ABSTRACT
Gametophytic male sterility (GMS) plays an important role in the study of pollen development and seed propagation of recessive nuclear male sterile lines insensitive to the environmental conditions in hybrid rice breeding. Since the inherent phenotypic and genetic characteristics of GMS, it is very difficult to find and identify the GMS mutants. However, due to the abundance of gene transcription data, a large number of pollen-specific genes have been found, and most of them may be associated with GMS. To promote the study of these genes in pollen development and heterosis utilization, in this study, an easy and efficient method of creating and identifying GMS was established using RNAi and OsMYB76R as a reporter. First, the OsC1/OsMYB76 gene involved in anthocyanin synthesis was modified, and we have validated that the modified OsMYB76R is workable as the same as the pre-modified OsMYB76 gene. Then, the ascorbic acid oxidase gene OsPTD1 was downregulated using RNAi, driven by its own promoter that resulted in abnormal pollen tube growth. Finally, the RNAi elements were linked with OsMYB76R and transformed into an osmyb76 mutant, and the distortion of purple color segregation was found in T1 and F1 generations. This indicates that the OsPTD1 GMS was prepared successfully. Compared to current methods, there are several advantages to this method. First, time is saved in material preparation, as one generation less needs to be compared than in the conventional method, and mutation screening can be avoided. In addition, for identification, the cost is lower; PCR, electrophoresis, and other processes are not needed; and no expensive chemicals or instruments are required. Finally, the results are more accurate, with much lower background effects, and no damage to the plant. The result is an easy, efficient, low-cost, and accurate method of preparing and identifying GMS genes.
ABSTRACT
BACKGROUND: Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required. RESULTS: Here, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene AtMyb75 in Arabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by A. rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max (L.) Merr.) and two model legume species, Lotus japonicas and Medicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of AtMyb75 as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation Rfg1 gene in the soybean PI377578 (Nod-) inoculated with Sinorhizobium fredii USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin. CONCLUSIONS: Anthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.
ABSTRACT
BACKGROUND: Agrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions. RESULTS: Here, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato. CONCLUSIONS: We established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.
