Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

Abstract

Abstract: The ‘double T-DNA’ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene (hpt) in one T-DNA region and three target genes (hLF, SB401, RZ10) in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Key words: anther culture, co-transformation, selectable marker-free transgenic plants, rice, double T-DNA binary vector

ZHU Li, FU Ya-ping, LIU Wen-zhen, HU Guo-cheng, SI Hua-min, TANG Ke-xuan, SUN Zong-xiu . Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture[J]. RICE SCIENCE, 2007, 14(4): 239-246 .

References

1 Goldsbrough A P, Lastrella C N, Yoder J I. Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Bio/Technology, 1993, 11: 1286–1292.
2 Dale E C, Ow D W. Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA, 1991, 88(23): 10558–10562.
3 Russell S H, Hoopes J L, Odell J T. Directed excision of a transgene from the plant genome. Mol Gen Genet, 1992, 243: 49–59.
4 Gleave A P, Mitra D S, Mudge S R, Morris B A M. Selectable marker-free transgenic plants without sexual crossing: Transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol, 1999, 40(2): 223–235.
5 Zuo J, Niu Q W, Moller S G, Chua N H. Chemical regulated, site-specific DNA excision in transgenic plants. Nat Biotech, 2001, 19(2): 157–161.
6 Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformations free from selection marker. Plant J, 1996, 10: 165–174.
7 McCormac A C, Fowler M R, Chen D F, Elliott M C. Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs，the effect of T-DNA size and implica- tions for genetic separation. Transg Res, 2001, 10: 143–155.
8 Darbani B, Eimanifar A, Stewart C N Jr, Camargo W N. Methods to produce marker-free transgenic plants. Biotechnol J, 2007, 2: 83–90.
9 Afolabi A S, Worland B, Snape J, Vain P. Novel pGreen/ pSoup dual-binary vector system in multiple T-DNA co-cultivation as a method of producing marker-free (clean gene) transgenic rice (Oryza sativa L.) plant. African J Biotechnol, 2005, 4: 531–540.
10 Yoder J I, Goldsbrough A P. Transformation systems for generating marker-free transgenic plants. Biol Tech, 1994, 12: 263–267.
11 Schocher R J, Shillito R D, Saul M W, Paszkowski J, Potrykus I. Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technology, 1986, 4: 1093–1096
12 Zhou H Y, Chen S B, Li X G, Xiao G F, Wei X L, Zhu Z. Generating marker-free transgenic tobacco plants by Agrobacterium-mediated transformation with double T-DNA binary vector. Acta Bot Sin, 2003, 45(9): 1103–1108.
13 Tang L, Liu Q Q, Deng X X, Wu X J, Sun S S M. LRP transgenic indica rice restorer line without resistance selection marker. Acta Agron Sin, 2006, 32(8): 1248–1251. (in Chinese with English abstract)
14 Zhang X C, Peng M, Wu K X, Guo L Q, Lin J Y, Lin J F. Generating marker-free transgenic soybean plants by Agrobacterium-mediated transformation with double T-DNA binary vector. Soybean Sci, 2006, 25(4): 369–372. (in Chinese with English abstract)
15 Yu H X, Liu Q Q, Wang L, Zhao Z P, Xu L, Huang B L, Tang S Z, Gu M H. Breeding of selectable marker-free transgenic rice lines containing AP1 gene with enhanced disease resistance. Sci Agric Sin, 2005, 38(12): 2373–2379. (in Chinese with English abstract)
16 Xing A, Zhang Z Y, Sato S, Staswik P, Clemente T. The use of the two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Biol Plant, 2000, 36: 456–463.
17 Si H M, Fu Y P, Xiao H, Hu G C, Cao J P, Huang D N, Sun Z X. Homozygous lines of transgenic rice (Oryza sativa L.) obtained via anther culture. Chinese J Rice Sci, 1999, 13 (1): 19–24. (in Chinese with English abstract)
18 Fu Y P, Si H M, Zhu Z G, Hu G C, Sun Z X. Homozygous transgenic plants regenerated from rice pollen derived calIus via Agrobacterium tumefaciens. J Zhejiang Univ: Agric ＆ Life Sci, 2001, 27(4): 407–410. (in Chinese with English abstract)
19 Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient trans- formation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6: 271–282.
20 Zhuo L S, Si H M, Cheng S H, Sun Z X. Phenylacetic acid stimulation of direct shoot formation in rice (Oryza sativa L.) anther and somatic tissue cultures. Plant Breeding, 1996, 115: 295–300.
21 Lu Y J, Zheng K L. A simple method for isolation of rice DNA. Chinese J Rice Sci, 1992, 6(1): 47–48. (in Chinese)
22 Widmer F, Seidler R J, Watrud L S. Sensitive detection of transgenic plant marker gene persistence in soil microcosms. Mol Eco1, 1996, 5: 603–613.
23 Widmer F, Seidler R J, Donegan K K, Reed G L. Quantifi- cation of transgenic plant marker gene persistence in the field. Mol Eco1, 1997, 6(l): 1–7.
24 Kohli A, Griffiths S, Palacios N, Twyman R M, Vain P, Laurie D A, Christou P. Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recom- bination hotspot in the CaMV35S promoter and confirms the predominance of microhomology mediated recombination. Plant J, 1999, 17: 591–601.
25 Cao M X, Huang J Q, Wei Z M, Yao Q H, Wan C Z, Lu J A. Agrobacterium-mediated multiple gene transformation in rice using a single vector. J Integr Plant Biol, 2005, 47(2): 233–242.
26 Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H. Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell, 2003, 15(8): 1771–1780.

[1]	Prathap V, Suresh KUMAR, Nand Lal MEENA, Chirag MAHESHWARI, Monika DALAL, Aruna TYAGI. Phosphorus Starvation Tolerance in Rice Through a Combined Physiological, Biochemical and Proteome Analysis [J]. Rice Science, 2023, 30(6): 8-.
[2]	Serena REGGI, Elisabetta ONELLI, Alessandra MOSCATELLI, Nadia STROPPA, Matteo Dell’ANNO, Kiril PERFANOV, Luciana ROSSI. Seed-Specific Expression of Apolipoprotein A-I_Milano Dimer in Rice Engineered Lines [J]. Rice Science, 2023, 30(6): 6-.
[3]	Sundus ZAFAR, XU Jianlong. Recent Advances to Enhance Nutritional Quality of Rice [J]. Rice Science, 2023, 30(6): 4-.
[4]	Kankunlanach KHAMPUANG, Nanthana CHAIWONG, Atilla YAZICI, Baris DEMIRER, Ismail CAKMAK, Chanakan PROM-U-THAI. Effect of Sulfur Fertilization on Productivity and Grain Zinc Yield of Rice Grown under Low and Adequate Soil Zinc Applications [J]. Rice Science, 2023, 30(6): 9-.
[5]	FAN Fengfeng, CAI Meng, LUO Xiong, LIU Manman, YUAN Huanran, CHENG Mingxing, Ayaz AHMAD, LI Nengwu, LI Shaoqing. Novel QTLs from Wild Rice Oryza longistaminata Confer Rice Strong Tolerance to High Temperature at Seedling Stage [J]. Rice Science, 2023, 30(6): 14-.
[6]	LIN Shaodan, YAO Yue, LI Jiayi, LI Xiaobin, MA Jie, WENG Haiyong, CHENG Zuxin, YE Dapeng. Application of UAV-Based Imaging and Deep Learning in Assessment of Rice Blast Resistance [J]. Rice Science, 2023, 30(6): 10-.
[7]	Md. Forshed DEWAN, Md. AHIDUZZAMAN, Md. Nahidul ISLAM, Habibul Bari SHOZIB. Potential Benefits of Bioactive Compounds of Traditional Rice Grown in South and South-East Asia: A Review [J]. Rice Science, 2023, 30(6): 5-.
[8]	Raja CHAKRABORTY, Pratap KALITA, Saikat SEN. Phenolic Profile, Antioxidant, Antihyperlipidemic and Cardiac Risk Preventive Effect of Chakhao Poireiton (A Pigmented Black Rice) in High-Fat High-Sugar induced Rats [J]. Rice Science, 2023, 30(6): 11-.
[9]	LI Qianlong, FENG Qi, WANG Heqin, KANG Yunhai, ZHANG Conghe, DU Ming, ZHANG Yunhu, WANG Hui, CHEN Jinjie, HAN Bin, FANG Yu, WANG Ahong. Genome-Wide Dissection of Quan 9311A Breeding Process and Application Advantages [J]. Rice Science, 2023, 30(6): 7-.
[10]	JI Dongling, XIAO Wenhui, SUN Zhiwei, LIU Lijun, GU Junfei, ZHANG Hao, Tom Matthew HARRISON, LIU Ke, WANG Zhiqin, WANG Weilu, YANG Jianchang. Translocation and Distribution of Carbon-Nitrogen in Relation to Rice Yield and Grain Quality as Affected by High Temperature at Early Panicle Initiation Stage [J]. Rice Science, 2023, 30(6): 12-.
[11]	Nazaratul Ashifa Abdullah Salim, Norlida Mat Daud, Julieta Griboff, Abdul Rahim Harun. Elemental Assessments in Paddy Soil for Geographical Traceability of Rice from Peninsular Malaysia [J]. Rice Science, 2023, 30(5): 486-498.
[12]	Monica Ruffini Castiglione, Stefania Bottega, Carlo Sorce, Carmelina SpanÒ. Effects of Zinc Oxide Particles with Different Sizes on Root Development in Oryza sativa [J]. Rice Science, 2023, 30(5): 449-458.
[13]	Tan Jingyi, Zhang Xiaobo, Shang Huihui, Li Panpan, Wang Zhonghao, Liao Xinwei, Xu Xia, Yang Shihua, Gong Junyi, Wu Jianli. ORYZA SATIVA SPOTTED-LEAF 41 (OsSPL41) Negatively Regulates Plant Immunity in Rice [J]. Rice Science, 2023, 30(5): 426-436.
[14]	Ammara Latif, Sun Ying, Pu Cuixia, Noman Ali. Rice Curled Its Leaves Either Adaxially or Abaxially to Combat Drought Stress [J]. Rice Science, 2023, 30(5): 405-416.
[15]	Liu Qiao, Qiu Linlin, Hua Yangguang, Li Jing, Pang Bo, Zhai Yufeng, Wang Dekai. LHD3 Encoding a J-Domain Protein Controls Heading Date in Rice [J]. Rice Science, 2023, 30(5): 437-448.