Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2012, Vol. 38 ›› Issue (12): 2185-2191.doi: 10.3724/SP.J.1006.2012.02185

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment of a Transformation System Using Inbred Line of Yellow-Seeded Brassica napus

LIN Na,LIU Lie-Zhao,YIN Jia-Ming,WANG Rui,CHAI You-Rong,LI Jia-Na*   

  1. College of Agronomy and Biotechnology, Southwest University / Chongqing Rapeseed Engineering & Technology Research Center / Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400716, China
  • Received:2011-12-01 Revised:2012-07-05 Online:2012-12-12 Published:2012-10-08
  • Contact: 李加纳, E-mail: ljn1950@swu.edu.cn

Abstract:

In this research, we established a transformation system using an inbred line of yellow-seeded Brassica napus. Hypocotyl explants precultured for 5–7 d on Murashige and Skoog medium containing 200 μmol L–1 acetosyringone were cocultured with Agrobacterium tumefaciens strain LBA4404 (pCNR) for 63–69 hours.The plasmid pCNR was constructed by inserting Δ6-fatty acid desaturase gene from Rhizopusstolonifer into plant high-efficient expression vector pCAMBIA2301G. Kanamycin-tolerant shoots were regenerated on shoot induction medium for three months after Agrobacterium inoculation. The average transformation efficiency was about 1.3% under optimal conditions. Results from GUS assay and PCR analysis of transformed plants indicated that the introduced genewas integrated into B. napus genomes.The Southern blot revealed that those transformants carried one or two copies of the goal gene. The fatty acids of the transgenic plant seeds were analyzed by GC, and the γ-linolenic content was 8.2%.

Key words: Brassica napus, Δ6-fatty acid desaturase, Transformation

[1]Knutzon D S, Thompson G A, Radke S E, JohnsonW B, Knauf V C, Kridl J C. Modification of Brassica seed oil by antisense expression of a stearoly acyl carrier protein desaturase gene. Proc Natl Acad Sci USA, 1992, 89: 2624–2628



[2]Shi S-W(石淑稳), Zhou Y-M(周永明), Sun X-C(孙学成), Zhang X-L(张献龙). Transformation of Brassica napus with herbicide resistance gene. J Huazhong Agric Univ (华中农业大学学报), 1998, 17(3): 205–210 (in Chinese with English abstract)



[3]Xu B-B(许本波), Xie L-L(谢伶俐), Tian Z-H(田志宏), Yan H(严寒), He Y(何勇). Study on genetic transformation system of yellow-seed rapeseed (Brassica napus L.). Acta Agric Jiangxi (江西农业学报), 2007, 19 (8): 4–6 (in Chinese with English abstract)



[4]Poulsen G B. Genetic transformation of Brassica. Plant Breed, 1996, 115: 209–225



[5]Khan M R, Rashid H, Ansar M, Chaudry Z. High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant Cell Tissue Organ Cult, 2003, 75: 223–231



[6]Leng H(冷虹), Li J N(李加纳), Lu H(陆合), Chai Y R(柴友荣), Yin J M(殷家明). Construction of seed-specific expression vector of Δ6-fatty acid desaturase gene. Chin Agric Sci Bull (中国农学通报), 2006, 22: 66–70 (in Chinese with English abstract)



[7]Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6, 271–282



[8]Hood E E, Chilton W S, Chilton M D, Fraley R T. T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants. J Bacteriol, 1986, 168: 1283–1290



[9]Ohta S, Mita S, Hattori T, Nakamura K. Construction and expression in tobacco of a beta-glucoronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol, 1990, 31: 805–813



[10]An G, Ebert R R, Mitra A. Binary vectors. In: Gelvin S B, Schilperroort R A, eds. Plant Molecular Biology Manual. Dordrecht: Kluwer Academic Publishers, 1988. A3: pp 1–19



[11]Jefferson R A. Asaaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep, 1987, 5: 387–405



[12]Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 1987, 19: 11–15



[13]Rücker B, Röbbelen G. Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.). Plant Breed, 1996, 115: 226–230



[14]Tsukazaki H, Kuginuki Y, Aida R, Suzuki T. Agrobacterium-mediated transformation of a doubled haploid line of cabbage. Plant Cell Rep, 2002, 21: 257–262



[15]Godwin I, Todd G, Lloyd B F, Newbury H J. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep, 1991, 9: 671–675



[16]Godwin A K, Testa J R, Handel L M, Liu Z, Vanderveer L A, Tracey P A, Hamilton T C. Spontaneous transformation of rat ovarian surface epithelial cells: association with cytogenetic changes and implications for repeated ovulation in the etiology of ovarian cancer. J Nat Cancer Inst, 1992, 84: 592–601



[17]Holford P, Hernandez N, Newbury H J. Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium bumefaciens. Plant Cell Rep, 1993, 11: 196–199



[18]Metz T D, Dixit R, Earle E D. Agrobacterium tumefaciens mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata). Plant Cell Rep, 1995, 15: 287–292



[19]Takasaki T, Hatakeyama K, Ojima K, Watanabe M, Toriyama K, Hinata K. Factors influencing Agrobacterium-mediated transformation of Brassica rapa L. Breed Sci, 1997, 47: 127–136



[20]Henzi M X, Christey M C, McNeil D L. Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica). Plant Cell Rep, 2000, 19: 994–999



[21]Deroles S C, Gardner R C. Analysis of T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol, 1988, 11: 365–377



[22]Stoger E, Fink C, Pfosser M, Heberle B E. Plant transformation by particle bombardment of embryogenic pollen. Plant Cell Rep, 1995, 14: 273–278

[1] FENG Ya, ZHU Xi, LUO Hong-Yu, LI Shi-Gui, ZHANG Ning, SI Huai-Jun. Functional analysis of StMAPK4 in response to low temperature stress in potato [J]. Acta Agronomica Sinica, 2022, 48(4): 896-907.
[2] TANG Rui-Min, JIA Xiao-Yun, ZHU Wen-Jiao, YIN Jing-Ming, YANG Qing. Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance [J]. Acta Agronomica Sinica, 2021, 47(4): 672-683.
[3] ZHOU Guan-Tong, LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong. Efficient screening system of effective sgRNA for cotton CRISPR/Cas9 gene editing [J]. Acta Agronomica Sinica, 2021, 47(3): 427-437.
[4] HAN Le,DU Ping-Ping,XIAO Kai. Functional characteristics of TaPYR1, an abscisic acid receptor family gene in mediating wheat tolerance to drought stress [J]. Acta Agronomica Sinica, 2020, 46(6): 809-818.
[5] ZHENG Yan-Yan, HUANG De-Hua, LI Ji-Long, ZHANG Hui-Fei, BAO Yin-Guang, NI Fei, WU Jia-Jie. Analysis of the stripe rust resistance in a wheat line CB037 with high regeneration and transformation efficiency [J]. Acta Agronomica Sinica, 2020, 46(11): 1743-1749.
[6] Huan TAN,Yu-Hui LIU,Li-Xia LI,Li WANG,Yuan-Ming LI,Jun-Lian ZHANG. Cloning and Functional Analysis of R2R3 MYB Genes Involved in Anthocyanin Biosynthesis in Potato Tuber [J]. Acta Agronomica Sinica, 2018, 44(7): 1021-1031.
[7] Yu-Jie ZHANG,Yuan-Yuan ZHANG,Hua-Ning ZHANG,Ning QIN,Guo-Liang LI,Xiu-Lin GUO. Characterization and Regulatory Roles in Thermotolerance of Wheat Heat Shock Transcription Factor Gene TaHsfA2e [J]. Acta Agronomica Sinica, 2018, 44(12): 1818-1828.
[8] Qian-Nan CHEN,Ke WANG,Sha TANG,Li-Pu DU,Hui ZHI,Guan-Qing JIA,Bao-Hua ZHAO,Xing-Guo YE,Xian-Min DIAO. Use of Bar Gene for the Stable Transformation of Herbicide-resistant Foxtail Millet Plants [J]. Acta Agronomica Sinica, 2018, 44(10): 1423-1432.
[9] YANG Jing,XING Guo-Jie,NIU Lu,HE Hong-Li,DU Qian,GUO Dong-Quan,YUAN Ying*,YANG Xiang-Dong*. Antisense RNA-Mediated GmFAD2-1B Gene Silencing Enhances Accumulation of Oleic Acid in Transgenic Soybean Seeds [J]. Acta Agron Sin, 2017, 43(11): 1588-1595.
[10] CHEN Xue-Ping**,JING Ling-Yun**,WANG Jia,JIAN Hong-Ju,MEI Jia-Qin,XU Xin-Fu,LI Jia-Na,LIU Lie-Zhao*. Correlation Analysis of Sclerotinia Resistance with Lignin Content and Monomer G/S and its QTL Mapping in Brassica napus L. [J]. Acta Agron Sin, 2017, 43(09): 1280-1289.
[11] HOU Lin-Tao,WANG Teng-Yue,JIAN Hong-Ju,WANG Jia,LI Jia-Na,LIU Lie-Zhao. QTL Mapping for Seedling Dry Weight and Fresh Weight under Salt Stress and Candidate Genes Analysis in Brassica napus L. [J]. Acta Agron Sin, 2017, 43(02): 179-189.
[12] ZHAO Pei,TENG Li-Jie,WANG Ke,DU Li-Pu,REN Xian,SHE Mao-Yun,YE Xing-Guo. Cloning, Molecular Characterization, and Functional Analysis of Wheat TaVIP1 Genes [J]. Acta Agron Sin, 2017, 43(02): 201-209.
[13] LU Kun,Shen Ge-Zi,LIANG Ying,FU Ming-Lian,HE Bin,TIE Lin-Mei,ZHANG Ye, PENG Liu,LI Jia-Na. Analysis of Yield Components with High Harvest Index in Brassica napusunder Environments Fitting Different Yield Levels [J]. Acta Agron Sin, 2017, 43(01): 82-96.
[14] KOU Ying-Ying,SONG Ying-Jin,YANG Shao-Hui*,WANG Jie-Hua. Codon Optimization and Expression of phyA Gene in Soybean (Glycine max Merr.) [J]. Acta Agron Sin, 2016, 42(12): 1798-1804.
[15] WANG Wen-Xiang,HU Qiong,MEI De-Sheng,LI Yun-Chang,ZHOU Ri-Jin,WANG Hui,CHENG Hong-Tao,FU Li,LIU Jia*. Genetic Effects of Branch Angle Using Mixture Model of Major Gene Plus Polygene in Brassica napus L. [J]. Acta Agron Sin, 2016, 42(08): 1103-1111.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!