R ESEARCH ARTICLE

doi: 10.2306/scienceasia1513-1874.2013.39.001

ScienceAsia 39 (2013): 1–11

Expression of drought tolerance in transgenic cottonZeeshan Shamim, Bushra Rashid∗, Shahid ur Rahman, Tayyab Husnain

Centre of Excellence in Molecular Biology, University of the Punjab, 87-W Canal Bank Road,Thokar Niaz Baig, Lahore-53700, Pakistan

∗Corresponding author, e-mail: bush rashid@yahoo.comReceived 13 Feb 2012Accepted 15 Nov 2012

ABSTRACT: Tolerance against drought in T1 progeny of transgenic cotton (Gossypium hirsutum) previously transformedwith GHSP26 (Heat Shock Protein Gene), GUSP1 (Universal Stress Protein Gene), and Phyto-B (Phytochrome-B Gene) wasinvestigated at the vegetative, squaring, and boll formation stage. Detection of transgenes into the progeny plants throughPCR showed a Mendelian inheritance pattern (3:1). Real-time PCR quantified the expression of GHSP26 as transgenic plantswhich tolerated drought stress for 10–12 days at an average of 18 fold more at the vegetative stage which was increased to 19fold at the squaring stage, while control plants withstood drought period only for 6 days. The gene expression was reducedto 13-fold more (13–15 days) in transgenic lines on an average basis as compared to control plants. Expression of GUSP1in transgenic progeny at the vegetative and squaring stage was quantified as 22 fold more (11–13 days) and decreased to15 fold when compared to the control plants which withstood the drought period only for 6 days. Average relative foldexpression of Phyto-B transgene as compared to the control was 0.67 more (8 days) at the vegetative stage. Thereafter,expression was elevated to 0.85 fold under drought stress conditions at squaring stage and continued to increase to 3.5 foldhigher (14–15 days) at boll formation stage when compared to that of control plants. Most notably, the number of bolls perplant, single boll weight, and seed cotton yield of our transgenic lines were greater than those of non-transgenic plants underdrought stress, which is of immense worth.

KEYWORDS: Gossypium hirsutum, water stress, transgenic inheritance, heat shock protein, universal stress protein,Phyto-B gene

INTRODUCTION

Cotton is an important crop in the world due to itsmost valuable fibre production and oilseeds1. Thegenus Gossypium contains about 50 diverse species,four of which are cultivated. G. hirsutum L. andG. barbadense L. are tetraploid (2n = 4x = 52)and G. arboreum L. and G. herbaceum L. are diploid(2n = 2x = 26). There is 5% reduction in the cottonyield due to various reasons, including shortage ofirrigation water2.

The diploid cotton species is not only the reservoirof important biotic and abiotic stress resistance genes,but it also offers better opportunities to study genestructure and function through techniques of geneknockouts3. As compared with classical breeding,transgenic technology not only introduced valuablegenes into cotton and other agriculturally importantcrops, but also made it possible to study function andregulation of such genes4, 5.

Abiotic stresses, such as drought, salinity, andheat, currently have a massive impact on crop produc-tivity and agricultural supply. Global water scarcitiesand quality issues are reaching crisis proportions not

only in developing countries but across the world6. Itis revealed that low yield of certain crops resultingfrom water scarcity demands the use of modern/ad-vanced biological techniques to resolve this problem.Recently, research into the molecular mechanisms ofstress responses has started to bear fruit and, in paral-lel, genetic modification of stress tolerance has alsoshown promising results that may ultimately applyto agriculturally and ecologically important plants7.Under drought stress, plants generally display manyphysiological responses which result in accumulationof certain differentially, expressed gene products8–10.Drought is also the major limiting factors for fibre andlint quality after flowering. Therefore it is importantto understand the genes expressed during droughtstress11 and ultimately we will require drought resis-tant cotton varieties.

Genes that have been successful in improvingdrought tolerance include those encoding heat-shockproteins (HSPs). These chaperones play a crucial rolein protecting plants against stress by re-establishingnormal protein conformation and thus normal cellularhomeostasis12, 13. The Universal Stress Protein (USP)super-family encompasses an ancient and conserved

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group of proteins that are found in bacteria, archaea,fungi, flies, and plants14. These proteins GUSP1and GUSP2 show specific expression patterns duringdrought stress15, 16.

Light play a fundamental role in the existence ofplants as a wide array of genes is transcriptionally reg-ulating the circadian clock in Arabidopsis thaliana17.So phytochrome controls the transition from vegeta-tive to reproductive growth, seedling establishment,and entrainment of circadian clock18, 19. The primaryphotoreceptors involved in regulating the red/far-redlight-induced responses are the phytochrome pig-ments. Phytochrome A (PHYA) is a light-liablephytochrome that predominates in dark-grown tissueswhereas PHYB is light stable and predominates inlight-grown tissue. The other Phytochromes (PHYC,PHYD, and PHYE) are also light stable and havecomplex overlapping and differential roles relative toPHYA and PHYB.

The present study has been conducted to as-sess the overexpression of the transgenes GUSP1,GHSP26 20, and Phytochrome-B21 which had beendriven under the CaMV 35S promoter and previouslytransformed in cotton (G. hirsutum). Over expressionof genes in the transgenic progeny plants at threedifferent growth stages (vegetative, squaring, and bollformation) has been studied under drought stress con-dition.

MATERIAL AND METHODS

Seed source, sterilization, and germination

Seeds of T1 transgenic progeny of G. hirsutumpreviously transformed with GUSP1, GHSP26, andPhyto-B genes were obtained from seed bank of Cen-tre of Excellence in Molecular Biology, Lahore, Pak-istan (GUSP1, GHSP26 20, and Phytochrome-B21).The plasmid construct used in this study was made aspCambia 1301 T-DNA region contained a GUS genealong with hygromycin plant selection gene drivenby the CaMV 35S promoter. Lint was removed fromthe seeds with concentrated H2SO4 and washed withtap water. The seeds which floated at the surface ofwater were discarded. Seeds sterilization was doneby using 0.1% HgCl2 for 5–10 min followed by 5washings with autoclaved distilled water and kept forgermination in dark at 30 °C on moist filter paper for72 h. After germination seedlings were grown incomposite soil (soil, sand, peat moss 1:1:1) for 40 daysin green house at 30± 2 °C and relative humidity near50%22. Metal halide illumination lamps (400 W) wereused to supplement natural radiation. Light radiationreached a maximum of 1500 µmol m2 s−1 at the top of

canopy at midday. Complete randomized design wasused for this study.

Amplification of foreign genes through PCR inprogeny of transgenic cotton

Transgenes (GUSP1, GHSP26, and Phyto-B) wereamplified through polymerase chain reaction (PCR) inthe T1 progeny of transgenic cotton plants. This wasdone only for the initial screening for amplificationof the foreign genes into the transgenic plants. Forthis purpose genomic DNA was isolated as describedearlier23 with some modifications. The sequence ofGUSP1 primer was F 5′-CTTCGACTGTATCTTGCTCATTTTC-3′ and R 5′-CCAAAGCTGGATTCCATATTAGAAG-3′, that of GHSP26 was F 5′-GGCTGAGCATCTGGTAGCTT-3′ and R 5′-AATCCAAACCGTGGACAATG-3′, and that of Phyto-B,F 5′-GGATCATGGTTTCCGGAGTCGG-3′ andR 5′-GGATCTAATATGGCATCATCAGCA-3′.The PCR was carried out with the above primers toamplify the 710 bp, 510 bp and 646 bp fragmentsof GHSP26, GUSP1 and Phyto-B genes, respectively.PCR reactions were performed in volume of 25 µl with2.5 U Taq DNA Polymerase, 0.2 mM dNTP’s, 10 pMof each primer, and 50 ng of DNA template. The PCRtemperature to amplify GHSP26 and GUSP1 geneswas kept at 94 °C for 4 min, 94 °C for 30 s 60 °Cfor 30 s, and 72 °C for 1 min followed by 40 times.PCR temperature to amplify Phyto-B gene was kept at95 °C for 4 min, 94 °C for 45 s, 56 °C for 45 s, and72 °C for 1 min followed by 40 times. PCR productswere resolved on 1% agarose gel and observed underUV light.

Drought stress treatment to transgenic plants andinheritance studies

To evaluate the temporal gene expression of can-didate genes for drought tolerance, transgenic andnon transgenic plants were kept without watering formaximum 15 days for drought stress treatments. Firstdrought stress was applied to the plants at vegetativegrowth stage (after 40–45 days of germination), sec-ond drought stress was applied at square formationstage (55–60 days old plants) and third drought stresswas applied at boll formation stage (120–130 daysold plants). Chi square (χ2) test was also applied forinheritance of the candidate genes in T1 progeny ofthe transgenic plants (Table 1).

Total RNA extraction and cDNA synthesis

Total RNA was extracted as the method describedearlier with some modifications24. Fresh young leafsamples were taken from the main terminal branch

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Table 1 Inheritance studies of foreign genes in T1 trans-genic cotton progeny after drought stress treatment.

Gene N O E χ2

GHSP26 21 14 15.75 0.78GUSP1 32 23 24.00 0.17Phyto-B 14 10 10.50 0.10

N : total number of plants. O: drought tolerant plants.E: expected drought tolerant plants.χ2 =

∑(O − E)2/E. The calculated value is in all

the cases less than the tabulated value for dof = 1 at5% level of probability (3.84), showing the inheritanceof these genes in accordance with Mendelian ratio forsingle gene inheritance (3:1).

parts and used for RNA extraction. RNA was quanti-fied with the help of Nanodrop ND-1000 spectropho-tometer by measuring absorbance at 260 nm and280 nm wavelength. RNA quality was checked byresolving the sample on 1% agarose gel. cDNA wassynthesized by using Fermentas cDNA synthesis kit(Cat # 1632).

Quantitative real-time PCR

The primers used for real-time PCR for GUSP1 wereF 5′-TCGGAGTTCAGAGAGAAGGAAG-3′ andR 5′-C TG G CA T CA C CC C AG T AA A T-3′, forGHSP26, F 5′-CCTAAACGGTTGGCTATGGA-3′

and R 5′-TGTCATTGCGTCCTCGAATA-3′, andfor Phyto-B, F 5′-CTCCTGGCTGAGTTTCTGCT-3′ and R 5′-GCTTGTCCACCTGCTGCTAT-3′.Real-time PCR reactions were carried out with iQ5cycler (BIO-RAD) in 96-well plate using the IQTMSYBR Green Super Mix. Different concentrations ofthe plasmid containing P2T1-4 were used as standardto validate the iQ5 Cycler reaction and to determinethe range of quantification (standard curve). cDNA(50 ng) from the transgenic cotton plants transformedwith GUSP1, GHSP26 and Phyto-B genes was used atvegetative, squaring and boll formation stages of plantgrowth. The reaction conditions for genes GHSP1 andGUSP26 were as follows: denaturation at 95 °C for5 min, followed by 40 cycles of denaturation at 94 °Cfor 30 s, annealing at 60 °C for 30 s, and extension at72 °C for 40 s, and final elongation step at 72 °C for10 min. For Phyto-B: denaturation at 95 °C for 3 min,followed by 40 cycles of denaturation at 94 °C for 45 s,annealing at 56 °C for 30 s, and extension at 72 °C for1 min, and final elongation step at 72 °C for 10 min.Melting curve was analysed by continuous monitoringof fluorescence between 60 °C and 95 °C with 0.5 °Cincrements after every 30 s. Cotton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) house-keeping

gene was used as a control. Gene specific primerswere designed using BIOEDIT software.

Effect of drought stress on yield

Productivity of transgenic cotton progeny was esti-mated as number of bolls per plant was counted,average single boll weight was measured and seedcotton yield per plant was hand collected. Seed cottonwas collected, dry petals and trash was removed andthe yield per plant was measured by weighing balance.

RESULTS AND DISCUSSION

Prevalence of drought stress is inconsistent underfield conditions and plants may perhaps be exposedto this abiotic stress at any time throughout theirlife. Cotton is comparatively drought-tolerant, butsevere water losses can slowdown plant maturity,affecting bolls and ultimately reduce yield. This studydemonstrates that cotton plants of transgenic linesof progeny transformed with GUSP1, GHSP26, andPhyto-B genes, showed improved drought toleranceat three plant growth stages (vegetative, squaring,and boll formation) that are key water stress periodsduring plant growth. All the transgenic lines showedgermination 60–100%.

Amplification of foreign genes in the T1 progenyof transgenic cotton plants

PCR showed the amplification of GHSP26 gene inT1 transgenic progeny of transgenic cotton (Fig. 1a).Out of 21 plants, 14 showed amplification of 710 bpfragment of this gene. GUSP1 gene was also ampli-fied in the progeny plants in the form of 510 bp withfull length primers (Fig. 1b). Out of 32 plants, 23were confirmed for the amplification of GUSP1 gene.Similarly PCR confirmed the amplification of Phyto-Bgene in T1 progeny (Fig. 1c) and out of 7 plants,6 amplified the 646 bp fragment of Phyto-B gene.Plasmid construct used in this study was made aspCambia 1301 T-DNA region contained a GUS genealong with hygromycin plant selection gene driven bythe CaMV 35S promoter.

Drought stress treatment to transgenic plants andinheritance studies

Although this study is based on the analysis of T1generation which is expected not to be homozygous,but it was done to investigate the initial studies re-lated to inheritance and integration of the transgenesinto the progeny plants. According to Mendelianinheritance pattern for gene integration, the ratio of

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710 bp

(a)M 1 2 3 4 5 6 -ve

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646 bp

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Fig. 1 PCR amplification of different drought tolerant genes in T1 transgenic cotton lines. (a) PCR product of GHSP26from transgenic plants. Lane M is a 1KB DNA ladder, lanes 1–6 transgenic plants, -ve negative control. (b) PCR product ofGUSP1 from transgenic plants. Lane M is a 50 bp DNA ladder, lanes 1–5 transgenic plants, -ve negative control. (c) PCRproduct of Phyto-B from transgenic plants. Lane M is a 1KB DNA ladder, lanes 1–4 transgenic plants, -ve negative control.

transgene containing plants to control or non trans-genic ones should fit the expected 3:1 in T1 gener-ation, which was also consistent with our statisticalresults. It is probably due to random chance orfor some other factors influencing the experimentalresults (Table 1). There are many reports in contextof inheritance and expression stability of foreign in-corporated genes in transgenic crops. The previousstudies revealed consistent Mendelian ratios for singlegene inheritance in certain crops like, alfalfa25, rice26,maize27, cotton28–31, and in cow pea32. However,in many cases, instead of Mendelian segregation, thecomplicated segregation profiles for the genes havealso been reported33–38. Nevertheless, in the presentstudy, Mendelian ratio of single gene inheritance wasfollowed.

GHSP26 gene expression

For temporal gene expression pattern studies, totalRNA was extracted from leaves under drought stresscondition at different growth stages, Vegetative stage(after 40–45 days germination), Square formation(after 55–60 days germination) and Boll formation(after 120–130 days germination). The productswere visualized as a smear along with two distinctribosomal RNA bands, 28S and 18S. Real-timePCR detected the expression level of foreign genesin the transgenic progeny. Different lines exhibiteddifferent gene expression in response to drought stressat vegetative stage. An optimum cotton crop at40 days after planting is thought to be a pictureof health. In addition to being drought stress-free,the crop would exhibit healthy leaves, with rootsextending into the row middles, and plants growingrapidly and uniformly. Among 6 lines of the GHSP26transformed lines, line 5 on average basis showedmaximum tolerance (12 days) to the drought stressshowing maximum expression (30 fold expression)of GHSP26 followed by line 3 and 6 which con-tended drought for 10 days each, while control (non-transgenic) withstood drought period only for 6 days

(Fig. 2a, b). Following the formation of first reproduc-tive branch, new branches will develop every 3 daysafter approximately. The first square is formed onthe lowest reproductive branch of the plant. Droughttolerance was continued to increase at squaring stage,line 5 on average basis showed maximum tolerance(12 days) and exhibited maximum expression (34fold expression) of HSP26 followed by line 3 and1 which endured for 10 days each as compared to6 days of control (non-transgenic under the aforesaidconditions) (Fig. 2c, d).

After fertilization, seeds are developed in a celllike structure, called boll. First bolls generally beginto open 125–130 days after sowing and irrigation dur-ing this period is thought to be critical. Transformedlines were once again subjected to drought stress andexpression of gene at boll formation revealed thattransgenic line 5 on average basis showed maximumdrought tolerance (15 days) showing maximum ex-pression (22 fold expression) of HSP26 followed byline 1 and 3 which endured for 14 and 13 days, respec-tively, (Fig. 2e, f). Control (non-transgenic) tolerateddrought stress for 7 days. Expression of GHSP26has been found reduced with the plant growth andfound at peak at earlier stage39–41, which is in contrastto previous reports7, 42 as the elevated expression ofTaldo1 gene at lateral stage of plant growth wasobserved as compared to juvenile stage where theexpression level was very less but this change in geneexpression could be genotype dependent.

GUSP1 gene expression

Universal stress protein play role in survival of cellsaffected by different abiotic stresses43. Progeny oftransformed lines consists of GUSP1 subjected todrought stress at vegetative stage and among 10 linesof the GUSP1 transformed lines, line 1 on averagebasis showed maximum tolerance (13 days) to thedrought showing maximum expression (70 fold ex-pression) of GUSP1 (Fig. 3a, b) followed by line 6 and8 which contended for 11 and 10 days, respectively,

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Fig. 2 Relative fold expression of GHSP26 in T1 progeny and transgenic cotton plants at different growth stages. Expressionof GHSP26 in leaves at (a) vegetative stage, (c) squaring stage, and (e) boll formation, and phenotype of transgenic (right)and control (left) plants at (b) vegetative stage, (d) squaring stage, and (f) boll formation stage. Plants were kept underwater withheld condition for 15 days at each developmental stage. The data was normalized with reference to GAPDH(Glyceraldehyde 3-phosphate dehydrogenase) as internal control. The solid bars are the standard deviation calculated fromthree repeats.

while control (non-transgenic) withstood drought pe-riod only for 6 days. After the vegetative stage,a temporal expression study of the drought tolerantGUSP1 gene was studied at the square formationstage. Different lines showed different level of geneexpression in response to drought stress at this stageas well but among 10 lines of the GUSP1 transformedlines, line 1 on average basis showed maximum toler-ance (12 days) to the drought and exhibited maximum

expression (82-fold expression) of GUSP1 followedby line 6 and 8 which endured for and 11 days eachas compared to 6 days of control (non-transgenic)under the aforesaid conditions (Fig. 3c, d). Transgeniclines exhibited different level of drought tolerance inresponse to drought stress at boll formation stage.Among 10 lines of the GUSP1 transformed lines,maximum drought tolerance (16 days) on averagebasis was observed in line 1 with maximum gene

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Fig. 3 Relative fold expression of GUSP1 in T1 progeny and transgenic cotton plants at different growth stages. Expressionof GUSP1 in leaves at (a) vegetative stage, (c) squaring stage, and (e) boll formation, and phenotype of transgenic (right)and control (left) plants at (b) vegetative stage, (d) squaring stage, and (f) boll formation stage. Plants were kept underwater withheld condition for 15 days at each developmental stage. The data was normalized with reference to GAPDH(Glyceraldehyde 3-phosphate dehydrogenase) as internal control. The solid bars are the standard deviation calculated fromthree repeats.

expression (47 fold expression) of GUSP1 followedby line 6 and 8 which endured for 14 days each(Fig. 3e, f). Control plants tolerated drought stressonly for 7 days. As the expression of GUSP1 hasfound to be decrease with the advancement in plantgrowth but even then it has expressed and helpedthe plant to tolerate the drought which indicate thatthis gene might function as a switch in adaptationof drought stress. Possible explanation for elevated

expression of GUSP1 gene at earlier stages may bethe meristematic activity of the plant44. Anotherreason may be the high copy number of transgene intransgenic plants45.

Phyto-B gene expression

Transgenic lines of Phyto-B were studied for geneexpression in response to drought stress at vegetativestage. Among 2 lines of the Phyto-B transformed

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Fig. 4 Relative fold expression of PHYTO-B in T1 progeny and transgenic cotton plants at different growth stages.Expression of GUSP1 in leaves at (a) vegetative stage, (c) squaring stage, and (e) boll formation, and phenotype of transgenic(right) and control (left) plants at (b) vegetative stage, (d) squaring stage, and (f) boll formation stage. Plant kept underwater withheld condition for 15 days at each developmental stage. The data was normalized with reference to GAPDH(Glyceraldehyde 3-phosphate dehydrogenase) as internal control. The solid bars are the standard deviation calculated fromthree repeats.

lines, line 2 showed maximum tolerance (8 days) tothe drought stress showing maximum expression (1.2fold expression) of Phyto-B (Fig. 4a, b) as comparedto control which withstood drought tolerance only for6 days.

Transgenic lines were subjected to drought stressat square formation stage and temporal analysis of thegene showed a little bit elevated expression patternbut almost equal expression as that of control under

drought stress condition (Fig. 4c, d). This elevatedexpression was continued to increase in transgeniclines in response to drought stress for 14–15 days atboll formation stage. Line 2 on average basis showed6.3 fold higher expressions as compared to that ofcontrol (non-transgene) (Fig. 4e, f).

Phytochrome may manipulate plant response todrought suggesting particular role in ABA regulationand stomatal conductance. Phyto-B gene restricts

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Fig. 5 Single boll weight of T1 cotton progeny transformedwith GHSP26, GUSP1 and Phyto-B genes. The solid barsare the standard deviation calculated from three repeats.

the fruit shedding in cotton crop and fruit sheddingis directly related to the production levels of ABA,therefore it is assumed that certain phytochrome genesmay suppress particular drought response under spe-cific environment21, 46. Gene expression of Phyto-Bwas higher at the latter plant growth stage and lowerat the earlier stage similar as reported earlier12, 42.Such difference in gene expression at different plantdevelopmental stages may be due to strong influenceof transcriptional activation of transgene, its site ofintegration and genotype dependence.

Effect of drought stress on yield

Application of plant biotechnology is one of the strate-gies for crop improvement and food production. Theultimate objective of this study is to get good yieldeven after the reduced irrigation and we observed thatthe number of bolls in transgenic plants have beenincreased as compared to non transgenic or controlplants. The boll weight has also been increased on thetransgenic lines as compared to the non-transgenic.Single boll weight of the transgenic progeny of plantstransformed with GHSP26 and GUSP1 was increasedby 23% and 36%, respectively (Fig. 5). Interestinglythe single boll weight of the transgenic progeny ofPhyto-B was found a little bit reduced on transgenicprogeny as compared to the control plants (Fig. 5).However, this reduction in boll weight is only 5%.Phyto-B gene is reported to increased photosyntheticactivity and enhancement of other physiological traitsin plants, therefore it is assumed that the increasedvegetative growth may have suppressed the boll for-mation process and ultimately the fold expression fordrought tolerance at vegetative and boll formation,

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Fig. 7 Yield/plant of T1 cotton progeny transformed withGHSP26, GUSP1, and Phyto-B genes. The solid bars arethe standard deviation calculated from three repeats.

respectively, is less (1.2 and 6.3) as compared to theGHSP26 and GUSP1. Number of bolls per plant of thetransgenic progeny of GHSP26, GUSP1, and Phyto-Bwas found to be increased by 25%, 36%, and 71%,respectively, as compared to non-transgenic plants(Fig. 6). The data collected for the seed cotton yieldper plant of the transgenic plants of GHSP26 of thesame progeny was also found increased by 54% whilecompared with non-transgenic plants. In the same wayseed cotton yield per plant for transgenic progeny ofGUSP1 was increased by 63%. Similarly the yielddata for transgenic progeny of PHYTO-B showed anincrease of 62% seedcotton yield per plant (Fig. 7).It has been observed that as the fold expression fordrought tolerance is increased, the yield is also foundto be increased which shows that the transgene isexpressing at its maximum.

Seasonal variations affect the transgene expres-sion in different cotton lines. This variation for thetransgene efficacy is correlated with the promoteractivity47, 48. The other factors reported for variation

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in the level of gene expression, might be alterationin nucleotide sequence, gene integration point, copynumber, cellular changes, and environmental fac-tors21, 49. Transgenic plants expressing the droughttolerant genes are morphologically better and growinglarger than the control plants under water withheldcondition. Moreover, roots of GHSP26, GUSP1, andPhyto-B expressing plants expected to be larger thannon transgenic plants. Therefore, fresh shoot androot biomass of transgenic plants may be higher thancontrol plants. Three types of transgenic plants, whencompared for the drought tolerance and the yield (interms of single boll weight, number of bolls and seedcotton yield), it is observed that the GUSP1 is thebest as showing 70–80 fold expression of droughttolerance for 13–12 days at vegetative and squaringstage, respectively, and 47 fold expression of droughttolerance for 16 days and yield was 63% in termsof seed cotton. Yield of other transgene expressingplants is also significantly higher than that of controlplants after drought treatment. These results are con-sistent with the other reports as more bolls and fibreformation in the transgenic plants expressing AVP1gene50. Transcription factors also play an importantrole in abiotic stress tolerance mechanisms and sofar a number of transcription factors have been foundto be involved in abiotic stress tolerance pathways.Genes encoding transcription factors may consider-ably increase the drought tolerance by regulating (overexpression or suppressing) the function of some genes.

Irrigation is very important after first bloomingand by regulating the irrigation period after bloomingyield could be increased51. Severe drought conditionsaffect the cotton plant’s development and may causesmall bolls and squares to shed. Improved main-tenance of stomatal conductance under water stressensures the higher photosynthesis, better growth andyield52. Exogenous application of Glycinebetaineimproves the growth and production of cotton plantsunder drought stress53.

In conclusion, most notably, the seed cotton yieldof our transgenic lines was greater than that of non-transgenic plants under drought stress, which is ofgreat worth. Therefore, it is expected that our resultsmay promote strategies to improve crop yields in aridand semiarid areas.

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