Li Yanjun, Tang Dan, Liu Zongrang, Chen Jianjun, Cheng Baoping, Kumar Rahul, Yer Huseyin, Li Yi
Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA.
Horticulture and Landscape College, Hunan Agricultural University, Changsha 410128, China.
Plants (Basel). 2022 May 30;11(11):1457. doi: 10.3390/plants11111457.
Although several protocols for genetic transformation of citrus have been published, it is highly desirable to further improve its efficiency. Here we report treatments of cells and citrus explants prior to and during co-cultivation process to enhance transformation efficiency using a commercially used rootstock 'Carrizo' citrange [ (L.) Osb. × (L.) Raf.] as a model plant. We found explants from light-grown seedlings exhibited higher transformation efficiency than those from etiolated seedlings. We pre-cultured cells in a 1/10 MS, 0.5 g/L 2-(-morpholino) ethanesulfonic acid (MES) and 100 µM acetosyringone liquid medium for 6 h at 25 °C before used to infect citrus explants. We incubated epicotyl segments in an MS liquid medium containing 13.2 µM 6-BA, 4.5 µM 2,4-D, 0.5 µM NAA for 3 h at 25 °C prior to infection. In the co-cultivation medium, we added 30 µM paclobutrazol and 10 µM lipoic acid. Each of these treatments significantly increased the efficiencies of transformation up to 30.4% (treating with acetosyringone), 31.8% (treating explants with cytokinin and auxin), 34.9% (paclobutrazol) and 38.6% (lipoic acid), respectively. When the three treatments were combined, we observed that the transformation efficiency was enhanced from 11.5% to 52.3%. The improvement of genetic transformation efficiency mediated by these three simple treatments may facilitate more efficient applications of transgenic and gene editing technologies for functional characterization of citrus genes and for genetic improvement of citrus cultivars.
尽管已经发表了几种柑橘遗传转化的方案，但进一步提高其效率仍非常必要。在这里，我们报告了在共培养过程之前和期间对细胞和柑橘外植体的处理，以提高转化效率，使用商业上使用的砧木“卡里佐”枳橙[（L.）Osb.×（L.）Raf.]作为模式植物。我们发现，来自光照生长幼苗的外植体比黄化幼苗的外植体表现出更高的转化效率。在用于感染柑橘外植体之前，我们将细胞在含有1/10 MS、0.5 g/L 2-（-吗啉代）乙磺酸（MES）和100 μM乙酰丁香酮的液体培养基中于25°C预培养6小时。在感染前，我们将上胚轴切段在含有13.2 μM 6-苄基腺嘌呤、4.5 μM 2,4-二氯苯氧乙酸、0.5 μM萘乙酸的MS液体培养基中于25°C孵育3小时。在共培养基中，我们添加了30 μM多效唑和10 μM硫辛酸。这些处理中的每一种都分别显著提高了转化效率，高达30.4%（用乙酰丁香酮处理细胞）、31.8%（用细胞分裂素和生长素处理外植体）、34.9%（多效唑）和38.6%（硫辛酸）。当将这三种处理组合时，我们观察到转化效率从11.5%提高到了52.3%。这三种简单处理介导的遗传转化效率的提高可能有助于更有效地应用转基因和基因编辑技术来进行柑橘基因的功能表征和柑橘品种的遗传改良。
