Panda Bhuban Mohan, Mehta Urmil J, Hazra Sulekha
Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Pune, India.
Ajeet Seed Ltd, Chitegaon, Paithan, Aurangabad, Maharashtra, 431105, India.
3 Biotech. 2017 May;7(1):21. doi: 10.1007/s13205-017-0608-x. Epub 2017 Apr 11.
Semecarpus anacardium L. is a tree species which produces secondary metabolites of medicinal importance. Roots of the plant have been traditionally used in folk medicines. Different strains of Agrobacterium rhizogenes (A4, ATCC15834 and LBA 9402) were used for induction of hairy roots in in vitro grown tissues of the plant. Hairy root initiation was observed after 25-30 days of infection. Optimum transformation frequency of 61% was achieved on leaf explants with ATCC15834 strain. Infection time of 30 min resulted in greater transformation frequency compared to 10 and 20 min, respectively. The hairy roots cultured in growth regulator-free semi-solid woody plant medium differentiated into callus. Whole shoots infected with ATCC 15834 were found to produce more transformants upon co-cultivation for 4 (65%) and 5 (67%) days. Induction of hairy roots in stem explants infected with ATCC 15834 was lower (52%) compared to leaves (62%) after 4 days of co-cultivation. In A4 and LBA9402 strains transformation efficiency was 49 ± 2.8% and 36 ± 5.7% in shoots after 4 days of co-cultivation. Transformation frequency was higher in ATCC15834 strain, irrespective of explants. The hairy roots of S. anacardium elongated slowly upon transfer to half-strength liquid medium. After 3-4 passages in liquid medium slender hairy roots started differentiating which were separated from the original explants. Visible growth of the roots was observed in hormone-free liquid medium after 2-3 months of culturing. Polymerase chain reaction with gene-specific primers from rol A, B and C genes confirms the positive transformation events.
印度楝是一种能产生具有药用价值的次生代谢产物的树种。该植物的根在传统民间药物中就有应用。使用不同菌株的发根农杆菌（A4、ATCC15834和LBA 9402）对该植物的离体培养组织诱导产生毛状根。感染25 - 30天后观察到毛状根起始。使用ATCC15834菌株在叶片外植体上实现了61%的最佳转化频率。与10分钟和20分钟相比，30分钟的感染时间导致更高的转化频率。在无生长调节剂的半固体木本植物培养基中培养的毛状根分化形成愈伤组织。发现用ATCC 15834感染的全株在共培养4天（65%）和5天（67%）后产生更多转化体。共培养4天后，用ATCC 15834感染的茎外植体中毛状根的诱导率（52%）低于叶片（62%）。在共培养4天后，A4和LBA9402菌株在芽中的转化效率分别为49±2.8%和36±5.7%。无论外植体如何，ATCC15834菌株的转化频率都更高。印度楝的毛状根转移到半强度液体培养基后生长缓慢。在液体培养基中传代3 - 4次后，细长的毛状根开始分化，并与原始外植体分离。在无激素的液体培养基中培养2 - 3个月后观察到根的明显生长。用来自rol A、B和C基因的基因特异性引物进行聚合酶链反应证实了阳性转化事件。
