An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus

Dai, Cheng; Li, Yuqing; Li, Long; Du, Zhuolin; Lin, Shengli; Xia, Tian; Li, Sijia; Yang, Bao; Yao, Wei; Wang, Jing; Guo, Liang; Lu, Shaoping

doi:10.1007/s11032-020-01174-0

Cited by 60 publications

(35 citation statements)

References 38 publications

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“…Two male sterile lines of rapeseed (Hau-A and pol CMS), two cultivars of tobacco (K326 and Yan97) and dozens of tomato varieties (described in Supplemental Table 4) were used as the female parents to evaluate the outcrossing HIR of dmp mutants. Rapeseed, tobacco and tomato transgenic plants were obtained by Agrobacterium-mediated transformation as previously described (Horsch et al, 1985; van Roekel et al, 1993; Dai et al, 2020). All plants were grown in the greenhouse under natural light.…”

Section: Methodsmentioning

confidence: 99%

A genotype independent DMP-HI system in dicot crops

Zhong

Chen

Wang

et al. 2021

Preprint

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Doubled haploid (DH) technology is used to obtain homozygous lines in a single generation, which significantly accelerates the crop breeding trajectory. Traditionally, in vitro culture is used to generate DHs, but is limited by species and genotype recalcitrance. In vivo haploid induction (HI) through seed is been widely and efficiently used in maize and was recently extended to several monocot crops. However, a similar generic and efficient HI system is still lacking in dicot crops. Here we show that genotype-independent in vivo HI can be triggered by mutation of DMP genes in tomato, rapeseed and tobacco with HI rates of ~1.9%, 2.4% and 1.2%, respectively. The DMP-HI system offers a robust DH technology to facilitate variety improvement in these crops. The success of this approach and the conservation of DMP genes paves the way for a generic and efficient genotype-independent HI system in other dicot crops.

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Section: Methodsmentioning

confidence: 99%

A genotype independent DMP-HI system in dicot crops

Zhong

Chen

Wang

et al. 2021

Preprint

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“…Restricted by mapping population possibly, we used a large group of 15,167 lethal seedlings to fine-map CYD1 to a 29 kb interval. Because 7-521y could not grow normally, it was impossible to obtain homozygous lethal mutants; therefore, we adjusted the traditional tissue culture method [ 23 ]. We planted a large number of heterozygous lines in M 0 , which were cultured in dark conditions for 5 d for the elongation of hypocotyls, and then transferred to light condition for 1 d to distinguish the mutant phenotype from the normal phenotype.…”

Section: Discussionmentioning

confidence: 99%

“…The hypocotyl of the mutants could be elongated in the dark. Tissue culture was performed as described previously [ 23 ]. We seeded the segregation population on M 0 solid medium and cultured in the dark for 5–6 d, and then cultured under light condition for 1 d. The yellow plants were selected for complementary transformation.…”

Section: Methodsmentioning

confidence: 99%

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

Song

et al. 2021

IJMS

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Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

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“…The procedure of Agrobaterium ‐mediated B. napus transformation was carried out as previously described (Dai et al., 2020 ). To genotype the CRISPR/Cas9‐mediated mutation, CTAB method was used to extract the genomic DNA (Molecular Cloning 4th edition).…”

Section: Methodsmentioning

confidence: 99%

DELLA proteins BnaA6.RGA and BnaC7.RGA negatively regulate fatty acid biosynthesis by interacting with BnaLEC1s in Brassica napus

Yan

Xia

et al. 2021

Plant Biotechnology Journal

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Seed oil content (SOC) and fatty acid (FA) composition determine the quality and economic value of rapeseed (Brassica napus). Little is known about the role of gibberellic acid (GA) in regulating FA biosynthesis in B. napus. Here, we discovered that four BnaRGAs (B. napus REPRESSOR OF GA), encoding negative regulators of GA signalling, were suppressed during seed development. Compared to the wild type, SOC was reduced in gain-of-function mutants bnaa6.rga-D and ds-3, which also showed reduced oleic acid and increased linoleic acid contents. By contrast, the loss-of-function quadruple mutant bnarga displayed higher SOC during early seed development than the wild type, with increased oleic acid and reduced linoleic acid contents. Notably, only BnaA6.RGA and BnaC7.RGA physically interacted with two BnaLEC1s, which function as essential transcription factors in FA biosynthesis. The FA composition did not significantly differ between bnarga bnalec1 sextuple mutants and bnalec1, suggesting that BnaLEC1s are epistatic to BnaRGAs in the regulation of FA composition. Furthermore, BnaLEC1-induced activation of BnaABI3 expression was repressed by BnaA6.RGA, indicating that GA triggers the degradation of BnaRGAs to relieve their repression of BnaLEC1s, thus promoting the transcription of downstream genes to facilitate oil biosynthesis. Therefore, we uncovered a developmental stagespecific role of GA in regulating oil biosynthesis via the GA-BnaRGA-BnaLEC1 signalling cascade, providing a novel mechanistic understanding of how phytohormones regulate FA biosynthesis in seeds. BnaRGAs represent promising targets for oil crop improvement.

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An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus

Cited by 60 publications

References 38 publications

A genotype independent DMP-HI system in dicot crops

A genotype independent DMP-HI system in dicot crops

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

DELLA proteins BnaA6.RGA and BnaC7.RGA negatively regulate fatty acid biosynthesis by interacting with BnaLEC1s in Brassica napus

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