Studies On Cryopreservation Of Germplasm Of Pomegranate

ABSTRACT Pomegranate germplasm resource is the original material of breeding and plays an important role in genetic breeding. The pollen, calli and shoot-tips of ’Tunisia soft-seed’,’Yudazi’and’Taishanhong’ pomegranate were used as test materials of cryopreservation, the acquisition of in vitro culture, pretreatment, thawing ways and regeneration were studied, and the cryopreservation system of pomegranate germplasm was set up. After the cryopreservation, pollen viability was tested by both dyeing and germination, while calli and shoot-tips were tested by TTC method. In the end, calli and shoot-tips being cryopreserved were regenerated. Main results are as follows:1. To determine the optimal method of the pomegranate pollen viability, Tunisia soft-seed’pomegranate pollen were tested by both dyeing and germination method. Results showed the10%glucose+100mg·L-1boric acid solution was the suitable solution to test the pomegranate pollen viability, while vigor was higher by using Benzidine dyeing method with good correlation, so they were both good motheds to test the pomegranate pollen viability.2. Fresh mature pomegranate pollen were gathered, the’Tunisia soft-seed’pollen were plunged into LN rapidly for storage following dehydrating for6-8h with silica gel at4℃or incubating in PVS4for60min at room temperature. After cryopreservation,40℃70s was the most suitable way for vitrification, while effects of different thawing ways on the survival of cryopreserved pollen were not significantly different for dehydration method. The viability of pollen was not influenced by the duration of storage in LN. The germination rate was significantly higher by using dehydrating. Words came to the’Yudazi’and’Taishanhong’ pomegranate pollen, suitable dehydrating time was6h, appropriate processing time was20～40min in PVS4and60min in PVS2respectively;40℃70s was the most suitable way for vitrification; dehydrating was better than vitrification, the poisoning of PVS might be the reason. The Preserving effect’Tunisia soft-seed’pomegranate pollen is better than’Yudazi’, and better than’Taishanhong’.3. Young leaves of the1-4node of pomegranate cutting seedlings were used to induce calli. Leaves were dipped in dishwashing for30min, washing out for2h in running water, disinfected by75%ethanol for30s and0.1%HgCl2for4min, rinsed for3-5times by sterile water, placed on the sterilizated filter paper after cutting injury, and inoculated on the MS+6-BA1.0mg-L-1+NAA0.1mg-L-1medium, cultured at18to22℃light condition after2weeks in dark, to induce calli growth.4. The top buds of pomegranate cutting seedlings were dipped in dishwashing for20min, washing out for1h in running water, disinfected by75%ethanol for30s and0.1%HgCl2for3min, rinsed for3-5times by sterile water, placed on the sterilizated filter paper, and inoculated on the first generation tissue culture medium(MS+6-BA0.5mg·L-1+NAA0.1mg·L-1),4weeks later,2～3shoots could be obtained from each bud, and they were subcultured on the MS+6-BA0.5mg·L-1+NAA0.1mg·L-1+KT1.0mg·L-1medium.5. The cryopreservation procedure of pomegranate callus tissue was as follows:cold harding for2-7d, pre-treated in5%DMSO liquid medium for120min, plunged into LN for24h after immersed in PVS2, then went though40℃water bath quickly, and washed by the improved MS (without Ca2+) medium which included1.2mol·L-1sucrose. The relative survival rates of three pomegranate varieties (’Tunisia soft-seed’,’Yudazi’,’Taishanhong’) after cryopreservation were83.1%,39.5%and88.0%respectively. Then the calli were cultured on the MS medium with6-BA1.0mg·L-1+NAA0.1mg·L-1for2weeks in dark and exposured to the light. The calli didn’t turn brown but dried out and lost the ability of differentiation.6. The procedure of vitrification was studied using the shoot-tips of pomegranate as materials. In the experiment, shoot-tips in length5mm were loaded by60%PVS2at5℃for2h, plunged into LN for24h after immersed in PVS2, then went though40℃water bath quickly, and washed by the improved MS (without Ca2+) medium which included1.2mol·L-1sucrose. The relative survival rates of three pomegranate varieties (’Tunisia soft-seed’,’Yudazi’,’Taishanhong’) after cryopreservation were77.4%,35.6%and80.5%respectively. Then the shoot-tips were cultured on the MS medium with6-BA0.5mg·L-1+NAA0.1mg·L-1+VC2.0mg·L-1. Some shoot-tips didn’t turn brown except small part but lost the ability of regeneration.