Evaluation of Beta-Lactam Antibiotics on the Regeneration of Peanut Plants and Their Inhibitory Effect on Agrobacterium Growth

The effect of beta-lactam antibiotics on shoot induction and plantlet regeneration from cotyledonary nodes was tested using two peanut cultivars.The culture media contained 4 mg/L 6-benzylaminopurine(BAP)as the main growth regulator.Various concentrations(100–600 mg/L)of cefotaxime,carbenicillin,and timentin were applied in the culture media.In all the tested media,there were no significant differences in the shoot induction as compared to the control.However,little phytotoxic effect was observed at higher concentrations of these antibiotics in the shoot elongation media.Under shoot elongation medium,shoots turned brownish and partly died at higher concentrations where shooting rates were not affected by the treatments.In cefotaxime,timentin,and carbenicillin-containing media,levels of antibiotics greater than 400,300,and 200 mg/L,respectively resulted in the brown coloration of plantlets.Moreover,the mean shoot number and shoot weight significantly decreased as their dosage increased.The results indicate that maximum levels of antibiotics have an adverse effect on the growth and development of peanuts.Also,cefotaxime(100–300 mg/L)and timentin(100–300 mg/L)will be sufficient in controlling Agrobacterium growth in the culture media with the least phytotoxic effect on the peanut plants.

Bioinformatics Analysis of Disease Resistance Gene PR1 and Its Genetic Transformation in Soybeans and Cultivation of Multi-resistant Materials

In agricultural production,a single insect-resistant and disease-resistant variety can no longer meet the demand.In this study,the expression vector pCAMBIA-3301-PR1 containing the disease-resistant gene PR1 was constructed by means of genetic engineering,and the PR1 gene was genetically transformed to contain the PR1 gene through the pollen tube method.In CryAb-8Like transgenic high-generation T7 receptor soybean,a new material that is resistant to insects and diseases is obtained.For T2 transformed plants,routine PCR detection,Southern Blot hybridization,fluorescence quantitative PCR detection,indoor and outdoor pest resistance identification and indoor disease resistance identification were performed.The results showed that there were 9 positive plants in the routine PCR test of T2 generation.In Southern Blot hybridization,both PR1 and CryAb-8Like genes are integrated in soybeans in the form of single copies.Fluorescence quantitative PCR showed that the expression levels of PR1 and CryAb-8Like genes are different in different tissues.The average expression levels of PR1 gene in plant roots,stems,and leaves are 2.88,1.54,and 5.26,respectively.CryAb-8Like genes are found in roots,stems,and leaves.The average expression levels were 1.36,1.39,and 4.25,respectively.The insectivorous rate of the CryAb-8Like gene in outdoor plants with positive insect resistance identification was 3.78%.The disc partition method was used indoors for pest resistance identification,and the bud length of transformed plants increased significantly.The average mortality rate of untransformed plants in indoor disease resistance identification was as high as 56.66%,and the average mortality rate of plants transformed with PR1 gene was 10.00%,and disease resistance was significantly improved.Therefore,a new material with resistance to diseases and insects is obtained.

Construction and Functional Analysis of CRISPR/Cas9 Vector of FAD2 Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil.In the synthesis pathway of soybean fatty acids,the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid.In this study,CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression.Firstly,the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed.Then,the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation,and the mutant plants were obtained.Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out.The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties.The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (Arachis hypogaea L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits.A combination of 6-benzylaminopurine(BAP)and thidiazuron(TDZ)plant growth regulators was applied to evaluate shoot regeneration capacity whereasα-naphthalene acetic acid(NAA)combination with 6-benzylaminopurine(BAP),and 2,4-dichlorophenoxyacetic acid(2,4-D)with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars.The result showed combination(BAP with TDZ)was found to be effective in promoting shoot.The highest shoot regeneration frequency(93%)was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency(87%)was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ.The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased.The highest rooting rate(93%)was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars.The rooting rate increased as the concentration of auxin to cytokinin ratio increased.The maximum rooting rate(83%)was obtained on MS medium supplemented with 0.3 mg/L 2,4-D with 0.2 mg/L BAP for the cultivar N3.The result indicated that BAP with NAA was much better than BAP with 2,4-D in rooting rate.Thus,the protocol developed was genotype independent and effective for peanut tissue culture.