Physiological changes in Gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation
Physiological changes in Gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation
复制标题
DOI：
10.1093/aob/mcl054
复制
发表时间：
2006-06-01
期刊：
影响因子：
4.2
通讯作者：
Akihama, Tomoya
中科院分区：
文献类型：
--
作者：
Suzuki, Mitsuteru;Ishikawa, Masaya;Akihama, Tomoya
背景和目的诱导植物的脱水耐受性是植物标本超低温保存中获得高存活率的关键。已有文献报道，两步蔗糖预培养能有效提高龙胆腋芽的脱水耐性，使其在超低温保存中存活。preculturing.center在标准的两步预培养中，将切下的龙胆腋芽在含有0.1 M蔗糖的MS培养基上于25 ℃孵育11 d（第一步：给予轻度渗透胁迫），随后在含有0.4 M和0.7 M蔗糖的MS培养基上各孵育1 d（第二步）。测定了龙胆芽预培养过程中脱落酸（阿坝）、脯氨酸和可溶性糖的含量。两步预培养和外源阿坝和脯氨酸的各种组合的效果是studied.center. Key结果在第一步预培养过程中，阿坝含量在第4天达到峰值，在第一步和第二步预培养结束时下降到背景水平。脯氨酸水平在第一预培养步骤中稳定增加，并在第二预培养步骤中进一步增加。用含有脯氨酸的培养基孵育芽，而不是两步预培养，无法使它们在干燥中存活。用阿坝代替0.1M蔗糖预培养能有效地提高芽的脱水耐性，但只有在第二步预培养后才能提高芽的脱水耐性。两步预培养培养基中含有阿坝合成抑制剂氟立酮，降低了芽的脱水耐性。正常的第一步预培养提高了可溶性糖的水平2.4倍，特别是蔗糖和棉子糖。用第二预培养步骤处理的芽大大增加了蔗糖levels.center dot结论这些观察结果导致假设第一预培养步骤涉及ABA介导的细胞变化，第二步骤诱导龙胆芽中蔗糖的加载。
center dot Background and Aims Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing.center dot Methods In standard two-step preculture, excised gentian axillary buds were incubated for 11d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied.center dot Key Results During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 M sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels.center dot Conclusions These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.