A highly efficient genetic transformation system for broccoli and subcellular localization

Abstract

IntroductionAgrobacterium-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.MethodsIn 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 DiscussionThis 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.

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis ‘Purpurea’

“…The transformation efficiency is comparable to that of other ornamental flower plants, such as Chrysanthemum morifolium Ramat (the transformation efficiency is between 1% and 8%) [ 47 , 48 , 49 ], Broccoli oleracea var. italica (2.7–6.4%) [ 27 ], B. oleraceae var Botrytis (7–13.6%) [ 50 ], and L. pumilum DC Fisch (5.7–13.0%) [ 51 ].…”

An Efficient Transformation System for Fast Production of VcCHS Transgenic Blueberry Callus and Its Expressional Analysis

“…It was reported that the Agrobacterium strain and concentration, genotype, explant type, and co-cultivation time significantly influence transformation efficiency [ 47 , 48 , 49 , 50 ]. There are different kinds of antibiotics to distinguish transgenic material from explants, such as kanamycin (Km) and hygromycin (Hyg), which were frequently applied to A. tumefaciens -mediated transformation methods for some woody plants [ 51 , 52 ].…”

Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli

“…It has been reported that the transformation efficiency of the Agrobacterium tumefaciens -mediated genetic transformation system for broccoli is generally approximately 2–26%, which is influenced by the explant type, receptor genotype, hormone concentration, culture time, and other factors [ 109 , 110 ]. In general, the broccoli genotype is the main factor affecting the efficiency of genetic transformation [ 44 , 109 ].…”

“…It has been reported that the transformation efficiency of the Agrobacterium tumefaciens -mediated genetic transformation system for broccoli is generally approximately 2–26%, which is influenced by the explant type, receptor genotype, hormone concentration, culture time, and other factors [ 109 , 110 ]. In general, the broccoli genotype is the main factor affecting the efficiency of genetic transformation [ 44 , 109 ]. It has been established that when the concentration of hygromycin B is 5 mg/L for the generation of young sprouts, significant differences in the differentiation rate are usually detected among broccoli genotypes.…”

“…Most research has shown that the selective antibiotic hygromycin B is most widely used in broccoli and has the highest and most stable selection efficiency. According to comparisons with other Brassica crops, such as Brassica napus and Brassica rapa , the optimal selection concentration is 5 mg/L hygromycin B in broccoli [ 109 ]. In addition, 8 mg/L hygromycin B is better for cabbage, whereas 50 mg/L hygromycin B is generally used for rape [ 113 ].…”