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Effects of Environmental Temperature on the Regeneration Frequency of the immature embryos of Wheat(Triticum aestivum L.)

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Journal of Integrative Agriculture 2014年第4期13卷 722-732页

作者： WANG Xin-min REN Xian YIN Gui-xiang WANG Ke LI Jia-rui DU Li-pu XU Hui-jun YE Xing-guo National Key Facility of Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops Ministry of Agriculture/Institute of Crop SciencesChinese Academy of Agricultural Sciences College of Life and Engineering Sciences Beifang University of Nationalities Department of Plant Pathology Kansas State University

The immature embryos （IEs） of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4℃ daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture （HSC） temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature （TAT） during the time of anthesis and sampling for regeneration of these tissues was around 280℃ for spring wheat type cv. CB037 and approximately 300℃ for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35℃ for 1 d or a high temperature between 30 and 33℃ lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37℃ in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37℃. Cryopreservation of-20℃ caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of-10℃ more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was -5℃, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at -5 or -10℃ inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

关键词： wheat immature embryos environmental temperature preservation plant regeneration

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Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

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Journal of Integrative Agriculture 2015年第1期14卷 11-19页

作者： ZHANG Wei WANG Xin-min FAN Rong YIN Gui-xiang WANG Ke DU Li-pu XIAO Le-le YE Xing-guo National Key Facility of Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences Chinese Academy of Agricultural Sciences Division of Agricultural and Environmental Science School of BiosciencesUniversity of Nottingham

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins （AGPs） and hydrogen peroxide （H202） have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 （KN 199） and Xinchun 9 （XC9）, together with low-frequency regeneration wheat line Chinese Spring （CS）, presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L-1 AGP or 0.005 to 0.01‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L-1 AGP application level in callus induction medium plant regeneration rates of 8.49,409.06 and 283.16% were achieved for Jimai 22 （JM22）, Jingdong 18 （JD18） and Yangmai 18 （YM18）, respectively; whereas at 100 mg L-1 AGP level, CS （105.44%）, Chuannong 16 （CN16）（80.60%） and Ningchun 4 （NC4）（62.87%） acted the best. Moreover CS （79.05%）, JM22 （7.55%）, CN16 （101.87%）, YM18 （365.56%）, Yangmai 20 （YM20）（10.48%）, and CB301 （187.40%） were more responsive to 0.005 %o of H2O2, and NC4 （35.37%） obtained the highest shoot regeneration rates at 0.01%o of H2O2. Overall, these two methods, inter-culture and AGP （or H2O2） application, can be further applied to wheat transgenic research.

关键词： wheat immature embryos plant regeneration inter-culture arabinogalactan proteins hydrogen peroxide

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Improvement of Plant Regeneration from immature embryos of Wheat Infected by Agrobacterium tumefaciens

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Agricultural Sciences in China 2011年第3期10卷 317-326页

作者： TAO Li-li YIN Gui-xiang DU Li-pu SHI Zheng-yuan SHE Mao-yun XU Hui-jun YE Xing-guo National Key Facility of Crop Gene Resources and Genetic Improvement Key Laboratory of Crop Genetics and Breeding Ministry ofAgriculture~Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 P.R.China

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

关键词： wheat immature embryos Agrobacterium tumefaciens transformation genotypes plant regeneration

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Optimization of Callus Induction Conditions from immature embryos in Maize and Plant Regeneration

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Phyton-International Journal of Experimental Botany 2020年第1期89卷 121-130页

作者： Peng Jiao Ruiqi Ma Zhuo Qi Zhenzhong Jiang Siyan Liu Jing Qu Shuyan Guan Yiyong Ma College of Life Sciences Jilin Agricultural UniversityChangchun130118China Northeast Normal University Environmental SchoolChangchun130118China College of Agronomy Jilin Agricultural UniversityChangchun130118China

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.

关键词： Maize optimization genetic regeneration immature embryos function verification regenerated plants

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Production of fertile transgenic wheat plants by micropro- jectile bombardment of immature embryos

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Progress in Natural Science:Materials International 1997年第3期 82-90页

作者：陈乐玫章力建郭宝燕贾士荣许宁赵南明

The plasmid pBI121 was introduced by microprojectile bombardment into immature embryos of four spring wheat cultivars, i.e. Jin 2148, 7506, 7606, 5926, and transformation parameters such as the bombardment velocity, tungsten particle diameter, DNA-coated particle loading way and DNA concentration were studied. Fertile transgenic plants were regenerated through the way of embryogenesis from the above transformed scutellar cells of the immature embryos of three (7506, 7606, 5926) out of four cultivars. The transformation frequency reached 0. 6% , 0.6% and 1.4%, respectively. The presence of the β-glucuronidase (GUS) gene in both R0 and R1 plants was verified by Southern analyses. G418 resistance in transformed Rl plants showed that the neomycin pbosphotransferase (NPTⅡ) gene segregated as a dominant Mendelian trait in Rl plants.

关键词： wheat immature embryos microprojectile bombardment fertile transgenic plants.

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Study on Callus Induction from immature embryos and Plant Regeneration of Different Genotypes of Maize

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Agricultural Biotechnology 2017年第6期6卷 29-31页

作者： Hao DONG Jing CHEN Tai'an Academy of Agricultural Sciences Tai' an 271000 China Daiyue Agricultural Bureau Tai' an 271000 China

The induction, subculture and differentiation of callus from immature embryos of maize （ Zea mays L. ） inbred line were studied and optimized. The re- suits revealed that, 2 mg/L 2,4-D was the optimal concentration to induce embryonic callus. Calli induced from inbred Qi319 and LY92 had good morphology and high regeneration. The embryos of the two inbred lines were selected as explants to establish efficient and stable tissue culture and transformation system.

关键词： Maize （Zea mays L. ） immature embryos Callus Subculture Differentiation

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Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

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Phyton-International Journal of Experimental Botany 2022年第2期91卷 363-374页

作者： Mengtong Liu Yangyang Zhou Tongyu Liu Jianyu Lu Jing Qu Shuyan Guan Yiyong Ma College of Life Sciences Jilin Agricultural UniversityChangchun130118China College of Agronomy Jilin Agricultural UniversityChangchun130118China

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.

关键词： Maize optimization genetic regeneration immature embryos Agrobacterium-mediated transformation

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Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

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Agricultural Sciences in China 2011年第4期10卷 490-498页

作者： ZHONG De-yi ZHU You-yin LIU qian ZHOU ti ZHAO De-gang Guizhou Key Laboratory ofAgro-Bioengineering Guizhou University Guiyang 550025 P.R. China Laboratory of Green Pesticide and Agricultural Biengineering Ministry of Education/Guizhou University Guiyang 550025 P.R.China College of Life Sciences Guizhou University Guiyang 550025 P.R.China

immature embryos from three elite Guizhou waxy maize inbred lines （W21019, B7, and QCL5036） were evaluated for their ability of forming callus and regeneration into plants. immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid （2,4-D）. The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect （P0.05） on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 （B7 and QCL5036） to 3.0 mg L-1 （W21019）. The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine （6-BA）. 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

关键词： waxy maize immature embryos embryogenic callus regeneration Zea mays L.

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Tissue culture capacities of different explants from Shandong elite common wheats with various genetic backgrounds

Tissue culture capacities of different explants from Shandon...

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2011全国植物生物学研讨会

作者： Jie Gao~1.Fengzhi Guo~2 Yu.lian Li~1.Qingqi Fan~1.Peng.Meng~1.Shuimei Liang~1. Chengyan Huang~1.Xiusheng Chu~1.Genying Li~(1*) 1.Crop Research Institute,Shandong Academy of Agricultural Sciences,Jinan,Shandong 250100, China 2.Crop Research Institute,Heze Academy of Agricultural Sciences,Heze,Shandong,274000, China

To screen out desirable cultivars for genetic transformation,immature embryos, inflorescences and anthers from 50 new released cultivars with competitive advantages in agronomic traits,were isolated at their proper development stages,and incubated on SD,W,C induction medium,respectively.The tissue culture capacities of those donor explants were evaluated after 5 weeks when the induced callus were transferred to their corresponding regeneration medium.The results showed that the callus induction frequencv of immature embrvos with the size of

关键词： common wheat inflorescences immature embryos isolated anthers tissue culture capacity correlations

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