A highly efficient genetic transformation system for broccoli and subcellular localization

by Selleckchem | Apr 12 2023

Introduction: Agrobacterium-mediated genetic transformation has been widely used for the identification of functional genes and regulatory and developmental mechanisms in plants. However, there are still some problems of low genetic transformation efficiency and high genotype dependence in cruciferous crops.

Methods: In this study, broccoli, a worldwide Brassica crop, was used to investigate the effects of genotype, explant type, concentration of hygromycin B used during seedling selection, overexpression vector type, RNAi and CRISPR/cas9 on the genetic transformation efficiency. At the same time, two vectors, PHG-031350 and PHG-CRa, were used for subcellular localization of the glucoraphanin synthesis-related gene FMOGS-OX5 and clubroot resistance gene by a PEG-Ca2+-mediated transient transformation system for broccoli protoplasts. Finally, the Agrobacterium-mediated genetic transformation system of broccoli was optimized and improved.

Results and discussion: This study showed that hypocotyl explants are more suitable for Agrobacterium-mediated transgene and CRISPR/Cas9 gene editing of broccoli. In contrast to previous studies, we found that 5 mg/L hygromycin B was more advantageous for the selection of resistant broccoli sprouts, and genotype 19B42 reached the highest transformation rate of 26.96%, which is higher than that in Brassica oleracea crops. In addition, the inbred line 19B42 successfully achieved high genetic transformation of overexpression, RNAi and CRISPR/Cas9 vectors; thus, it is powerful recipient material for the genetic transformation of broccoli. Subcellular localization proved that the glucoraphanin metabolism-related gene Bol031350 and clubroot resistance gene CRa were both expressed in the cytoplasm and nucleus, which provided a scientific basis for studying the regulation of glucosinolate metabolism and clubroot resistance in cruciferous crops. Therefore, these findings will provide new insight into the improvement of the genetic transformation and molecular breeding of Brassica oleracea crops.

Comments：

As per the study, broccoli was used to investigate the effects of various factors on the genetic transformation efficiency, including genotype, explant type, concentration of hygromycin B, overexpression vector type, RNAi, and CRISPR/Cas9. The results showed that hypocotyl explants were more suitable for Agrobacterium-mediated transgene and CRISPR/Cas9 gene editing of broccoli. Additionally, the study found that 5 mg/L hygromycin B was more advantageous for the selection of resistant broccoli sprouts, and genotype 19B42 achieved the highest transformation rate of 26.96%, which is higher than that in Brassica oleracea crops.

Furthermore, the inbred line 19B42 was found to be a powerful recipient material for the genetic transformation of broccoli, achieving high genetic transformation of overexpression, RNAi, and CRISPR/Cas9 vectors. The study also used two vectors, PHG-031350 and PHG-CRa, for subcellular localization of the glucoraphanin synthesis-related gene FMOGS-OX5 and clubroot resistance gene by a PEG-Ca2+-mediated transient transformation system for broccoli protoplasts. The study found that both genes were expressed in the cytoplasm and nucleus, providing a scientific basis for studying the regulation of glucosinolate metabolism and clubroot resistance in cruciferous crops.

In summary, the study provides new insights into the improvement of the genetic transformation and molecular breeding of Brassica oleracea crops, particularly broccoli, which is a widely grown crop worldwide. The findings can potentially contribute to the development of improved crop varieties with enhanced traits such as increased resistance to diseases or higher nutritional value.