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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (5): 1172-1180.doi: 10.3724/SP.J.1006.2024.34167

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-05-12 Published:2024-02-07
  • Contact: E-mail: linzhongxu@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32172025)

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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

Fig. 1

Embryogenesis and plant regeneration process of cotton in IK system A: hypocotyl; B: the induced callus tissue of 1-2 months; C: the embryonic callus with cotyledons; D: a large amount of embryoid formation; E: plant regeneration; F: hydroponics can be transplanted."

Fig. 2

Embryogenesis and plant regeneration process of cotton in DK system A: hypocotyl; B: the induced callus tissue of 1-2 months; C: the embryonic callus with cotyledons; D: a large amount of embryoid formation; E: plant regeneration; F: hydroponics can be transplanted."

Fig. 3

Callus proliferation frequency profile in IK and DK systems CSC: callus secondary fecundity. "

Fig. 4

Line chart of culture-related traits of regenerated lines in IK and DK systems A: CET, callus embryo time; B: CRE, the embryo rate of callus."

Table 1

Comparison of CSC, CRE, and CET in regenerated YE lines"

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

Fig. 5

Some regenerated plants"

Fig. 6

Identification of positive transformation plants in YE3 A and E: embryonic callus; B and F: embryo; C, G, D, and H: leaf. Bar: 1000 μm."

Table 2

Agronomic traits of 12 regenerated broad leaf lines"

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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