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L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis).

ABSTRACT: Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant's recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD₆₀₀ of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO₃ combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T₀ transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T₀) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

SUBMITTER: Sivanandhan G

PROVIDER: S-EPMC8576496 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis).

Sivanandhan Ganeshan G Moon Jiae J Sung Chaemin C Bae Solhee S Yang Zhi Hong ZH Jeong So Young SY Choi Su Ryun SR Kim Sang-Gyu SG Lim Yong Pyo YP

Frontiers in plant science 20211026

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant's recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regener ...[more]

PMID: 34764973

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Project description:BackgroundChinese cabbage (Brassica rapa ssp. pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence from Arabidopsis thaliana (13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species. Arabidopsis is therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed in Brassica species. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms in Brassica rapa ssp. pekinensis.ResultsIn the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage and Arabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively.ConclusionsThe evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.

Dataset Information

L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis).

Publications

L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (<i>Brassica rapa</i> ssp. <i>pekinensis</i>).

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