香蕉茎尖的玻璃化法超低温保存及其植株再生

Cryopreservation of in Vitro Shoot Tips of Banana by Vitrification and Its Regeneration

以香蕉（Musa spp.）为试材,对其离体培养茎尖玻璃化法超低温保存影响因素进行研究.结果表明,不定芽在MS＋3.0～5.0 mg/L 6-BA＋0.1 mg/L NAA的培养基上分化较好.香蕉茎尖超低温保存较佳体系是：2.0～3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d,剥取带1～2个叶原基的茎尖（长1.0～1.5 mm）,室温（25℃）下装载液（MS＋2 mol/L甘油＋0.4 mol/L蔗糖）装载20～30 min,然后用玻璃化溶液（PVS2）于0℃下处理40 min,换1次PVS2后迅速投入液氮.保存至少1 h后,在40℃水浴中化冻90 s,用1.2 mol/L蔗糖培养液洗涤2次,每次10 min,然后转入含0.3 mol/L蔗糖的MS培养基上,暗培养10～15 h后转移到含0.5 mg/L 6-BA的MS培养基中,暗培养1周后转移到正常光下,3个香蕉品种（巴西蕉、广东香蕉2号、广东粉蕉1号）的成活率分别为75.9%、40.0%和69.6%,再生率分别为63.4%、35.0%和63.4%.再生植株生长和分化正常,生根后可移栽成活.

Shoot tips excised from healthy in vitro plants of three Banana cuhivars were successfully cryopreserved with the vitrification technique. A suitable procedure was established as follows： 3 -4 weeks after subculture, shoot tips about 2 -3 cm in length were precultured for 2 days on MS medium supplemented with 0. 4 mol/L sucrose. Dissected shoot apices about 1.0 - 1.5 mm in length with one or two leaf primordium were loaded with a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 20 - 30 min at room temperature. These excised shoot tips were sufficiently dehydrated in a highly concentrated plant vitrification solution for 40 min at 0℃. And then plunged directly into liquid nitrogen and conserved for at least 1 h. After rapid thawing in water at 40℃ for 90 s, the tips were rinsed with 1.2 mol/L sucrose solution for 20 min at room temperature and then plated on MS medium supplemented with 0. 5 mg/L 6-BA. The cryopreserved materials were cultured in darkness for one week so that they could grow quickly. Successfully cryopreserved shoot tips resumed growth within 10 days of plating and developed shoots within 1 month without intermediary callus formation. This simple protocol was successfully applied to the three cultivars belonging to two genomic groups of banana. The survival rate of shoot tips was 75.9%, 40. 0%, 69. 6%, and regrowth rate reached 63.4%, 35.0%, 63.4% 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.