Abstract: Hairy roots were induced by infecting leaf explants of Scrophularia buergeriana with Agrobacterium rhizo- genes strains A4 and R1601. Hairy roots could grow rapidly and autonomously in liquid or solid plant-growth regulator-free MS medium. The transformation was confirmed by PCR amplification of the rolB gene of the Ri plasmid from S. buergeriana hairy roots. Hairy roots could form green callus in MS medium with 0.5 mg·L-1 6-BA and 0.1 mg·L-1 NAA, then regenerated plantlets were induced from the callus. A whole transgenic plantlet was obtained. Adventitious buds could be directly induced from hairy roots in MS medium with 0.5 mg·L-1 6-BA and 0.02 mg·L-1 NAA after about 15-20 d, with the induction rate of buds up to 85%.
3.2 毛状根与再生植株的rolB基因检测rolB基因是Ri质粒T-DNA上与毛状根形成密切相关的基因之一。图2为北玄参毛状根(A4) rolB基因PCR扩增产物的凝胶电泳。由图2可知, 毛状根均能扩增出预期的423 bp的特异性条带, 而非转化根不能扩增出特异性条带, 表明Ri质粒含rolB基因的T-DNA片段已插入且整合至北玄参毛状根基因组中并且表达。实验证明所获得的可自主生长的毛状根为Ri质粒T- DNA转化毛状根。 图2https://www.chinbullbotany.com/article/2017/1674-3466/1674-3466-52-6-783/img_2.png<b>图2</b> 北玄参毛状根(A4) <i>rolB</i>基因PCR扩增产物的凝胶电泳<br/>1-4: 毛状根DNA扩增片段; 5, 6: 非转化根DNA扩增结果; 7: 发根农杆菌菌落扩增的片段; M: DL2000 marker<br/><b>Figure 2</b> Gel electrophoresis analysis of PCR fragments of<i> rolB</i> gene amplified from the genome DNA of hairy roots (A4)<br/>1-4: Fragments amplified from genome DNA of hairy roots; 5, 6: Amplification products from genome DNA of untransformed roots; 7: Fragments amplified from the colony of <i>Agrobacte- rium rhizogenes</i>; M: DL2000 marker Figure 2https://www.chinbullbotany.com/article/2017/1674-3466/1674-3466-52-6-783/img_2.png<b>图2</b> 北玄参毛状根(A4) <i>rolB</i>基因PCR扩增产物的凝胶电泳<br/>1-4: 毛状根DNA扩增片段; 5, 6: 非转化根DNA扩增结果; 7: 发根农杆菌菌落扩增的片段; M: DL2000 marker<br/><b>Figure 2</b> Gel electrophoresis analysis of PCR fragments of<i> rolB</i> gene amplified from the genome DNA of hairy roots (A4)<br/>1-4: Fragments amplified from genome DNA of hairy roots; 5, 6: Amplification products from genome DNA of untransformed roots; 7: Fragments amplified from the colony of <i>Agrobacte- rium rhizogenes</i>; M: DL2000 marker
图2 北玄参毛状根(A4) rolB基因PCR扩增产物的凝胶电泳 1-4: 毛状根DNA扩增片段; 5, 6: 非转化根DNA扩增结果; 7: 发根农杆菌菌落扩增的片段; M: DL2000 marker Figure 2 Gel electrophoresis analysis of PCR fragments of rolB gene amplified from the genome DNA of hairy roots (A4) 1-4: Fragments amplified from genome DNA of hairy roots; 5, 6: Amplification products from genome DNA of untransformed roots; 7: Fragments amplified from the colony of Agrobacte- rium rhizogenes; M: DL2000 marker
3.3 植物生长调节剂对毛状根愈伤组织及不定芽的诱导将快速生长且转化鉴定呈阳性的毛状根剪切成2-3 cm的根段, 分别转入表1所示的附加不同植物生长调节剂的MS培养基中培养。从表1可以看出, 培养基中生长素的浓度越高, 毛状根产生愈伤组织越快(图1B); 并且随着培养基中细胞分裂素浓度的升高, 芽点出现的速度也越快, 愈伤组织由淡黄、白色逐渐变成绿色。毛状根在MS+0.5 mg·L-1 6-BA+0.1 mg·L-1NAA培养基上形成的愈伤组织呈绿色, 继代1-2次就可以出现芽点(图1C); 在MS+0.5 mg·L-1 6-BA+0.02 mg·L-1 NAA培养基上培养约15-20天, 毛状根可直接形成不定芽, 且不定芽诱导率较高, 约为85% (图1D)。 表1 Table 1 表1 表1 6-BA和NAA组合对北玄参毛状根愈伤组织诱导及植株再生的影响 Table 1 Effect of 6-BA and NAA on callus induction and adventitious shoots formation of hairy roots in Scrophularia buergeriana
Med- ium
6-BA (mg·L-1)
NAA (mg·L-1)
Change of hairy roots
1
0.1
0.02
Few adventitious buds induced
2
0.1
0.1
Formation of light yellow callus
3
0.2
0.02
Few adventitious buds induced
4
0.2
0.1
Formation of green callus
5
0.5
0.02
More adventitious buds developed
6
0.5
0.1
Firstly formed green callus, then developed more adventitious buds
表1 6-BA和NAA组合对北玄参毛状根愈伤组织诱导及植株再生的影响 Table 1 Effect of 6-BA and NAA on callus induction and adventitious shoots formation of hairy roots in Scrophularia buergeriana
刘莉莉, 卢淑波, 徐佳萍, 张庆田, 李昌禹 (2015). 以黄花乌头发根为外植体的再生培养体系建立. 植物学报 50, 623-627. DOI:10.11983/CBB14174URL6-BA and 0–0.5 mg·L NAA, then regeneration plantlets were induced from the callus. A whole transgenetic plantlet was obtained. The optimal medium for inducing callus and adventitious buds was 1/2MS+2.0 mg·L 6-BA+0.2 mg·L NAA+3% sugar. The optimal medium for inducing roots was 1/2MS+0.5 mg·L 6-BA+0.1 mg·L NAA+5 mg·L GA+3% sugar. [本文引用: 1]
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张继栋, 杨雪清, 乔爱民, 孙敏, 何生根, 雷栀, 尹彩霞 (2008). 木本曼陀罗毛状根植株再生体系的建立. 热带亚热带植物学报 16, 480-485. DOI:10.3969/j.issn.1005-3395.2008.5.1979URLPlant regeneration system derived from hairy roots of Datura arborea L. was established. The optimum medium for inducing adventitious buds by ‘one-step method’ was MS+6-BA 2.0 mg L-1+NAA 0.2 mg L-1. Using ‘two-step method’, callus was induced on MS medium supplement with 6-BA 4.0 mg L-1+KT 0.5 mg L-1+2,4-D 0.5 mg L-1 at first, then adventitious buds were differentiated from the induced callus on MS medium supplemented with 6-BA 4.0 mg L-1+NAA 0.2 mg L-1. The optimum rooting medium for adventitious buds was 1/2MS + IBA 0.1 mg L-1. By using PCR technique, the gene segments rolB and rolC were obtained from the leaves of regeneration plant. The results of HPLC showed that the contents of tropane alkaloids (TA) in regeneration plants was much more than those in the wild plants.
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表1 6-BA和NAA组合对北玄参毛状根愈伤组织诱导及植株再生的影响 Table 1 Effect of 6-BA and NAA on callus induction and adventitious shoots formation of hairy roots in Scrophularia buergeriana
