红龙草的组织培养及发根农杆菌对红龙草的遗传转化研究
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摘要
本文研究了红龙草(Altemanthera Ficoidea cv．"Ruliginosa")叶片的组织培养、发根农杆菌(Agrobacterium rhizogenes)ATCC 15834对红龙草的遗传转化以及毛状根液体培养过程中培养液某些营养成分的消耗变化。结果如下：
红龙草叶片外植体在MS+6-BA1.0mg／L+4-PU 1.0mg／L+NAA 0.1mg／L的培养基上形成浅绿色愈伤组织，20d后愈伤组织诱导率达100％。而在添加6-BA1.0 mg／L和NAA0.4 mg／L的MS培养基中，约45.31％的愈伤组织分化出不定芽。不定芽在1／2MS+NAA0.4mg／L的培养基上可全部生根，长成完整植株。
红龙草叶片外植体被发根农杆菌ATCC 15834感染8d后，从形态学下端叶脉处陆续分化出乳白色的不定根，21d后，叶片外植体的生根率高达92.5％。其中转化毛状根的频率为53.8％，毛状根呈密集丛生状、多分枝，被大量白色细绒毛。红龙草毛状根能在无激素的固体或液体培养基上快速自主生长。PCR扩增结果证实，Ri质粒的T-DNA部分已在红龙草毛状根的基因组中整合及表达。
红龙草毛状根在MS+6-BA 2.0mg／L+4-PU2.0 mg／L+NAA0.5 mg／L的培养基上形成愈伤组织，其愈伤组织诱导率达68.75％。而在添加6-BA2.0mg／L和NAA0.8mg／L的MS培养基上，部分愈伤组织能分化出幼芽。
红龙草毛状根液体培养的生长过程分为三个时期：生长迟滞期、快速生长期、生长平台期。对毛状根液体培养过程中培养基内蔗糖、硝态氮、氨态氮以及磷酸盐含量的分析结果表明：培养基中蔗糖的消耗速率与毛状根的生长曲线呈正相关。在毛状根生长迟滞期内，培养基中的硝态氮和磷酸盐的消耗较快，其消耗速率分别为16.71 μg／ml·d和1.67μg／ml·d；但在快速生长期，其消耗速率反而减慢。而在生长迟滞期内，培养液中氨态氮的消耗高达35.29μg／ml·d，但进入快速生长期后，氨态氮含量先明显回升，然后又逐渐下降。培养基的pH值随毛状根培养时间的延长而逐渐降低。
Tissue culture and plantlet regeneration from leaf explants of A.Ficoidea cv."Ruliginosa" and its genetic transformation with Agrobacterium rhizogenes ATCC 15834 were investigated as well as changes in the contents of sucrose, nitrogen and phosphate in liquid medium during the hairy root cultures. The main results were as follows:
Light green calli could be formed from all the leaf explants of A.Ficoidea cv."Ruliginosa" when cultured for 20 days on MS+6-BA1.0mg/L+4-PU 1.0mg/L+ NAA 0.lmg/L. Adventitious shoots could be developed from the calli at the frequency of 45.31% on MS medium supplemented with 1.0 mg/L 6-BA and 0.4 mg/L NAA. Adventitious shoots could be rooted and formed regenerated plants on 1/2 MS medium supplemented with NAA0.4mg/L.
Hairy roots could be induced from the basal surface of the explants 8 days after inoculation of A.Ficoidea cv."Ruliginosa" leaf explants with A.rhizogenes ATCC 15834. The percentage of rooted leaf explants was 92.5% 21 days after infection, but the inducing frequency of hairy roots was 53.8%. Hairy roots were highly branched and had a plenty of white root hairs. All the hairy roots of A.Ficoidea cv."Ruliginosa" could have a rapid autonomous growth on phytohormone-free liquid or solid MS medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A.rhizogenes.
The green callus was developed at the induction rate of 68.75% from hairy roots cultured onto MS medium supplemented with 6-BA 2.0mg/L,4-PU 2.0mg/L and NAA 0.5mg/L.Adventitious buds can be observed to form gradually from the calli on MS medium supplemented with 6-BA2.0mg/L and NAA0.8mg/L.
When cultured in liquid medium, the growth period of A.Ficoidea cv."Ruliginosa" hairy roots could be divided into three phases: lag phase, rapid-growth phase and plateau
phase. Changes in contents of sucrose, nitrogen and phosphate in liquid medium were determined during the whole culture of hairy roots. The results showed that the consumption rate of sucrose in the medium was positively related to the growth rate of hairy roots. Nicrate nitrogen and phosphate in liquid medium were quickly consumed at the lag phase of the growth, the consumption rate was 16.71 μ g/ml d and 1.67μ g/m) d, respectively; but during the rapid-growth phase, its consumption rate was decreased. The content of ammonium nitrogen in the medium reduced rapidly during the lag phase, but showed a slightly increase during the rapid-growth phase. The pH value in the medium was reduced gradually with time during the whole culture of hairy roots.
红龙草叶片外植体在MS+6-BA1.0mg／L+4-PU 1.0mg／L+NAA 0.1mg／L的培养基上形成浅绿色愈伤组织，20d后愈伤组织诱导率达100％。而在添加6-BA1.0 mg／L和NAA0.4 mg／L的MS培养基中，约45.31％的愈伤组织分化出不定芽。不定芽在1／2MS+NAA0.4mg／L的培养基上可全部生根，长成完整植株。
红龙草叶片外植体被发根农杆菌ATCC 15834感染8d后，从形态学下端叶脉处陆续分化出乳白色的不定根，21d后，叶片外植体的生根率高达92.5％。其中转化毛状根的频率为53.8％，毛状根呈密集丛生状、多分枝，被大量白色细绒毛。红龙草毛状根能在无激素的固体或液体培养基上快速自主生长。PCR扩增结果证实，Ri质粒的T-DNA部分已在红龙草毛状根的基因组中整合及表达。
红龙草毛状根在MS+6-BA 2.0mg／L+4-PU2.0 mg／L+NAA0.5 mg／L的培养基上形成愈伤组织，其愈伤组织诱导率达68.75％。而在添加6-BA2.0mg／L和NAA0.8mg／L的MS培养基上，部分愈伤组织能分化出幼芽。
红龙草毛状根液体培养的生长过程分为三个时期：生长迟滞期、快速生长期、生长平台期。对毛状根液体培养过程中培养基内蔗糖、硝态氮、氨态氮以及磷酸盐含量的分析结果表明：培养基中蔗糖的消耗速率与毛状根的生长曲线呈正相关。在毛状根生长迟滞期内，培养基中的硝态氮和磷酸盐的消耗较快，其消耗速率分别为16.71 μg／ml·d和1.67μg／ml·d；但在快速生长期，其消耗速率反而减慢。而在生长迟滞期内，培养液中氨态氮的消耗高达35.29μg／ml·d，但进入快速生长期后，氨态氮含量先明显回升，然后又逐渐下降。培养基的pH值随毛状根培养时间的延长而逐渐降低。
Tissue culture and plantlet regeneration from leaf explants of A.Ficoidea cv."Ruliginosa" and its genetic transformation with Agrobacterium rhizogenes ATCC 15834 were investigated as well as changes in the contents of sucrose, nitrogen and phosphate in liquid medium during the hairy root cultures. The main results were as follows:
Light green calli could be formed from all the leaf explants of A.Ficoidea cv."Ruliginosa" when cultured for 20 days on MS+6-BA1.0mg/L+4-PU 1.0mg/L+ NAA 0.lmg/L. Adventitious shoots could be developed from the calli at the frequency of 45.31% on MS medium supplemented with 1.0 mg/L 6-BA and 0.4 mg/L NAA. Adventitious shoots could be rooted and formed regenerated plants on 1/2 MS medium supplemented with NAA0.4mg/L.
Hairy roots could be induced from the basal surface of the explants 8 days after inoculation of A.Ficoidea cv."Ruliginosa" leaf explants with A.rhizogenes ATCC 15834. The percentage of rooted leaf explants was 92.5% 21 days after infection, but the inducing frequency of hairy roots was 53.8%. Hairy roots were highly branched and had a plenty of white root hairs. All the hairy roots of A.Ficoidea cv."Ruliginosa" could have a rapid autonomous growth on phytohormone-free liquid or solid MS medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A.rhizogenes.
The green callus was developed at the induction rate of 68.75% from hairy roots cultured onto MS medium supplemented with 6-BA 2.0mg/L,4-PU 2.0mg/L and NAA 0.5mg/L.Adventitious buds can be observed to form gradually from the calli on MS medium supplemented with 6-BA2.0mg/L and NAA0.8mg/L.
When cultured in liquid medium, the growth period of A.Ficoidea cv."Ruliginosa" hairy roots could be divided into three phases: lag phase, rapid-growth phase and plateau
phase. Changes in contents of sucrose, nitrogen and phosphate in liquid medium were determined during the whole culture of hairy roots. The results showed that the consumption rate of sucrose in the medium was positively related to the growth rate of hairy roots. Nicrate nitrogen and phosphate in liquid medium were quickly consumed at the lag phase of the growth, the consumption rate was 16.71 μ g/ml d and 1.67μ g/m) d, respectively; but during the rapid-growth phase, its consumption rate was decreased. The content of ammonium nitrogen in the medium reduced rapidly during the lag phase, but showed a slightly increase during the rapid-growth phase. The pH value in the medium was reduced gradually with time during the whole culture of hairy roots.
引文
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