Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

被引:0
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作者
Jesse Jones
Elaine Zhang
Dominick Tucker
Daniel Rietz
Douglas Dahlbeck
Michael Gomez
Claudia Garcia
Jean-Philippe Marelli
Donald Livingstone
Ray Schnell
Brian Staskawicz
Myeong-Je Cho
机构
[1] University of California,Innovative Genomics Institute
[2] University of California,Department of Plant and Microbial Biology
[3] Mars Center for Cocoa Science,undefined
[4] Mars Wrigley Plant Science Laboratory,undefined
[5] Mars Cacao Laboratory,undefined
来源
In Vitro Cellular & Developmental Biology - Plant | 2022年 / 58卷
关键词
Genetic transformation; Somatic embryogenesis; Regeneration; Grafting;
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学科分类号
摘要
A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.
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页码:133 / 145
页数:12
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