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Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa.

ABSTRACT: The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg/L BAP + 0.4 mg/L NAA + 0.01 mg/L GA₃ + 5 mg/L AgNO₃ + 0.75 mg/L Potassium Iodide (KI), MS salt supplemented with 1 mg/L IBA and 0.37 mg/L KI produced an equal number of roots (3) in UAF11 and Toria. For the establishment of transformation protocols, Agrobacterium-mediated floral dip transformation was attempted using different induction media, infection time, and flower stages. The induction medium III yielded a maximum of 7.2% transformants on half-opened flowers and 5.2% transformants on fully opened flowers in UAF11 and Toria, respectively, with 15 min of inoculation. This study would provide the basis for the improvement of tissue culture and transformation protocols in multilocular and bilocular Brassica genotypes.

SUBMITTER: Khan UM

PROVIDER: S-EPMC9824786 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa.

Khan Uzair Muhammad UM Shaheen Nabeel N Farooq Ayesha A Maqbool Rizwana R Khan Sultan Habibullah SH Azhar Muhammad Tehseen MT Rana Iqrar Ahmad IA Seo Hyojin H

Plants (Basel, Switzerland) 20221229 1

The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg ...[more]

PMID: 36616290

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Project description:High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide (glufosinate) painting, and QuickStix Kit for Liberty Link (bar) were used to verify the positive transgenic plants, and absolute quantification PCR confirmed the exogenous gene existed as one to three copies in the soybean genome. This study provides an improved protocol for Agrobacterium-mediated transformation in soybean and a useful reference to improve the transformation efficiency in other plant species.

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Dataset Information

Optimization of Regeneration and Agrobacterium-Mediated Transformation Protocols for Bi and Multilocular Varieties of Brassica rapa.

Publications

Optimization of Regeneration and <i>Agrobacterium</i>-Mediated Transformation Protocols for Bi and Multilocular Varieties of <i>Brassica rapa</i>.

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