农杆菌介导的籼稻9311和华占遗传转化体系的研究

doi:10.16819/j.1001-7216.2023.220305

摘要/Abstract

摘要：

【目的】水稻转基因技术是推动水稻分子生物学研究和精准育种的重要工具。农杆菌介导法是水稻遗传转化的主流方法。然而受基因型限制，典型籼稻的转化效率仍然偏低，需提高其转化效率。【方法】以顽拗型籼稻品种9311和华占为受体材料，研究了不同大量元素、微量元素、有机物和碳源的组合，植物激素浓度，侵染培养基，乙酰丁香酮（AS）浓度和光周期对遗传转化效率的影响。【结果】在成熟种子组织培养试验中，MS大量元素、B5微量元素、N6有机物和麦芽糖为9311最优组合，华占最优组合为MS大量元素、MS微量元素、MS有机物和麦芽糖。诱导培养基中添加0.5 mg/L的BAP或1.5 mg/L的KT可显著提高9311和华占的组织培养再生率，可达70.0%以上。在侵染过程中，添加100 μmol/L AS的转化效率高于200 μmol/L AS。光周期实验显示9311和华占宜采用不同的光周期策略，9311在全黑暗条件下进行愈伤诱导和筛选，在全光照条件下进行分化，转化效率最高，达6.0%~6.4%，而华占在12 h光照/12 h黑暗条件下进行诱导、筛选和分化，获得的转化效率最高（5.0%~7.5%）。【结论】本研究优化了顽拗型籼稻9311和华占的组织培养和遗传转化体系，该体系也可以应用于其他相关籼稻基因型的遗传转化。

关键词: 顽拗型籼稻, 根癌农杆菌, 组织培养, 遗传转化

Abstract:

【Objective】 The rice transgenic technology is an important tool to promote rice molecular biology research and precision breeding. Agrobacterium-mediated transgenic technology has become the mainstream method for rice genetic transformation. Nevertheless, due to its genotype, the genetic transformation efficiency of typical indica rice is still quite low. It is urgent to improve its transformation efficiency. 【Method】 The effects of medium composition and photoperiod on genetic transformation efficiency of recalcitrated indica rice varieties 9311 and Huazhan were investigated. 【Result】 The results showed that MS macro element, B5 micro element, N6 organic matter and maltose were the best combination for 9311 tissue culture. MS macro element, MS micro element, MS organic matter and maltose were the best combination for Huazhan tissue culture. Adding 0.5 mg/L BAP or 1.5 mg/L KT into the induction medium could significantly improve the regeneration rate of 9311 and Huazhan tissue culture, up to more than 70.0%. The transformation efficiency with 100 μmol/L AS in the infection solution was higher than that with 200 μmol/L AS. Photoperiod experiments suggested that different photoperiod strategies should be adopted for 9311 and Huazhan transformation. 9311 transformation efficiency peaked, 6.0%?6.4%, under continuous darkness at the induction and selection stages and continuous light at the differentiation stage. Huazhan transformation efficiency reached the highest, 5.0%?7.5%, under 12 h photoperiod at all stages. 【Conclusion】 Collectively, the present study optimized tissue culture and genetic transformation system of recalcitrant indica rice varieties 9311 and Huazhan. Our methods could also be applied to the genetic transformation of other related indica rice genotypes.

Key words: recalcitrated indica rice, Agrobacterium tumefaciens, tissue culture, genetic transformation

张佳, 王慧杰, 何正权, 刘文真. 农杆菌介导的籼稻9311和华占遗传转化体系的研究[J]. 中国水稻科学, 2023, 37(2): 213-224.

ZHANG Jia, WANG Huijie, HE Zhengquan, LIU Wenzhen. Analysis of Agrobacterium-Mediated Genetic Transformation System of indica Rice 9311 and Huazhan[J]. Chinese Journal OF Rice Science, 2023, 37(2): 213-224.

图/表 11

参考文献 49

[1]	Ray D K, Mueller N D, West P C, Foley J A, Hart J P. Yield trends are insufficient to double global crop production by 2050[J]. PLoS ONE, 2013, 8(6): e66428.
[2]	Amini M, Deljou A, Nabiabad H S. Improvement of in vitro embryo maturation, plantlet regeneration and transformation efficiency from alfalfa somatic embryos using Cuscuta campestris extract[J]. Physiology & Molecular Biology of Plants, 2016, 22(3): 1-10.
[3]	Arun M, Chinnathambi A, Subramanyam K, Ganapathi K S. Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean[J]. 3 Biotech, 2016, 6(2): 1-12.
[4]	He Y, Jones H D, Chen S, Chen X M, Wang D S, Xia L Q. Agrobacterium -mediated transformation of durum wheat with improved efficiency[J]. Journal of Experimental Botany, 2010, 61(6): 1567-1581. PMID
[5]	Hiei Y, Komari T. Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens[J]. Plant Cell, Tissue and Organ Culture, 2006, 85(3): 271.
[6]	Hiei Y, Ohta S, Toshihiro K, Komari T. Efficient transformation of rice mediated by Agrobacterium and sequences analysis of the boundaries of the T-DNA[J]. Plant Journal, 1994, 6(2): 271-282. PMID
[7]	Daigen M, Kawakami O, Nagasawa Y. Efficient anther culture method of the japonica rice cultivar Koshihikari[J]. Breeding Science, 2000, 50(3): 197-202.
[8]	Ozawa K. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice[J]. Plant Science, 2009, 176(4): 522-527.
[9]	Ozawa K. A high-efficiency Agrobacterium-mediated transformation system of rice[J]. Methods in Molecular Biology, 2012, 847: 51-57.
[10]	Sallaud C, Meynard D, van Boxtel J, Gay C, Bès M, Brizard J P, Larmande P, Ortega D, Portefaix M R, Al E. Highly efficient production and characterization of T-DNA plants for rice functional genomics[J]. Theoretical and Applied Genetics, 2003, 106(8): 1396-1408. PMID
[11]	Chakraborty M, Reddy P S, Narasu M L, Rana K. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker[J]. Physiology and Molecular Biology of Plants, 2016, 22(1): 51-60. PMID
[12]	Hoque M E, Mansfield J W. Effect of genotype and explant age on callus induction and subsequent plant regeneration from root-derived callus of indica rice genotypes[J]. Plant Cell Tissue and Organ Culture, 2004, 78(3): 217-223.
[13]	Torrizo L B, Zapata F J. Anther culture in rice: IV. The effect of abscisic acid on plant regeneration[J]. Plant Cell Reports, 1986, 5(2): 136-139. PMID
[14]	Ge X J, Chu Z H, Lin Y J, Wang S. A tissue culture system for different germplasms of indica rice[J]. Plant Cell Reports, 2006, 25(5): 392-402. PMID
[15]	Tan L W, Rahman Z A, Goh H H, Hwang D J, Zainal Z. Production of transgenic rice overexpressing Abp57 gene through Agrobacterium-mediated transformation[J]. Sains Malaysiana, 2017, 46(5): 703-711.
[16]	Chu C C. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources[J]. Science in China, 1975, 18(5): 659.
[17]	Gamborg O L, Ojima K. Nutrient requirements of suspension cultures of soybean root cells[J]. Experimental Cell Research, 1968, 50(1): 151-158. PMID
[18]	Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures[J]. Physiologia Plantarum, 1962, 15(3): 473-497.
[19]	Dewir Y H, Naidoo Y, Dasilva J A T. Thidiazuron-induced abnormalities in plant tissue cultures[J]. Plant Cell Reports, 2018, 37(11): 1451-1470. PMID
[20]	Jimenez V M, Bangerth F. Hormonal status of maize initial explants and of the embryogenic and non-embryogenic callus cultures derived from them as related to morphogenesis in vitro[J]. Plant Science, 2001, 160(2): 247-257. PMID
[21]	Ramesh M, Gupta A K. Transient expression of β-glucuronidase gene in indica and japonica rice callus cultures after different stages of co-bombardment[J]. African Journal of Biotechnology, 2005, 4(7): 596-600.
[22]	Wani S H, Sanghera G S, Gosal S S. An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice variety PAU 201[J]. New Biotechnology, 2011, 28(4): 418-422.
[23]	别晓敏, 杜丽璞, 佘茂云, 徐惠君, 叶兴国. 不同生长素类型及ABA搭配对小麦幼胚再生效果的影响[J]. 核农学报, 2011, 25(5): 1023-1028.
	Bie X M, Du L P, She M Y, Xu H J, Ye X G. Effects of Different auxins and combining application with ABA on regeneration of immature embryos of wheat[J]. Journal of Nuclear Agricultural Sciences, 2011, 25(5): 1023-1028. (in Chinese with English abstract)
[24]	田文忠. 提高籼稻愈伤组织再生频率的研究[J]. 遗传学报, 1994, 21(3): 215-221.
	Tian W Z. Improvement of plant regeneration frequency in vitro in indica rice[J]. Journal of Genetics and Genomics, 1994, 21(3): 215-221. (in Chinese with English abstract)
[25]	Aldemita R R, Hodges T K. Agrobacterium tumefaciens -mediated transformation of japonica and indica rice varieties[J]. Planta, 1996, 199(4): 612-617.
[26]	Godwin I, Todd G, Ford-Lloyd B, Newbury H J. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species[J]. Plant Cell Reports, 1991, 9(12): 671-675. PMID
[27]	Smith R H, Hood E E. Agrobacterium tumefaciens transformation of monocotyledons[J]. Crop Science, 1995, 35(2): 301-309.
[28]	Clement W K F, Lai K S, Wong M Y, Maziah M. Heat and hydrolytic enzymes treatment improved the Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.)[J]. Plant Cell, Tissue and Organ Culture, 2015, 125(1): 183-190.
[29]	Yaqoob U, Kaul T, Nawchoo I A. Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties[J]. Indian Journal of Plant Physiology, 2017, 22(3): 346-353.
[30]	Lin Y J, Zhang Q. Optimising the tissue culture conditions for high efficiency transformation of indica rice[J]. Plant Cell Reports, 2005, 23(8): 540-547. PMID
[31]	Patel M, Dewey R E, Qu R. Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection[J]. Plant Cell, Tissue and Organ Culture, 2013, 114(1): 19-29.
[32]	Amoah B K, Wu H, Sparks C, Jones D H. Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue[J]. Journal of Experimental Botany, 2001, 52(358): 1135-1142. PMID
[33]	Shri M, Rai A, Verma P K, Misra P, Dubey S, Kumar S, Verma S, Gautam N. An improved Agrobacterium-mediated transformation of recalcitrant indica rice cultivars[J]. Protoplasma, 2013, 250(2): 631-636.
[34]	Toriyama K, Hinata K. Cell suspension and protoplast culture in rice[J]. Plant Science, 1985, 41(3): 179-183.
[35]	Basu A, Ray A, Sarkar S, Chaudhuri T R, Kundu S. Agrobacterium mediated genetic transformation of popular Indica rice Ratna (IET 1411) [J]. African Journal of Biotechnology, 2014, 13(31): 3187-3197.
[36]	Burman N, Chandran D, Khurana J P. A rapid and highly efficient method for transient gene expression in rice Plants[J]. Front Plant Science, 2020, 11: 584011-584022.
[37]	Sundararajan S, Sivaraman B, Rajendran V, Ramalingam S. Tissue culture and Agrobacterium-mediated genetic transformation studies in four commercially important indica rice cultivars[J]. Journal of Crop Science and Biotechnology, 2017, 20(3): 175-183.
[38]	Nguyen V C, Nguyen V K, Singh C H, Devi G S. Fast recovery of transgenic submergence tolerant rice cultivars of North-East India by early co-cultivation of Agrobacterium with pre-cultured callus[J]. Physiolgy and Molecular Biology of Plants, 2017, 23(1): 115-123.
[39]	郝建琴, 许语辉, 黄敏, 张伟漫, 刘伟利, 方标, 王振南, 王化琪. 山梨醇对栽培稻成熟胚愈伤组织分化的影响[J]. 农业生物技术学报, 2014, 22(8): 983-991.
	Hao J Q, Xu Y H, Huang M, Zhang W M, Liu W L, Fang B, Wang Z N, Wang H Q. Effect of sorbitol on callus regeneration from mature embryos of rice[J]. Journal of Agricultural Biotechnology, 2014, 22(8): 983-991. (in Chinese with English abstract)
[40]	沈秋平, 严孙艺, 薛宇彤, 张立朋, 吕尊富, 李飞飞. 籼稻9311成熟胚高效再生体系的建立[J]. 杂交水稻, 2019, 34(2): 49-52.
	Shen Q P, Yan S Y, Xue Y T, Zhang L P, Lü Z F, Li F F. Establishment of high-efficiency regeneration system from mature embryo as explants of indica rice 9311[J]. Hybrid Rice, 2019, 34(2): 49-52.
[41]	周永国, 尹中明, 沈忠伟, 李建粤. 籼稻9311成熟胚再生体系[J]. 上海师范大学学报, 2007, 36(4): 71-77.
	Zhou Y G, Yin Z M, Shen Z W, Li J Y. Study on the regeneration system from mature embryo of the indica rice 9311[J]. Journal of Shanghai Normal University, 2007, 36(4): 71-77. (in Chinese with English abstract)
[42]	Doyle J F, Dickson E E. Preservation of plant samples for DNA restriction endonuclease analysis[J]. Taxon, 1987, 36(4): 715-722.
[43]	Zaidi M A, Narayanan M, Sardana R, Taga I, Postel S, Johns R, Mcnulty M, Mottiar M, Mao J, Loit E. Optimizing tissue culture media for efficient transformation of different indica rice genotypes[J]. Agronomy Research, 2006, 4(2): 563-575.
[44]	易自力, 曹守云, 王力, 储成才, 李祥, 何锶洁, 唐祚舜, 周朴华, 田文忠. 提高农杆菌转化水稻频率的研究[J]. 遗传学报, 2001, 28(4): 352-358.
	Yi Z L, Cao S Y, Wang L, Chu C C, Li X, He S J, Tang Z S, Zhou P H, Tian W Z. Improvement of transformation frequency of rice mediated by Agrobacterium[J]. Journal of Genetics and Genomics, 2001, 28(4): 352-358. (in Chinese with English abstract)
[45]	李双成, 王世全, 尹福强, 邹良平, 起登风, 江洪, 李平. 提高农杆菌介导转化水稻效率的因素[J]. 中国水稻科学, 2005, 19(3): 231-237.
	Li S C, Wang S Q, Yin F Q, Zou L P, Qi D F, Jiang H, Li P. Some factors affecting Agrobacterium-mediated transformation frequencyin rice[J]. Chinese Journal of Rice Science, 2005, 19(3): 231-237. (in Chinese with English abstract)
[46]	Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho M J, Scelonge C, Lenderts B, Chamberlin M, Cushatt J. Morphogenic regulators baby boom and wuschel improve monocot transformation[J]. Plant Cell, 2016, 28(9): 1998-2015.
[47]	Wang K, Shi L, Liang X, Zhao P, Guo Y. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation[J]. Nature Plants, 2022. https://doi.org/10.1038/s41477-021-01085-8.
[48]	Debernardi J M, Tricoli D M, Ercoli M F, Hayta S, Dubcovsky J. A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants[J]. Nature Biotechnology, 2020, 38(11): 1-6.
[49]	Xiao K Z, Mao X H, Wang Y H, Wang J L, Wei Y D, Cai Q H, Xie H, Zhang J F. Transcript profiling reveals abscisic acid, salicylic acid and jasmonic-isoleucine pathways involved in high regenerative capacities of immature embryos compared with mature seeds in japonica rice[J]. Rice Science, 2018, 25(4): 57-64.

培养基 Medium	成分 Composition
愈伤诱导培养基 Callus induction medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L Phytagel
侵染培养基 Infection medium	AA大量+AA微量+AA有机+20 g/L蔗糖+10 g/L葡萄糖+铁盐+0.5 g/L酸水解酪蛋白+876 mg/L L-谷氨酰胺+174 mg/L 精氨酸+260 mg/L天冬氨酸 AA Macro+AA Micro+AA Vitamins+20 g/L sucrose+10 g/L glucose+Fe-Na-EDTA+0.5 g/L acid hydrolyzed casein +876 mg/L L-glutamine +174 mg/L arginine +260 mg/L aspartic acid
选择培养基 Selection medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+30 g/L甘露醇+2.5 mg/L 2,4-D+0.5 mg/L BAP +5 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+30 g/L mannitol+2.5 mg/L 2,4-D+0.5 mg/L BAP+5 g/L Phytagel
分化培养基 Regeneration medium	MS大量+MS微量+B5有机+麦芽糖+铁盐+2 g/L酸水解酪蛋白+30 g/L山梨醇+0.2 mg/L NAA +4 mg/L BAP+5 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Maltose+Fe-Na-EDTA+2 g/L acid hydrolyzed casein+30 g/L sorbitol +0.2 mg/L NAA+4 mg/L BAP+5 g/L Phytagel MS大量+MS微量+B5有机+铁盐+30 g/L葡萄糖+0.5 mg/L IBA+4 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Fe-Na-EDTA+30 g/L glucose+0.5 mg/L IBA+4 g/L Phytagel
生根培养基 Rooting medium
YEM培养基 YEM medium	10 g/L甘露醇+2 g/L C-谷氨酰胺+0.5 g/L磷酸二氢钾+0.2 g/L氯化钠+0.2 g/L硫酸镁+0.3 g/L酵母提取物+15 g/L Agar 10 g/L mannitol +2 g/L C-glutamine +0.5 g/L monopotassium phosphate+0.2 g/L sodium chloride+0.2 g/L magnesium sulfate +0.3 g/L yeast extract +15 g/L Agar

培养基 Medium	成分 Composition
愈伤诱导培养基 Callus induction medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+2.5 mg/L 2, 4-D+0.5 mg/L BAP+1.5 mg/L ABA +3.9 g/L Phytagel
侵染培养基 Infection medium	AA大量+AA微量+AA有机+20 g/L蔗糖+10 g/L葡萄糖+铁盐+0.5 g/L酸水解酪蛋白+876 mg/L L-谷氨酰胺+174 mg/L 精氨酸+260 mg/L天冬氨酸 AA Macro+AA Micro+AA Vitamins+20 g/L sucrose+10 g/L glucose+Fe-Na-EDTA+0.5 g/L acid hydrolyzed casein +876 mg/L L-glutamine +174 mg/L arginine +260 mg/L aspartic acid
选择培养基 Selection medium	MS大量+B5微量+N6有机+麦芽糖+铁盐+0.3 g/L酸水解酪蛋白+2.87 g/L脯氨酸+30 g/L甘露醇+2.5 mg/L 2,4-D+0.5 mg/L BAP +5 g/L植物凝胶 MS Macro+B5 Micro+N6 Vitamins+Maltose+Fe-Na-EDTA+0.3 g/L acid hydrolyzed casein+2.87 g/L Proline+30 g/L mannitol+2.5 mg/L 2,4-D+0.5 mg/L BAP+5 g/L Phytagel
分化培养基 Regeneration medium	MS大量+MS微量+B5有机+麦芽糖+铁盐+2 g/L酸水解酪蛋白+30 g/L山梨醇+0.2 mg/L NAA +4 mg/L BAP+5 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Maltose+Fe-Na-EDTA+2 g/L acid hydrolyzed casein+30 g/L sorbitol +0.2 mg/L NAA+4 mg/L BAP+5 g/L Phytagel MS大量+MS微量+B5有机+铁盐+30 g/L葡萄糖+0.5 mg/L IBA+4 g/L植物凝胶 MS Macro+MS Micro+B5 Vitamins+Fe-Na-EDTA+30 g/L glucose+0.5 mg/L IBA+4 g/L Phytagel
生根培养基 Rooting medium
YEM培养基 YEM medium	10 g/L甘露醇+2 g/L C-谷氨酰胺+0.5 g/L磷酸二氢钾+0.2 g/L氯化钠+0.2 g/L硫酸镁+0.3 g/L酵母提取物+15 g/L Agar 10 g/L mannitol +2 g/L C-glutamine +0.5 g/L monopotassium phosphate+0.2 g/L sodium chloride+0.2 g/L magnesium sulfate +0.3 g/L yeast extract +15 g/L Agar

培养基名称 Media	基本培养基 Basal media（BM）			碳源 Carbon source	平均愈伤诱导率 Average frequency of callus induction / %		组织培养再生率 Regeneration rate of tissue culture / %
培养基名称 Media	大量 Macro	微量 Micro	有机 Vitamin	碳源 Carbon source	9311	华占Huazhan	9311	华占 Huazhan
RBM₁	N6	N6	N6	蔗糖Sucrose	76.0±4.0	81.3±1.2	14.6±2.1 ab	2.7±2.7 bc
RBM₂	N6	MS×5	MS	麦芽糖Maltose	78.3±0.7	75.7±1.7	2.7±2.7 c	7.8±2.2 ab
RBM₃	N6	B5	B5	混合 Mixed	69.7±3.5	79.5±5.5	7.2±2.9 abc	2.4±2.4 bc
RBM₄	MS	N6	MS	混合 Mixed	69.3±4.8	77.0±1.0	15.9±4.1 ab	13.0±2.4 a
RBM₅	MS	MS×5	B5	蔗糖Sucrose	82.7±2.0	71.3±2.6	5.2±0.4 bc	8.9±2.2 ab
RBM₆	MS	B5	N6	麦芽糖Maltose	78.0±5.3	68.0±3.0	20.0±5.5 a	8.5±2.6 ab
RBM₇	DKN	N6	B5	麦芽糖Maltose	48.7±3.5	56.7±3.4	0.0±0.0 c	0.0±0.0 c
RBM₈	DKN	MS×5	N6	混合 Mixed	49.3±3.2	53.7±2.3	0.0±0.0 c	0.0±0.0 c
RBM₉	DKN	B5	MS	蔗糖Sucrose	49.0±1.5	57.0±1.0	0.0±0.0 c	0.0±0.0 c

培养基名称 Media	基本培养基 Basal media（BM）			碳源 Carbon source	平均愈伤诱导率 Average frequency of callus induction / %		组织培养再生率 Regeneration rate of tissue culture / %
培养基名称 Media	大量 Macro	微量 Micro	有机 Vitamin	碳源 Carbon source	9311	华占Huazhan	9311	华占 Huazhan
RBM₁	N6	N6	N6	蔗糖Sucrose	76.0±4.0	81.3±1.2	14.6±2.1 ab	2.7±2.7 bc
RBM₂	N6	MS×5	MS	麦芽糖Maltose	78.3±0.7	75.7±1.7	2.7±2.7 c	7.8±2.2 ab
RBM₃	N6	B5	B5	混合 Mixed	69.7±3.5	79.5±5.5	7.2±2.9 abc	2.4±2.4 bc
RBM₄	MS	N6	MS	混合 Mixed	69.3±4.8	77.0±1.0	15.9±4.1 ab	13.0±2.4 a
RBM₅	MS	MS×5	B5	蔗糖Sucrose	82.7±2.0	71.3±2.6	5.2±0.4 bc	8.9±2.2 ab
RBM₆	MS	B5	N6	麦芽糖Maltose	78.0±5.3	68.0±3.0	20.0±5.5 a	8.5±2.6 ab
RBM₇	DKN	N6	B5	麦芽糖Maltose	48.7±3.5	56.7±3.4	0.0±0.0 c	0.0±0.0 c
RBM₈	DKN	MS×5	N6	混合 Mixed	49.3±3.2	53.7±2.3	0.0±0.0 c	0.0±0.0 c
RBM₉	DKN	B5	MS	蔗糖Sucrose	49.0±1.5	57.0±1.0	0.0±0.0 c	0.0±0.0 c

基本培养基 Basal medium	处理 Treatment	植物激素浓度 Phytohormone concentration / (mg L⁻¹)				组织培养再生率 Regeneration rate of tissue culture / %
基本培养基 Basal medium	处理 Treatment	BAP	KT	TDZ	ABA	9311	华占 Huazhan
BM₂	T₁	0.25	0.00	0.10	0.00	11.3±3.0 bc	6.0±1.7 d
	T₂	0.25	0.75	0.00	1.50	12.6±3.5 bc	18.5±3.5 bc
	T₃	0.25	1.50	0.20	3.00	39.0±8.2 a	31.9±2.7 a
	T₄	0.00	0.00	0.00	3.00	1.7±1.7 c	0.0±0.0 e
	T₅	0.00	0.75	0.20	0.00	2.4±2.4 c	8.9±1.8 cd
	T₆	0.00	1.50	0.10	1.50	34.6±5.4 a	24.4±4.8 ab
	T₇	0.50	0.00	0.20	1.50	22.2±4.1 b	14.9±3.6 cd
	T₈	0.50	0.75	0.10	3.00	9.2±1.5 c	9.4±3.0 cd
	T₉	0.50	1.50	0.00	0.00	1.5±1.5 c	26.2±0.4 ab