Studies On Cryopreservation And Genetic Transformation Of Embryogenic Cell Suspension Of Banana

Based on pre-researches,further studies were carried out on the establishment of regeneration system,cryopreservation and genetic transformation of Musa AA Pisang Mas cv.Mas.The main results are as follows:1.Using immature male flower of Musa AA Pisang Mas cv.Mas as explant,the embryogenic calli were induced with different concentration of 2,4-D and picloram. The results showed that the induction percentage of embryogenic calli was doubled when 4 mg/L 2,4-D in the callus induction medium was substituted by 4 mg/L picloram.It was difficult to establish embryogenic cell suspension by picloram, because the moisture content of embryogenic calli is too high.But embryogenic cell suspension was easy to establish if induced by 2,4-D.The effect of different concentration of ABA was compared on induction,muturation and germination of somatic embryo.The results were that 0.5 mg/L ABA could promote the induction, maturation and germination of somatic embryo.The number of mature embryo somatic was 37600/mL PCV.The percentage of germination and the plant conversion were 30.73%and 23.42%,respectively.2.Embryogenic cell suspension of Musa AA Pisang Mas cv.Mas was successfully cryopreserved with the vitrification technique.A suitable procedure was established as follows:the embryonic cell suspensions in the logarithmic phase were precultured for 2 days on liquid medium with 0.5 mol/L sucrose.Then cells were loaded in liquid medium with a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 40 min at room temperature.These exercised cells were sufficiently dehydrated in a highly concentrated vitrification solution for 30 min at 0℃,then plunged directly into liquid nitrogen and conserved for at least 24h.Then rapid thawing in water at 37℃for 90 s, the cells were rinsed in a 1.2 mol/L sucrose cleaning solution for three times.The highest rate of cell viability detected by TTC method is 80.46%,then plated on a transition culture medium supplemented with 0.5 mol/L sucrose.After 24 h,the cells were transferred to recovery culture medium.The plantlets can be obtained after further culturing. 3.Ultrastructure of embryogenic cell suspension of banana during vitrification cryopreservaton was observed by using the transmission electron microscopy(TEM). The results showed that the extent of cell plasmolysis became more and more severe during the pretreatment and dehydration after cells were precultured.Meanwhile,cells with numerous smaller vacuoles and a few organelles intumescentiae.Cell walls,cell membranes and organelles were lethally injured after liquid nitrogen preservation, which may account for the death of some cells.And although there were some extent changes in many other cells structure,these were kinds of reversible damage,which may be rehabilitated automatically during recovery culture and regenerable plantlets.4.Embryogenic cell suspension(Musa AA Pisang Mas cv.Mas) was used as the receptors for transformation by Agrobacterium tumefaciens containing pCAMBIA1301. By detecting the transient expression of GUS gene,the factors affecting the early phase of Agrobacterium-mediated transformation of banana were investigated.The results showed that the the highest rate of transient expression is 25.30%,when mixing the bacterium OD₆₀₀ 0.2 concentration with the embryonic cell suspension in the logarithmic phase,then centrifugating at room temperature,8000 r/min for 5 minutes, and then shaking at 110 rpm in orbital shaker for 5 minutes,finally co-culturing for 8 days in somatic embryo induction and maturation medium.The transgentic resistance buds can be obtained after the somatic embryo was reelected and cultured.