School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
J Biotechnol. 2009 Sep 10;143(3):198-206. doi: 10.1016/j.jbiotec.2009.07.007. Epub 2009 Jul 17.
Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82+/-0.14 mg g(-1) dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80-90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 microg g(-1) dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.
采用三种不同的植物组织培养形式，对离体植物病毒积累进行了研究。用相同的初始病毒：生物量比，用烟草花叶病毒（TMV）感染悬浮细胞、发根和丛生愈伤组织。在这三种培养系统中，病毒感染都没有影响组织生长或形态。在发根和丛生愈伤组织中的平均最大病毒浓度相似，约比悬浮细胞高一个数量级。发根因其在液体培养基中的形态稳定性和相对容易培养而被认为是首选宿主。发根中的平均最大病毒浓度为 0.82+/-0.14 mg g(-1) 干重；病毒外壳蛋白占生物量中总可溶性蛋白的最大比例约为 6%。通过不同的半连续培养操作模式进一步研究了发根中的病毒积累，目的是延长根生长阶段并提供营养补充；然而，与简单的分批培养相比，根中的病毒浓度并没有提高。在所有测试的培养中，病毒在生物量中的相对感染力下降了 80-90%，无论使用何种植物组织形式或培养操作模式。在分批培养中，用转基因 TMV 载体接种的发根积累了绿色荧光蛋白（GFP）；然而，生物量中 GFP 的最大浓度相对较低，为 39 microg g(-1) 干重，可能是由于载体的遗传不稳定性。这项工作突出了与丛生愈伤组织和悬浮植物细胞相比，使用发根进行 TMV 体外繁殖的优势，并证明与半连续操作相比，批量根培养更有利于在生物量中积累高浓度的病毒。
