新疆海岛棉胚性愈伤组织培养体系

doi:10.6048/j.issn.1001-4330.2022.06.003

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (6): 1321-1329.DOI: 10.6048/j.issn.1001-4330.2022.06.003

• 作物道传育种·耕作栽培·生理生化 • 上一篇下一篇

新疆海岛棉胚性愈伤组织培养体系

杨静¹(), 王为然¹, 王萌¹, 朱家辉¹, 宁新民¹, 阿里甫·艾尔西¹, 闵玲²(), 孔杰¹^,³()

1.新疆农业科学院经济作物研究所,乌鲁木齐 830091
2.华中农业大学作物遗传改良国家重点实验室,武汉 430070
3.国家棉花工程技术研究中心,乌鲁木齐 830091

收稿日期:2021-10-11 出版日期:2022-06-20 发布日期:2022-07-07
通信作者: 闵玲,孔杰
作者简介:杨静(1989-),女,陕西宁强人,助理研究员,研究方向为长绒棉遗传育种. (E-mail) hzjingy@126.com
基金资助:
国家自然科学基金新疆联合基金(U2003209);新疆维吾尔自治区重大科技专项(2021A02001-4);新疆农科院青年科技骨干培养(xjnkq-2019008);新疆长绒棉育种联合攻关项目(2022-2-2);棉花产业体系(CARS-15-09);天山英才计划(2021)

Preliminary Study on the Embryogenic Callus Culture System of Xinjiang Island Cotton (G. barbadense L. )

YANG Jing¹(), WANG Weiran¹, WANG Meng¹, ZHU Jiahui¹, NING Xinmin¹, Alifu Aierxi¹, MIN Ling²(), KONG Jie¹^,³()

1. Institute of Economic Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
2. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
3. National Engineering Research Center of Cotton, Urumqi 830091, China

Received:2021-10-11 Online:2022-06-20 Published:2022-07-07
Correspondence author: MIN Ling, KONG Jie
Supported by:
NSFC-United fund of Xinjiang(U2003209);Xinjiang Major Science and Technology Projects(2021A02001-4);Young Sci-Tech Talents Projects of XAAS(xjnkq-2019008);XinJiang Joint Research on the breeding of long-staple cotton(2022-2-2);China Agriculture Research System(CARS-15-09);Tianshan Talents Project(2021)

摘要/Abstract

摘要：

【目的】 研究新疆海岛棉不同材料愈伤组织培养体系,筛选再生能力强的海岛棉材料,为新疆海岛棉基因工程育种提供参考。【方法】 以105份新疆海岛棉为材料,利用改良后的MS培养基对下胚轴共培养,在2,4-D诱导培养基中诱导分化产生愈伤组织,浸染农杆菌介导的红色荧光蛋白,验证转化效果。【结果】 海岛棉材料愈伤组织的增殖速率与其基因型有关,改良后的MS共培养基、2,4-D诱导培养基可有效用于新疆海岛棉共培养和胚性愈伤组织增殖;愈伤组织增殖的适宜培养时间为4个月,时间过长不利于其增殖;愈伤增殖速度与转化率呈正相关,增殖快的材料其红色荧光蛋白的转化率较高;筛选出2份增殖快且转化率好的海岛棉材料。【结论】 建立了新疆海岛棉胚性愈伤组织培养体系与鉴定方法,筛选出9份愈伤增殖好、且能够有效转化的海岛棉材料。

关键词: 海岛棉; 胚性愈伤组织; 组织培养; 农杆菌

Abstract:

【Objective】 To explore the callus culture system of different materials of Xinjiang island cotton and to select variety with strong regeneration ability.【Method】 With 105 accessions taken as research materials, hypodermal axis was cocultured with modified MS media, callus differentiation was induced in the 2,4-d-induced medium. After that, the conversion effect was verified by immersion-mediated red fluorescent protein.【Result】 The proliferation rate of the callus in island cotton was related to different genotype. The improved MS co-medium and 2,4-D induction medium could be effective for the co-culture and embryogenic callus proliferation of Xinjiang island cotton. The appropriate culture time was 4 months for callus proliferation, and long time was not benefit for proliferation. Callus proliferation speed was positively correlated with the conversion rate, showing the material had a higher conversion rate of red fluorescent protein with the fast proliferation. Altogether, two island cotton accessions with fast multiplication and good conversion rate were selected. 【Conclusion】 In this study, the embryogenic callus culture system and identification method of Xinjiang island cotton are initially established, island cotton materials that have 9 good callus proliferation are screened out and that can be effectively transformed.

Key words: G. barbadense L.; embryogenic callus; tissue culture; agrobacterium

中图分类号:

S562

杨静, 王为然, 王萌, 朱家辉, 宁新民, 阿里甫·艾尔西, 闵玲, 孔杰. 新疆海岛棉胚性愈伤组织培养体系[J]. 新疆农业科学, 2022, 59(6): 1321-1329.

YANG Jing, WANG Weiran, WANG Meng, ZHU Jiahui, NING Xinmin, Alifu Aierxi, MIN Ling, KONG Jie. Preliminary Study on the Embryogenic Callus Culture System of Xinjiang Island Cotton (G. barbadense L. )[J]. Xinjiang Agricultural Sciences, 2022, 59(6): 1321-1329.

图/表 8

表1 海岛棉试验材料

Table 1 The experiment accessions of island cotton

材料编号Material code
907	947	966	974	984	1011	1028	1046	1061	1074	1086	1094	1108	1119
920	954	967	975	987	1012	1036	1050	1062	1075	1087	1098	1109
921	955	968	976	995	1013	1040	1051	1063	1076	1088	1099	1111
922	960	969	977	998	1014	1041	1052	1064	1080	1089	1101	1112
923	961	970	979	999	1016	1042	1056	1065	1081	1090	1102	1113
926	962	971	980	1007	1017	1043	1058	1067	1083	1091	1103	1115
927	964	972	981	1008	1024	1044	1059	1070	1084	1092	1104	1116
933	965	973	982	1010	1025	1045	1060	1073	1085	1093	1105	1118

图1 海岛棉愈伤组织诱导转化过程注:A.陆地棉Jin668无菌苗(CK);B.Jin668下胚轴;C.海岛棉无菌苗;D~F.923、971、962下胚轴。Bars 值均为1 cm

Fig.1 Process of callus induction and transformin island cotton Note: A.The aseptic seedling of Jin668; B.Jin668 hypocotyl; C.The aseptic seedling of island cotton; D-F.Hypocotyl of 923,971,962.Bars value is 1 cm

图2 愈伤组织增殖注:A.未浸染农杆菌的陆地棉Jin668(CK)的愈伤组织;B~C,E~F.培养4个月后的愈伤组织;D.Jin668浸染农杆菌后培养4个月的愈伤组织。Bars值均为1 cm

Fig.2 Callus induction and proliferation Note: A.Callus of upland cotton Jin668 (CK) not infested with agrobacterium; B-C,E-F, Callus of 923,986,1007 and 972 after 4 months of culture,respectively.D.The callus of Jin668 after 4 months of cultivation inoculation with agrobacterium.Bars value is 1 cm

图3 海岛棉材料下胚轴增殖变化

Fig.3 Variation of hypocotyledonary axis proliferation different accessions of island cotton

表2 不同诱导时间下胚轴愈伤组织质量(部分)

Table 2 Callus quality of hypocotyl at different induction time

材料编号 Code	诱导4个月增重 Add weight after 4 months(g)	愈伤颜色质地 Callus of color and phenotype	愈伤细胞形态 Callus of cell morphology	诱导6个月增重 Add weight after 6 months(g)	愈伤颜色质地 Callus of color and phenotype	愈伤细胞形态 Callus of cell morphology
Jin668	0.26±0.08	浅黄,疏松	长柱、近球形	0.35±0.04	浅棕,疏松	长柱形
962	0.13±0.02	浅黄色,疏松	同上	0.64±0.15	浅黄,疏松	近球、长柱形
1010	0.22±0.04	粉白色,硬	同上	0.83±0.15	浅绿,硬	同上
1025	0.35±0.06	浅绿色,疏松	同上	0.47±0.10	浅绿色,疏松	同上
1052	0.24±0.07	浅绿色,硬	同上	0.35±0.13	红褐色,疏松	同上
964	0.20±0.06	绿色,疏松	近球形	1.10±0.14	浅黄,软	近球形
986	0.12±0.03	绿色,硬	同上	0.45±0.10	浅黄,疏松	同上
1011	0.16±0.06	浅绿,硬	同上	0.29±0.04	浅黄,硬	同上
1086	0.10±0.01	浅绿,疏松	同上	0.18±0.03	浅绿,硬	同上
1103	0.18±0.03	同上	同上	0.39±0.05	浅绿,硬	同上

图4 愈伤组织增殖较好的海岛棉注:A~H分别为962,964,1010,1011,1025,1052,1086和1103培养4个月后的愈伤组织。Bars值均为1 cm

Fig.4 The better callus induction and proliferation of accessions Note:A.The callus of 962,964,1010,1011,1025,1052,1086 and 1103 after 4 months of cultivation,respectively.Bars value is 1 cm

图5 海岛棉红色荧光蛋白转化注:A~D.Jin668、964、1086和1052培育4个月后的愈伤组织;E~H.Jin668、964、1086和1052培育4个月愈伤组织中的红色荧光蛋白;I~L.Jin668、964、1086和1052培育4个月后愈伤组织的细胞形态(甲苯胺蓝染色).A,B,C,D的bars值为1cm,E、F、G、H、I、J、K、L为100 μm

Fig.5 Observation of red fluorescence protein transform in island cotton Note:A-D.Callus of Jin668,964,1086 and 1052 cultivated 4 Callus after 4 months, respectively; E-H.Jin668,964,1086 and 1052 after 4 months of cultivation of red fluorescent protein in callus; I-L.Jin668, 964,1086 and 1052 after 4 months of cultivation tissue cell morphology (toluidine blue staining), respectively.Bars value of A, B, C, D is 1 cm, and E, F, G, H,I,J,K, L is 100 μm

图6 红色荧光蛋白转化率较低的下胚轴增殖变化注:A,D.海岛棉1010和1103培育4个月后的愈伤组织;B,E.1010和1103培育4个月后愈伤组织中的红色荧光蛋白;C,F:1010和1103培育4个月后愈伤组织的细胞形态(甲苯胺蓝染色).A和D的bars值为1 cm,B、C、E、F为100 μm

Fig.6 Observation of lower transform ratio of red fluorescence protein in island cotton Note:A, D: Island cotton 1010and 1103 callus after 4 months of cultivation; B, E: 1010 and1103 callus after 4 months of cultivation of red fluorescence protein; C, F: 1010 and 1103 callus cell morphology after 4 months of cultivation (toluidine blue staining).Bars value of A and D is 1cm, B, C, E, and F is 100 μm

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新疆海岛棉胚性愈伤组织培养体系

Preliminary Study on the Embryogenic Callus Culture System of Xinjiang Island Cotton (G. barbadense L. )

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