减少培养基中大量元素进行菊花离体保存的方法技术

本发明专利技术涉及通过减少培养基中大量元素进行菊花离体保存的方法，专用于菊花种质资源离体保存。取菊花脚芽为外植体，以腋芽进行继代增殖。在１／２ＭＳ或１／２ＭＳ（１／２ＮＨ↓［４］↑［＋］）或１／４ＭＳ，附加６－ＢＡ０．３ｍｇ／Ｌ和ＮＡＡ０．１ｍｇ／Ｌ的培养基上进行保存，１２个月后存活率均为１００％，比正常ＭＳ培养基上的试管苗延长存活８个月。保存材料恢复生长正常，其后代经过氧化物酶（ＰＯＤ）及酯酶（ＥＳＴ）同工酶、ＩＳＳＲ分子标记检测未发生遗传变异。本发明专利技术首次利用减少培养基中大量元素保存菊花试管苗并对其后代进行遗传稳定性鉴定，不但保存１年后植株生长正常，而且保存材料的后代未发生遗传性变异。

Method for in vitro conservation of Chrysanthemum by reducing a large number of elements in culture medium

The invention relates to a method for in vitro conservation of Chrysanthemum by reducing a large number of elements in a culture medium. The buds were taken as explants and the axillary buds were subcultured. In 1 / 1 or 2MS / 2MS (1 / 2NH 4 + = \down\) or 1 / 4MS medium supplemented with 6BA0.3mg / L and NAA0.1mg / L were preserved after 12 months survival rate was 100%, compared to normal MS plantlets based on the survival of 8 april. The storage materials recovered and grew normally, and their offspring were detected by POD and esterase (EST) isozyme and ISSR markers. The invention is used for the first time to reduce the major element in the medium and preservation of Chrysanthemum Plantlets in vitro identification of genetic stability of their offspring, not only kept for 1 years after the plants grow normally and keep the descendant has no genetic variation.

【技术实现步骤摘要】

【技术保护点】
通过减少培养基中大量元素进行菊花离体保存的方法，包括：以菊花脚芽为外植体，用７５％乙醇表面消毒３０ｓ，无菌水冲洗２次，再用０．１％升汞灭菌８ｍｉｎ，无菌水冲洗５次后，将其切割成去叶带１个节的茎段接入ＭＳ培养基上；２０ｄ后将萌发的腋芽 转接到ＭＳ＋０．３ｍｇ．Ｌ↑［－１］６－ＢＡ＋０．１ｍｇ．Ｌ↑［－１］ＮＡＡ培养基上进行增殖培养；３０ｄ后将增殖的不定芽切割成去叶带２个节的茎段转入大量元素均减少１／２量的ＭＳ或ＮＨ↓［４］ＮＯ↓［３］减少３／４量，其余大量元素减少１／２量的ＭＳ或大量元素均减少３／４量的ＭＳ，均附加６－ＢＡ０．３ｍｇ／Ｌ和ＮＡＡ０．１ｍｇ／Ｌ的培养基上进行保存；以上培养基中含蔗糖３％、琼脂０．７％，ｐＨ为６．０，培养条件２３±２℃、光照强度２０００～３０００ｌｕｘ、光照时间１２ｈ／ｄ 。

【技术特征摘要】

【专利技术属性】
技术研发人员：房伟民，王艳芳，陈发棣，管志勇，刘兆磊，滕年军，陈素梅，赵宏波，许高娟，缪恒彬，姜贝贝，丁玲，
申请(专利权)人：南京农业大学，
类型：发明
国别省市：84[中国|南京]