Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa
Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa
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DOI：
10.1007/s11240-019-01582-x
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发表时间：
2018-08
期刊：
影响因子：
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通讯作者：
Kerry Swartwood;J. Eck
中科院分区：
文献类型：
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作者：
Kerry Swartwood;J. Eck
酸浆，也被称为地面樱桃，结出一种黄色的、营养丰富的小果实，外面包裹着一层纸质的外壳。以期实现OFP的大规模生产。为了实现这一目标，需要通过利用植物育种、基因工程和基因编辑等现有方法来改善不良特征，如难以管理的杂乱无章的生长习性和广泛的落果。在这项研究中，我们建立了植物再生和农杆菌介导的方法，使基因工程和基因编辑OFP的应用成为可能。普鲁伊诺萨。对7-8天大的试管苗的子叶和下胚轴外植体进行了植株再生的研究。外植体在含有2 mg/L玉米素的Murashige和Skoog盐基培养基上培养2周，然后转移到含有1 mg/L玉米素的培养基上。只有下胚轴外植体能再生不定芽。用含有绿色荧光蛋白报告基因和新霉素磷酸转移酶II(NptII)选择标记基因的pJL33双元载体的根癌农杆菌AGL1感染下胚轴外植体。共培养后，外植体在含有50、100、200、250、300 mg/L卡那霉素的选择性再生培养基上培养，以确定高效回收转基因株系的最有效水平。对pttⅡ基因进行聚合酶链式反应分析，结果表明，含200 mg/L卡那霉素的转化效率最高，为24%。本研究为今后改进OFP的基因工程和基因编辑方法奠定了基础。普鲁伊诺萨。
Physalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production ofP. pruinosafruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration andAgrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing ofP. pruinosa. Cotyledon and hypocotyl explants from 7 to 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected withAgrobacterium tumefaciensstrain AGL1 containing the pJL33 binary vector that has the green fluorescent protein reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on PCR analysis for the presence of thenptIIgene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement ofP. pruinosa.