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农杆菌介导的难转化的万代卡森喜悦兰花的高效转化。

Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem's Delight orchid with higher efficiency.

作者信息

Gnasekaran Pavallekoodi, Antony Jessica Jeyanthi James, Uddain Jasim, Subramaniam Sreeramanan

机构信息

School of Biological Sciences, Universiti Sains Malaysia (USM), 11800 Minden Heights, Penang, Malaysia.

出版信息

ScientificWorldJournal. 2014;2014:583934. doi: 10.1155/2014/583934. Epub 2014 Apr 8.

DOI:10.1155/2014/583934
PMID:24977213
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4000978/
Abstract

The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600 nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 μM acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

摘要

本研究建立了利用类原球茎（PLB）通过农杆菌介导的遗传转化来生产转基因万代卡森的喜悦汤姆·博伊金（VKD）兰花的方法。使用gusA基因表达对几个参数进行了测试和量化，如PLB大小、浸泡时间、创伤程度、农杆菌密度、共培养时间和乙酰丁香酮浓度，以优化农杆菌介导的VKD的PLB遗传转化效率。根据结果，用手术刀划伤的3-4毫米PLB在600纳米波长下于0.8单位的农杆菌悬浮液中浸泡30分钟，产生了最高的GUS表达。此外，将感染的PLB在含有200μM乙酰丁香酮的瓦辛和温特共培养基上黑暗中共培养4天，增强了GUS表达。对在250毫克/升头孢噻肟和30毫克/升遗传霉素存在下选择的推定转化体进行PCR分析，证明了小麦win1、小麦win2和nptII基因的存在。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/b44b3a5554c8/TSWJ2014-583934.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/16c0ef3c7c50/TSWJ2014-583934.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/c91abe6f938e/TSWJ2014-583934.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/02ce4bcba018/TSWJ2014-583934.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/ddd806c23958/TSWJ2014-583934.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/b44b3a5554c8/TSWJ2014-583934.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/16c0ef3c7c50/TSWJ2014-583934.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/c91abe6f938e/TSWJ2014-583934.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/02ce4bcba018/TSWJ2014-583934.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/ddd806c23958/TSWJ2014-583934.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ab0f/4000978/b44b3a5554c8/TSWJ2014-583934.005.jpg

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