In Vitro Conservation And Proteomics Of Tube Plantlets In Fortunella Crassifolia And Citrus Microcarpa

In this experiment, the tube plantlets of Fortunella crassifolia and Citrus microcarpa were used to investingate the approaches of in vitro conservation and the related proteomics. The main results were as follows:1. The in vitro regeneration techniques were established in Fortunella crassifolia and Citrus microcarpa.The media for seed germination, micropropagation, growth and rooting of tube plantlets were optimized in Fortunella crassifolia and Citrus microcarpa. The optimum medium for inducing rapid germination of seeds was 1/4 MS medium supplemented with sucrose 30 g·L^·1, on which the seeds germinated completely within 15 days. The optimum medium for multiplication of aseptic shoots was MS medium adding BA 1.0 mg·L^·1, NAA 0.05 mg·L^·1 and Sucrose 20 g·L^·1. The optimum medium for growth of plantlets was MS medium adding BA 0.1 mg·L^·1, NAA 0.01 mg·L^·1 and Sucrose 20 g·L^·1. The optimum medium for rooting was 1/2 MS medium adding IBA 0.2 mg·L^·1, NAA 0.3 mg·L^·1 and Sucrose 20 g·L^·1, on which the plantlets rooted completely within 45 days. Materials for the studies on in vitro conservation and proteomics could be provided by the in vitro regeneration system.2. The technique determing the plantlets from the nucellar embryos were established by RAPD-PCR in Fortunella crassifolia and Citrus microcarpa.After iterative experiments, the optimal amplification system and program for Fortunella crassifolia and Citrus microcarpa RAPD-PCR were established and applied to identify all the plantlets from a seed. The RAPD analysis of plantlets from 20 seeds indicated that the embryo with the biggest cotyledons was generally the nucellar embryo in a seed and the probability was alomost up to 100%. Therefore, the embryos with the biggest cotyledons were selected as the materials for in vitro conservation in this experiment, which could ensure that the conserved materials were indentical to the mother trees in genetics.3. The restricting growth methods of in vitro conservation were established in Fortunella crassifolia and Citrus microcarpa. The nucellar plantlets or the regenerated plants could be conserved on 40 mL hormones-free MS medium in the container of glass canning jar(9cm×5.5cm)with plastics lid without continuous subculture for 12 months. Adding the hormones-free blank medium with 3 g·L^·1 agar if the water in the medium was almost lost could keep the tube plantlets strong and prolong the conserved time, and the conserved time had been up to 28 months at present. It was concluded that the most important factor affecting the time for in vitro conservation was the moisture in Fortunella crassifolia and Citrus microcarpa.4. The examining system of genetic stability of the conserved plantlets by RAPD-PCR was established.The tube plantlets conserved without subculture for 1 year could be restored to grow. The analysis of RAPD-PCR of the materials from the conserved plantlets and the normal plantlets showed that the patterns of RAPD were completely identical, which suggested that there be no variance in the DNA level.5. The studies on the proteomics of the in vitro conservation plantlets were carried out.The tube plantlets of Citrus microcarpa were used as materials for the studies on the proteomics during the in vitro conservation. The results showed that as the conserved time lasted, most of the proteins in the tube plantlets decreasd or disappeared; a few of the proteins increased remarkably and several new proteins appeared, which suggested the increasing proteins should be the key proteins for maintaining the life activity and basic metabolism of "dormant" conserved plantlets. Five distinguishing protein spots were chose for the analysis of Peptide Mass Fingerprint by MALDI-TOF-MS, and the matich analysis through the database of Mascot showed that the two proteins accumulating largly during the conservation were very similar to putative miraculin-like protein 2 and considered as the miraculin-like protein, which might be not only of the similar function of miraculin, but also of the anti-hungry or anti-aging functions; the protein appeared during conservation was an unknown protein; one of the two proteins appeared after restoring culture was considered as a HSP70 protein, and the other was an unknown protein.