糜子愈伤诱导及再分化最适条件研究

doi:10.11924/j.issn.1000-6850.casb18120078

摘要/Abstract

摘要：

研究旨在应用组织培养技术探究愈伤诱导及再分化的最适条件,以期为糜子建立遗传转化体系奠定基础。采用5个糜子品种研究愈伤诱导及再分化的最适条件。利用植物激素2,4-二氯苯氧乙酸(2,4-D)诱导茎尖愈伤组织,筛选出发芽率高、愈伤组织诱导率高、染菌率低的品种,并对诱导愈伤最适激素浓度及配比进行了鉴定。结果显示,‘冀黍2号’出芽率高、愈伤组织诱导率高、染菌率低,适宜进行组织培养;2,4-D对‘冀黍2号’愈伤诱导效果最好,最适浓度为2.5 mg/L,诱导率达86.67%,淡黄色块状,结构紧密,质地较硬,继代培养后形成的胚性愈伤组织状态更好。再分化过程中,2.5 mg/L 2,4-D+3.5 mg/L TDZ时,出现明显的嫩芽。该试验获得‘冀黍2号’愈伤诱导及再分化的最适条件,可为糜子再生体系的构建提供参考。

关键词: 糜子, 愈伤诱导, 再分化, 茎尖, 植物激素, 组织培养

Abstract:

The aim is to explore the optimum condition for callus induction and redifferentiation by tissue culture technology, so as to lay a foundation for the establishment of genetic transformation system of Panicum miliaceum L.. Five Panicum miliaceum L. varieties were used to study the optimal condition for callus induction and redifferentiation. Plant hormone 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to induce shoot tip callus. Varieties with high germination rate, high callus induction rate and low bacterial infection rate were screened. The optimum hormone concentration and ratio for inducing callus were identified. The results showed that ‘Jishu No. 2’ had high germination and callus induction rates and low bacterial infection rate, showing it was suitable for tissue culture. 2,4-D had the best induction effect on ‘Jishu No. 2’, with the optimum concentration of 2.5 mg/L and the induction rate of 86.67%. The callus was light yellow block, compact in structure and hard in texture. The embryogenic callus formed after subculture was better than that of other varieties. In the process of redifferentiation, when the culture condition was 2.5 mg/L 2,4-D + 3.5 mg/L TDZ, tender buds appeared obviously. The optimum condition for callus induction and redifferentiation of ‘Jishu No. 2’ are obtained, which could provide reference for constructing the regeneration system of Panicum miliaceum L..

Key words: Panicum miliaceum, callus induction, redifferentiation, shoot tip, plant hormones, tissue culture

中图分类号:

S516

王春芳, 刘晶晶, 李伟, 栾素荣, 史慎奎, 刘国庆. 糜子愈伤诱导及再分化最适条件研究[J]. 中国农学通报, 2020, 36(9): 94-99.

Chunfang Wang, Jingjing Liu, Wei Li, Surong Luan, Shenkui Shi, Guoqing Liu. The Optimal Condition for Callus Induction and Redifferentiation of Panicum miliaceum L.[J]. Chinese Agricultural Science Bulletin, 2020, 36(9): 94-99.

图/表 5

参考文献 28

[1]	Lu H, Zhang J, Liu K, et al.Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009,106:7367-7372.
[2]	Lu C, Vasil I K.Somatic embryogenesis and plant regeneration from freely suspended cells and cell groups of Panicum maximum Jacq.[J]. Ann. Bot., 1981,48:543-548.
[3]	曹晓宁,王君杰,王海岗,等. 糜子栽培研究进展.[J]. 安徽农业科学,2015(31):79-81,84.
[4]	曹孜义,郭彩月,郝建军.糜子组织培养生成植株.[J]. 甘肃农业科技, 1980(06):13,41,12.
[5]	孙宇慧,刘天相,石善党,等.小麦穗粒数及千粒重主效QTL共定位区QC-7AL的精细定位及遗传效应分析[J].麦类作物学报,2018,38(11):1288-1292.
[6]	Hunt H V, Badakshi F, Romanova O, et al.Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P. miliaceum[J]. Journal of experimental botany, 2014, 65(12):3165-3175.
[7]	Santosh G R, Dipak S, James S.Mapping QTLs for morpho-agronomic traits in proso millet (Panicum miliaceum L.)[J]. Molecular Breeding,2016,15(09):1955-1967.
[8]	Cho Y I, Chung J W, Lee G A, et al.Development and characterization of twenty-five new polymorphic microsatellite markers in proso millet (Panicum miliaceum L.)[J]. Genes & Genomics, 2010,32(3):267-273.
[9]	Rajput S G, Santra D K.Evaluation of genetic diversity of proso millet germplasm available in the United States using simple-sequence repeat markers[J]. Crop Science, 2016, 56(5):2401-2409.
[10]	Rajput S G, Plyler-Harveson T, Santra D K.Development and characterization of SSR markers in proso millet based on switchgrass genomics[J]. American Journal of Plant Sciences, 2014,5(01):175.
[11]	Wang R Y, Ji X, Lu P, et al.Analysis of genetic diversity in common millet (Panicum miliaceum) using fluorescent SSR in China[J]. Acta Agronomica Sinica, 2017,43(4):530-548.
[12]	Wang R, Liu X, Wang H, et al.Evaluation of genetic diversity of common millet (Panicum miliaceum) germplasm available in China using high motif nucleotide repeat SSR markers[J]. Scientia Agricultura Sinica, 2017,50(20):3848-3870.
[13]	Wang R Y, Wang H G, Liu X H, et al.Waxy allelic diversity in common millet (Panicum miliaceum L.) in China[J]. The Crop Journal, 2018, 6(04):377-385.
[14]	罗士韦.植物组织培养在花卉上的应用.[J]. 植物生理学通讯,1979(03):1-9.
[15]	罗士韦.第七讲植物组织与细胞培养概况.[J]. 植物生理学通讯,1979(03):47-49,44.
[16]	罗士韦.中国植物组织细胞培养的应用.[J]. 陕西林业科技,1979(Z1):63.
[17]	Rangan T S, Vasil I K.Somatic embryogenesis and plant regeneration in tissue cultures of Panicum miliaceum L. and Panicum miliare Lamk[J]. Zeitschrift für Pflanzenphysiologie, 1983,109(1):49-53.
[18]	张树录,郑国锠.糜子离体体细胞胚胎发生和植株再生Ⅱ.各种因素的影响.[J]. 西北植物学报,1987(04):239-245.
[19]	袁进成,刘颖慧.谷子的组织培养和遗传转化研究进展.[J]. 安徽农业科学,2009(23):10889-10890.
[20]	丰锋,陈建国,李洪波.植物组织培养无菌操作技术.[J]. 中国花卉盆景, 2002(01): 30-31.
[21]	王节之,张海风,郑向阳,等. 常用激素对谷子幼穗组培的影响.[J]. 山西农业科学,1998(03):40-43.
[22]	许智宏,卫志明,杨丽君.谷子和狗尾草的幼穗培养.[J]. 植物生理学通讯,1983(05):40.
[23]	袁进成,刘颖慧,董志平.谷子成熟胚诱导愈伤组织及植株再生的研究和条件的优化.[J]. 生物技术通报,2013(03):77-82.
[24]	王寒玉. 谷子愈伤诱导优化及转化影响因素分析[D].保定:河北农业大学,2012.
[25]	李惠. 谷子不同外植体再生体系建立及优化的研究[D].太谷:山西农业大学,2015.
[26]	刁现民,段胜军,陈振玲,等. 谷子体细胞无性系变异分析.[J]. 中国农业科学,1999(03):23-24,26-28.
[27]	Lu C Y, Chandler, Stephen F, Vasil I K. Somatic Embryogenesis and Plant Regeneration from Cultured Immature Embryos of Rye(Secale cereale L.) 1984, 237-244.
[28]	王寒玉,杜艳伟,赵晋锋,等.谷子茎尖愈伤诱导优化及转化因素浅析[J].中国农业科技导报,2012,14(3):36-42.

名称	发芽率/%	愈伤组织诱导率/%	愈伤生长状况	染菌率/%
冀黍1号	80.00	55.00	淡黄色颗粒状	2.80
冀黍2号	92.00	89.00	淡黄色块状,结构紧密,质地较硬	1.00
龙黍18	78.00	71.00	淡黄色块状,质地松软	2.10
龙黍21	83.00	80.00	白色块状,质地紧密	3.33
龙黍23	88.00	37.00	淡黄色块状,结构紧密,质地较硬	4.20

名称	发芽率/%	愈伤组织诱导率/%	愈伤生长状况	染菌率/%
冀黍1号	80.00	55.00	淡黄色颗粒状	2.80
冀黍2号	92.00	89.00	淡黄色块状,结构紧密,质地较硬	1.00
龙黍18	78.00	71.00	淡黄色块状,质地松软	2.10
龙黍21	83.00	80.00	白色块状,质地紧密	3.33
龙黍23	88.00	37.00	淡黄色块状,结构紧密,质地较硬	4.20

处理	激素配比	处理	激素配比
T1	2,4-D	T4	2,4-D+ZT (1:1)
T2	NAA	T5	2,4-D+TDZ (1:1)
T3	ZT	T6	NAA+6BA (1:1)

处理	激素配比	处理	激素配比
T1	2,4-D	T4	2,4-D+ZT (1:1)
T2	NAA	T5	2,4-D+TDZ (1:1)
T3	ZT	T6	NAA+6BA (1:1)

处理	浓度/(mg/L)	愈伤生长量	愈伤生长状况	愈伤组织诱导率/%	生根率/%	染菌率/%
2,4D	0.5	+++	黄绿色颗粒状,结构致密	46.67	—	—
	1.0	+++	黄绿色颗粒状,结构较致密	50	—	—
	1.5	+++	黄绿色块状,结构致密	60	—	—
	2.0	+++	绿色和暗黄色块状,结构较致密	68.33	—	—
	2.5	++++	淡绿色块状,结构致密	86.67	—	—
	3.0	+++	黄绿色和红色块状,结构致密	54.67	—	—
NAA	0.5	++	白色小颗粒状,结构疏松,生根	43.67	26.33	—
	1.0	+++	白色小颗粒状,结构疏松,生根	53.00	33.00	—
	1.5	+++	黄绿色分散状,愈伤较大,生根	93.00	53.67	—
	2.0	++++	淡黄色分散状,结构疏松,生根	80.67	43.00	—
	2.5	+++	绿色和暗黄色块状,结构较致密	88.00	98.00	—
2,4-D+TDZ	0.5	++	白色小颗粒状,结构疏松	12.33	—	0.00
	1.0	++	白色小颗粒状,结构疏松	10.33	—	5.67
	1.5	++	黄绿色分块状,愈伤较大,	45.00	—	13.00
	2.0	++	淡黄色块状,结构紧密,叶片生长	21.00	—	14.33
	2.5	++	绿色和暗黄色块状,结构较致密,有叶片生长	18.33	—	8.00
	3.0	++	绿黄色块状,结构较致密,有部分褐化	25.67	—	14.00