贵州珍珠半夏的离体培养及遗传稳定性的分子检测
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摘要
本文以贵州珍贵半夏品种—珍珠半夏为材料,对其离体快繁、适宜于遗传改良的再生体系、愈伤组织培养,以及离体材料的体细胞遗传变异等方面进行了探讨,以期为半夏试管苗的大规模工厂化生产,种质资源的离体保存及遗传转化等提供技术平台。取得以下结果:
1建立并优化了珍珠半夏离体快繁体系
采用珍珠半夏球茎为外植体,接种在MS+KT(1.0mg/L)+NAA(0.2-0.4mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基中,可在30d内完成芽的萌发、增殖以及诱导生根等离体快繁的全过程。炼苗约15d后即可定植于大田。
2建立并优化了叶柄和叶片的离体培养体系
外植体类型、年龄,培养基中生长调节物质(PGR)种类及含量等因子对珍珠半夏再生有很大影响。叶柄接种于MS+BA(0.5 mg/L)+IBA(0.2mg/L)+蔗糖(3.0%)+琼脂(0.7%)的培养基中,获得了较高的不定芽再生频率;暗培养和AgNO_3对其不定芽再生率影响不大。
珍珠半夏叶片再生的最适培养基为MS+TDZ(0.2mg/L)+IBA(0.2mg/L)+蔗糖(3.0%)+琼脂(0.7%)。
3愈伤组织培养
外植体类型、基本培养基、生长素种类及浓度对珍珠半夏愈伤组织的诱导有一定影响。以球茎接种在MS+BA(1.0mg/L)+2,4-D(2.0mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基上,出愈率最高,且可以有效的防止褐化,愈伤组织质量较好,可用于长期继代培养。
TDZ对愈伤组织的再分化效果优于BA、KT及ZT,在MS+TDZ(0.2 mg/L)+IBA(0.5mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基中,分化的不定芽生长状况良好,且能正常生根。
4组培材料遗传稳定性的分子检测
采用96个随机引物产生的RAPD标记,对田间材料、组培材料第5代、10代和15代的珍珠半夏试管苗DNA进行检测,没有发现变异;采用78个随机引物对珍珠半夏继代第15代内20个株系的DNA进行检测,也未检测到变异。因此在所用引物检测范围内,珍珠半夏试管苗在15代以内DNA分子上没有发现变异。
To satisfy the increasing demand of intensive production,germplasm conservation and genetic improvement,the efficient in vitro regeneration for micropropagation and genetic engineering from various explants were established and optimized.Somaclonal variation of pinellia ternate Breit 'zhenzhubanxia' in vitro plants was further assessed using RAPD markers in this present study.The main results are as follows:
1 Establishment and optimization of aseptic cultures
The tuber showed the best germination,multiplication and root induction in MS supplemented with BA(0.5 mg/L)+IBA(0.2 mg/L),from which just 30 d were needed to the regeneration of an in vitro plant.With acclimatization of 15 d in autoclaved soil,the plants showed 90%of survival after transplantation.Using just-mentioned procedure,the optimized in vitro micropropagation system for the penillia was established.
2 Establishment and optimization of in vitro culture of petiol and lamina
Explant types,age and plant growth regulators,had affects on the regeneration rate and quality of adventitious buds.The petiol showed the best regeneration rate and quality of adventitious buds in MS+BA(0.5 mg/L)+IBA(0.2 mg/L)+sucrose(3.0%) +agar(0.7%);AgNO_3 and dark duration could not significantly affect the regeneration rate and quality of adventitious buds.
MS medium supplemented with 0.2 mg/L TDZ and 0.2 mg/L IBA gave the best regeneration from lamina.
3 Establishment and optimization of callus culture
Explant types,basal medium and concentrations of auxins diversely influenced callus formation.Tuber gave the best callus formation rate in MS added with BA(1.0 mg/L) and IBA(2.0 mg/L).The callus showed the the best vigour and quality.
TDZ showed the best effect on callus redifferentiation.With the supplement of 0.2 mg/L TDZ and 0.5 mg/L IBA in the medium,the most regenerated bud demonstrated vigorness,and rooted easily.
4 Assessment of genetic stability using molecular marker
To evaluate the genetic stability of in vitro material,the stock plant,as well as in vitro shoots collected at the 5~(th),10~(th) and 15~(th) cycle of subculture were employed to carried out PCR using 96 arbitrary primer.Further,RAPD markers amplified with 78 primers were also adopted to assess the somaclonal variation of in vitro materials randomly selected from the 15~(th) cycle of transfer.There was no DNA variation in the plants that had been transferred as many as 15 cycles in pinellia.
1建立并优化了珍珠半夏离体快繁体系
采用珍珠半夏球茎为外植体,接种在MS+KT(1.0mg/L)+NAA(0.2-0.4mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基中,可在30d内完成芽的萌发、增殖以及诱导生根等离体快繁的全过程。炼苗约15d后即可定植于大田。
2建立并优化了叶柄和叶片的离体培养体系
外植体类型、年龄,培养基中生长调节物质(PGR)种类及含量等因子对珍珠半夏再生有很大影响。叶柄接种于MS+BA(0.5 mg/L)+IBA(0.2mg/L)+蔗糖(3.0%)+琼脂(0.7%)的培养基中,获得了较高的不定芽再生频率;暗培养和AgNO_3对其不定芽再生率影响不大。
珍珠半夏叶片再生的最适培养基为MS+TDZ(0.2mg/L)+IBA(0.2mg/L)+蔗糖(3.0%)+琼脂(0.7%)。
3愈伤组织培养
外植体类型、基本培养基、生长素种类及浓度对珍珠半夏愈伤组织的诱导有一定影响。以球茎接种在MS+BA(1.0mg/L)+2,4-D(2.0mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基上,出愈率最高,且可以有效的防止褐化,愈伤组织质量较好,可用于长期继代培养。
TDZ对愈伤组织的再分化效果优于BA、KT及ZT,在MS+TDZ(0.2 mg/L)+IBA(0.5mg/L)+蔗糖(3.0%)+琼脂(0.7%)培养基中,分化的不定芽生长状况良好,且能正常生根。
4组培材料遗传稳定性的分子检测
采用96个随机引物产生的RAPD标记,对田间材料、组培材料第5代、10代和15代的珍珠半夏试管苗DNA进行检测,没有发现变异;采用78个随机引物对珍珠半夏继代第15代内20个株系的DNA进行检测,也未检测到变异。因此在所用引物检测范围内,珍珠半夏试管苗在15代以内DNA分子上没有发现变异。
To satisfy the increasing demand of intensive production,germplasm conservation and genetic improvement,the efficient in vitro regeneration for micropropagation and genetic engineering from various explants were established and optimized.Somaclonal variation of pinellia ternate Breit 'zhenzhubanxia' in vitro plants was further assessed using RAPD markers in this present study.The main results are as follows:
1 Establishment and optimization of aseptic cultures
The tuber showed the best germination,multiplication and root induction in MS supplemented with BA(0.5 mg/L)+IBA(0.2 mg/L),from which just 30 d were needed to the regeneration of an in vitro plant.With acclimatization of 15 d in autoclaved soil,the plants showed 90%of survival after transplantation.Using just-mentioned procedure,the optimized in vitro micropropagation system for the penillia was established.
2 Establishment and optimization of in vitro culture of petiol and lamina
Explant types,age and plant growth regulators,had affects on the regeneration rate and quality of adventitious buds.The petiol showed the best regeneration rate and quality of adventitious buds in MS+BA(0.5 mg/L)+IBA(0.2 mg/L)+sucrose(3.0%) +agar(0.7%);AgNO_3 and dark duration could not significantly affect the regeneration rate and quality of adventitious buds.
MS medium supplemented with 0.2 mg/L TDZ and 0.2 mg/L IBA gave the best regeneration from lamina.
3 Establishment and optimization of callus culture
Explant types,basal medium and concentrations of auxins diversely influenced callus formation.Tuber gave the best callus formation rate in MS added with BA(1.0 mg/L) and IBA(2.0 mg/L).The callus showed the the best vigour and quality.
TDZ showed the best effect on callus redifferentiation.With the supplement of 0.2 mg/L TDZ and 0.5 mg/L IBA in the medium,the most regenerated bud demonstrated vigorness,and rooted easily.
4 Assessment of genetic stability using molecular marker
To evaluate the genetic stability of in vitro material,the stock plant,as well as in vitro shoots collected at the 5~(th),10~(th) and 15~(th) cycle of subculture were employed to carried out PCR using 96 arbitrary primer.Further,RAPD markers amplified with 78 primers were also adopted to assess the somaclonal variation of in vitro materials randomly selected from the 15~(th) cycle of transfer.There was no DNA variation in the plants that had been transferred as many as 15 cycles in pinellia.
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