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作物学报 ›› 2024, Vol. 50 ›› Issue (5): 1172-1180.doi: 10.3724/SP.J.1006.2024.34167

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

陆地棉重组自交系再生能力和遗传转化效率筛选

乐愉(), 王涛, 张献龙, 林忠旭*()   

  1. 华中农业大学植物科学技术学院 / 作物遗传改良全国重点实验室, 湖北武汉 430070
  • 收稿日期:2023-10-16 接受日期:2024-01-12 出版日期:2024-05-12 网络出版日期:2024-02-07
  • 通讯作者: 林忠旭, E-mail: linzhongxu@mail.hzau.edu.cn
  • 作者简介:E-mail: leyu_hzau@163.com
  • 基金资助:
    国家自然科学基金项目(32172025)

Screening of regeneration capacity and genetic transformation efficiency in recombinant inbred lines of Gossypium hirsutum L.

LE Yu(), WANG Tao, ZHANG Xian-Long, LIN Zhong-Xu*()   

  1. College of Plant Science & Technology, Huazhong Agricultural University / National Key Laboratory of Crop Genetic Improvement, Wuhan 430070, Hubei, China
  • Received:2023-10-16 Accepted:2024-01-12 Published:2024-05-12 Published online:2024-02-07
  • Contact: E-mail: linzhongxu@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32172025)

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摘要：

转基因工程育种是棉花种质创新的有效手段, 为了拓展陆地棉可再生基因型, 丰富棉花转基因受体, 本研究以湖北省高产阔叶棉品种‘鄂棉22’ (E22)为母本、高再生能力的鸡脚叶品种‘豫早1号’ (YZ1)为父本, 通过单籽传法构建了F9重组自交系群体(YE); 利用棉花中比较成熟的IBA+KT (IK)和2,4-D+KT (DK) 2种不同激素组合的培养体系, 分别对重组自交系群体的164个家系的愈伤组织诱导率(CIF)、愈伤组织继代繁殖力(CSC)、愈伤组织的出胚率(CRE)以及愈伤组织出胚时间(CET)进行比较, 共获得12个可再生的阔叶棉家系; 利用目前成熟的棉花遗传转化体系对可再生阔叶棉家系进行遗传转化效率分析, 同时对其进行田间农艺性状考察, 最终获得了一个遗传转化效率为82.9%并且农艺性状更优良的家系YE3。本研究为棉花的遗传转化及基因功能研究拓展了陆地棉基因型种质资源。

关键词: 棉花, 重组自交系, 体细胞胚胎发生, 再生能力, 遗传转化效率

Abstract:

Transgenic engineered breeding is an effective way of cotton germplasm innovations. In order to expand the renewable genotypes of upland cotton and to enrich cotton transgenic receptors, we constructed an F9 recombinant inbred population (YE) by the single-seed descent method using the high-yielding broad leaf cotton variety Emian 22 (E22) from Hubei province as the male parent and the high regeneration-capable okra leaf variety Yuzao 1 (YZ1) as the female parent. We compared the callus induction rate (CIF), callus secondary fecundity (CSC), embryo rate of callus (CRE), and callus embryo time (CET) of 164 recombinant inbred lines using two different hormone combinations of IBA+KT (IK) and 2,4-D+KT (DK), respectively, which were mature tissue culture systems in cotton. A total of 12 regenerable broad leaf lines were obtained and evaluated for the genetic transformation efficiency using the current mature genetic transformation system in cotton; meanwhile, agronomic traits of these lines were investigated. Finally, a family line YE3, with a genetic transformation efficiency of 82.9% and better agronomic traits, was obtained. This study develops a new genotype of upland cotton, which will facilitate the genetic transformation and gene function research of cotton.

Key words: cotton, RILs, somatic embryogenesis, regeneration capacity, transformation efficiency

图1

IK体系下棉花体细胞胚胎发生和植株再生流程 A: 棉花下胚轴; B: 1~2个月的愈伤组织; C: 胚性愈伤组织, 伴有子叶型胚状体; D: 大量胚状体形成; E: 植株再生; F: 水培后可移栽。"

图2

DK体系下棉花体细胞胚胎发生和植株再生流程 A: 棉花下胚轴; B: 1~2个月的愈伤组织; C: 胚性愈伤组织, 无胚状体; D: 大量胚状体形成; E: 植株再生; F: 水培后可移栽。"

图3

IK和DK体系下愈伤组织增殖的频率分布图 CSC: 愈伤组织继代繁殖力。"

图4

IK和DK体系下可出胚家系再生相关性状的频率分布图 A: CET, 愈伤组织最早出现的时间; B: CRE, 愈伤组织出胚率。"

表1

再生YE家系CSC、CRE和CET比较"

家系a
Lines a		 叶形
Shape of leaves		 培养体系b
Culture system b		 愈伤组织继代繁殖力
CSC (g)		 愈伤组织出胚率CRE (%) 愈伤组织出胚时间
CET (d)
90 d 180 d
E22 阔叶
Broad leaf		 IBA+KT 3.03 0 0 -
2,4-D+KT 5.56 - 33.3 189
YZ1 鸡脚叶
Okra leaf		 IBA+KT 4.14 70 75 70
2,4-D+KT 4.51 67.5 100 61
YE1 鸡脚叶
Okra leaf		 IBA+KT 3.70 56.25 100 70
2,4-D+KT 5.34 25 100 80
YE3 阔叶
Broad leaf		 IBA+KT 2.80 50 100 67
2,4-D+KT 4.23 37.5 100 80
YE20 鸡脚叶
Okra leaf		 IBA+KT 4.62 45.83 100 85
2,4-D+KT 4.83 37.5 85 87
YE21 阔叶
Broad leaf		 IBA+KT 4.93 37.5 100 80
2,4-D+KT 4.37 29.17 100 87
YE22 阔叶
Broad leaf		 IBA+KT 3.98 45.83 100 90
2,4-D+KT 6.02 37.5 100 90
YE32 鸡脚叶
Okra leaf		 IBA+KT 4.00 33.33 100 75
2,4-D+KT 4.46 - 100 95
YE33 阔叶
Broad leaf		 IBA+KT 3.92 31.25 67 70
2,4-D+KT 7.28 - 100 102
YE38 阔叶
Broad leaf		 IBA+KT 3.48 42.5 100 67
2,4-D+KT 6.26 50 100 80
YE54 阔叶
Broad leaf		 IBA+KT 4.77 43.75 67 75
2,4-D+KT 4.46 - 67 130
YE81 阔叶
Broad leaf		 IBA+KT 3.94 25 50 80
2,4-D+KT 5.24 - 67 154
YE86 阔叶
Broad leaf		 IBA+KT 3.74 67.5 100 80
2,4-D+KT 3.88 67.5 100 80
YE87 阔叶
Broad leaf		 IBA+KT 4.23 50 100 69
2,4-D+KT 4.84 50 100 85
YE108 鸡脚叶
Okra leaf		 IBA+KT 4.74 81.25 67 80
2,4-D+KT 5.03 37.5 67.5 80
YE110 鸡脚叶
Okra leaf		 IBA+KT 5.01 43.75 50 71
2,4-D+KT 3.66 37.5 100 75
YE111 阔叶
Broad leaf		 IBA+KT 3.58 60 100 69
2,4-D+KT 4.53 50 67.5 86
YE120 鸡脚叶
Okra leaf		 IBA+KT 3.62 31.25 67 80
2,4-D+KT 4.88 37.5 100 80
YE122 鸡脚叶
Okra leaf		 IBA+KT 3.86 70.83 100 69
2,4-D+KT 5.60 50 100 85
YE127 鸡脚叶
Okra leaf		 IBA+KT 3.22 43.75 100 67
2,4-D+KT 5.76 - 100 95
YE130 鸡脚叶
Okra leaf		 IBA+KT 5.42 84.38 100 69
2,4-D+KT 5.53 41.67 100 79
YE137 鸡脚叶
Okra leaf		 IBA+KT 5.42 - 25 110
2,4-D+KT 5.53 31.25 67 90
YE138 阔叶
Broad leaf		 IBA+KT 4.51 56.25 67 67
2,4-D+KT 7.44 - 67.7 130
YE142 阔叶
Broad leaf		 IBA+KT 5.68 - 75 116
2,4-D+KT 5.13 - 67.7 154
YE144 鸡脚叶
Okra leaf		 IBA+KT 5.19 - 25 110
2,4-D+KT 5.75 - 67 154
YE148 鸡脚叶
Okra leaf		 IBA+KT 3.81 50 100 78
2,4-D+KT 3.81 37.5 100 75
YE164 鸡脚叶
Okra leaf		 IBA+KT 3.24 62.5 67 71
2,4-D+KT 5.31 37.5 100 75

图5

部分再生植株"

图6

YE3转基因植株阳性鉴定 A和E: 胚性愈伤组织; B和F: 体细胞胚; C、G、D和F: 叶片; 标尺为1000 μm。"

表2

12个可再生阔叶家系农艺性状考察"

家系a
Lines a		 纤维长度
Fiber length (mm)		 纤维强度
Fiber strength
(g tex-1)		 马克隆值
Micronaire
(μg Inch-1)		 整齐度
Uniformity index
(%)		 伸长率
Elongation rate
(%)		 衣分
Lint percentage
(%)
E22 26.37±2.19 28.27±2.57 5.84±0.45 84.36±1.42 6.64±0.14 43.72±0.02
YZ1 27.01±2.51 25.63±1.61 5.83±0.16 86.02±2.67 6.57±0.23 35.43±0.05
YE3 27.89±1.37 28.76±0.55 5.57±0.36 86.39±1.15 6.72±0.07 40.53±0.02
YE21 27.05±0.80 28.71±1.63 6.14±0.12 86.79±2.28 6.67±0.12 40.84±0.02
YE22 25.95±1.44 27.18±2.66 5.90±0.25 84.83±2.34 6.56±0.15 39.78±0.04
YE33 27.19±2.00 26.29±1.89 5.84±0.33 85.54±1.78 6.57±0.15 39.93±0.01
YE38 26.67±1.34 26.19±2.81 5.37±0.31 85.77±1.57 6.59±0.18 38.87±0.01
YE54 27.33±0.95 28.88±1.23 5.95±0.25 85.78±1.76 6.76±0.13 39.94±0.04
YE81 28.80±0.53 31.20±4.52 5.46±0.26 87.06±0.98 6.72±0.16 35.71±0.00
YE86 26.34±0.46 25.81±1.06 6.05±0.38 85.44±1.58 6.58±0.11 39.13±0.00
YE87 27.36±1.48 28.57±2.30 6.31±0.78 85.59±3.11 6.64±0.10 40.85±0.06
YE111 27.71±1.65 27.06±2.19 5.77±0.21 85.87±1.62 6.76±0.05 39.09±0.02
YE138 27.78±1.80 29.36±3.11 5.59±0.18 86.10±1.49 6.67±0.15 39.45±0.01
YE142 28.05±1.32 27.32±1.92 5.41±0.20 86.64±1.44 6.61±0.16 39.42±0.01
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