Cloning of the GhACTIN1 gene
The codon-optimized GhACTIN1 cassette ligated into the pUC-57 cloning vector, was subjected to bacterial transformation using Top10 of E. coli. The restriction-digested band of 2 kb using PstI and SacI enzymes confirmed the ligation of the GhACTIN1 gene cassette into the PUC-57 vector. (Supplementary material: Fig. S1). The GhACTIN1 cassette was ligated in the pCAMBIA-1301 vector (Fig. 1). The amplification of the 577 bp fragment, resolved on 1.5% agarose gel, confirmed the successful ligation of the GhACTIN1 cassette in the pCAMBIA-1301 vector construct (Supplementary material: Figs. S2A, S3A, S3B). The compactness of the construct was confirmed through the restriction digestion method. The excision of a 2 kb fragment, resolved at 0.8% agarose gel, confirmed the compactness of the construct pCAMBIA-1301_GhACTIN1 (Supplementary material: Fig. S2). The successful transformation of pCAMBIA-1301_GhACTIN1 Construct into Agrobacterium strain LBA4404 by electroporation method was confirmed by colony PCR. The amplification of the 577 bp fragment, resolved on 1.5% agarose gel, confirmed the successful introduction of pCAMBIA-1301_GhACTIN1 into Agrobacterium strain LBA4404 (Supplementary material: Fig. S3).
Transformation of the GhACTIN1 gene in cotton plants
CEMB-88 variety was selected for transformation. A total of 7,500 isolated embryos from sterilized germinated cotton seeds were subjected to excision at the shoot apex region by a sharp scalpel and treated with Agrobacterium inoculum containing the GhACTIN1 gene and co-cultivated on zero MS media plates. After a week, the root-sprouting embryos were shifted to culture tubes containing MS selection media. A total of 78 plants survived on the selection MS media, and the transformation efficiency was 1.04%. After 5–6 weeks, the surviving plantlets were shifted to pots containing autoclaved soil (Fig. 2a–f).
Establishment of putative transgenic cotton plants in the field
Out of 78 putative transgenic cotton plants, shifted into pots for acclimatization, only 27 plants survived. The well-established pot plants were then shifted to the CEMB field to grow in the natural environment.
Molecular analyses of putative transgenic cotton plants in T0 progeny
Putative transgenic cotton plants were subjected to molecular analyses such as PCR, transient expression of GUS through GUS assay, and relative expression of the GhACTIN1 gene at mRNA level to confirm the integration and expression of the transgene in cotton plants.
Confirmation of transgene through amplification by PCR
PCR analyses were performed using extracted genomic DNA as described in 3.7 using gene-specific primers. The amplification of the 577 bp fragment confirmed the successful introduction of the transgene in the cotton variety CEMB-88. No PCR amplification was observed in control non-transgenic cotton plants (Fig. 3A).
Histochemical GUS assay
Histochemical GUS expression assay of young developing fiber attached to the ovules of both putative transgenic and non-transgenic control cotton plants was done by application of substrate to the cotton fibers, respectively. The appearance of blue color in transgenic cotton plant fiber confirmed the initial screening of the GhACTIN1 gene in Agrobacterium-inoculated cotton plants for their successful introduction and expression of the cassette in fibers. However, no GUS activity (appearance of blue color) was observed in non-transgenic cotton plant fibers (Fig. 3B).
Development of advanced generation of GhACTIN1 transgenic cotton plants
The seeds from the transgenic cotton plants (A-08, A-15, A-24, and A-36) analyzed by qRT-PCR (Supplementary material: Fig. S4) were raised to their T1 generation in the form of four respective lines. Each transgenic cotton line was comprised of 7 plants. Non-transgenic cotton plants were also raised in a separate line as a control line to study molecular, biochemical, and physiological characteristics comparatively.
Molecular analyses of T1 generation of transgenic cotton plants
Confirmation of advanced generation of transgenic cotton lines through PCR amplification
All four transgenic cotton lines’ advanced-generation cotton plants were subjected to PCR amplification by using gene-specific primers and isolated DNA as a template. The amplification of the 577 bp band resolved on 0.8% agarose gel in T1 transgenic cotton plants confirmed the successfully integrated transgene after being segregated in the advanced generation of cotton. However, no amplification of the DNA band was observed in the non-transgenic control cotton line (Supplementary material: Fig. S5).
Quantitative real-time PCR (qRT-PCR) of transgenic cotton plants (T1 Generation)
To quantify the GhACTIN1 gene mRNA transcript level, total mRNA was isolated from different developmental stages of cotton fiber such as initiation (4DPA), elongation (15DPA), along with secondary wall synthesis (25DPA), and reverse transcribed into cDNA. Exponential amplification of fiber cDNAs through real-time PCR revealed that GhACTIN1 gene mRNA level was very low at the end of initiation or the beginning of elongation (4DPA) and was maximum during elongation (15DPA) while gradually decreased at the end of elongation or the beginning of secondary wall synthesis (25DPA). The transgenic line A-36 showed a maximum increase in mRNA expression of GhACTIN1 during the fiber elongation phase, which was calculated to be 18.09 folds, while during initiation and secondary wall synthesis, it was estimated to be 3.2 and 4.6 folds, respectively when compared to the non-transgenic control line. The mRNA expression patterns were recorded in other transgenic cotton lines. Statistical analysis, two-way ANOVA, of the group data, indicated a significant difference in transgene mRNA expression during elongation time compared to initiation and secondary wall synthesis time (Fig. 4).
Determination of Transgene Integration Location and Copy Numbers at Chromosomal Level of Transgenic Cotton Plants
FISH (Fluorescent in situ hybridization) analysis
GhACTIN1 gene integration location on chromosome and transgene copy numbers in transgenic cotton lines was determined through Fluorescent In Situ Hybridization in advanced generation (T2). Transgenic cotton line A-36, which showed the maximum improvement in fiber characteristics, including fiber strength, length, micronaire, and maturity ratio along with higher expression of the GhACTIN1 gene was selected for Fluorescent In Situ Hybridization analysis. The FISH analysis revealed that the GhACTIN1 gene-specific probe hybridized at chromosome number 8 in hemizygous form (Fig. 5B). The single bright fluorescent signal on chromosome number 8 indicates a single copy number in transgenic cotton line A-36 while FISH analysis of the non-transgenic control line determined no fluorescent signal (Fig. 5A).
Plant biochemical and physiological analyses
Quantification of F-actin
F-actin filament quantification analyses of 16DPA cotton fibers of transgenic cotton lines compared to the non-transgenic control line revealed a significant increase in the quantity of F-actin filament. Transgenic lines A-08, A-15, A-24, and A-36 showed fluorescence intensity of 32.1, 26.4, 27.3, and 35.7au with increments of 7.8%, 6.4%, 6.6%, and 8.7%, respectively, when compared to 24.4au fluorescence intensity of non-transgenic control cotton fiber (Fig. 6A).
Measurement of cellulose contents
Cellulose contents comparative analyses result of the transgenic and control cotton lines showed higher values in transgenic cotton lines than control. A maximum of 4.7% increment in cellulose contents was observed in transgenic cotton line A-36 while A-08, A-15, and A-24 showed an increment of 2.2%, 3.2%, and 1.3% compared to non-transgenic cotton fibers (Fig. 6B).
Stomatal conductance measures CO2 absorption rate with the evaporation of H2O through the stomatal aperture. The stomatal conductance of transgenic cotton lines A-08, A-15, A-24, and A-36 was calculated to be 173.6, 185.3, 170.2, and 206.5 mmol m-2 s-1 values correspondingly compared to non-transgenic control line 146.6 mmol m-2 s-1 (Fig. 6C). One-way ANOVA analysis indicates that transgenic lines A-15 and A-36 significantly differed from the non-transgenic control line in stomatal conductance.
Photosynthetic rate measurement
The photosynthetic rate (PN) of transgenic and non-transgenic control plants was measured through IRGA CIRUS3. The photosynthetic rate was 9.0, 9.5, 8.4, and 10.8 µmol CO2 m-2 s-1 for transgenic lines A-08, A-15, A-24 and A-36 while 7.6 µmol CO2 m-2 s-1 for non-transgenic control cotton plants. The photosynthetic rate in cotton plant lines A-15 and A-36 was significantly higher in PN values than non-transgenic control cotton plant line when analyzed through ANOVA (Fig. 6D).
Water use efficiency
The WUE of transgenic cotton lines A-08, A-15, A-24, and A-36 was found to be 4.7, 5.0, 4.3, and 5.2 mmolCO2 mol-1 H2O, respectively when compared to non-transgenic control cotton line with 4.2 mmol CO2 mol-1 H2O. No significant difference in water use efficiency was observed except in transgenic cotton line A-36 (Fig. 6E).
Rate of transpiration
A positive correlation between transpiration rate and photosynthetic rate (CO2 assimilation rate) was observed in transgenic cotton lines. Transpiration rate in cotton lines A-08, A-15, A-24, and A-36 was found to be 2.4, 2.5, 2.0, and 3.18 mmol m−2 s-1 in a sequential order compared to the control line where it is recorded to be 1.5 mmol m−2 s-1. A 0.5 to 1.68 mmol m−2 s−1 increment in transpiration rate was recorded (Fig. 6F).
Determination of cotton fiber quality and its microscopic examination
Cotton fiber length
Fiber length is one of the most significant quantitative traits from the commercial point of view. The High-Volume Instrument analysis of cotton fiber of transgenic cotton lines A-08, A-15, A-24, and A-36 showed the lengths as 27.1, 27.3, 26.6, and 27.6 mm compared to 26.2 mm of non-transgenic control cotton line. Transgenic cotton lines A-08, A-15, and A-36 significantly increased fiber length, while transgenic cotton line A-24 showed a constant value with no significant impact on fiber length in contrast to the non-transgenic control cotton line. A maximum of 5.3% increment in the transgenic cotton line was recorded compared to the non-transgenic control cotton (Fig. 7A,B).
Cotton fiber strength
Cotton fiber strength is one of the important traits among fiber quality determination parameters for the textile industry as the strength of fiber further affects neps production and the spinning performance. The fiber of the selected transgenic cotton line along with the non-transgenic control line analyzed by CCRI, labs revealed that the strength of transgenic cotton lines namely A-08, A-15, A-24, and A-36 was determined to be 27.3, 29.5, 29.4 and 30.2 g/tex sequentially in comparison to the non-transgenic control cotton line which was 24.3 g/tex. When the data was statistically evaluated, all transgenic cotton lines showed a significant increase in fiber strength compared to the non-transgenic control line. Overall, a maximum of 24.2% increment in fiber strength of transgenic cotton fiber was observed (Fig. 7E). Scanning electron microscopic analysis, zoomed at × 400 of the transgenic cotton fiber showed a higher number of twists per unit area compared to non-transgenic control cotton fiber. The higher number of twists can directly be correlated with the higher strength of cotton fiber (Fig. 7E,F).
Maturity ratio and uniformity index of cotton fiber
Fiber maturity is a ratio of cell wall thickness to the diameter or the cell wall thickness compared to the size of the lumen, and its values of 0.7 to 0.9 are considered to be optimum. Similarly, the uniformity index (UI%) is the ratio of mean length to the UHML (upper half mean length). The maturity ratio of transgenic cotton lines, namely A-08, A-15, A-24, and A-36 was found to be 0.83, 0.86, 0.84, and 0.87, respectively, relative to the maturity ratio of non-transgenic control cotton line, which was recorded to be 0.76. Statistically, all the transgenic cotton lines showed significant improvement in maturity ratio compared to the non-transgenic control cotton line. A maximum increase of 10.5% was observed in the maturity ratio of transgenic cotton lines (Fig. 7C). However, no significant difference was obtained in the uniformity index of transgenic cotton lines when compared with non-transgenic control cotton lines. The uniformity index of transgenic cotton lines A-08, A-15, A-24, and A-36 was recorded to be 84, 83.5, 83.1 and 85.6%, respectively compared to 82.7% of the non-transgenic control line (Fig. 7C,D).
Micronaire values of cotton fiber
Micronaire is defined as the combination of fiber fineness and maturity. The lower the micronaire values, the better the fiber fineness and maturity ratio. The fiber samples from four transgenic cotton lines along with non-transgenic control line samples were subjected to the air-flow resistance measuring method at CCRI lab, and maicronaire values calculated to be 3.6, 3.4, 3.6, and 3.1 of A-08, A-15, A-24, and A-36 transgenic cotton lines respectively in comparison to 4.1 of the non-transgenic control line. Transgenic cotton line A-36 showed a maximum value of 24.3% (Fig. 8A). Further scanning electron microscopic analysis of transgenic and control cotton fiber, observed at × 4000 revealed that the smoothness of the transgenic cotton fiber surface as compared to the non-transgenic control line, which is directly proportional to the cotton fiber fineness (Fig. 8B).
Agronomical characteristics of transgenic and non-transgenic cotton plants in T2 generation
Agronomical characteristics of transgenic cotton plants compared to the non-transgenic control line were taken into account to define any comparable change in both group of plants which may be attributed to insertional change or any contribution from genetic modification.
Seed index, ginning out turn (GOT), and seed cotton yield
The preferable expression of the ACTIN1 gene in the embryo sac can also influence the seed weight. When the seed index of the transgenic cotton line in comparison to the non-transgenic control line was calculated by weighing 100 healthy disease-free seeds from each The seeds index was found to be 10.6, 11, 9.8, and 12.1 g sequentially in the transgenic cotton line A-08, A-15, A-24, and A-36 respectively while non-transgenic control cotton line the seed weight was recorded to be 8.4 g. Overall, a 1.2 to 3.7 g increment in seed index was recorded (Fig. 9A). Figure 9B(a,b) is a pictorial representation of the seed index of non-transgenic control and transgenic seeds. Ginning out turn (GOT) or lint percentage (lint %) of transgenic and control cotton lines was calculated using the lint-to-seed weight ratio. The lint percentage of transgenic cotton plants lines A-08, A-15, A-24, and A-36 was found to be 37.5, 38.4, 36.3, and 39.7% while the control line showed 35.1% of GOT. Three transgenic cotton lines, namely A-08, A-15, and A-36, significantly improved their lint percentage compared to the control cotton line (Fig. 9C). Seed cotton yield is an important parameter. The increase in seed cotton yield of 24.9 to 64.6 g was calculated in transgenic cotton lines compared to 218.8 g of the control line. The transgenic cotton line A-08, A-15, A-24, and A-36 were found to have 245.7, 255.5, 243.7, and 283.4 g of seed cotton yield respectively, while in the control cotton line, the seed cotton yield remained to be 218.8 g (Fig. 9D).
Fresh and dry cotton bolls weight
The fresh and dry boll weight analysis of transgenic cotton plants showed an increase in average weight as compared to non-transgenic control cotton plants. Fresh weight was 17.6, 18.0, 17.2, and 18.4 g in transgenic cotton lines A-08, A-15, A-24, and A-36 compared to 14.3 g in the control line. The dry boll weight of these transgenic cotton lines was recorded to be 4.9, 5, 4.6, and 5.2 g in contrast to 3.3 g of the non-transgenic control line. A maximum of 28.6% and 57.5% increment in fresh and dry boll weight was recorded, respectively, in transgenic cotton lines (Fig. 10A). Figure 10B(a–d) represents fresh and dry cotton bolls taken from transgenic and non-transgenic control plants.