建立银杏遗传转化受体系统的研究
摘要
银杏(Ginkgo biloba)又名白果、公孙树，是我国特有的珍贵孑遗树种，也是目前世界上珍稀裸子植物之一，现仅存一科一属一种，素有裸子植物“活化石”之称，在植物学上具有重要的科学价值。
1980年以来，银杏在食品、保健、医药、木材，绿化等领域得到广泛的开发利用，对银杏的繁殖和育种提出了新的要求。但是，银杏是雌雄异株植物，实生苗定植后一般需要20～30年才能开花结果，银杏常规育种时间长，效率低，严重制约了早期经济效益的提高和生产的发展。另外，杂交育种和实生选种时，童期长、开花结果晚更是木本果树品种改良的主要限制因素。
开展银杏组织培养和遗传转化，研究银杏成花的分子机理，进行银杏分子育种有重要的理论和现实意义。随着现代生物技术的发展，植物组织培养作为一种重要的研究手段取得了可喜的进步，裸子植物银杏组织培养尤其困难。但目前银杏组织培养和遗传转化的研究并不多，成功的例子还没有。建立一个银杏的高频高效再生体系已是刻不容缓的事。
1．银杏组织培养过程中，尤其是在愈伤组织的继代培养中，褐变现象特别严重，曾有不少的人做过这方面的研究，但都没有成功，而本研究通过对不同糖类物质、抗氧化剂、吸附剂以及不同的培养基对褐变的影响和控制效果，探索出有效控制褐化现象发生的最佳培养条件，试验结果表明：MS+ZT1.0mg/L+NAA1.0mg/L+蔗糖50g/L+琼脂8g/L+AC2g/L培养基上的继代效果最好，继代时间最好在15d左右。
2．银杏的不同器官和组织都能够诱导出愈伤组织来，其中，子叶和胚轴10d左右全部愈伤化，诱导速度和诱导率均最高，根则很难诱导，愈伤组织很少，褐化很快；叶片诱导的愈伤组织，生长慢，褐化也慢，在培养基上保持两三个月而不褐化；胚乳的诱导时间也较长，需要30d左右。诱导的难易分别为：根段、叶片、胚乳、胚根、叶柄、茎段、胚轴、子叶。
3．银杏不同外植体对丛生芽的诱导不同，银杏茎段培养可以直接得到丛生芽，但数量极少，而且大部分属于腋芽萌发，分化率20％左右。也可以利用银杏优良无性系的不同器官为外植体，离体培养产生愈伤组织，经分化获得丛
生芽。但是不同器官诱导的愈伤组织对于丛生芽的诱导也是不同的，子叶诱导的
愈伤组织的分化率较高，有36％左右，并且继代次数的增加也能够增加分化的
几率．而茎段和胚诱导的愈伤组织基本上没有丛生芽发生，这可能和不同愈伤组
织的内部结构有关。成熟胚的胚状体诱导率较低，诱导相当困难，在
N6＋BAI．smg／L＋NAAO．sing／L＋蔗糖 30g／L＋琼脂 sg／L＋AcZg／L上诱导效果相对
较好，可以有＊．5％的诱导率。
4＊的含量对茎段诱导根的发生有着很大的影响，含量高不利于根的诱导，
IBA和 KT的配合使用利于根的诱导。实验结果表明，在培养基 1／ZMS＋蔗糖
309几＋琼脂 sg／L＋IBAO．sing／L＋KTO．sing／L＋AcZg／L上诱导效果相对较好．
Ginkgo biloba.L,locally known as Baiguo and Gongsun tree,is special rare plan
in China. At presellt,only one section, one category,one genus exists,so it is called
"living fossil" of gymnospenn and be of vital scientific value in botany.
Ginkgo biIoba.L was exploited and uti1ized widely as foodstuff, health care
prote ction, medicine, lumber and vire scence since 1980's, so new demand was
put forward on breed of Ginkgo .Bot Ginkgo is a dioecous tree and takes 20-30 years
that its seedlings come into bearing. Routine breeding time is very long and its
productivity also is low so that it restricts seriously early economy benefit and
development of Gingo. In addition, long juvenile period and late fruit are become
primary restrictive factors in crossbreed and seed selection.
It is very importat in theory and practice to study its molecular principle and to
develop molecular breeding system by establishing Ginkgo tissue cultore and genetic
transformation. With the development of modem biological science and
technology,great progress has been made in tissue culthe as an importam means of
science study Ginkgo is a gymnosperm, so its tissue culture is especially hard .At
present,the research aboot Ginkgo tissue culture and genetic transformation was
limited and it has not been reported abollt its success, so it is very necessary to
establish Ginkgo regeneration system.
1 .Browning is serious problem in tissue culture of Gingho biloba L. especially,
subculturing of medium. Many researchers have conducted experimellts about it, but
not succeeded. Culture media were compared to find the best medium of gingkgo
culture for controlling callus browning by different sugars, antioxidants and sorbents.
The result showed that the medium with MS+ZT 1 .0mg/L +NAA 1 .0mg/L+ sucrose
50g/L+agar 8g/L+AC 2g/L was the best medium, at l5 days subculturing intervals.
2. Different Gingkgo organs and tissues can all induce callus. Cotyledon and
hypocotyl's rate and quamity are the most among these explams, and callus can
be obtained in 10 days by cotyledon and hypocotyl. Reversely it is difficult to
indue callus with root,and the callus from root is lnde and easy to become
browning. The calIus obtained from leaf grows very slow and does not become
browning uniill in 2 or 3 months. It takes about 30 days to induce callus with
endosperm. So, the rank of inducing callus from easy to hard is cotyledon, hypocotyls, stem, leafstalk, radicel, endosperm,leaf and root.
3. Rate of cluster buds induced by different Gingkgo explants is not the same. Among them , cluster buds can be obtained directly from stems and mostly from axillar bud, but the amount is little and differentiation rate is only 20%. There are also different in inducing cluster buds from callus of clone Gingkgo and its other tissues and organs , some callus can induce cluster buds, but others can not . Differentiation rate from the callus of cotyledon can be up to 36% and the number of cluster buds is positive related to the times of subculturing. But cluster buds can not be obtained from the callus of stems and embryo .It has likely relate to the inner structure of callus.lt is very difficult to get embryoid from mature embryo and its inducing rate is very low , and only 11.5% in the media containing N6+BA 1.5mg/L+NAA 0.5mg/L+sucrose 30g/L+agar 8g/L+Ac 2g/L which is the best media for Gingo tissue culture.
4. The content of nitrogen has a considerable influence on inducing roots from stems and it is negative effect on inducing roots if the content of nitrogen is too high. IBA +KT is the best of all hormone combinations in inducing roots. The result shows that medium with 1/2 MS +IBA 0.8mg/L+KT 0.50.8mg/L+sucrose 30g/L+agar 8g/L+Ac 2g/L is the most suitable for inducing root.
1980年以来，银杏在食品、保健、医药、木材，绿化等领域得到广泛的开发利用，对银杏的繁殖和育种提出了新的要求。但是，银杏是雌雄异株植物，实生苗定植后一般需要20～30年才能开花结果，银杏常规育种时间长，效率低，严重制约了早期经济效益的提高和生产的发展。另外，杂交育种和实生选种时，童期长、开花结果晚更是木本果树品种改良的主要限制因素。
开展银杏组织培养和遗传转化，研究银杏成花的分子机理，进行银杏分子育种有重要的理论和现实意义。随着现代生物技术的发展，植物组织培养作为一种重要的研究手段取得了可喜的进步，裸子植物银杏组织培养尤其困难。但目前银杏组织培养和遗传转化的研究并不多，成功的例子还没有。建立一个银杏的高频高效再生体系已是刻不容缓的事。
1．银杏组织培养过程中，尤其是在愈伤组织的继代培养中，褐变现象特别严重，曾有不少的人做过这方面的研究，但都没有成功，而本研究通过对不同糖类物质、抗氧化剂、吸附剂以及不同的培养基对褐变的影响和控制效果，探索出有效控制褐化现象发生的最佳培养条件，试验结果表明：MS+ZT1.0mg/L+NAA1.0mg/L+蔗糖50g/L+琼脂8g/L+AC2g/L培养基上的继代效果最好，继代时间最好在15d左右。
2．银杏的不同器官和组织都能够诱导出愈伤组织来，其中，子叶和胚轴10d左右全部愈伤化，诱导速度和诱导率均最高，根则很难诱导，愈伤组织很少，褐化很快；叶片诱导的愈伤组织，生长慢，褐化也慢，在培养基上保持两三个月而不褐化；胚乳的诱导时间也较长，需要30d左右。诱导的难易分别为：根段、叶片、胚乳、胚根、叶柄、茎段、胚轴、子叶。
3．银杏不同外植体对丛生芽的诱导不同，银杏茎段培养可以直接得到丛生芽，但数量极少，而且大部分属于腋芽萌发，分化率20％左右。也可以利用银杏优良无性系的不同器官为外植体，离体培养产生愈伤组织，经分化获得丛
生芽。但是不同器官诱导的愈伤组织对于丛生芽的诱导也是不同的，子叶诱导的
愈伤组织的分化率较高，有36％左右，并且继代次数的增加也能够增加分化的
几率．而茎段和胚诱导的愈伤组织基本上没有丛生芽发生，这可能和不同愈伤组
织的内部结构有关。成熟胚的胚状体诱导率较低，诱导相当困难，在
N6＋BAI．smg／L＋NAAO．sing／L＋蔗糖 30g／L＋琼脂 sg／L＋AcZg／L上诱导效果相对
较好，可以有＊．5％的诱导率。
4＊的含量对茎段诱导根的发生有着很大的影响，含量高不利于根的诱导，
IBA和 KT的配合使用利于根的诱导。实验结果表明，在培养基 1／ZMS＋蔗糖
309几＋琼脂 sg／L＋IBAO．sing／L＋KTO．sing／L＋AcZg／L上诱导效果相对较好．
Ginkgo biloba.L,locally known as Baiguo and Gongsun tree,is special rare plan
in China. At presellt,only one section, one category,one genus exists,so it is called
"living fossil" of gymnospenn and be of vital scientific value in botany.
Ginkgo biIoba.L was exploited and uti1ized widely as foodstuff, health care
prote ction, medicine, lumber and vire scence since 1980's, so new demand was
put forward on breed of Ginkgo .Bot Ginkgo is a dioecous tree and takes 20-30 years
that its seedlings come into bearing. Routine breeding time is very long and its
productivity also is low so that it restricts seriously early economy benefit and
development of Gingo. In addition, long juvenile period and late fruit are become
primary restrictive factors in crossbreed and seed selection.
It is very importat in theory and practice to study its molecular principle and to
develop molecular breeding system by establishing Ginkgo tissue cultore and genetic
transformation. With the development of modem biological science and
technology,great progress has been made in tissue culthe as an importam means of
science study Ginkgo is a gymnosperm, so its tissue culture is especially hard .At
present,the research aboot Ginkgo tissue culture and genetic transformation was
limited and it has not been reported abollt its success, so it is very necessary to
establish Ginkgo regeneration system.
1 .Browning is serious problem in tissue culture of Gingho biloba L. especially,
subculturing of medium. Many researchers have conducted experimellts about it, but
not succeeded. Culture media were compared to find the best medium of gingkgo
culture for controlling callus browning by different sugars, antioxidants and sorbents.
The result showed that the medium with MS+ZT 1 .0mg/L +NAA 1 .0mg/L+ sucrose
50g/L+agar 8g/L+AC 2g/L was the best medium, at l5 days subculturing intervals.
2. Different Gingkgo organs and tissues can all induce callus. Cotyledon and
hypocotyl's rate and quamity are the most among these explams, and callus can
be obtained in 10 days by cotyledon and hypocotyl. Reversely it is difficult to
indue callus with root,and the callus from root is lnde and easy to become
browning. The calIus obtained from leaf grows very slow and does not become
browning uniill in 2 or 3 months. It takes about 30 days to induce callus with
endosperm. So, the rank of inducing callus from easy to hard is cotyledon, hypocotyls, stem, leafstalk, radicel, endosperm,leaf and root.
3. Rate of cluster buds induced by different Gingkgo explants is not the same. Among them , cluster buds can be obtained directly from stems and mostly from axillar bud, but the amount is little and differentiation rate is only 20%. There are also different in inducing cluster buds from callus of clone Gingkgo and its other tissues and organs , some callus can induce cluster buds, but others can not . Differentiation rate from the callus of cotyledon can be up to 36% and the number of cluster buds is positive related to the times of subculturing. But cluster buds can not be obtained from the callus of stems and embryo .It has likely relate to the inner structure of callus.lt is very difficult to get embryoid from mature embryo and its inducing rate is very low , and only 11.5% in the media containing N6+BA 1.5mg/L+NAA 0.5mg/L+sucrose 30g/L+agar 8g/L+Ac 2g/L which is the best media for Gingo tissue culture.
4. The content of nitrogen has a considerable influence on inducing roots from stems and it is negative effect on inducing roots if the content of nitrogen is too high. IBA +KT is the best of all hormone combinations in inducing roots. The result shows that medium with 1/2 MS +IBA 0.8mg/L+KT 0.50.8mg/L+sucrose 30g/L+agar 8g/L+Ac 2g/L is the most suitable for inducing root.
引文
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