An efficient sorghum transformation system using embryogenic calli derived from mature seeds

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.

Establishment of a Bivector Genetic Transformation System in Recalcitrant Maize Inbred Lines

Maize is an important grain crop with high nutritional value. An effective transformation system is crucial for the genetic improvement of maize traits, but many important maize inbred lines remained recalcitrant to transformation. In this study, we developed a bivector transformation system that worked well in two recalcitrant maize inbred lines. This system included an induction vector (ZmBBM-ZmWUS) and an indicator vector (GFP), using microprojectile bombardment technology combined with Agrobacterium-mediated transformation. We found that the Zheng58 and Mo17 recalcitrant inbred lines could be transformed with this system. The whole transformation cycle lasted only 52 days, 38 days less than the traditional transformation cycle. Additionally, it was possible to eliminate inference of the induction vector and obtained progenies with only the target gene. Our results suggested that the bivector system was an optimization of the current maize transformation methods and could potentially be used in genetic improvement of maize inbred lines.

A highly efficient regeneration, genetic transformation system and induction of targeted mutations using CRISPR/Cas9 in Lycium ruthenicum

Background CRISPR/Cas9 is a rapidly developing genome editing technology in various biological systems due to its efficiency, portability, simplicity and versatility. This editing technology has been successfully applied in in several important plants of Solanaceae such as tomato, tobacco, potato, petunia and groundcherry. Wolfberry ranked the sixth among solanaceous crops of outstanding importance in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome sequencing and the lack of efficient regeneration and genetic transformation systems. Results In this study, we have established an efficientregeneration and genetic transformation system of Lycium ruthenicum. We have used this system to validate target sites for fw2.2, a major fruit weight quantitative trait locus first identified from tomato and accounted for 30% of the variation in fruit size. In our experiments, the editing efficiency was very high, with 95.45% of the transgenic lines containing mutations in the fw2.2 target site. We obtained transgenic wolfberry plants containing four homozygous mutations and nine biallelic mutations in the fw2.2 gene. Conclusions These results suggest that CRISPR-based gene editing is effective for the improvement of black wolfberry traits, and we expect this approach to be routinely applied to this important economic fruit.

Screening of Vietnamese soybean genotypes for Agrobacterium-mediated transgenic transformation

Soybean [Glycine max (L) Merr.] is one of the most important crops used for human food and animal feed globally. Transgenic soybean covers more than 74% of the global soybean production area, which is an achievement of genetically modified programs. The Agrobacterium-mediated method is commonly used for soybean transformation, but the efficiency of this method is affected by various factors including genotypes. Screening of the soybean genotypes suitable for Agrobacterium-infection and plant regeneration is the most important step to establish an efficient genetic transformation system. In this study, we screened thirty Vietnamese soybean genotypes including seventeen cultivated soybean genotypes (CSG) and thirteen local soybean genotypes (LCG) for shoot regeneration ability and transient infection via Agrobacterium tumefaciens method. Two CSG cultivars, DT22 and VX93, had significantly high efficiencies for shoot regeneration and transient infection compared with the control genotypes Jack and William 82. The shoot regeneration of DT22 and VX93 was 92.32% with 5.75 shoots/explant and 93.35% with 5.92 shoots/explant, respectively, whereas the control genotypes Jack and William 82 had 91.35% with 4.6 shoots/explant and 82.64% with 5.7 shoots/explant. Similarly, the transient infection of DT22 and VX93 was 84% and 86%, respectively, which was comparable with that of Jack (86%) William (82%). The success of transgenic development was confirmed by the β-Glucuronidase staining, PCR, and Basta leaf painting. The results indicated that cultivars DT22 and VX93 could be used for stable Agrobacterium-media transformation. Keywords: Soybean, Agrobacterium, transformation, transient infection, transgenic.

An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae)

The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.

A Highly Efficient Protocol To Establish Regeneration And Genetic Transformation Systems And Create Targeted Mutations Using CRISPR/Cas9 in Lycium Ruthenicum

Background: The CRISPR/Cas9 system has rapidly become the preferred tool for various biological sequencing projects due to its high efficiency, specificity, simplicity and versatility, and it has been utilized for targeted genomic alternations in several important plants of Solanaceae, including tomato, tobacco, potato, petunia and groundcherry. Wolfberry is the sixth most important solanaceous crop in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome and the lack of efficient regeneration and genetic transformation systems.Results: In this study, we established a suitable regeneration and genetic transformation system of Lycium ruthenicum, the fw2.2 gene was identified, which was the first fruit weight gene identifified from tomato and accounted for 30% of the variation in fruit size. The gene editing of black wolfberry were carried out by CRISPR/ Cas9 for the first time here with a very high editing efficiency (95.45%) in fw2.2. Four homozygous mutations and nine biallelic mutations were obtained from T0 generation plants. Conclusions: These results suggest that the CRISPR/Cas9 system is effective for gene editing study of black wolfberry, and we expect this approach to be routinely applied to this important economic fruit.