菊花茎尖外植体高效超低温保存技术建立的分析

Analysis on the Establishment of Efficient Cryopreservation Technology for Chrysanthemum Stem Tip Explants

菊花是全球最重要的鲜切花之一,建立高效的菊花茎尖超低温保存体系具有重要意义。本研究以菊花‘Sei Hillary’试管苗为材料,研究了茎尖大小、炼苗期光照条件、六种再生培养基配方、超低温保存方法、母株感染病害情况对超低温保存效率的影响,结果发现,上述参数均显著影响茎尖超低温保存效率,组织切片观察揭示了超低温保存方法影响再生效率的原因。本研究建立了一个高效的菊花茎尖超低温保存体系,主要技术步骤包括:健康试管苗在16 h的光周期下4℃炼苗6周、切取携带3~4片叶原基的茎尖(1.5 mm),利用小滴玻璃化法进行超低温保存,快速解冻后于添加0.05 mg/L GA3的1/2Knop培养基上再生培养。该体系可获得85.5%成活率和53.7%的再生率,为菊花种质资源提供了一种简单高效的长期保存方法。

Chrysanthemum is one of the most important fresh cut flowers in the world,it is of great significance to establish an efficient cryopreservation system for chrysanthemum shoot tips.We here described an efficient cryopreservation method for shoot tip cryopreservation of chrysanthemum ‘Sei Hillary’.In this study,effects of several key parameters on recovery of cryopreserved shoot tips were investigated,including shoot tip size,light regimes used for cold-hardening of in vitro stock shoots,six post-culture mediums,cryopreservation methods,as well as virus infection types of in vitro stock shoots.Histological observation was conducted to explain the differences of shoot regeneration patterns between encapsulation-vitrification and droplet-vitrification.Major steps for efficient shoot tip cryopreservation of chrysanthemum’Sei Hillary’was summarized as followings:in vitro healthy stock shoots were cold-hardened at 4℃ for 6 weeks under 16 h photoperiod;shoot tips 1.5 mm,with 3~4 leaf primordia were excised and subject to droplet-vitrification.Following rapid thawing,shoot tips were post-cultured on 1/2 Knop medium supplemented with 0.05 mg/L GA3.This droplet-vitrification produced 85.5%survival and 53.7%shoot regeneration,it provides a simple and efficient method for long-term preservation of chrysanthemum germplasm resources.