Standardized Genetic Transformation Protocol for Chrysanthemum cv. 'Jinba' with TERMINAL FLOWER 1 HomologCmTFL1a
Standardized Genetic Transformation Protocol for Chrysanthemum cv. 'Jinba' with TERMINAL FLOWER 1 HomologCmTFL1a
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DOI：
10.3390/genes11080860
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发表时间：
2020-08-01
期刊：
影响因子：
3.5
通讯作者：
Gao, Yike
中科院分区：
文献类型：
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作者：
Haider, Saba;Gao, Yaohui;Gao, Yike
菊花(菊花xmorifoliumRamat)金巴是一种独特的短日照菊花，可以作为研究开花分子机制的模式生物。通过建立合适的再生和遗传转化体系，可以提高金巴在全球花卉市场上的商业价值。针对农杆菌介导法转化菊花存在的转化效率低、品种特异性高等典型问题，设计了一套高效、稳定的转化体系。在此，我们以CmTFL1a为外源基因，对影响金坝遗传转化的主要性状进行了筛选，并对其转化方案进行了标准化。确定了不同的抗生素(卡那霉素、美罗培南和羧青霉素)和生长调节剂(6-BA、2，4-D和NAA)进行遗传转化的适宜浓度，以考察它们对金坝叶片组织培养再生植株的影响，从而通过农杆菌EHA105对转化方案进行了标准化。此外，通过聚合酶链式反应(PCR)分析，证实了转基因植株中转基因的存在和稳定表达。在转基因植株中组成性表达的CmTFL1反义基因在茎尖中的表达高于茎和叶。CmTFL1基因的过表达延缓了向生殖阶段的过渡，并显著影响了植株的形态。本研究有助于了解菊花中TFL1同源基因的生物学现象。此外，我们的发现还可以为其他菊花品种的基因工程和育种探索创新的可能性。
Chrysanthemum (ChrysanthemumxmorifoliumRamat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated withAgrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by usingCmTFL1aas a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized throughAgrobacterium tumefaciens(EHA105). In addition, the presence of the transgene and its stable expression inCmTFL1atransgenic plants were confirmed by polymerase chain reaction (PCR) analysis. TheCmTFL1atransgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression ofCmTFL1aled to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon ofTFL1homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.