Fig 1.
(a) pKGW-RR. DS-Red gene in pKGW-RR is transcribed by AtUbi10 (Ler) promoter and Nos terminator for plant expression. pKGW-RR was isolated from E. coli cells and digested for confirmation before Agro-transformation. pKGW-RR vector was used for optimizing Coker-312 transformation protocol for visualizing each step of transformation procedure. (b) pHSE-401-Rep. pHSE-401-Rep carried Cas9 gene (transcribed by CaMV 35S promoter) and a gRNA to target conservative region of Rep gene for Cotton Leaf Curl Khokran Virus (CLCuKV). This construct carried hygromycin antibiotic resistance gene for plant selection and kanamycin (NptII) antibiotic resistance gene for bacterial selection. (c) pG-Rec. Rec-cassette as NptII gene flanked between two directly oriented Cre mediated LoxP sites and a PhiC31 recombinase mediated attP57 site. (d) pCas-Rec. Whole Rec cassette combined with Cas9 gene to specify the locus of Cas 9 to be integrated.
Fig 2.
(A) Coker-312 delinted seeds, (B) Seeds sown in moist sunshine mix soil in darkness, (C) Emergence of cotyledonary leaves and whitish hypocotyls, (D) Hypocotyls growth in dark (yellowish appearance), (E) Hypocotyls turned green when subjected to sunlight for 48 hours.
Fig 3.
(A) Agro-transformed hypocotyls, (B) Embryos regenerated from callus, (C) Shoot development, (D) Root development, (E) In-Vitro plant.
Table 1.
Primers used for transgene analysis.
Fig 4.
Cotton embryos comparable under white light and DS-Red.
(Portion of embryos expressing strong DS-Red fluoresce to red color under stereomicroscope. Fluorescent embryos growing from highly fluorescent calli were considered as true transgenic embryos (D, E and F). DS-Red (transgene) was observed transferring from callus to embryos and ultimately transferred to roots and shoots).
Fig 5.
PCR amplification of pKGW-RR, pG-Rec, pHSE-401-Rep and pCas9-Rec.
(5A) Kanamycin gene amplification (557 bp) in DNA samples transformed with DS-Red vector. (5B) Kanamycin gene amplification (557 bp) in DNA samples transformed with pG-Rec vector. (5C) Complete DS-Red cassette amplification (2489 bp) in DNA samples putatively transformed with pKGW-RR vector. (5D) Whole Cas9 cassette amplification (7019 bp) in plants transformed with pHSE-401-Rep. (5E) Cas9 gene amplification (4266 bp) in plants transformed with pHSE-401-Rep. (5F) Whole Rec cassette amplification in plants transformed with pG-Rec (1493 bp). (5G) Whole Rec cassette amplification (1449 bp) in plants transformed with pCas-Rec vector.
Table 2.
Number of independent transformation events, regenerated embryos, positively transformed plantlets and transformation percentage per batch measured for four plasmids (1, 2, 3 and 4 for pKGW-RR, pG-Rec, pHSE-401–Rep and pCas-Rec respectively).
Fig 6.
Copy number measurement in cotton transgenics.
(Blot hybridization analysis in transgenic cotton genomic DNA hybridized with labeled probe (nptII, hptII and Cas9 genes). The approximate DNA length is shown to the right and cassettes integrated in particular transgenic are shown above the autoradiograph).
Fig 7.
Transformation effeciency: Transformation effeciency depicted in four distinct batches of all the four plasmids transformed (pKGWRR, pHSE-401, pG-Rec and pCas-Rec).
Transformation percentage per batch is calculated on the basis of positively transformed plants.
Fig 8.
Way forward to cotton projects.