中文摘要：本发明公开了一种快速创制转基因玉米双单倍体纯合后代的方法，与现有技术相比，本发明用玉米单倍体诱导系东诱1号为父本给转基因玉米杂交种授粉，挑取单倍体幼胚进行组织培养，利用秋水仙素给单倍体幼胚进行染色体组加倍，进而培养转基因双单倍体玉米植株，并收获转基因双单倍体种子。本发明可用于转基因玉米回交转育后代的快速纯合，也可用于玉米双单倍体群体的快速建立，进行玉米重要性状的遗传分析。
外文摘要：NOVELTY - Method for obtaining homozygous progeny of transgenic haploid corn involves using test materials to obtain haploid filial (F1) hybrid, planting the test hybrid material, performing field management, inducing F1 hybrid pollination, sterilizing 20 days old young spike, placing immature embryo in a culture dish, doubling haploid embryo, growing immature embryos, transplanting seedling to soil matrix, selfing, extracting total DNA, performing PCR amplification, screening marker genes for herbicide-tolerant glyphosate genes, and performing statistical analysis.
USE - The method is useful for obtaining homozygous progeny of transgenic haploid corn (claimed).
ADVANTAGE - The method is rapid.
DETAILED DESCRIPTION - Method for obtaining homozygous progeny of transgenic haploid corn involves using test materials comprising corn induced haploid No: 1, L7 and JY3, for and Northeast Agricultural University corn breeding research group labeled with Rnj marker to obtain haploid filial (F1) hybrid, where the female parent is transgenic corn arginase ZmARG gene corn HiII T2 generation lines, and the male parent is 344, planting the test hybrid material by designing random block in double-row area of 5 m in length with planting distance of 30 cm, and width of 70 cm, where 17 planting strains are placed in each row, performing field management consistent with the standard, and common corn production field of 5 m in length, row spacing of 30 cm, and plant spacing of 70 cm, sowing hybrid seed, inducing F1 hybrid pollination, recording the pollination time, where each induces at least 10 strain, sterilizing 20 days old young spike after pollinating for 18 days in 0.1% mercuric chloride for 8 minutes, stripping bracts, washing the soaked young spike thrice with 75% ethanol, and sterile water, placing immature embryo on a sterile filter paper in culture dish containing selective culture medium, culturing at 28 degrees C for 24 hours with light! intensi ty of 16 hours and under dark condition for 8 hours, doubling haploid of young embryo by selecting haploid immature embryo shield, inoculating into culture dish for doubling at 26 degrees C under dark condition for 24 hours, transferring the immature embryos to the growth culture flask, where each flask comprises 15 immature embryos, culturing at 26 degrees C for 1 week under dark condition, and for 2-3 weeks under light condition until roots are well-development, transplanting the seedling to soil matrix in greenhouse, identifying leaf sheath color of haploid plants until mature, selfing, harvesting seeds, extracting total DNA of corn seedling leaves using cetyltrimethylammonium bromide (CTAB), where the ZmARG gene is a corn endogenous gene, performing PCR detection of diploid by performing PCR amplification reaction by predenaturing at 94 degrees C for 5 minutes, denaturing at 94 degrees C for 30 seconds, annealing at 60 degrees C for 30 seconds, extending at 72 degrees C for 1 minute, for 32 cycles, and extending again for 8 minutes, using primers, where the primers are designed based on the promoter ubiquitin (Ubi) sequence and the target gene sequence, primer ARG-F is located inside the promoter Ubi sequence, and primer ARG-R is located within the target gene sequence, obtaining PCR amplified product of 775 base pair in size, screening marker genes for herbicide-tolerant glyphosate genes, and 522 base pair size product, subjecting the PCR amplified product to 1% agarose gel electrophoresis, performing statistical analysis using formula: haploid induction rate=haploid embryo count divide by seedling embryo numberx 100% (I), and doubling rate=seed number divide by number of regenerated seedlingsx 100% (II), and then analyzing the result using Excel 2010 to initialize the test data, and independent sample T test and variance analysis program in statistical package for the social sciences (SPSS) statistics 17.0 software. The selective culture medium is Murashige and Skoog (MS) liqui! d cultur e medium comprising 100 uM abscisic acid (ABA). The proliferation culture medium is MS liquid culture medium comprising 2 mg/l asparagines, 1 mg/l 6-benzylaminopurine (BAP), and 0.05% colchicine. The growth culture medium is MS solid culture medium. The primer comprises ARG-F base pair sequence of (SEQ ID NO: Not defined), and ARG-R base pair sequence of (SEQ ID NO: Not defined). cgcccttctttggtgat (SEQ ID NO: Not defined), and ccctgccttcatacgct (SEQ ID NO: Not defined).
主权项：一种快速创制转基因玉米双单倍体纯合后代的方法，其特征在于：包括试验材料：玉米单倍体诱导系东诱1号、L7和JY3，为东北农业大学农学院玉米课题组选育，具有R?nj标记；用于创建单倍体的F1杂交种，其母本为转玉米精氨酸酶ZmARG基因玉米HiII的T2代株系，父本为合344；上述常规玉米材料公众可从东北农业大学农学院玉米课题组获得；试验方法：材料的种植：采用随机区组设计，3次重复，双行区，行长5m，株距30cm，行宽70cm，每行17株；两地田间管理标准一致，同普通玉米生产田；5米行长，株距30cm，行距70cm；杂交种一次播种，时间为当年4月29日，诱导系分3期播种，时间分别为当年4月29日，5月7日和5月11日；授粉：用诱导系给F1杂交种授粉，挂牌记录授粉时间，每个诱导系至少授10株；组织培养的试验流程：培养基：(1)筛选培养基：MS液体培养基+100uM?ABA；(2)加倍培养
申请号：CN201611244164.4
专利号：CN106613985-A
公开/公告号：CN106613985-A
申请日：2016-12-29
公开/公告日：2017-05-10
申请/专利权人：东北农业大学
发明/设计人：邸宏 ; 周羽 ; 曾兴 ; 王振华 ; 祖红月
分类号：A01H4/00
主分类号：A01H4/00
法律状态：公开发明
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