食用百合种质资源离体保存技术初步研究
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摘要
食用百合属无性繁殖蔬菜，传统的种质保存方法需每年进行种质更新，不仅耗费大量的人力、物力和财力，还容易受到病虫害和自然灾害的影响。为了克服传统保存方法所面临的问题，本研究以兰州百合为试材，在建立百合快繁体系的基础上，进行了种质资源缓慢生长和超低温保存初步研究。主要结果如下：
以百合鳞片微繁为基础的百合试管苗保存研究，以MS为基本培养基添加不同浓度的ABA和MNT配制24种培养基在4种不同的温度下保存，结果显示：试管苗在2±1℃和5±1℃条件下，弱光保存6个月，各种处理的成活率均为100%，且试管苗生长健壮。相比之下，添加MNT的试管苗中，SN7为最好，添加ABA的试管苗中，以SA5最好。25±1℃和10±1℃下，8h/d光照条件下保存6个月，凡在培养基中加有ABA的试管苗基本死亡，加有MNT的试管苗需要进行继代培养。4种不同温度下，ABA和MNT对试管苗均有很强的生长抑制作用。但ABA的抑制性较强，试管苗生长弱小，加有MNT的试管苗生长正常。
百合茎尖玻璃化超低温保存基本条件的研究，发现前处理和解冻方式是影响超低温保存存活率的主要因素。本研究得出百合茎尖超低温保存的程序和基本条件为：切取百合茎尖2～3mm，在0.5mol·L-1蔗糖的MS培养基上预培养1～2d后，0℃下用100%PVS2脱水处理20min后，换用新鲜的100%PVS2，然后将材料直接投入液氮。48h后取出，先在40℃水浴中化冻2min，再在25℃水浴中化冻10min，在MS+6-BA 0.5mg·L-1 +NAA 0.1mg·L-1+GA 0.3mg·L-1+蔗糖30g·L-1+琼脂7g·L-1上恢复培养，冻后存活率最高可达到52.6%。
Edible lily is one of vegetative vegetables. The conservation of edible lily germplasm with the traditional method needs to replant every year. This method not only needs a lot of labor and money, but also is easily affected by diseases and insect pests, natural disasters. In order to settle those problems, the preservation in vitro of edible lily was explored in this paper. The establishment of propagation system and the effects of some key factors affecting the preservation quanlity of edible lily germplasm were involved in the preservation in vitro. The cryopreservation method of edible lily germplasm was also studied. The main results were as follows:
The conservation of lily plantlets based on micropropagation of edible lily squama was tired on in 24 different MS medium under different temperatures (2,5,10,25). The results sho- wed: the plantlets that were conserved for 6 months at low temperature(2±1℃ and 5±1℃) all can survive.The SN7 that was appended with MNT was the best medium for the plant- lets and the SA5 that was appended with ABA was the best medium for the plantlets at 2±1℃ and 5±1℃.All plantlets conservered on the medium appended with ABA at 25±1℃and 10 ±1℃with 8h/d photoperiod 6 months almost died. The plantlets conserved on the medium appended with MNT must be step-culture. ABA and MNT was well growath depr- essor for lily preservation in vitro at 4 different temperatures. MNT was better than ABA. This is bec- ause the plantlets conserved in the medium appended with ABA were too depre- ssed and the plantlets in the medium appended with MNT are stuggy.
The basic condition for the cryopreservation of lily shoot tips by vitrification was stu- died. The results indicated that the major factors determining whether the lily cryopreserved were successful or not were pre-teament and thawing methods of the shoot tips. The proced- ure and corresponding conditions obtained in the study were as follows: the shoot-tips were precultured on MS medium with 0.5M sucrose for 2 days and detached growing tip tissue with the size of 2～3mm was dehyrated with a vitrification solution (100%PVS2 Solu tion) for 20 minutes at 0℃. After new vitrification solution was replaced. Then plunge into liquid nitrogen. We got the highest survival rate (52.6%) after cryopreservation. The materials were plunged into liquid nitrogen. After 48h,The material was taken off from liquid nitrogen and
thawed in 40℃ was cultivated on MS + 6-BA 0.5mg·L-1+NAA 0.1mg·L-1 +GA 0.3 mg·L-1+ sucrose 30g·L-1 +agar 7g·L-1The highest survival rate of tips after cryopreservation was 52.6%.
以百合鳞片微繁为基础的百合试管苗保存研究，以MS为基本培养基添加不同浓度的ABA和MNT配制24种培养基在4种不同的温度下保存，结果显示：试管苗在2±1℃和5±1℃条件下，弱光保存6个月，各种处理的成活率均为100%，且试管苗生长健壮。相比之下，添加MNT的试管苗中，SN7为最好，添加ABA的试管苗中，以SA5最好。25±1℃和10±1℃下，8h/d光照条件下保存6个月，凡在培养基中加有ABA的试管苗基本死亡，加有MNT的试管苗需要进行继代培养。4种不同温度下，ABA和MNT对试管苗均有很强的生长抑制作用。但ABA的抑制性较强，试管苗生长弱小，加有MNT的试管苗生长正常。
百合茎尖玻璃化超低温保存基本条件的研究，发现前处理和解冻方式是影响超低温保存存活率的主要因素。本研究得出百合茎尖超低温保存的程序和基本条件为：切取百合茎尖2～3mm，在0.5mol·L-1蔗糖的MS培养基上预培养1～2d后，0℃下用100%PVS2脱水处理20min后，换用新鲜的100%PVS2，然后将材料直接投入液氮。48h后取出，先在40℃水浴中化冻2min，再在25℃水浴中化冻10min，在MS+6-BA 0.5mg·L-1 +NAA 0.1mg·L-1+GA 0.3mg·L-1+蔗糖30g·L-1+琼脂7g·L-1上恢复培养，冻后存活率最高可达到52.6%。
Edible lily is one of vegetative vegetables. The conservation of edible lily germplasm with the traditional method needs to replant every year. This method not only needs a lot of labor and money, but also is easily affected by diseases and insect pests, natural disasters. In order to settle those problems, the preservation in vitro of edible lily was explored in this paper. The establishment of propagation system and the effects of some key factors affecting the preservation quanlity of edible lily germplasm were involved in the preservation in vitro. The cryopreservation method of edible lily germplasm was also studied. The main results were as follows:
The conservation of lily plantlets based on micropropagation of edible lily squama was tired on in 24 different MS medium under different temperatures (2,5,10,25). The results sho- wed: the plantlets that were conserved for 6 months at low temperature(2±1℃ and 5±1℃) all can survive.The SN7 that was appended with MNT was the best medium for the plant- lets and the SA5 that was appended with ABA was the best medium for the plantlets at 2±1℃ and 5±1℃.All plantlets conservered on the medium appended with ABA at 25±1℃and 10 ±1℃with 8h/d photoperiod 6 months almost died. The plantlets conserved on the medium appended with MNT must be step-culture. ABA and MNT was well growath depr- essor for lily preservation in vitro at 4 different temperatures. MNT was better than ABA. This is bec- ause the plantlets conserved in the medium appended with ABA were too depre- ssed and the plantlets in the medium appended with MNT are stuggy.
The basic condition for the cryopreservation of lily shoot tips by vitrification was stu- died. The results indicated that the major factors determining whether the lily cryopreserved were successful or not were pre-teament and thawing methods of the shoot tips. The proced- ure and corresponding conditions obtained in the study were as follows: the shoot-tips were precultured on MS medium with 0.5M sucrose for 2 days and detached growing tip tissue with the size of 2～3mm was dehyrated with a vitrification solution (100%PVS2 Solu tion) for 20 minutes at 0℃. After new vitrification solution was replaced. Then plunge into liquid nitrogen. We got the highest survival rate (52.6%) after cryopreservation. The materials were plunged into liquid nitrogen. After 48h,The material was taken off from liquid nitrogen and
thawed in 40℃ was cultivated on MS + 6-BA 0.5mg·L-1+NAA 0.1mg·L-1 +GA 0.3 mg·L-1+ sucrose 30g·L-1 +agar 7g·L-1The highest survival rate of tips after cryopreservation was 52.6%.
引文
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VillalobosVM，EngelmanF：Exsituconservation of plantgerm plasmusing biotechnology [J]．Biotechol， 1995，11 (4)：37 5-382
MarinMI，Duran-VilaN．Conservation of citrusgermplasminvitro [J] ．J.Amer．Soc.
Hort．Sci，1991，116(4)：740～746
TonyHH，ChenandKuttyK，Kartha.．Cryopreservation of plant cellsand organs [A].
In :Forestand Crop Biotechnology Progressand prospects (FredrickA． Valentine
Ed)[C]．Neidelberg,London，Paris，Tokyo：Sprigner-Verlag，1988：217～240
Ford，Lloyd BV， Jackson MT．Biotechnolog yand methods of conservation of plantgenetic resouces [J]．J .Biotechol，1991，43(1)：247～256
Mantell SH， Smith H． Plant Biotechnology [M]．Cambridge：Cambridge Universitypress，1983：168～218
Mullin RH，Schligel DE． Cold storage maintenance of strawberry meristem plantlets [J]．Hortscience， 1976，11(1)：100～101
Lundergan C，Janick J．Low temperature storage of invitro apples hoots[A]．Hortscience，1979，14(4)：5～14
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