| Papaya(Carica papaya Z,.)is an important fruit tree in many tropical and subtropical countries. Shoot-tips micropropagation,somatic embryogenesis and cropreservation by vitrification were studied in this experiment to investigate mayor influencing factors. A practical method was also amended and adopted for propagation and germplasm conservation of high-quality papaya cultivars. The main results are as follow:1. Medium for Papaya shoot-tips culture was optimized and selected. The cluster buds were removed from the shoots and subcultured on the proliferation medium containing 0.5mg/L BA, 0.lmg/L NAA, 40mg/LADS and l.0mg/LGAs, which resulted a 6.8-fold increase in the total number of plantlets during every 4-week period. Additionally, the root-inducing medium containing 2.0mg/LIBA and 2% AC was found to be better than any others used in this experiment.2.The effects of explants type and medium components on callus induction, somatic embryogenesis and plants regeneration of Papaya were investigated. Data showed that cotyledons and immature embryos were all suitable materials for callus induction, somatic embryogenesis and plants regeneration. However, the capacity of callus- induction varied with different varieties of explants. The medium containing 1.00mg/L2,4-D, 0.30mg/LBA and 0.05 mg/L NAA was found optimum for callus- induction.Addition of ADS and CH(casein hydrolysate) to this medium showed beneficial effects on somatic embryogenesis. All regenerated plantlets in this experiment could normally root and survive after transplantation.3.Shoot tips of Papaya were successfully cryopreserved using the vitrification method for the first time. Shoot tips about 3~5cm in length were precultured for 3 days in MS medium supplemented with 5% DMSO. Shoot tips about 1.5~2.5mm in length with one or two primordial leaves were pretreated in a solution containing 60% PVSi for 40-50 min at room temperature ,and then treated with another solution containing 100%PVSa at 0 for 30 min. After changing with fresh PVS2, shoot tips were immersed into liquid nitrogen directly and conserved for 24 hour at least. After being rapidly thawed in a water bath at 40 ,shoot tips were washed twice with sucrose solution (1.2mol/L)and transferred onto the same MS medium as the previous one. Survival rate of shoot tips was 53.3%, and regeneration rate reached 51.6%. No morphological abnormality was observed in the plants regenerated from cryopreserved shoot tips. The ploidy analysis of the cryopreserved shoot-tips showed that the ploidyconstitution of chromosome remained relatively stable during cryopreservation period. Plantlets rooted normally and survived after transplanting.4.Ultrastructure of cryopreserved cells was observed by using the transmission electron microscopy (TEM). The results showed that the plasmolysis became more and more severe during the pretreatment and dehydration after the shoot-tips were precultured. A few cell walls, cell membranes and nucleus envelopes were lethally injured during cryopreservation period,which may account for the death of some cells after cryopreservation. On the other hand, there were also many cells, which were basically similer to that of control material. They could survive and regenerate plantlets after cryopreservation.
|
PDF Full Text Request