenesis from the conserved EC at high frequency in 15 cultivars, physiological changes of minimal growth conservation of longan EC, the chromosome variations of conserved longan EC as well as somatic embryogenesis(SE), RAPD analysis of minimal growth conservation of embryogenic cultures during SE and plantlets regenerating from the conserved EC.The main results were as follows:1. The optimization of the conditions for minimal growth conservation of longan ECThe effects of the concentrations of mannitol and inositol, the amount of inoculation, mannitol and inositol cross culture, sucrose, agar, salt, PP333 and light quality on the growth of the embryogenic callus(EC) were compared in this experiment in order to provide a scientific basis for prolonging the subculture cycle of longan EC.The obtained results showed that at the scope of this study, agar, PP333, salt concentrations and light quality did not significantly affected the subculture cycle, and adding 20g/L mannitol and 0.1g/L inositol together with controlling the inoculation amount 0.1g/bottle,t he subculture cycle could be prolonged to more than 70d.2. Plant regeneration at high frequency via somatic embryogenesis from longan EC after minimal growth conservationIn this experiment, longan EC of 15 cultivars which were randomly selected from 52 accessions of in vitro conserved longan EC (Fenglisui, SongfenbenII, Jidanben, Miaoqiao, Yunben,Youtanben, Fuyan, Qingkejiao, Pumingan, Xuesi, Honghezi, Gongmaben, Lidongben, Julong105, Shuangzimu)were used as the materials for the studies on the induction, maturation, regeneration of somatic embryos and transplanting. During the induction of somatic embryos, the sucrose concentration, organic substances, plant growth regulators and light quality affected the time, quantity and quality in different degrees of somatic embryogenesis; somatic embryogenesis occurred from 15d to 35d, many of the embryoids were white, the frequency of somatic embryogenesis was all 100%, and the number of differentiated embryoids was from 2550-13220 per gram. During embryoid maturation, the concentrations of sucrose, organic supplemental substances and light quality affected the size and the shape of matured embryoids, most of which were white and larger, from 5 mm to 15mm in size. The research results of plant regeneration showed that different maturation time, different types of embryoid and light quality had great effects on the germination rates of somatic embryos and plantlet growth. Most of the normal matured somatic embryos germinated after 15d, and the germination rates were different from 50% to 93.3%. And the rate of transplanting seedlings transplanted on sandy soil(peat soil:sand=1:2) was up to 83.33%. All the above showed that 15 cultivars of longan EC kept strong capacity of somatic embryogenesis after minimal growth conservation. 3. Changes of total sugar contents, starch contents and organic acid contents of longan EC after minimal growth conservationThe changes of total sugar contents and starch contents of longan EC from Honghezi, Songfengben and Miaoqiao were tested during conservation in this experiment. The results showed that the accumulation peak time of the total sugar contents of 3 cultivars cultured on four kinds of media were different.The accumulation peaks of Honghezi and Miaoqiao EC cultured in Medium A and B were earlier than those on Medium C and D, and Honghezi EC cultured on Medium D, Miaoqiao EC cultured on Medium C and D presented three peaks. Songfenben cultured on Medium A and D presented one peak, while cultured on Medium B and C presented two peaks. And the changes of starch contents were basically the same as the total sugar contents. The changes of the organic acid contents of Honghezi, Songfengben and Miaoqiao EC were also tested during conservation in this experiment.The results showed that the organic acid contents presented the downward trend, but the accumulation peak of acid contents were different, the accumulation peak meant organic acid reached a high level, and with the EC gradually aging, the organic acids contents appeared decline.4. Variations of chromosome number of longan EC after minimal growth conservationIn this experiment, the variations of chromosome number of longan EC after minimal growth conservation was studied to provide a scientific approach for lowering variant rate. The obtained results showed that the diploid cells accounted for 85.0%, and the polyploid cells accounted for 12.4% in EC; the diploid cells account for 89.4% and polyploid cells accounted for 8.4% in globular embryos; the diploid cells accounted for 94.4% and polyploid cells accounted for 4.8% in cotyledon embryos, and the diploid cells accounted for 96.6% and the polyploid cells accounted for 2.8% in root tips of seedlings.The results showed that in the process of SE, the proportion of the polyploid cells in the embryoid and plantlet was lower than that in EC, and the polyploid cells were higher than nullisomic cells.The proportion of diploid cells gradually increased, which showed that it was an elimination process of variant cells.At the same time, the chromosome variation was found in Honghezi EC clultured for 5 mo in this experiment, and the variation rate accounted for 8.6% cultured for 5mo, 10% cultured for 3a,15.0% cultured for 13a. And the variation rate of Songfenben EC and Julong 105 EC cutured for 3a accounted for 9.0% and 9.8% respectively. All the above results showed that the prophase variations of chromosome number of longan EC were probably larger, but it was likely to tend stable in the process of long-term subculture.5. RAPD analysis on longan EC in the process of somatic embryogenesis after minimal growth conservation and plant regeneration via somatic embryogenesisIn this experiment, the hereditary stability tested on longan EC in the process of somatic embryogenesis after minimal growth conservation and plant regeneration via somatic embryogenesis by randomly amplified polymorphic DNAs (RAPD) technique on DNA molecular level.The results of RAPD analysisi by 10 primers proved that hereditary stability of longan EC which were cultured in different times in the process of somatic embryogenesis after minimal growth conservation had some differences, but they kept in lower variability (less than 5.1 %) . On the other hand, the results of RAPD analysis by 10 primers proved that hereditary stability of plantlets regenerating via somatic embryogenesis had some differences.The variation rate of plantlets was 9.70% for EC conserved for 5 months, 13.07% for EC conserved for 3a, 15.07% for EC conserved for 13a.The obtained results showed that genetic variability of plantlets regenerating via somatic embryogenesis after minimal growth conservation tended stable, which were beneficial to germplasm conservation in vitro.
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