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Plant Physiology and Biochemistry 69 (2013) 90e100

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Plant Physiology and Biochemistry


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Research article

Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants
Govind Kumar Rai a, b, Neha Prakash Rai a, Sushma Rathaur b, Sanjeev Kumar a, Major Singh a, *
a b

Indian Institute of Vegetable Research, P.O. Jakhini-Shahanshahpur, Varanasi 221 305, India Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India

a r t i c l e i n f o
Article history: Received 28 December 2012 Accepted 3 May 2013 Available online 15 May 2013 Keywords: Antioxidant Ascorbic acid DREB1A HalliwelleAsada cycle Reactive oxygen species Solanum lycopersicum

a b s t r a c t
Transgenic tomato lines (cv. Kashi Vishesh) over-expressing AtDREB1A/CBF3 driven by stress-inducible rd29A promoter showed signicantly higher activities of key antioxidant enzymes when exposed to water-decit for 7, 14, and 21 days. Transgenic tomato plants exposed to water-decit recorded lower levels of hydrogen peroxide and superoxide anion formation compared to the non-transgenic plants, suggesting alleviation of reactive oxygen species (ROS). A signicant increase in activities of enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) was observed in response to the different durations of water-decit conditions. In contrast, enzyme guaiacol peroxidase (POD) activity was lower in the transgenic lines and showed a negative correlation with ROS, ascorbic acid (AsA), and glutathione levels. The concentrations of AsA, glutathione and their reduced forms were higher in the transgenic plants and increased with ROS levels. These results indicate that AtDREB1A transgenic tomato lines are better adapted to water-decit as they showed lower drought-induced oxidative stress due to activation of the antioxidant response. 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction Plants frequently encounter water-decit due to drought, cold, and/or salinity at some stage of their life. In many regions of the globe, sufcient water is not always available for cultivation and only 14.51% agricultural area is equipped for irrigation [1]; this alarming situation makes drought one of the most adverse environmental factors [2]. Water-decit strongly affects plants by altering their physiological functions, mainly due to the imbalanced water relationships and formation of reactive oxygen species, ultimately leading to reduced plant growth and overall fruit yield so

Abbreviations: APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDHAR, monodehydroascorbate reductase; POD, guaiacol peroxidase; qRT-PCR, quantitative real-time PCR; WT, wildtype; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. Tel.: 91 542 2635236/237, 91 9451579735 (mobile); fax: 91 5443 229007. E-mail addresses: govindrai25@yahoo.in (G.K. Rai), singhvns@gmail.com (M. Singh). 0981-9428/$ e see front matter 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.05.002

that plants are unable to achieve the genetically determined maximum theoretical yield potential [2]. Therefore, the capacity of plants to withstand water-decit stress assumes a great economic signicance in ever-changing climate. Reactive oxygen species (ROS) are critical regulatory molecules for vital processes of cells, produced at low levels (w240 mM s1  O2 , and 0.5 mM H2O2 in chloroplasts) under normal growth conditions [3]. Abiotic stresses alter the cellular homoeostasis and enhance ROS production [3,4], which is thought to act as signals for the activation of stresseresponse [5]. In response to water-decit, plants also produce abscisic acid (ABA) that stimulates closure of the stomatal guard cells to reduce water loss. This process generally results in an imbalance between the use and the generation of electrons due to decreased availability of CO2 for photosynthesis, leading to overproduction of ROS [6]. Due to highly reactive and toxic nature, the over-production of ROS results in the welldocumented phenomenon of oxidative stress [4,6,7]. Thus ROS aggravate the effects of drought stress in plants by affecting cell membrane properties and cause oxidative damage to chlorophylls, lipids, proteins and nucleic acids, and make them non-functional [5,8,9]. The antioxidant defence systems of plants alleviate the

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oxidative stress by detoxifying the ROS, and thus play an important role in drought tolerance [7]. Such antioxidant defence systems include low-molecular mass non-enzymatic antioxidants (e.g., ascorbic acid, glutathione, and carotenoids) as well as enzymes such as catalase (CAT), superoxide dismutase (SOD), and enzymes that are involved in HalliwelleAsada cycle, also known as ascorbateeglutathione cycle [10]. There is clear evidence for an increase in total foliar antioxidant activity under many stress conditions [4,11,12]. A simultaneous increase in many different components of the antioxidant defence systems is known to occur in tolerant cultivars when subjected to drought stress [4,7,13]. Tomato (Solanum lycopersicum), a member of the family Solanaceae, is a major vegetable crop consumed all over the world. It has also been extensively utilized as a model plant system for various genetic and molecular biology investigations. Although tomato can be successfully cultivated around the world, its growth and development is rather sensitive to a number of environmental stresses, especially drought, salinity, and extreme temperatures [14]. Water-decit has a profound effect on tomato production worldwide [15], and tomato plants fail to produce high yields in a fragile ecosystem [14]. Furthermore, most of the commercial tomato cultivars are moderately to highly sensitive to drought stress during seed germination, seedling emergence, vegetative growth, and reproduction phases [14]. At molecular level, drought adaptation process in plants is regulated at the level of transcription. These regulatory genes provide novel opportunities for improvement of stress tolerance as well as for understanding the signalling and tolerance pathways [4,16,17]. The transcription factor encoding gene AtDREB1A/CBF3 (from Arabidopsis thaliana) regulates drought adaptation response and is an attractive tool to engineer plants for achieving enhanced drought tolerance [17e19]. The AtDREB1A/CBF3 protein binds to a 5 bp cis-acting core DRE sequence (CCGAC), also known as C-repeat (CRT), which is present in the promoter region of many downstream water-decit stress-inducible genes in A. thaliana [18] as well as in heterologous plant systems such as chrysanthemum [20], peanut [17], and Lolium perenne [19]. Earlier, we had developed A. thaliana rd29A promoter-driven AtDREB1A/CBF3 cDNA (GenBank accession no. AF07460) harbouring transgenic tomato cv. Kashi Vishesh lines (Fig. 1A) through Agrobacterium-mediated transformation [21]. The transgenic tomato lines expressing AtDREB1A were evaluated for drought tolerance and the best ve lines were

selected for further experimentation on the basis of overexpression of AtDREB1A transcripts, relative water content, electrolyte leakage, and survival frequency of drought exposed plants. The present study examines the antioxidant mechanisms that may have a role in alleviating drought-induced oxidative stress in the AtDREB1A transgenic tomato lines, by evaluating the effect of rd29A::AtDREB1A over-expression on key antioxidant enzymes and non-enzymatic antioxidant ascorbic acid (AsA) and glutathione under a progressive drought period. 2. Results Wilting symptoms in the transgenic tomato plants were delayed; they remained green and continued to grow after 14 d of water-decit stress (Fig. 1B, C), while leaf yellowing was recorded in the non-transgenic (WT) plants. Even after 21 d of water-decit, the upper 4e5 leaves of the transgenic plants remained green, while all the leaves, except the upper two, of the WT plants became yellow (Fig. 1D). An increase in relative expression levels of the AtDREB1A gene in transgenic tomato lines was recorded, following drought exposure. At the 14th d of drought exposure, expression increased to 206e241-folds of that without water-decit; the expression level was 66e89 folds at 7th d and 36e65 folds at the 21st d (Fig. 2). 2.1. Reactive oxygen species (ROS) formation under water-decit Leaves of drought exposed AtDREB1A transgenics and WT tomato plants showed a higher degree of superoxide anion formation and hydrogen peroxide (H2O2) accumulation (Table 1). Under wellwatered conditions (i.e., before the start of the water-decit), the ROS values of transgenic lines were almost similar to those of the WT plants. The levels of ROS increased with increasing duration of water-decit in both transgenic and WT plants. H2O2 concentration in both transgenic as well as WT plants increased during water stress; this increase was signicantly higher (2.2e3.2 times) in WT plants as compared to that in the transgenic plants (1.5e1.9 times) during the 7e14 d of drought exposure. In the case of superoxide  ) formation, a gradual increase of 5.5% (7 d) to 89.2% anion (O2 (21 d) over the well-watered conditions was recorded for transgenic plants. Contrary to this, a substantially higher increase of 21.7% (7 d), to 112.1% (21 d) was registered in WT plants (Table 1).

Fig. 1. Drought exposed rd29A::AtDREB1A/CBF3 transgenic tomato (cv. Kashi Vishesh) plants and their comparison with non-transgenic (wild-type) control plants. (A) Well-watered (0 d), (B) plants exposed to 7 d, (C) 14 d, and (D) 21 d of water-decit.

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WT plants, which further increased to w260% (P of drought exposure. 2.5. Glutathione reductase (GR) activity

0.01) on the 14 d

Fig. 2. Expression analysis for AtDREB1A gene by RT-PCR using gene-specic primers in T2 generation transgenic tomato plants after exposure to 0, 7, 14, or 21 days of drought. Lanes: WT Amplicon from non-transformed plant; D53, D76, D86 Amplicon from T2 generation transgenic lines; 0 well-watered treatment; 7, 14, 21 days of waterdecit exposure.
The O2 generated by 7 d drought treated AtDREB1A tomato plants was 25.7% lower than that in 7 d stressed WT plants.


2.2. Catalase (CAT) activity The CAT activity increased up to 14 d of water-decit in both transgenic as well as WT plants, and thereafter a gradual decrease was observed (Fig. 3A). At 7 d of water-decit, transgenic tomato lines showed a 1.5-fold (P 0.05) increase compared to WT plants, which further increased to 2-fold (P 0.01) at 14 d of stress. 2.3. Superoxide dismutase (SOD) activity SOD activities in the transgenic and WT plants of tomato were comparable under well-watered conditions. But under waterdecit, the transgenic tomato lines exhibited signicantly higher SOD activities than those of WT plants (Fig. 3B); the magnitude of this increase became progressively higher with the duration of moisture stress (57% at 7 d, 141% at 14 d, and 326% at 21 d in D86 transgenic line). The SOD activity was the highest on 14 d of stress in both transgenic as well as WT plants (Fig. 3B). 2.4. Ascorbate peroxidase (APX) activity The specic activity of enzyme APX in transgenic lines increased signicantly up to 14 d of water-decit, and thereafter a sharp decline was noted (Fig. 3C). The transgenic and WT lines did not differ signicantly in APX specic activity under well-watered conditions. At 7 d of water-decit, the transgenic plants showed a signicantly elevated (w85%; P 0.05) APX activity than that of
Table 1 Effect of water-decit on hydrogen peroxide and superoxide anion formation on rd29A::AtDREB1A/CBF3 transgenic tomato lines after exposure to different durations of water-decit. Line Duration of water-decit exposure (days) 0 (well-watered) H2O2 (mmol WT D41 D53 D55 D76 D86 Superoxide WT D41 D53 D55 D76 D86 g1 fresh weight) 2.20 1.82 2.14 2.15 2.69 2.15 anion (DA540 min1 mg1 0.087 0.084 0.075 0.079 0.070 0.065 7 4.83 3.40* 3.63* 3.81* 2.85** 2.72* protein) 0.105 0.078* 0.080* 0.070* 0.084* 0.079* 14 6.94 3.23*** 4.73* 4.46** 4.81* 3.67* 0.157 0.099** 0.100* 0.092** 0.103** 0.083*** 21

GR catalyzes NADPH-dependent reduction of oxidized glutathione disulde (GSSG) to reduced glutathione (GSH). Under non-stress conditions, the activity of GR in transgenic lines did not differ signicantly than that of the non-transgenic control plants. However, GR activity in transgenic plants increased till 14 d of stress and thereafter it declined (Fig. 3D). In contrast, the WT plants showed a gradual increase in GR activity with the stress durations. On the day 7 of water-decit, the GR activity in D76 transgenic line was recorded to be w36% more than that of WT plants, which increased to w50% (P 0.01) at 14 d. At 21 d of water-decit, the D76 and D86 transgenic lines recorded 9 and 13% higher GR activity, respectively, compared with their non-transgenic counterparts. 2.6. Dehydroascorbate reductase (DHAR) activity Under well-watered conditions, the DHAR activity did not differ signicantly between transgenic and WT tomato plants (Fig. 3E). The DHAR activity increased in both transgenic as well as WT plants till 14 d of water-decit, but it registered a sharp decline at 21 d (Fig. 3E). The transgenic plants, however, exhibited signicantly higher DHAR activity than WT plants, and this superiority increased with the duration of the stress (64% at 7 d, 74% at 14 d, and 365% at 21 d in D86 transgenic line). 2.7. Monodehydroascorbate reductase (MDHAR) activity The specic activity of MDHAR increased with the durations of water-decit in both transgenic as well as WT plants (Fig. 3F). At 7 d of water-decit, transgenic lines recorded w81% (P 0.05) higher activity as compared to that of WT plants; this difference increased to w99% at 14 d, but declined to merely w63% at 21 d. On 14 d of water-decit, the MDHAR activity in transgenic lines was in the order D86 > D76 > D53 > D41 > D55. 2.8. Guaiacol peroxidase (POD) activity The transgenic plants had w18% lower POD activity than the WT plants under well-watered conditions. The increasing duration of water-decit effected a progressive decline in POD activity in both transgenic and WT plants. The transgenic lines D76 and D86 had 57% and 59% lower (P 0.05) POD activity, respectively, than WT plants regardless of the water-decit durations, with the lowest value on 21 d of water-decit (Fig. 3G). 2.9. Forms of ascorbate (AsA) accumulation under water-decit

10.43 7.58* 7.79* 7.29** 6.70* 6.38** 0.184 0.132* 0.156* 0.139* 0.151* 0.127*

Total AsA was signicantly higher (29e72%) in the transgenic lines compared to that of WT plants (Fig. 4A). The reduced AsA, however, registered a rise of 66e70% at all the three periods of stress in the transgenic lines over that of WT plants (Fig. 4B). The dehydroascorbate (DHA) level was comparable between transgenic and WT plants at 7 and 14 d of water-decit (Fig. 4C). Increases of 75%, 80%, and 29% were recorded in the reduced AsA/DHA coefcient in the transgenic lines than in WT lines after 7, 14 and 21 d of drought exposure, respectively (Fig. 4D). 2.10. Glutathione accumulation during water-decit The accumulation of glutathione was observed to be signicantly higher in the transgenic lines as compared to that in WT

WT, non-transformed plant. D41, D53, D55, D76, D86 are transgenic lines. * indicate statistically signicant change in transgenic lines compared to nontransgenic (WT) plants; *P 0.05, **P 0.01, and ***P 0.001.

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Fig. 3. Activities of key antioxidant enzymes in rd29A::AtDREB1A/CBF3 transgenic and non-transformed (WT) tomato at increasing days of water-decit. (A) Catalase activity, (B) superoxide dismutase activity, (C) ascorbate peroxidase activity, (D) glutathione reductase activity, (E) dehydroascorbate reductase activity, (F) monodehydroascorbate reductase activity, and (G) guaiacol peroxidase activity. The data are mean of three replicates SE. Asterisks represent signicantly different values in transgenic lines relative to WT plants; *P 0.05, **P 0.01, and ***P 0.001.

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Fig. 4. Concentration of (A) total ascorbic acid (AsA), (B) reduced AsA, (C) dehydroascorbate (DHA), and (D) the relationship of reduced AsA/DHA in transgenic and non-transgenic (WT) tomato lines exposed to different periods of water-decit. The data are mean of three replicates SE. Asterisks represent signicantly different values in transgenic lines relative to WT plants; *P 0.05, and **P 0.01.

plants during the water-decit period, and it increased gradually with increasing days of water-decit (60% higher at 7 d, and 68% more at 14 and 21 d; Fig. 5A). The GSH concentration in transgenic plants followed a similar trend, and drastically increased at 21 d (103%) from the moderate increase of 39% at 7 d and of 37%, at 14 d of water-decit (Fig. 5B). In contrast, the GSSG magnitude in the transgenic lines presented a different trend and decreased with the severity of water-decit. The GSSG values were 93% (7 d) and 163% (14 d) augmented than that of the WT plants, and thereafter it showed a decline (Fig. 5C). 3. Discussion Drought and other abiotic stresses are often associated with increased ROS levels. Prolonged drought inevitably causes ROS over-production, which disturbs plant metabolism, leads to oxidative damage to the cellular components [22], and has a major contribution in reduced productivity under stress conditions. The ROS also act as secondary messengers in the stresseresponse signal transduction pathway and as a cellular indicator of stress [5]. Plants have evolved enzymatic and non-enzymatic systems for ROS scavenging and detoxication. These antioxidants are activated in abiotic stresses and alleviate oxidative stress before it becomes lethal [22]. The key enzymatic antioxidants include SOD, CAT, APX, MDHAR, DHAR, GR, and POD, which play a major role in protecting plant tissues against oxidative stress. The non-enzymatic systems comprise antioxidant compounds, such as water-soluble antioxidants, ascorbic acid (AsA), and glutathione [4,12].

There are evidences of enhanced antioxidant activities in transgenic plants expressing stress tolerance genes [4,16,17,23]. Studies on the effect of DREB1A gene on enzymatic antioxidant system under water decit stress, however, are limited and responses of antioxidant systems to AtDREB1A expression is yet to be explored. In addition, the involvement of non-enzymatic antioxidants AsA and glutathione in drought tolerance generated by AtDREB1A gene have not received much attention. Therefore, the present study investigates differences in antioxidant mechanisms of transgenic tomato plants carrying AtDREB1A/CBF3 transcription factor and WT plants exposed for 7, 14, and 21 d to water-decit. The rd29A promoter of A. thaliana, used to drive the AtDREB1A/CBF3 gene, was found to be an effective drought inducible promoter in tomato. The results indicate that the ROS accumulation was drought dependent, and the rd29A::AtDREB1A/ CBF3 expression in transgenic plants signicantly reduced the accumulation of ROS. These ndings also suggest effective detoxication of superoxide anion and H2O2 in the transgenic tomato plants. The transgenic tomato plants had lower H2O2 concentration and  formation as compared to their non-transgenic counterparts, O2 indicating a balanced ROS formation in the transgenic lines under  accumulation was a water-decit. The increase in H2O2 and O2 function of increasing durations of water-decit in the both  is transgenic as well as WT tomato lines. It has been noted that O2 generally the rst ROS to be generated [4]. In view of the formation   , the presence of O2 and H2O2 of more reactive ROS triggered by O2 is considered to be an index of the reactive oxygen species. The half-

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Fig. 5. Concentration of (A) total glutathione, (B) reduced glutathione (GSH), and (C) oxidized glutathione (GSSG) in transgenic and non-transgenic (WT) tomato plants exposed to different days of water-decit. The data are mean of three replicates SE. Asterisks represent signicantly different values in transgenic lines relative to WT plants; *P 0.05, and **P 0.01.

life of H2O2 is relatively longer than the half-life of other ROS, and the excess H2O2 in plant cells may lead to oxidative stress. In the case of ZAT12 transgenic tomato, the level of H2O2 has been shown to be dependent upon drought severity [23]. At low concentrations, H2O2 acts as a signal molecule in tolerance systems to various biotic

and abiotic stresses, and high concentration of H2O2 ultimately lead to programmed cell death [4]. Changes in key antioxidant enzyme activities are required to avoid, adjust, or neutralize oxidative stress [9]. There are evidences that a rise in total foliar antioxidant activity occurs in many stress situations including drought [4,7,23,24]. The water-decit exposed rd29A::AtDREB1A transgenic tomato plants registered signicantly higher SOD and CAT activity compared with their non-transgenic counterparts. The metallo-enzyme SOD activity is responsible for the elimination of superoxide radicals in cells, especially after exposure to stress. SOD is the most effective intracellular enzymatic  by catalyzing its dismutation, one antioxidant, which removes O2  O2 being reduced to H2O2 and another oxidized to O2. The H2O2 is converted to water molecule during the enzymatic reaction involving CAT, APX, and POD [4]. The upregulation of SOD is implicated in combating oxidative stress due to abiotic stresses and plays a critical role in the survival of plants. A signicant increase in SOD activity under drought stress conditions has been observed in some of the drought tolerant genotypes of rice [24] and wheat [22], transgenic tomato plants over-expressing BcZAT12 [23], as well as drought tolerant transgenic peanut [17], chrysanthemum [20], and Lolium perenne plants [19] over-expressing AtDREB1A. Furthermore, Bo and co-workers [20] observed that drought exposure caused an increased SOD activity in rd29A::AtDREB1A chrysanthemum plants compared to that of constitutively regulated CaMV35S::AtDREB1A expressing chrysanthemum plants. CAT has a higher turnover rate among the antioxidant enzymes with the potential to directly dismutate H2O2 into H2O and O2, and is essential for ROS detoxication during stress [4]. Increased CAT activity has been reported in cherry tomato, especially in tolerant varieties exposed to drought [17]. Expression of CATALASE1(CAT1) gene in response to heterologous over-expression of AtCBF1 in transgenic tomato plants has been characterized [16]. Due to CAT1 expression, catalase activity was increased, and hydrogen peroxide concentration decreased in constitutively over-expressing AtCBF1/DREB1B transgenic tomato plants compared with the wild-type plants with or without water-decit [16]. In the case of B. campestris, a simultaneous activation of SOD and CAT is required for enhanced oxidative stress tolerance and enhancement of either SOD or CAT activity had only a minor effect [25]. In addition, a higher SOD and CAT activities have been reported in drought tolerant sesame cultivars [13]. In the present study, a higher activity of APX was noted in drought exposed transgenic tomato plants. The APX is known to be involved in scavenging of H2O2 under waterewater and AsAeGSH cycles, and utilizes AsA as an electron donor. APX has a higher afnity for H2O2 than CAT and POD, and thus may have a crucial role in ROS regulation during stress [4]. Enhanced APX expression has been reported in many plants during stress conditions, viz., drought tolerant tomato lines [7,23], and AtDREB1A over-expressing transgenic peanut plants [17]. It has been reported that plants exposed to mild stress show a higher chloroplasticeAPX activity, which declined in severe drought conditions [24]. In the case of Nicotiana tabacum, over-expression of APX in chloroplasts enhanced tolerance to salt and water-decit [26]. GR is a potential enzyme of the AsAeGSH cycle and plays an essential role in defence against oxidative stress by sustaining the reduced state of glutathione and maintaining the AsA pool [5,27]. In our study, the drought exposed transgenic tomato plants had signicantly higher activities of GR. Transgenic tobacco plants having low GR activity showed enhanced sensitivity to oxidative stress [27]. Drought exposed rice [24] and transgenic tomato [23] plants possessed higher GR activity. A higher accumulation of SOD, APX, and GR has also been reported in drought exposed transgenic peanut plants over-expressing rd29A::AtDREB1A [17].

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In the present study, enhanced activities of DHAR and MDHAR were recorded in the transgenic tomato lines during drought period compared to the corresponding WT plants. DHAR expression was the highest after 14 d of water-decit, while the MDHAR activity increased with the drought duration. DHAR regenerates AsA from the oxidized state and regulates the redox state of cellular AsA, which is critical for ROS-mediated tolerance to oxidative stress. It has been reported that transgenic plants over-expressing DHAR showed a higher level of AsA with or without aluminium (Al) stress, whereas, plants over-expressing MDHAR showed a higher AsA level only in the absence of Al [28]. Over-expression of DHAR enhanced drought tolerance in tobacco [29], while over-expression of MDHAR in transgenic tobacco enhanced tolerance against salt and osmotic stresses [30]. The increase in MDHAR activity contributed to drought tolerance in cherry tomato [7]. The AtDREB1A overexpressing transgenic tomato plants showed lower POD activity under the drought treatments as compare to the WT plants. A lower POD activity was surmised in drought exposed wheat plants [22]. In contrary, a higher POD activity was reported in drought exposed transgenic Lolium perenne plants over-expressing AtDREB1A [19].  scavenging enzyme SOD The balance between the activity of O2 and the activities of H2O2 scavenging enzymes APX/CAT is consid ered to be essential for determining the steady-state level of O2 and H2O2 in cells [17]. Ectopic over-expression of SOD results in excess SOD activity relative to the H2O2 scavenging enzyme activities [9]. This phenomenon is reported to be deleterious to the cells in a variety of organisms since under such conditions the SOD ac , but may be tivity may not be adequately high to scavenge all O2 abundant to produce more H2O2 than in the plants not overexpressing SOD [9]. This may lead to formation of more highly  reactive hydroxyl radicals by reaction involving the remaining O2 with H2O2. An increase in the ratio of SOD to APX or CAT activity (H2O2 scavenging enzymes; Table 2), rather than individual changes in the activity of each enzyme decides the level of oxidative stress [9,31]. In the present study, the ratio of SOD:CAT and SOD:APX activity decreased during 7e14 d of water-decit in the both transgenic as well as WT tomato plants, but in all the three drought regimes, SOD:CAT ratio of AtDREB1A transgenic lines was always lower, whereas the SOD:APX ratio was lower only up to 14 d of stress (Table 2). These enzymatic alterations support the proposition that AtDREB1A over-expression in transgenic tomato plants alleviates drought-induced oxidative stress and helps them adapt better to the water-decit. These ndings are similar to the report that drought induced oxidative stress in transgenic tomato plants expressing BcZAT12 gene is controlled primarily by the enzymes SOD, APX and CAT [23]. The levels of non-enzymatic antioxidants of a plant are good indicators of the redox state, and their accumulation is indispensable for stress tolerance. Within the non-enzymatic systems, AsA is the most abundant and powerful water soluble antioxidant

synthesized in the plant cells. It reacts chemically with ROS to minimize the damage, has the ability to donate electrons in a number of enzymatic and non-enzymatic reactions, and also acts as a natural substrate for many plant peroxidases [4,8,32]. It is believed that the level of AsA increases in plants exposed to drought stress, while the buffering capacity generated by AsA contributes to stress tolerance [7]. For example, drought tolerant genotypes of tobacco, poplar, and cherry tomato contain a higher foliar AsA and show improved tolerance to oxidative stress under drought conditions [7,33]. The AsA redox system consists of Lascorbic acid, monodehydroascorbate (MDHA) and DHA. A high ratio of reduced to oxidized AsA is essential to eliminate ROS in cells [7]. The regeneration of AsA is extremely important because fully oxidized DHA has a short half-life and would be lost unless it is brought to its reduced state. In agreement to this, AtDREB1A transgenic tomato lines registered higher AsA and increased ratios of reduced to oxidized AsA compared to the WT plants, and appear to possess a greater capacity to eliminate ROS. The H2O2 scavenging under moderate drought is selectively performed by AsA through AsAeGSH cycle [34]. The regeneration of oxidized AsA (MDHA or DHA) to reduced AsA is also an important process in the antioxidant response involving the enzymes MDHAR, DHAR, and GR [25]. Evidence from transformed poplar plants over-expressing GR supported the role of DHAR, GSH and GR in maintaining the foliar AsA pool [33]. Glutathione is one of the crucial metabolites in plants, and its reduced form (GSH) is considered as an important component for intra-cellular defence against ROS-induced oxidative damage, and also a potential scavenger of OH, 1O2 and H2O2. It also plays an important role in growth and development of plants, including cell differentiation, cell death and senescence, and enzymatic regulation [4]. With the imposition of water-decit for 7e21 d, the rd29A::AtDREB1A transgenic tomato lines exhibited higher glutathione level compared with the WT plants. GSH plays a key role in the antioxidative defence system by regenerating another potential water soluble antioxidant like AsA, via the AsA-GSH cycle [10]. It has been reported that when the intensity of a stress increases, GSH concentrations usually decline and redox state becomes more oxidized, leading to deterioration of the system [11]. The balance between the GSH and GSSG is a central component in maintaining cellular redox state. The role of GSH in the antioxidant defence system provides a strong basis for its use as a stress marker. Furthermore, an elevated GSH concentration has been correlated with the ability of plants to withstand metal-induced oxidative stress [4]. Maintenance of a high reduced to oxidized ratio of AsA and glutathione is essential for proper scavenging of the ROS in cells [5]. An increase in the AsA and glutathione contents in wheat and maize plants subjected to water-decit has been reported [35]. The overall balance between these antioxidants has to be tightly controlled. This

Table 2 Comparison of activity ratios of antioxidant enzymes of rd29A::AtDREB1A/CBF3 plants of tomato cv. Kashi Vishesh after 7, 14, and 21 d of exposure to water-decit. Line Duration of water-decit exposure (days) 0 SOD/CAT WT D41 D53 D55 D76 D86 0.199 0.140 0.199 0.211 0.114 0.120 SOD/APX 1.682 1.622 2.023 1.736 2.055 1.338 7 SOD/CAT 0.168 0.039 0.053 0.044 0.067 0.097 SOD/APX 0.721 0.446 0.675 0.432 0.584 0.605 14 SOD/CAT 0.096 0.048 0.055 0.046 0.075 0.078 SOD/APX 0.640 0.279 0.346 0.289 0.383 0.506 21 SOD/CAT 0.098 0.078 0.087 0.049 0.124 0.087 SOD/APX 1.647 1.817 2.486 1.167 2.434 3.148

WT, non-transformed plant. D41, D53, D55, D76, D86 are transgenic lines.

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ratio is maintained by MDHAR, DHAR, and GR utilizing the reducing power of NADPH [5]. The components of the HalliwelleAsada cycle, which involves AsA, GSH, APX, MDHAR, and GR, represent the primary H2O2 detoxication mechanism in plant cells [7]. In the present study, drought exposed transgenic tomato lines showed markedly higher activities of these enzymes than their non-transgenic counterparts. In case of cherry tomato, it has been reported that the drought tolerant genotypes recorded higher activities of the enzymes (GR, DHAR, MDHAR, and APX) of HalliwelleAsada cycle [7]. In short, the results of present study showed that AtDREB1A overexpression in transgenic tomato resulted in a greater activation of the components of HalliwelleAsada cycle under drought conditions. This response would maintain the levels of reduced AsA, and GSH, facilitating the detoxication of ROS during the drought stress episode. Previous studies established the association between detoxication of ROS and capacity to tolerate drought in plants overexpressing regulatory genes [16,17,23]. The present results are in agreement to the previous reports that induced over-expression of AtDREB1A/CBF3 increases drought tolerance through the scavenging of ROS and the stabilization of macromolecular structures [17,19]. In case of the rd29A::AtDREB1A/CBF3 transgenic tomato lines, there was a close relationship among the mean values of components of antioxidant system (SOD, CAT, APX, GR, and DHAR) in response to the drought-induced oxidative stress (Table 3). A signicant relationship was reported between the SOD and APX in rd29A::AtDREB1A transgenic peanut plants exposed to drought, but not between SOD-GR and GR-APX [17]. In contrast, the MDHAR activity and the levels of AsA, reduced AsA, total glutathione, and GSH in the rd29A::AtDREB1A/CBF3 transgenic tomato lines showed a positive correlation with ROS level. A signicant negative correlation was noted between POD activity and H2O2 level in the transgenic lines. Interestingly, the increased activities of CAT, SOD, APX, GR, and DHAR coincided with AtDREB1A expression in the transgenic tomato lines (Table 3) and were at the highest on 14th d of water-decit. Therefore, it may be concluded that the enhanced oxidative stress tolerance under drought stress was associated with the over-expression of AtDREB1A gene, which increased the activities of these enzymes. This supports the opinion that the higher antioxidant levels in AtDREB1A transgenic peanut contributed to lower ROS and a better osmotic adjustment leading to improved drought tolerance [17]. It has also been shown that heterologous AtDREB1A expression in chrysanthemum confers improved drought tolerance, which could be attributed in part to

enhanced SOD activity [20]. A similar increase in activities of superoxide anion and H2O2 scavenging (SOD and POD) enzymes was also reported in Lolium perenne expressing AtDREB1A [19]. Expression of AtDREB1A also manifests a differential gene expression under water-decit, thereby acting as a master-switch to neutralize the effects of drought [36]. In the present study, the activities of the key antioxidant enzymes, except that of POD, were signicantly higher in all the ve rd29A::AtDREB1A/CBF3 transgenic tomato lines. The drought exposed transgenic tomato plants experienced less oxidative stress than the WT plants. Further, the levels of antioxidative enzymes as well as non-enzymatic antioxidants of HalliwelleAsada cycle were greater in the transgenic lines exposed to drought. These results indicate that the AtDREB1A transgenic tomato plants had better tolerance to drought-induced oxidative stress due to the improved antioxidant response. The up-regulation of genes responsible for antioxidants might be a consequence of the over-expression of AtDREB1A under drought making the transgenic tomato plants useful in drought-prone areas. 4. Materials and methods 4.1. Plant materials and drought stress conditions Homozygous T2 plants of ve independent transgenic tomato (cv. Kashi Vishesh) lines (viz., D41, D53, D55, D76, and D86) harbouring promoter Atrd29A regulated AtDREB1A/CBF3 gene were used in this study. The presence of single copy insertion of AtDREB1A/CBF3 gene in these transgenic lines was conrmed through PCR, Southern blot, and segregation analyses [21]. To select homozygous lines, approximately 100 T2 seedlings each from individual T1 plants were raised and screened for kanamycin resistance using ve foliar sprays of kanamycin (400 mg l1). Kanamycin foliar spray was also applied on control seedlings of wild-type (WT, non-transgenic tomato plants of the same cultivar). The homozygous T2 plants were selected only when all the tested T2 seedlings, derived from the seeds harvested from a single T1 plant, were scored as kanamycin resistant. The homozygous T2 as well as the WT (control) seedlings were transplanted into earthen pots lled with soil and farm yard manure (5:1). The plants were put in a growth chamber having controlled conditions with 50% relative humidity, 25  C/15  C (day/night) temperature, and a 16 h/8 h photoperiod with a photon ux density of 350 mmol m2 s1.

Table 3 Correlation coefcient values (r) between the different biochemical parameters of rd29A::AtDREB1A/CBF3 transgenic tomato lines under water-decit and well-watered conditions. For each parameter, average value of the ve transgenic tomato lines was used. SOD SOD CAT APX GR DHAR MDHAR POD  O2 H2O2 AsA RAsA Glut GSH DREB1A DDs 1 0.87** 0.86** 0.77** 0.79** 0.26 0.19 0.02 0.07 0.01 0.11 0.36 0.29 0.95** 0.37 CAT 1 0.97** 0.85** 0.76** 0.14 0.03 0.02 0.06 0.09 0.03 0.29 0.20 0.97** 0.34 APX GR DHAR MDHAR POD
O2


H2O2

AsA

RAsA

Glut

GSH

DREB1A

DDs

1 0.78** 0.86** 0.01 0.12 0.18 0.10 0.20 0.14 0.18 0.06 0.96** 0.18

1 0.53* 0.47* 0.43 0.30 0.39 0.19 0.31 0.57** 0.52** 0.85* 0.60*

1 0.29 0.34 0.52* 0.47* 0.38 0.4 0.13 0.27 0.80** 0.2

1 0.83** 0.88** 0.88** 0.58** 0.82** 0.93** 0.97** 0.23 0.93**

1 0.72** 0.78** 0.70** 0.80** 0.75** 0.84** 0.09 0.79**

1 0.92** 0.46** 0.72** 0.82** 0.91** 0 0.88**

1 0.49** 0.75** 0.83** 0.91** 0.08 0.93**

1 0.83** 0.59** 0.60** 0.1 0.53**

1 0.77** 0.82** 0.04 0.78**

1 0.95** 0.36 0.91**

1 0.26 0.97**

1 0.38

SOD, superoxide dismutase; CAT, catalase; APX, ascorbate peroxidase; GR, glutathione reductase; DHAR, dehydroascorbate reductase; MDHAR, monodehydroascorbate  , superoxide anion; H2O2, hydrogen peroxide; AsA, total ascorbic acid; RAsA, reduced ascorbic acid; Glut, glutathione; GSH, reduced peroxidase; POD, guaiacol peroxidase; O2 glutathione; DREB1A, AtDREB1A/CBF3 expression level; DDs, days of drought stress. * indicates a signicant correlation: *P 0.05, and **P 0.01.

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The water-decit treatments began after 50 d of germination when plants were in the late-vegetative stage, by withholding irrigation for 7, 14, or 21 d (Fig. 1). The control treatment (0 d) included well-watered plants, which received w80% eld capacity irrigation, whereas the 7, 14, or 21 d of water-decit corresponded to about 40, 25, or 15% eld capacity soil moisture, respectively. The fully-expanded fourth leaf (from the tip, excluding petiole) was harvested on completion of 0, 7, 14, or 21 d of drought exposure, frozen immediately in liquid N2, and kept at 80  C until they were used for the experiments. At each harvest, three replicates of each tomato line were taken, and each replication had three plants. The expression of AtDREB1A mRNA was checked using qRT-PCR. 4.2. Detection of hydrogen peroxide (H2O2) The H2O2 content in leaf samples was measured as per the procedure described earlier [23]. Leaf samples (200 mg) were crushed in 5 ml of 50 mM sodium phosphate buffer (pH 6.5). An aliquot (3 ml) of this solution was mixed with 1 ml of 0.1% titanium sulphate in 20% (v/v) H2SO4 and centrifuged at 6000 g for 15 min. The intensity of yellow colour of the supernatant was measured at 410 nm. The H2O2 concentration was calculated using extinction coefcient 0.28 mmol1 cm1 and it was expressed in mmol g1 FW.
) measurement 4.3. Superoxide anion (O2


extraction buffer for 4 h. The assay mixture contained 50 mM sodium carbonate-bicarbonate buffer (pH 9.8), containing 0.1 mM EDTA, 100 ml enzyme extract and 0.6 mM epinephrine in a nal volume of 3 ml. Epinephrine was the last component to be added. The adrenochrome formation in the next 4 min was recorded at 470 nm. One unit of SOD activity is equal to the amount of enzyme required to cause 50% inhibition of epinephrine oxidation under the experimental conditions. 4.6. Assay of ascorbate peroxidase (APX) activity Ascorbate peroxidase (APX; EC 1.11.1.11) was assayed as per previously published procedure [37]. About 200 mg leaf sample was homogenized in 5 ml of 50 mM potassium phosphate buffer (pH 7.8) containing 1.0 mM EDTA, 1% PVP, 1 mM ascorbic acid, and 1 mM phenylmethylsulfonyl uoride at 4  C. After centrifugation at 22,000 g for 10 min at 4  C, the supernatant was dialyzed against the same extraction buffer. Reaction mixture in a nal volume of 3 ml contained 50 mM potassium phosphate buffer (pH 7.0), 0.2 mM EDTA, 0.5 mM AsA, 0.2 mM H2O2, and the enzyme at 25  C. H2O2 was the last component to be added and the rate of ascorbate oxidation was monitored by the decrease in absorbance at 290 nm (extinction coefcient of 2.8 mM1 cm1) up to 5 min. Correction was made for the low, non-enzymic oxidation of ascorbic acid by H2O2 and the specic activity of enzyme was expressed as mmol ascorbate oxidized min1 mg1 protein. 4.7. Assay of glutathione reductase (GR) activity Glutathione reductase (GR; EC 1.6.4.2) was assayed as per the method described earlier [7]. Leaf sample (200 mg) was homogenized in 5 ml of 100 mM TriseHCl buffer (pH 7.8) at 4  C. The homogenate was centrifuged at 22,000 g for 20 min at 4  C and the supernatant was used for enzyme assay. The reaction mixture in a nal volume of 2 ml contains 100 mM TriseHCl buffer (pH 7.8), 0.2 mM NADPH, 0.5 mM oxidized glutathione (GSSG), 3 mM MgCl2 and 200 ml of the enzyme extract. The assay was initiated by the addition of NADPH at ambient temperature. The reaction was monitored by decrease in absorbance of NADPH at 340 nm (extinction coefcient 6.2 mM1 cm1) for 5 min. Corrections were made for NADPH oxidation in the absence of GSSG, and the specic activity of enzyme was expressed as mmol NADPH oxidized min1 mg1 protein. 4.8. Assay of dehydroascorbate reductase (DHAR) activity Dehydroascorbate reductase (EC 1.8.5.1) activity was assayed as per previously published methods [7,37]. Leaf samples (200 mg) were homogenized in 5 ml of 25 mM sodium phosphate buffer (pH 7.0) at 4  C, and after centrifugation at 22,000 g for 10 min at 4  C the supernatant was used for enzyme assay. The reaction mixture (2.0 ml) contained 25 mM sodium phosphate buffer (pH 7.0), 2.5 mM GSH, 0.4 mM DHA, and 0.2 ml enzyme extract. The assay was initiated by the addition of DHA at room temperature. The DHAR activity was measured by monitoring increase in absorbance for 3 min at 265 nm (extinction coefcient of 14 mM1 cm1) due to the formation of ascorbate. The rate of reaction was corrected for the nonenzymatic reduction of DHA by GSH. The specic activity of enzyme was expressed as mmol NADPH oxidized min1 mg1 protein. 4.9. Assay of monodehydroascorbate reductase (MDHAR) activity The enzyme monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) activity was assayed as per the method described earlier [7]. About 200 mg leaf sample was homogenized in 5 ml of 100 mM

) production was measured The rate of superoxide anion (O2 following an earlier published method [9]. About 200 mg fresh leaf sample was homogenized under a N2 atmosphere in cold (0e4  C) in 100 mM sodium phosphate buffer (pH 7.2) containing 1 mM sodium diethyl dithiocarbamate to inhibit SOD activity. After  ) was centrifugation at 22,000 g for 20 min, superoxide anion (O2 measured in the supernatant, as its capacity to reduce nitro blue tetrazolium (NBT). The assay mixture in a nal volume of 3 ml contained 100 mM sodium phosphate buffer (pH 7.2) with 1 mM sodium diethyl dithiocarbamate and 0.25 mM NBT. The absorbance of the end product was measured at 540 nm and superoxide anion formation was expressed as DA540 min1 mg1 protein.


4.4. Assay of catalase (CAT) activity Catalase (CAT, EC 1.11.1.6) activity was determined as per the method described earlier [23]. Fresh leaf sample (200 mg) was homogenized in 5 ml of 50 mM TriseNaOH buffer (pH 8.0) containing 0.5 mM EDTA, 2% (w/v) polyvinyl pyrrolidone (PVP), and 0.5% (v/v) Triton X-100. The homogenate was centrifuged at 22,000 g for 10 min at 4  C, and after dialysis the supernatant was used for enzyme assay. The assay mixture contained 100 mM potassium phosphate buffer (pH 7.0), 50 mM H2O2, and 200 ml enzyme extract in a nal volume of 3 ml. The decomposition of H2O2 was followed at 240 nm (extinction coefcient of 0.036 mM1 cm1) by decrease in absorbance for 5 min at 25  C. Catalase activity was expressed as mmol of H2O2 oxidized min1 (mg protein)1. 4.5. Assay of superoxide dismutase (SOD) activity The superoxide dismutase (SOD; EC 1.15.1.1) was assayed according to the method described earlier [9]. For enzyme extraction, w200 mg fresh leaf sample was homogenized using chilled mortar and pestle in 5 ml of 100 mM potassium-phosphate buffer (pH 7.5), containing 0.5 mM EDTA, 0.1% Triton X-100, and 2% PVP. The extract was centrifuged at 22,000 g for 10 min at 4  C. The supernatant was dialyzed in a cellophane membrane tube against the cold

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HEPES-HCl buffer (pH 7.6) at 4  C, and after centrifugation at 22,000 g for 10 min at 4  C, the supernatant was used as enzyme extract. The reaction mixture (2 ml) contained 100 mM HEPES-HCl buffer (pH 7.6), 2.5 mM AsA, 0.15 mM NADPH (added at last), and 0.2 ml enzyme extract. The rate of NADPH oxidation was monitored by recording the change in absorbance at 340 nm (extinction coefcient of 6.2 mM1 cm1) up to 3 min and the specic activity of enzyme was expressed as mmol ascorbate oxidized min1 mg1 protein. 4.10. Assay of guaiacol peroxidase (POD) activity Guaiacol peroxidase (EC 1.11.1.7) activity was assayed as per the procedure published previously [9]. Leaf samples (w200 mg) were homogenized in 5 ml of 60 mM sodium phosphate buffer (pH 7.0) at 4  C. The homogenates were centrifuged at 22,000 g for 10 min and after dialysis, it served as enzyme preparation. Assay mixture in a nal volume of 2.5 ml contained 40 mM sodium phosphate buffer (pH 6.0), 2 mM H2O2, 9 mM guaiacol and the reaction was initiated by adding 50 ml enzyme extract. Increase in absorbance was measured at 470 nm (extinction coefcient of 26.6 mM1 cm1) up to 5 min and the specic activity was expressed as mmol of H2O2 reduced min1 mg1 protein. 4.11. Protein estimation In all the enzyme preparations, protein was estimated by the Lowrys method using bovine serum albumin (BSA) as the standard [38]. 4.12. Ascorbate (AsA) assay About 200 mg of leaf sample was ground with liquid nitrogen and homogenized in 5 ml of 5% (w/v) meta-phosphoric acid at 4  C. The homogenate was centrifuged at 15,000 g for 15 min at 4  C, and the supernatant was used for ascorbate and glutathione assay. The extraction and quantication of total AsA, reduced AsA, and DHA followed the previously described method [39]. Total AsA was determined through a reduction of DHA to AsA by dithiothreitol (DTT). The leaf extract (0.2 ml) in 5% (w/v) metaphosphoric acid was added to 0.5 ml of 150 mM sodium phosphate buffer (pH 7.5) and 0.1 ml of 10 mM DTT. The mixture was incubated at ambient temperature in dark for 10 min. Further, 0.1 ml of 0.5% (w/v) Nethylmaleimide was added together with 0.4 ml of 44% (v/v) orthophosphoric acid, 0.4 ml of 4% (w/v) 2,20 -bipyridyl in 70% (v/v) ethanol, and 0.2 ml of 3% (w/v) FeCl3. The resultant reaction mixture was incubated at 40  C in dark for 40 min. Finally, the absorbance was measured at 525 nm and the total AsA concentration was quantied with reference to a standard AsA curve prepared following the same procedure as above. The reduced AsA was quantied in the same way as the previous procedure, replacing 0.1 ml of DTT with 0.1 ml of distilled H2O. Finally, the DHA concentration was deduced from the difference between total AsA and reduced AsA and it was expressed as mg g1 DW. 4.13. Glutathione assay The method described by Owens and Belcher was used for the analysis of reduced and total glutathione [40]. This assay is based on oxidation of the reduced glutathione (GSH) by 5,50 -dithio-bisnitrobenzoic acid (DTNB), and oxidized glutathione (GSSG) is reduced to GSH by the action of GR and NADPH. The GSH was estimated in a reaction mixture (2 ml) of 100 mM potassium phosphate buffer (pH 8.0) containing 50 ml of the extract, 1 mM EDTA, and 30 ml of 4% (w/v) DTNB. The reaction mixture was

incubated at room temperature for 3 min and absorbance was recorded at 412 nm. Total glutathione was measured in the same reaction mixture with addition of 0.25 mM NADPH and 0.5 units of glutathione reductase. Concentration of total glutathione and GSH was calculated by referring to a standard curve of 1e5 mg of GSH. 4.14. Statistical analysis Each experiment was set up with three replications, and the data were represented as mean standard error. Data were analysed by analysis of variance (ANOVA) appropriate for the design, and the statistical signicance of differences (2-tailed) among the treatment means of transgenic and WT plants was determined by the students t-test. Acknowledgements The authors are grateful to Dr. K.C. Bansal, Director, National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India for providing AtDREB1A gene construct, and to Dr. B.D. Singh, Emeritus Professor, School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India for critical reading of the manuscript. The authors are also thankful to The Director, Indian Institute of Vegetable Research (IIVR), Varanasi, India for providing infrastructure facilities. The support from National Project on Transgenic Crops (NPTC), ICAR, New Delhi, India is gratefully acknowledged. References
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