Studies On Pineapple Germplasm Cryopreservation By Vitrification

Pineapple (Ananas comosus) is an important herbaceous fruit trees in many tropical and subtropical countries. Shoot-tips cryopreservation by vitrification of four pineapple cultivars: Aoka(Smooth Cayenne Group), Hawaaii(Smooth Cayenne Group), Comte de Paris(Queen Group), Tainong-16(crossbreed) and ultrastructure of cryopreserved cells were studied in this experiment. The main results of this reserch are as follows:1. Shoot tips of four Pineapple cultivars were successfully cryopreserved with the vitrification technique. A suitable procedure was established as follows: shoot tips about 2～3cm in length were precultured for 1～2 days on MS medium supplemented with 0.5mol/L sucrose. Dissected shoot apices about 1.0-2.5mm in length with one or two primordium leaves were pretreated in a solution containing 2mol/L glycerol and 0.4mol/L sucrose for 50 min at room temperature, and then treated with solution of PVS2 at 0℃ for 40 min. After changing with fresh PVS2, shoot tips were immersed into liquid nitrogen directly and conserved for at least 1h. After being rapidly thawed in a water bath at 40℃ for 90s, shoot tips were washed with sucrose solution (1.2mol/L) for 20min at room temperature. Then the shoot tips were plated on MS medium supplemented with 0.3 mg/L sucrose, after 48h of culture in darkness, the cryopreserved materials were transferred onto MS medium supplemented with 0.5 mg/L BA and cultured in darkness for one week. The heighest survival rate and regeneration rate was 96.2% and 93.3% respectively. Almost all the shoot tips formed roots and were successfully transferred to soil in pots. The regenerate plantlets could normally root and survive after transplantation.2. The differences of cryopreservation between different pineapple cultivars were compared. The survival rate of four pineapple cultivars was 95.5%, 95.3%,78.6% and 87.7% respectively,and regrowth rate was 93.2%, 92.6%, 76.7% and 87.7% respectively. Almost all the shoot tips formed roots and were successfully transferred to soil in pots. The plantlets could normally root and survive after transplantation.3. The suitable resuming medium for pineapple shoot tips after cryopreservation was selected. Solid MS medium supplemented with 0.5 mg/L BA is optimum for the growth of shoot tips of pineapple after cryopreservation. In that condition, survival rate and regeneration rate are the highest, 95.6% and 93.0% respectively.4. Ultrastructure of cryopreserved cells was observed by using the transmission electron micryoscopy (TEM). The results showed that the plasmolysis became more and more severe during induced dehydration of pineapple shoot-tips. Some cells protoplasts condensed, and cell walls, cell membranes and nucleus envelopes were badly injured...