RESUMO
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.
Assuntos
Agrobacterium/fisiologia , Glycine max/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Rhizobium , Glycine max/microbiologia , Transformação GenéticaRESUMO
We investigated the effects of different drip irrigation quotas on leaf area index (LAI), diffuse non-interceptance (DIFN), light interception rate (LIR), canopy apparent photosynthetic rate (CAP), and cotton yield of Xinluzao 45 in field of north Xinjiang. There were five drip irrigation treatments, i.e. W1: 600 m3·hm-2, W2: 540 m3·hm-2, W3: 480 m3·hm-2, W4: 420 m3·hm-2, W5: 360 m3·hm-2. The results showed that with the decrease in drip irrigation quota, LAI, LIR and CAP of cotton substantially decreased, with the difference between W1 and W2 being not statistically significant from the full squaring to boll opening stage. Moreover, DIFN was enhanced with the reduction in drip irrigation quota. Highest seed and lint yield (6549 kg·hm-2 and 2677 kg·hm-2, respectively) presented in W1. W2 got a 6.5% reduction of seed cotton yield compared with W1, but the irrigation water use efficiency was enhanced by 3.9%. LAI, LIR and CAP were positively correlated with seed cotton yield from full flowering stage to full boll stage. Therefore, drip irrigation quota at 540 m3·hm-2 could increase irrigation water use efficiency, maintain higher leaf area index, increase diffuse non-interceptance, and ensure light interception rate from full flowering stage to full boll stage, which could enhance canopy apparent photosynthetic rate and water use efficiency without sacrificing yield.