Abstract
Key message
We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target.
Abstract
Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59–1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T2 transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.
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Abbreviations
- As:
-
Acetosyringone
- BAP:
-
6-Benzyl amino purine
- CCM:
-
Co-cultivation medium
- CDS:
-
Coding sequence
- CMS:
-
Cytoplasmic male sterility
- ESEM:
-
Environmental scanning electron microscopy
- MES:
-
Morpholino ethane sulfonic acid
- MS:
-
Murashige and Skoog medium
- GUS:
-
β-Glucuronidase
- Kan:
-
Kanamycin
- Rif:
-
Rifampicin
- SM:
-
Selection medium
- WT:
-
Wild type
References
Baskar V, Gangadhar BH, Park SW, Nile SH (2016) A simple and efficient Agrobacterium tumefaciens-mediated plant transformation of Brassica rapa ssp. pekinensis. 3 Biotech 6:88. https://doi.org/10.1007/s13205-016-0402-1
Bastaki NK, Cullis CA (2014) Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate. J Vis Exp 94:e52189. https://doi.org/10.3791/52189
Chen GH, Yan W, Yang LF, Gai JY, Zhu YL (2014) Overexpression of StNHX1, a novel vacuolar Na+/H+ antiporter gene from Solanum torvum, enhances salt tolerance in transgenic vegetable soybean. Hortic Environ Biotechnol 55:213–221. https://doi.org/10.1007/s13580-014-0003-z
Chen GH, Yan W, Yang SP, Wang A, Gai JY, Zhu YL (2015) Overexpression of rice phosphate transporter gene OsPT2 enhances tolerance to low phosphorus stress in soybean. J Agric Sci Technol 17:469–482. https://doi.org/10.1016/j.scienta.2014.07.037
Chen G, Ye X, Zhang S, Yuan L, Zhu S, Hou J, Wang C (2018) Comparative transcriptome analysis between fertile and CMS flower buds in Wucai (Brassica campestris L.). BMC Genom 19:908. https://doi.org/10.1186/s12864-018-5331-4
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x
Curtis IS, Hong GN (2001) Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method-plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res 10:363–371. https://doi.org/10.1023/A:1016600517293
Das SC, Hassan L, Quddus MA (2016) Agrobacterium-mediated genetic transformation in Brassica species. J Biosci Agric Res 7:647–658. https://doi.org/10.18801/jbar.070216.77
Desfeux C, Clough S, Bent A (2000) Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol 123:895–904. https://doi.org/10.1104/pp.123.3.895
Dong X, Kim WK, Lim YP, Kim YK, Hur Y (2013) Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes. Plant Sci 199–200:7–17. https://doi.org/10.1016/j.plantsci.2012.11.001
Gerszberg A (2018) Tissue culture and genetic transformation of cabbage (Brassica oleracea var. capitata): an overview. Planta 248:1037–1048. https://doi.org/10.1007/s00425-018-2961-3
Han Y, Han S, Ban Q, He Y, Jin M, Rao J (2017) Overexpression of persimmon DkXTH1 enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants. Plant Cell Rep 36:583–596. https://doi.org/10.1007/s00299-017-2105-4
Kowalczyk T, Gerszberg A, Durańska P, Biłas R, Hnatuszko-Konka K (2018) High efficiency transformation of Brassica oleracea var. botrytis plants by Rhizobium rhizogenes. AMB Express 8:125. https://doi.org/10.1186/s13568-018-0656-6
Lee H, Zhang ZJ (2014) Agrobacterium-mediated transformation of maize (Zea mays) immature embryos. Cereal Genom 1099:273–280. https://doi.org/10.1007/978-1-62703-715-0_22
Li L, Zhang L, Wang C, Zhu S, Yuan L, Hou J, Fang R, Zhu C, Chen G (2018) Molecular cloning and expression analysis of FLOWERING LOCUS C gene BcFLC2 in Wucai (Brassica campestris L.). Mol Plant Breed 16:5199–5027. https://doi.org/10.13271/j.mpb.016.005199 (in Chinese)
Liu W, Yang Y, Liu Q (2018) Establishment of an efficient regeneration system using heading leaves of Chinese cabbage (Brassica rapa L.) and its application in genetic transformation. Hortic Environ Biotechnol 59:583–596. https://doi.org/10.1007/s13580-018-0064-5
Naeem I, Munir I, Iqbal A, Ullah F (2016) Optimization of an efficient non-tissue culture transformation method for Brassica juncea. Pak J Bot 48:319–324 (WOS: 000373392200040)
Rani S, Sharma V, Hada A, Bhattacharya RC, Koundal KR (2017) Fusion gene construct preparation with lectin and protease inhibitor genes against aphids and efficient genetic transformation of Brassica juncea using cotyledons explants. Acta Physiol Plant 39:115. https://doi.org/10.1007/s11738-017-2415-8
Ravanfar SA, Orbovic V, Moradpour M, Abdul AM, Karan R, Wallace S, Parajuli S (2017) Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica. Biotechnol Genet Eng Rev 33(1):1–25. https://doi.org/10.1080/02648725.2017.1309821
Rodin W, Sujipuli K, Ratanasut K (2014) The floral-dip method for rice (Oryza sativa) transformation. Int J Agric Technol 10:467–474. http://www.ijat-aatsea.com (Record Number: 20153071350)
Sarker RH, Akter S, Hoque MI, Mollika SR (2016) Agrobacterium-mediated genetic transformation of two varieties of Brassica juncea (L.) using marker genes. Plant Tissue Cult Biotechnol 26:55–65
Wang WC, Menon G, Hansen G (2003) Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants. Plant Cell Rep 22:274–281. https://doi.org/10.1007/s00299-003-0691-9
Xu H, Wang X, Zhao H, Liu F (2008) An intensive understanding of vacuum infiltration transformation of pakchoi (Brassica rapa ssp. chinensis). Plant Cell Rep 27:1369–1376. https://doi.org/10.1007/s00299-008-0564-3
Ye GN, Stone D, Pang SZ, Creely W, Gonzalez K, Hinchee M (1999) Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J 19:249–257. https://doi.org/10.1046/j.1365-313X.1999.00520.x
Zou M, Yuan L, Zhu S, Liu S, Ge J, Wang C (2016) Response of osmotic adjustment and ascorbate-glutathione cycle to heat stress in a heat-sensitive and a heat-tolerant genotype of wucai (Brassica campestris L.). Sci Hortic 211:87–94. https://doi.org/10.1016/j.scienta.2016.08.011
Zou M, Yuan L, Zhu S, Liu S, Ge J, Wang C (2017) Effects of heat stress on photosynthetic characteristics and chloroplast ultrastructure of a heat-sensitive and heat-tolerant cultivar of wucai (Brassica campestris L.). Acta Physiol Plant 39:30. https://doi.org/10.1007/s11738-016-2319-z
Acknowledgements
The National Natural Science Foundation of China (31801853); National Key R & D Program of China (2017YFD0101803); Provincial Science and Technology Major Project of Anhui (17030701013); and Major (KJ2017ZD15) and Key (KJ2017A153) Projects of Natural Science Research Funds in Support of Colleges supported this work.
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Communicated by Kan Wang.
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Chen, G., Zeng, F., Wang, J. et al. Transgenic Wucai (Brassica campestris L.) produced via Agrobacterium-mediated anther transformation in planta. Plant Cell Rep 38, 577–586 (2019). https://doi.org/10.1007/s00299-019-02387-0
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DOI: https://doi.org/10.1007/s00299-019-02387-0