Optimization of Callus Induction Conditions from Immature Embryos in Maize and Plant Regeneration

This research uses the immature embryos of inbred maize lines(GSH9901,Hi01,Hi02,and Chang 7-2)as receptor materials to establish the callus induction system.These inbred lines provide the receptor materials for the genetic regeneration of maize and the verification of the genetic functions of maize.The factor experiment and orthogonal experiments were used to investigate the impacts of different genotypes,immature embryo size,shield orientation,2,4-D concentration,proline concentration,and folic acid concentration on the induction rate of embryogenic callus tissue.A sensitivity experiment testing glyphosate(Bar)and an antibiotic(Cefotaxime sodium)were also conducted.The results indicate that the immature embryos of inbred maize line GSH9901 were the most effective for callus tissue induction,and the immature embryos with a length of 1.6-2.0 mm produce the best result.The upward shield face is more successful for the formation of induced callus.Using orthogonal analysis,we found that the optimal combination for the induction system was A_(3)(2,4-D concentration 0.25 mg mL^(-1)),B_(1)C_(3)(proline concentration 0.8 mg mL^(-1)),and D 2(folate Concentration 0.5 mg mL^(-1))and the induction rate reached 84%.We found that cold storage at 4℃ for 1 d is more conducive for the formation of embryogenic callus than the other treatments tested.The sensitivity experiment for callus tissue screening revealed the critical concentration of glyphosate to be 10 mg ml^(-1),and the critical concentration of antibiotic is 250 mg ml^(-1).Using this combination of glyphosate and antibiotic resulted in regenerated plants.This study established the optimal conditions for immature embryo callus tissue induction in maize.

Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize(Zea mays)lines(H8183,H8184,and H8185)were used for Agrobacterium infection.We used theβ-glucuronidase gene(GUS)as the target gene and the glufosinate resistance gene(bar)as the selection marker.We conducted research on several aspects,such as different genotypes,coculture conditions,screening agent concentrations,and concentrations of indole-3-butytric acid(IBA),6-benzylaminopurine(6-BA),and ascorbic acid(Vc)in the differentiation medium.We optimized the genetic transformation system,and the obtained results indicated that among the three lines studied,the induction rate of H8185 was the highest at 93.2%,followed by H8184,with H8183 having the lowest induction rate(80.1%).The best coculture method was that using the N6 coculture medium layered with a sterile filter paper.Using orthogonal analysis,we found that the optimal combination of the three factors in the differentiation medium was A_(3)(1 mg mL^(−1)IBA),B_(3)C_(1)(1.6 mg mL^(−1)6-BA),and D_(3)(1.5 mg mL^(−1)Vc).Through GUS staining analysis,Bar test-strip analysis,and polymerase chain reaction,five transgenic plants were finally obtained.This study established the optimal conditions for genetic transformation in maize.

Meta-Analysis of Flowering-Related Traits and Mining of Candidate Genes in Maize

Maize flowering is an important agronomic character,which is controlled by quantitative trait loci(QTL).Over the years,a large number of flowering-related QTL have been found in maize and exist in public databases.However,combining these data,re-analyzing and mining candidate loci and fine mapping of flowering-related traits to reduce confidence intervals has become a hot issue in maize.In this study,the QTL of 6 important agronomic traits of maize flowering were collected from 15 published articles,including flowering period(DA),Days to tasseling(DTT),Days to silking(DS),Days to pollen shedding(DTP),anthesis-silking interval(ASI)and the photosensitive(PS).Through meta-analysis,622 QTL were integrated into 26 meta-QTLs(MQTL).Finally,the candidate genes related to maize flowering(Gene IDs:ZM00001D005791,ZM00001D019045,ZM00001D050697,ZM00001D011139)were identified by Gene Ontology(GO)enrichment and hierarchical cluster analysis of expression profile.Based on the results of this study,the genetic characteristics of maize flowering traits will be further analyzed,which is of great significance to guide the improvement of important agronomic characters and improve the efficiency of breeding.

Development of a New Cold-Tolerant Maize(Zea mays L.)Germplasm Using the ICE1 Gene from Arabidopsis thaliana

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION(ICE1)expression in response to cold stress,RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar.Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations.From the regenerated plantlets,three T1 lines were screened and identi-fied by PCR.A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize(Zea mays L.)genomes of the three T1 generations.Under low temperature-stress conditions(4°C),the relative conductivity levels decreased by 27.51%–31.44%,the proline concentrations increased by 12.50%–17.50%,the malondialdehyde concentrations decreased by 16.78%–18.37%,and the peroxidase activities increased by 19.60%–22.89%in the T1 lines compared with those of the control.A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots,stems,and leaves of the T1 lines.ICE1 positively regulates the expression of the CBF genes in response to cold stress.Thus,this study showed the successful transformation of maize with the ICE1 gene,resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.

Optimization of Callus Induction Conditions from Immature Embryos of Maize under Stress

The embryos of maize(Zea mays L.)inbred lines GS02,GS07,GS08,GS11 and GS15 were used as receptor materials to optimize the receptor system from the aspects of genotype,medium components and stress(PEG6000,mannitol,salt and low phosphorus).The results showed that GS07 had the highest induction rate(95.2%).Orthogonal test analysis showed that the best combination of medium components in induction was A2B3C1D3(2),namely,the concentration of 2,4-dichlorophenoxy acetic acid(2,4-D)was 4 mg·mL^(-1),the concentration of L-Proline(L-Pro)was 0.8 mg·mL^(-1),and the concentration of silver nitrate(AgNO3)was 10 mg·mL^(-1)(or 5 mg·mL).Interestingly,we found that the optimal medium supplemented with 30 g·L^(-1)PEG6000 or 80 g·L^(-1)mannitol was suitable for antioxidant enzyme activity and malondialdehyde(MDA)content in GS07 callus.Exogenous 10 mmol·L^(-1)Ca^(2+)in the medium components with 100 mmol·L^(-1)sodium chloride(NaCl)could improve the activity of antioxidant enzymes in GS07 callus.Callus of GS07 could divide normally and grow well in medium components with 27 mg·L^(-1)KH_(2)PO_(4).This study enhanced the adaptability of maize callus to stress and optimized the culture conditions.

Character Identification of an Early Flowering Mutant

The concept of gene-function-genetic trait was introduced to explore the effects of early flowering on the growth and development of maize at the jointing stage and to obtain early flowering mutants using ethyl methanesulfonate mutagenesis.First,we studied gene expression,phytohormones,and lignin content to explore the physiological peculiarities of the early flowering mutant.Then we analyzed the genetic features of the mutants during the jointing stage by measuring physiological and biochemical indices of drought tolerance.The results showed that the photosynthetic rate of the mutant was significantly higher than that of the control and the rate of accumulation of dry matter was rapid.In addition,the lignin content increased while drought resistance diminished.Therefore,we concluded that early flowering leads to faster overall growth and development.