Extended Research on the Application of VIGS Technology in the Whole Growth Cycle of Cotton

doi:10.13560/j.cnki.biotech.bull.1985.2024-0729

Abstract

Abstract:

Objective To break through the time limit bottleneck that cotton VIGS technology can only be applied in cotton cotyledon stage, and make up for the application deficiency of VIGS technology in cotton reproductive growth stage. Method Using Nongdamian 8 as the material and GhCLA1 as the verification gene, 4-day-old cotton seedlings' stem (cotyledon folded) were injected, 7-day-old cotton seedlings cotyledon (cotyledon fully unfolded) were injected, also flowering and bolling stages cotton stem and base of new fruit branches and new leaves were injected with VIGS respectively. Phenotype observation and RT-PCR were used to verify the silencing efficiency of target genes and photobleaching duration. Result The leaf photobleaching rate of 4-day-old cotton seedlings was 80% by VIGS injected to stems, that of 7-day-old cotton seedlings was 100% by VIGS injected to cotyledon, and that of flowering stage and bolling stage cotton was 90% and 68.75% by VIGS injection at the base of new fruit branches, respectively. Conclusion A stable VIGS application technology system for the whole growth cycle of cotton is obtained.

Key words: VIGS, cotton, cotyledon, stem, growing period

DING Ruo-xi, DOU Shuo, AN Ye-zhi, KONG Wen-hui, GUO Wen-jing, ZHANG Dong-mei, WANG Xing-fen, MA Zhi-ying, WU Li-zhu. Extended Research on the Application of VIGS Technology in the Whole Growth Cycle of Cotton[J]. Biotechnology Bulletin, 2025, 41(2): 58-64.

Figures/Tables 6

Table 1 Statistics of leaf photobleaching rates after VIGS treatment at different tissue sites of cotton

注射部位和时期 Injection site and period		注射株数 Number of injected cotton seedlings	白化株数 Number of photobleaching cotton seedlings	白化率 Photobleaching rate/%	操作难易程度 Degree of difficulty for operation
4日龄幼苗茎注射		50	40	80.0±0.1	较难
7日龄幼苗子叶注射		45	45	100±0.1	容易
新生果枝基部（茎）注射	开花期	30	27	90.0±0.0	较难
新生果枝基部（茎）注射	结铃期	32	22	68.8±0.1	较难
节间注射	开花期	40	16	40.0±0.1	难
节间注射	结铃期	31	12	38.7±0.1	难
叶片注射	开花期	30	0	0±0.0	无法注入
叶片注射	结铃期	31	0	0±0.0	无法注入

Fig. 1 Photobleaching phenotype of cotton seedling after VIGS treatmentA: Uninfected cotton seedling. B: Cotton plants of 10 d after stem injection at 4 d of age. C: Cotton plants of 10 d after cotyledon injection at 7 d of age. Bar=2 cm

Fig. 2 Photobleaching phenotype of cotton plants after VIGS treatmentA: Phenotype of cotton plants at 3 weeks after VIGS injected to the cotton stem. B: Phenotype of cotton at 6 weeks after VIGS injected to cotton stem. C: The contrast between photobleaching leaves and old green leaves. D: Photobleaching downward. E: Coexistence of photobleaching and green on stem. Bar=10 cm

Fig. 3 Photobleaching phenotype of cotton bollA: Caducous photobleaching cotton bolls. B: Un-withered photobleaching cotton bolls. C: Mature photobleaching cotton bolls. D: The comparation of photobleaching cotton bolls and normal bolls. Bar=2 cm

Fig. 4 Phenotype observation of photobleaching durationA: Cotton phenotype at 3 weeks after injection. B: Cotton phenotype at 6 weeks after injection. C: Cotton phenotype at 12 weeks after injection. D: Cotton phenotype at 19 weeks after infection. E: Cotton phenotype at 22 weeks after infection. Bar=10 cm

Fig. 5 Identification of the GhCLA1 gene silence in cottonA: The electrophoresis result of cotton total RNA. B: The GhCLA1 gene silenced identification results by RT-RCR. C: The GhCLA1 gene silenced identification results by RT-qRCR. The capital letters indicate that the differences reached the highly significant level (P<0.01). M: 2 000 bp marker. 1: True leaf of cotton seedling untreated by VIGS. 2: The true photobleaching leaf at 10 d after VIGS treatment to seedling stems. 3: Coexisting photobleaching and green leaf at 10 d after VIGS treatment to seedling stems. 4: Normal leaf of cotton plant untreated by VIGS. 5: Completely photobleaching leaf at 2 months after VIGS treatment of plant stems. 6: Completely photobleaching stem at 2 months after VIGS treatment of plant stems. 7: Stems while coexisting of photobleaching and green leaves at 2 months after VIGS treatment of plant stems. 8: Blank control

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