Selection of Maize Inbred Lines with High Regeneration and Susceptibility to Agrobacterium tumifacien

Ten-maize inbred lines of maize （Zea mays L.） with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-β-glucuronidase （gus） gene, and GV3301 carrying the green fluorescent protein （gfp） gene to study the susceptibility of different genotypes in maize to A. tumifaciens. All embryonic calli initiated from 10-maize inbred lines were able to regenerate into plantlets, and the regeneration frequencies of inbred lines 6010, 6038, 6015, 6051, and 6060 were 61.11%, 31.94%, 45%, 33.33%, and 56.94%, respectively, which were higher than that of other lines. Analysis of variance indicated that the susceptibility of the various genotypes in maize to A. tumifacien C58 showed a significant difference among each other, and the inbred lines 6010, 6015, 6051, 6050, 6058, 6060, 6069, 6077 were susceptible to A. tumifacien C58, of which frequency of gus expression were over 70%. Expression of GFP was observed in six-inbred lines （6050, 6015, 6051, 6058, 6069, 6077）. The inbred lines 6051, 6010, 6015, 6060, and 6050 had the high regeneration and the susceptibility to A. tumifaciens C58; and the inbred lines 6051, 6015, and 6060 had the high regeneration and the susceptibility to Agrobacterium tumifaciens GV3301.

Genetic Diversity of Chinese Temperate and Exotic Tropical, Subtropical Quality Protein Maize Inbreds by SSR Markers

Information on genetic relationship is of great value to maize (Zea mays L.) breeding. Theobjectives of this study were: 1) to classify 22 quality protein maize (QPM) inbreds intodifferent groups by using simple sequence repeats (SSR) markers, which included exotictropical, subtropical and domestic temperate QPM and normal maize inbreds; 2) to examine theconsistency of grouping results obtained from SSR, specific combining ability (SCA) analysis,and genetic backgrounds of these inbreds. A set of 39 polymorphic SSR primers was selected from70 primer pairs, which detected 136 alleles among the 22 lines. The mean polymorphisminformation content was 0.55. Based on analysis of genetic similarities, five groups wereidentified including Luda Red Cob, Sipingtou, Reid, Lancaster and a miscellaneous group withseveral tropical inbreds which could not be classified into the above four groups. The resultsgenerally agreed with previous results based on analysis of yield combining ability andpedigree data.

A Dynamic Model of Heavy Ion ~7Li Irradiation Mutagenesis Based on Maize Inbred Line Nutrition Difference

To reveal the saddle-type dose effect relationship, we propose a radiation mutagenesis model based on maize nutrition difference resulting from heavy ion ~7Li radiation. Through irradiation mutagenesis, apparent trait selection, amino acids and fatty acids content determination, and modeling, dynamic evolution from microscopic damage and repair initiation to the final macroscopic biological effects are considered simultaneously. The results show that the steady state nature is independent of evolution time and only relates to different radiation doses.Heavy ion ~7Li radiation could effectively cause maize phenotypic variation and could improve nutritional quality.This model not only gives a good fit to the experimental results on most types of amino acids and fatty acids, but also offers an adequate explanation of the experimental phenomenon underlying the saddle-type bimodal dose effect. By combining experimental results with theoretical analyses, we suggest that the synergy of the stimulus effect and momentum transfer is the main cause of the saddle-type dose effect bimodal curve. This provides an effective strategy for conducting maize germplasm innovation.

Media Appraisal for Somatic Embryogenesis of Elite Inbred Lines of Maize

Several protocols have been published for somatic embryogenesis in maize. It is essential to compare different protocols on selected germplasm at initial stages of tissue culture to identify a protocol that would yield optimum results. The ultimate goal is to use in vitro system for selecting and generating stress tolerant maize germplasm. Immature embryos of the elite inbred lines of maize （87014 ×Z28-11 and POP43SRS5-57） were cultured on three different semi-solid media （0.5% agar） and the effectiveness of the media for somatic embryogenesis assessed over a period two weeks of sub-culturing. A significant difference was observed among the media in proportion of potentially embryogenic calli and callus cluster size at 1 and 2 weeks after culturing. N6 medium supplemented with casaminoacid （100 mg/L）, 2, 4-dichlorophenoxy acetic acid （1 mg/L）, L-proline （25 mM） and sucrose （2%）, and Murashige and Skoog （MS） medium supplemented with 2, 4-D （2 mg/L） and sucrose （3%） gave higher proportion of potentially embryogenic calli and callus size than MS medium supplemented with casaminoacids （100 mg/L）, 2, 4-D （2 mg/L）, abscissic acid （3.3μM）, and silver nitrate （195 μM） and sucrose （3%）. The difference between genotypes is not significant

Evaluation of drought tolerance in ZmVPP1-overexpressing transgenic inbred maize lines and their hybrids

The vacuolar proton-pumping pyrophosphatase gene(VPP)is often used to enhance plant drought tolerance via genetic engineering.In this study,the drought tolerance of four transgenic inbred maize lines overexpressing ZmVPP1(PH4CV-T,PH6WC-T,Chang7-2-T,and Zheng58-T)and their transgenic hybrids was evaluated at various stages.Under normal and drought conditions,the height and fresh weight were greater for the four transgenic inbred maize lines than for the wild-type(WT)controls at the germination and seedling stages.Additionally,the transgenic plants exhibited enhanced photosynthetic efficiency at the seedling stage.In irrigated and non-irrigated fields,the four transgenic lines grew normally,but with increased ear weight and yield compared with the WT plants.Moreover,the ear weight and yield of the transgenic hybrids resulting from the PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W crosses increased in the non-irrigated field.Our results demonstrated that the growth and drought tolerance of four transgenic inbred maize lines with improved photosynthesis were enhanced by the overexpression of ZmVPP1.Moreover,the Chang7-2 and PH4CV transgenic lines may be useful for future genetic improvements of maize hybrids to increase drought tolerance.

Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles

Current gene delivery methods for maize are limited to specific genotypes and depend on timeconsuming and labor-intensive tissue culture techniques.Here,we report a new method to transfect maize that is culture-free and genotype independent.To enhance efficiency of DNA entry and maintain high pollen viability of 32%-55%,transfection was performed at cool temperature using pollen pretreated to open the germination aperture(40%–55%).Magnetic nanoparticles(MNPs)coated with DNA encoding either red fluorescent protein(RFP),β-glucuronidase gene(GUS),enhanced green fluorescent protein(EGFP)or bialaphos resistance(bar)was delivered into pollen grains,and female florets of maize inbred lines were pollinated.Red fluorescence was detected in 22%transfected pollen grains,and GUS stained 55%embryos at 18 d after pollination.Green fluorescence was detected in both silk filaments and immature kernels.The T1 generation of six inbred lines showed considerable EGFP or GUS transcripts(29%–74%)quantitated by polymerase chain reaction,and 5%–16%of the T1 seedlings showed immunologically active EGFP or GUS protein.Moreover,1.41%of the bar transfected T1 plants were glufosinate resistant,and heritable bar gene was integrated into the maize genome effectively as verified by DNA hybridization.These results demonstrate that exogenous DNA could be delivered efficiently into elite maize inbred lines recalcitrant to tissue culture-mediated transformation and expressed normally through our genotype-independent pollen transfection system.

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.