Source of plant material
The G.arboreumFBD-1variety of cotton was selected basis on its high germination percentage and yield. Seeds of FBD1 were obtained from the Four Brothers Group of Pakistan on a research collaboration basis. Recombinant plasmid pGA482 containing the 35SCpPIP2 gene construct was provided by Forman Christian College, Lahore, Pakistan.
Computational analysis of the PIP2 construct
The nucleotide sequence of theCpPIP2 gene construct (that was used to transform G.arboreum) was translated into a protein sequence using the online tool EMBOSS-Transeq (https://www.ebi.ac.uk/Tools/st/emboss_transeq/). The molecular structure of PIP2 protein was then modelled from the amino acid sequence using the SWISS-MODEL Server (https://swissmodel.expasy.org/). Since there is not a 3D structure available for C. proceraPIP2 protein in the Protein Data Bank (PDB),a 3D structure of spinach aquaporin SoPIP2 (PDB ID: 4IA4) was used as a template for protein modelling. Structures of SoPIP2 and CpPIP2 were compared using the FATCAT server (http://fatcat.sanfordburnham.org/fatcat-cgi/cgi/fatcat.pl?-func=pairwise) to determine the confidence of the newly built protein structure.
Amplification of the PIP2 construct in E. coli and transformation of Agrobacterium tumefaciens
The pre-cloned35SCpPIP2construct was amplified in E. coli strain TOP10. The purified recombinant plasmid pGA482 containing the CpPIP2 gene was confirmed by polymerase chain reaction (PCR) amplification with the gene-specific primers (forward:5’-CCACCCCTACTCCAAAAATG-3’; reverse: 5’-AATCCCACACCGCAGATAG-3’). The PCR mix was comprised of 1µL of plasmid, 2µL of 10x PCR buffer (Fermentas cat# B34), 2µL of 2mM dNTPs, 1µL of 25mMMgCl2, 2µL of each of forward and reverse primers (10pM) and 0.5 µL of Taq Polymerase enzyme 5U (Fermentas cat# EP0071) and deionized water to a 20µLfinal reaction volume. PCR was performed at 95ᴼCfor 5min for 1cycle, and then 35 cycles of 95ᴼCfor 45s, 63ᴼCfor 45 s and 72ᴼC for 1min, and then a final amplification at 72ᴼC for 10 min. The recombinant plasmid was then transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation using an electroporator (Bio-Rad, California, USA). The transformed A. tumefaciens cells were then incubated on ice for 2 minutes and diluted with 1 mLYeast extract peptone (YEP) broth and incubated at 30ᴼC for 3 hours at 200 rpm. Then, 100µLof cultured cells were plated on YEP medium supplemented with 50µg/mL kanamycin and grown at 30ᴼC.Transformed A. tumefaciens colonies were isolated and screened again to confirm the gene insert by PCR, using the gene-specific primers.
Transformation of the CpPIP2 gene into G. arboretum
Cotton seeds were de-linted with concentrated sulphuric acid, and then surface sterilized with0.1% HgCl2 and 0.1% sodium dodecyl sulphate and rinsed thoroughly with sterilized distilled water. The sterilized cotton seeds were then germinated in the dark at 30ᴼC. The germinated embryos of 30-36 hr were used for Agrobacterium-mediated transformation as per the method of Rao et al.(2011).The explants were incubated with Agrobacterium (overnight grown and suspended in Murashige and Skoog(MS) broth) for 30-60 min at 28ᴼC in the dark. The shoot apex explants were then blot-dried on sterile filter paper and placed into semi-solid MS (minimal growth medium) plates supplemented with cefotaxime (100µg/mL). Several untreated shoot apexes injured similarly were Agrobacterium-treated and plated as controls. The healthy plantlets were then transferred to test tubes containing MS and supplemented with 50µg/mL kanamycin and 100µg/mL cefotaxime and allowed to grow under 60μE m−2s−1 light for 16/8hrlight/dark cycle, for in vitro growth at 28ᴼC+ 2ᴼC for 2-3months.
Acclimatization of tissue-cultured cotton plants
After attaining a height of approximately 6inches,G. arboreum seedlings in glass tubes were transferred to sterilized soil pots of 6-inch diameter. They were then covered with polythene bags for gradual acclimatization to a culture room temperature environment, starting from 15 min and gradually increasing to a full day in a culture room. The plants were further acclimatized to sunlight for hardening in a similar fashion as above and then shifted to a field tunnel.
Detection of transgenic cotton plants
Genomic DNA from putative transgenic cotton plants was isolated using a Favor Prep Plant Genomic DNA Extraction Kit (cat #FAPGK 001, Favorgen Biotech Corp.). All the primers used in this study were designed using Primer Premier v3.0. The gene-specific primers were:forward primer 5’-CCACCCCTACTCCAAAAATG-3’ and reverse primer 5’-AATCCCACACCGCAGATAG-3’.
Gene expression analysis of transgenic cotton plants
Transgenic cotton plants from the T1 generation were used for CpPIP2gene expression analyses. Young leaves from transgenic cotton plants were used for total RNA extraction according to the protocol of Gul et al. (2020). The cDNA was synthesized using aRevertAidFirst Strand cDNA Synthesis Kit (cat # K1621Thermo Fischer Scientific) in a reaction mixture comprised of 4 µL 5x reaction buffer, 2 µL 10 mM dNTP mix, 1 µLRiboLock RNase inhibitor (20 U/µL) and 1 µL RevertAid M-MuLV Reverse Transcriptase (200 U/µL), in a final reaction volume of 20 µL. The reaction was incubated at 42ᴼC for 60 minutes and then terminated by heating at 70ᴼC for 5 minutes. Quantitative real-time PCR (qRT-PCR) was performed using MaximaSYBR Green/ROX (cat # K0229 Thermo Fischer Scientific) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal (reference) standard. For each reaction, 7.5 µL of Maxima®SYBR Green/ROX qPCR Master Mix (2x), 1 µL forward primer, and 1 µL reverse primer was mixed and made up to a final volume of 15 µl by the addition of nuclease-free water. Real-time PCR was performed after an initial denaturation at 95ᴼC for 5 min, and then performed at 95ᴼC for 30s, 51ᴼC for 45s,and 72ᴼC for 1min, for a total of 35 cycles. The forward primer 5’-AGGAATTGCTTGGGCTTTCG-3’ and reverse primer 5’-TGGAATGCCTTCACGAATCC-3’ were used for gene amplification. The qPCR analysis was run on PikoReal real-time PCR system (Thermo Fisher Scientific).
Fiber length analysis
Mature cotton bolls were harvested upon maturity and fibers were separated from cotton seeds. Fiber samples from transgenic and non-transgenic cotton plants were dispatched to the Central Cotton Research Institute, Multan, Pakistan, for fiber length, strength, micronair value, uniformity index and ginning out turn percentage (GOT%) analyses.
Fluorescent in situ Hybridization (FISH)
FISH was employed to determine the chromosomal location of the CpPIP2 gene in transgenic plants as described by Gul et al. (2020) .
Statistical comparisons between plants were performed by one-way analysis of variance (ANOVA), with Dunnett’s multiple comparison test, using GraphPad Prism (version 7)(GraphPad Software Inc., San Diego, California, USA).Significant differences were set at a p-value of < 0.05. Data presented as histograms represent experimental means ± SD.