J. Agr. Sci. Tech. (2015) Vol. 17: 1805-1814

Genetic Transformation of Using Bt Cry2A Gene and Characterization in Indian Cultivar Arka Vikas

V. S. Hanur 1, B. Reddy 1* , V. V. Arya 1, and P. V. Rami Reddy 2

ABSTRACT

Transgenic tomato plants of south Indian cultivar Arka Vikas were developed using Agrobacterium strain EHA 105, harbouring Bt Cry2A gene with a construct containing 35S CaMV promoter, OCS terminator and nptII selectable marker, through Agrobacterium -mediated transformation. This study was conducted to improve the regeneration and transformation protocol for south Indian cultivar Arka Vikas. Hypocotyl was used as explant source for transformation due to high regeneration efficiency, molecular analysis through PCR for putative transformants in T 0 generation and qualitative ELISA method was performed for Bt protein expression followed by bioassays. Insect bioassay studies was conducted using neonate larva of armigera to screen the plants and the plants expressing good resistance with molecular and phenotypic characters were carried further for successive generations. The experimental results concluded that Bt gene was deployed in tomato cultivar successfully and had developed resistance to neonate larva of Helicoverpa armigera at laboratory conditions. These results signified that transgenic lines expressed substantial quantity of Bt Cry2A protein efficient in management of Helicoverpa armigera . Precise screening of transgenic T 1 lines is highly important to obtain single copy number plants since the expression of Bt protein in successive generations promotes effective management of this in the future.

Keywords: Agrobacterium, Bt Cry2A, Helicoverpa armigera, Transformation.

INTRODUCTION affecting many agricultural and horticultural crops (Zalcucki et al., 2002) across the Tomato ( lycopersicum ) is a world, causing huge economic failure in major vegetable crop cultivated in India for tomato production. The loss incurred due to this pest alone in various other crops like

Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021 its nutritional and commercial values (Mueller et al., 2005). It has been proved , okra, pigeon pea including tomato is that lycopene, a major constituent in tomato, estimated to be 10,000 million rupees is known for its medicinal value in reducing (Raheja, 1996). Control of this pest is the risk of cancer (Giovannucci, 1999), becoming difficult even after spending lowers the cholesterol levels (Sesso et al., nearly $600 million worth chemical 2004) and improving human diet. The major pesticides (Ghosh, 2001). In India, chemical limiting factor in tomato crop productivity is control of this pest is not always effective due to lepidopteron pest Helicoverpa due to resistance development to all major armigera (Hubner) (Atwal, 1986). This pesticides. Biotechnology has become a polyphagus pest is abundant in nature powerful tool in developing transgenic crops (Chandrashekara et al., 2012) by introducing ______1Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore, 560089, India. * Corresponding author , e-mail: [email protected] 2 Division of Entomology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore, 560089, India.

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specific genes to combat against the method and insect bioassays were carried lepidopteron pests. Bt transgenic out for phenotypic resistant characters. technology, a suitable alternative option for Screening was also done for successive efficient control of this pest, was proved generations and the data obtained was earlier in case of (Gurinder and recorded. Data on individual plant was Chhabra, 2013). The techniques in genetic recorded regarding Bt protein expression engineering highly promotes to alter various and phenotypic resistance (Vageeshbabu et crops with biotechnology approach (Sidhu et al., 2012) towards challenge inoculation al., 2010) with additional benefits.. The with first instar neonate larvae and later transformation studies in tomato were done instar larvae on leaves of tomato fruit borer earlier by many researchers for lycopene H. armigera were performed for further (Fraser et al., 2002), β-caratone (Romer et progress in development of insect resistant al., 2000) and other cry genes (Paramesh et plants. Bioassays conducted on Bt lines al., 2010). Tomato plant is normally chosen showed moderate to high levels of resistance among dicotyledonous for gene introduction to tomato fruit borers. (Wing et al., 1994) since in vitro plant regeneration protocol has been standardized MATERIALS AND METHODS with Agrobacterium -mediated transformation (McCormick, 1991). The hypocotyl explants for regeneration and Seed Sterilization transformation of Cry2A gene in tomato through Agrobacterium-mediated The genuine breeder seeds obtained from transformation were attempted Division of Vegetable Crops, Indian (Vageeshbabu et al., 2011) for obtaining Institute of Horticultural Research (IIHR), successful transformants. Regenerating Bangalore, and were pre-soaked in Captoff, explants on specific media (Madhulatha et a commercially available fungicide (CAP- al., 2007) is the routine procedure used in 50), at a concentration of 0.2% for ten obtaining transformants in many crops. minutes. The seeds were dried and then Tomato cultivar Arka Vikas is a major treated with GA 3 (250 ppm) concentration hybrid variety developed by IIHR Bangalore for ten minutes, then used for surface and extensively studied in transformation sterilization, seeds were treated with studies due to its adaptability to various absolute ethanol for 30-45 sec followed by stress conditions. The Agrobacterium - double distilled water wash twice, a drop of

Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021 mediated transformation is preferred due to tween 20 was added along with NaClO its success rate and simplicity in dicot (Sodium hypochlorite) and was let for 6-8 species (Atkinson, 2002) and was minutes followed by doubled distilled water standardized in south Indian tomato cultivar wash twice. The seeds were blot dried on Arka vikas (Vageeshbabu et al., 2009). autoclaved sterile tissue paper and placed in Insect resistant transgenic tomato plants with ½MS medium (Shadang et al., 2007) for other cry genes have been reported earlier seed germination. (Mandaokar et al., 2000) and the present activity is involved in development of Bt tomato plants containing Cry2A gene being Agrobacterium Strains effective against lepidopteron pests and resistance phenotype screening of The Agrobacterium strain EHA 105 used transformed plants were done respectively. was obtained from Dr. P. Ananda Kumar, Molecular analysis including PCR (using Principal Scientist, NRCPB, New Delhi. nptII and Cry2A gene specific primers), This strain harboring binary vector pBinBt bt protein expression analysis Cry2A gene along with 35S CaMV confirmation using dipstick ELISA promoter, OCS terminator sequences and

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Neomycin phosphotransferase (nptII) gene bud initiation, and later transferred to SEM resistant to kanamycin was used as selection (Shoot Elongation Medium) for shoot marker. elongation. These explants were further subjected to RIM (Root Induction Medium) Agrobacterium C ulture and Plant for inducing roots and were maintained for 2 Transformation weeks. Then, the plants were subjected to hardening by placing in autoclaved soilrite mixture and were transferred to greenhouse A single colony of Agrobacterium for acclimatization (Figure1). containing Cry2A gene was taken for inoculation with autoclaved 5 mL YEP medium (Yeast extract, Peptone and NaCl) Molecular Characterization of containing 50 mg L -1 kanamycin and was Transformants allowed to grow overnight in an incubator shaker at 28°C. The culture was diluted to Molecular characterization was performed obtain optimization for co-cultivation and for putative transformants and its subsequent hypocotyls from PreConditioning (PC) generations (Rashid et al., 1996). Genomic media selected were suspended in YEP DNA was isolated using C-TAB method medium containing Agrobacterium and left (Sambrook et al., 1989). Fresh leaf sample for 10-12 minutes. The hypocotyls were then from T 1 plants grown in greenhouse was blot dried on autoclaved sterile tissue paper taken for DNA isolation and the same DNA and were placed back into PC media. isolated from leaf samples was used for PCR analysis with nptII and gene specific Regeneration of the Transformed primers. Explants Enzyme Linked Immunosorbent Assay The co-cultivated explants after agrobacterium mediated infection were Qualitative dot ELISA (Lateral flow transferred to SIM (Shoot Initiation immuno diagnostic assay) was used for Medium), subcultured twice every 2 weeks testing the presence of Cry protein in continuously onto fresh medium for shoot transgenic plants. Qualitative ELISA on Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021

(a) (b)

Figure 1 . Callus initiation from hypocotyls after transformation (a), Multiple shoots from transformed Calli (b).

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different samples were conducted at plants were sown and maintained in laboratory conditions from plants growing in greenhouse conditions. The grown plants insect proof transgenic nethouse, for were labeled individually. Confirmation of determining the Bt protein expression. insertion of nptII and cry2A genes was Cry2A Bt Xpresstrips TM (Desigen, Jalna) performed using PCR specific primers and were used for accessing the Bt protein the results were tabulated. The expression by following manufacturer’s oligonucleotide primers were obtained from instructions. The sample extraction was Sigma ® and the complete gene sequence for individually carried out for one single plant Cry2A gene was provided by Dr. P. Ananda in each line, and leaf discs were obtained Kumar (Principal Scientist NRCPB), primer using the same micro centrifuge tube cap sequence is given below. and discs were dropped directly into the tubes. Around 500 µL sample extraction Primer Sequence for NPT II Gene buffer was added to each tube and using autoclaved separate micropestles, the leaf tissue was thoroughly crushed for 30 Forward primer: 5’– seconds to 1 minute. One Bt Xpresstrip TM AGAAGAACTCGTCAAGAAGGC –3’. was kept in the sample extract for 10 Reverse primer: 5’– minutes incubation. A purple color band in GAACAAGATGGATTGCACGCA –3’. the of Bt Xpresstrip TM indicates that strip is working, band in bottom line of strip with Primer Sequence for Cry2A Gene green color indicates the positive line, the results were recorded for different Bt lines, which varied in the protein expression (Figure2). Forward primer: 5’– ATGAACAACGTGCTCAACTCCGGGAG GACA–3’. PCR Analysis of T 1 Transformants Reverse primer: 5’– TTAGTAGAGTGCGGAAGGGTTGGTCG Individual plant were considered as GCAC–3’ discrete line during seed collection and (See Figure3.) maintained separately. The seeds of tomato Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021

Figure 2. Qualitative dot ELISA for samples 1-23, two controls and water as negative samples.

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(a) (b) Figure 3. PCR for NPTII (A), and CRY2A (B) genes.

moderate to high levels of resistance to Insect Bioassays tomato fruit borers. Neonate larvas of H. armigera were utilized for insect bioassays Insect bioassays were performed to assess and for insect cultures a standardized the potential of transformed tomato plants protocol developed in our lab had been for cry2A gene presence and its gene selected. The evaluation of resistance expression phenotypically. Leaves from the phenotype under in vitro conditions is more control and transformed plants were beneficial in laboratory bioassays collected for insect bioassays. Successful in (Vageeshbabu et al., 2012) and was vitro rearing of H. armigera was done using attempted during this bioassays. For in vitro modified semi synthetic diet (Ahmed and bioassay, the challenge inoculation using McCaffery, 1991). Screening was also done freshly hatched neonate larva were for successive generations and the data performed on 45-day old tomato leaves obtained was recorded. Data on individual (Figure 4), since the expression of Bt protein plant was recorded regarding Bt protein in leaves is crucial by contributing more in expression and phenotypic resistance the control of this pest (Manjunath 2006). (Vageeshbabu et al., 2012) towards Regularly used petri dishes of tissue culture challenge inoculation with first instar specimen plates (Tarsons) were utilized for neonate larvae and later instar larvae on detached leaf bioassay. The bottom surface leaves of tomato fruit borer H. armigera , was covered with autoclaved Whatman filter

Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021 were performed for further progress in paper presoaked in sterile water was placed development of insect resistant plants. to maintain moisture in leaf content. Bioassays conducted on Bt lines showed Observations on mortality of dead larva were recorded.

(a) (b) Figure 4. Leaf Bioassay for Cry2A gene , (a), Dead larva from leaf bioassay (b).

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Statistical Analysis quality of the crop, and reducing risk of environmental pollution caused by Standard deviations along with one way application of chemical pesticides. Insect ANOVA for mean values were performed resistant plants are an integral component of for all the mortality values (Table 1) (Figure integrated pest management and Bt genes 5). are effective sources against tomato fruit borer H. armigera . In developing transgenic plants for insect resistance, the Cry2A gene RESULTS AND DISCUSSION is expansively considered due to its strict binding action on brush border membrane In the present study, synthetic Cry2A gene vesicles of H.armigera (Hernandez- was introduced to tomato cultivar Arka Rodriguez et al., 2008). Introduction of this Vikas and screened initially for molecular Cry2A gene to local cultivars requires in- and phenotypic traits of resistance. depth study in producing transgenic plants Subsequent generations of transgenic plants that are effective on lepidopteron and were also screened for molecular and dipteran species (Saleem and Shakoori, phenotypic traits of resistance. In general, 2010). Many researchers have reported transgenic plants are favorable in transformation in many other diverse tomato comparison with orthodox cultivation cultivars already, but still an attempt made procedures by reducing the application of to transform south Indian cultivar Arka chemical pesticides, improving yield and Vikas was due to its preference in tomato

Table 1. Percentage mortality of neonate larvae during detached leaf bioassay under lab conditions.

Cry2A Day 1 Day 2 Day 3 Day 4 Day 5 Control Rep 1 50 83.3 100 100 100 0 Rep 2 50 66.6 100 100 100 0 Rep 3 50 83.3 83.3 100 100 0 Rep 4 33.3 66.6 83.3 100 100 0 Rep 5 33.3 50 83.3 100 100 0 Rep 6 33.3 66.6 83.3 100 100 0 SEM 41.65±3.7 69.40±5.1 88.87±3.5 100±0.0 100±0.0 0 Downloaded from jast.modares.ac.ir at 16:24 IRST on Monday September 27th 2021 CD at 5% 9.991 7.445 5.381 NS NS 0

Figure 5. Daily data on percentage mortality of neonate larva H. armigera (Hubner).

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cultivation across India. Various other CONCLUSION cultivars like Pusa Ruby the outcome of transformation frequency was about 6% The wide involvement of Agrobacterium et al., (Vidya 2000) whereas in Micro-Tom for implementing transgenics through gene cultivar around 40% transformations was insertion has gained importance in et al., reported (Sun 2006) and agriculture to attain better transgenic plants. transformation was studied in Arka vikas This method of gene delivery needs to be cultivars by which 18% transformation rate tuned highly for achieving success with was attained (Figure 1). Transformation good transformation rate. Tomato cultivar success is heavily dependent on gene Arka Vikas was transformed with Cry2A integration and its optimum expression in gene through this technique and was plant and its successive generations. Results standardized for efficient regeneration of obtained from qualitative ELISA proved transformants. Molecular screening and sufficient protein was expressed in phenotype screening using H. armigera transgenic lines (Figure 2) that showed good larva showed significant results in gene H. armigera mortality against . Confirmation expression and insect mortality. The results of Cry2A gene integration was analyzed obtained are a significant step in using PCR in T 0 and T 1 generations using development of potential insect resistant gene specific primers (Figures 3-A and -B). cultivars in tomato. Several transgenic lines Analysis of variance was performed with having BT Cry2A gene showed varietal mean values of mortality data. The larval expression. The future prospects of this mortality of challenged neonate larva was study, like histopathological effects and 85% to 90% on Bt leaves, except those protein expression studies on larval feeding, expressed less protein and, in the control will be investigated in detail and the output plant leaves, the larva entered into next will be published elsewhere. instar stages. A considerable discrepancy was observed regarding mortality between the control and treated larval groups and a ACKNOWLEDGEMENTS Critical Difference (CD) at 5% showed a st rd range from 5-9% during 1 day to 3 day of Authors are highly grateful for ICAR post-treatment in case of transgenic leaves “Network Project on Bt-Transgenic fed larval groups in comparison with control Tomato” for providing financial support to plates (Figure 5). The initial larval stages of carry this research work.

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ﺗﺮارﻳﺨﺖ ژﻧﺘﻴﻜﻲ ﮔﻮﺟﻪ ﻓﺮﻧﮕﻲ ﺑﺎ ﻛﺎر ﺑﺮد ژن Bt Cry2A و ﻣﺸﺨﺺ ﻛﺮدن وﻳﮋﮔﻴﻬﺎي آن در ﻛﻮﻟﺘﻴﻮار Arka Vikas

و. س. ﻫﺎﻧﻮر، ب . ردي ، و. و. آري، و پ. و. راﻣﻲ ردي

ﭼﻜﻴﺪه

در اﻳﻦ ﭘﮋوﻫﺶ، ﺑﺎ اﺳﺘﻔﺎده از رﻳﺴﻪ EHA 105 اﮔﺮوﺑﺎﻛﺘﺮﻳﻮم ﻛﻪ ﺣﺎﻣﻞ ژن Bt Cry2A ﺑﺎ ﺳﺎزواره ( construct ) ﺣﺎوي آﻏﺎزﮔﺮ 35S CaMV و ﭘﺎﻳﺎن دﻫﻨﺪه OCS و ﻣﺎرﻛﺮ اﻧﺘﺨﺎﺑﻲ nptII ﺑﻮد،

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ﻛﺎﻟﺘﻴﻮار راﻳﺞ ﮔﻮﺟﻪ ﻓﺮﻧﮕﻲ در ﺟﻨﻮب ﻫﻨﺪ ﺑﻪ ﻧﺎم Arka Vikas ﺗﺮارﻳﺨﺘﻪ ﺷﺪ. ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ﺑﻪ اﻳﻦ ﻣﻨﻈﻮر اﻧﺠﺎم ﺷﺪ ﻛﻪ روش ﺑﺎززاﻳﻲ و ﺗﺮارﻳﺨﺘﻲ ﻛﺎﻟﺘﻴﻮار راﻳﺞ ﮔﻮﺟﻪ ﻓﺮﻧﮕﻲ در ﺟﻨﻮب ﻫﻨﺪ ﺑﻪ ﻧﺎم Arka Vikas ﺑﻬﺒﻮد ﻳﺎﺑﺪ. ﺑﺮاي اﻳﻦ ﻛﺎر ازﻫﻴﭙﻮﻛﻮﺗﻴﻞ ﻛﻪ ﻛﺎرآﻳﻲ ﺑﺎززاﻳﻲ ﺑﺎﻻﻳﻲ دارد ﺑﻪ ﻋﻨﻮان ﻣﻨﺒﻊ رﻳﺰ ﻧﻤﻮﻧﻪ ﺑﺮاي ﻋﻤﻞ ﺗﺮارﻳﺨﺖ اﺳﺘﻔﺎده ﺷﺪ و ﺗﺠﺰﻳﻪ ﻣﻠﻜﻮﻟﻲ از ﻃﺮﻳﻖ ﭘﻲ ﺳﻲ آر ﺑﺮاي ﺗﺮارﻳﺨﺖ ﺳﺎز ﻫﺎي ﻣﻔﺮوض در ﻧﺴﻞ To و روش ﻛﻴﻔﻲ اﻻﻳﺰا ﺑﺮاي ﺑﻴﺎن ﭘﺮوﺗﺌﻴﻦ Bt و ﺑﻪ دﻧﺒﺎل آن زﻳﺴﺖ آزﻣﻮن ﺣﺸﺮه اي اﺟﺮا ﺷﺪ. ﻣﻄﺎﻟﻌﻪ زﻳﺴﺖ آزﻣﻮن ﺣﺸﺮه اي ﺑﺎ اﺳﺘﻔﺎده از ﻻرو ﭘﻮره اي ( helicoverpa armigera (neonate ﺑﺮاي ﻏﺮﺑﺎل ﻛﺮدن ﮔﻴﺎﻫﺎن اﻧﺠﺎم ﺷﺪ و ﮔﻴﺎﻫﺎﻧﻲ ﻛﻪ ﻣﻘﺎوﻣﺖ ﺧﻮﺑﻲ ﺑﺎ ﻣﺸﺨﺼﺎت ﻣﻠﻜﻮﻟﻲ و ﻓﻨﻮﺗﻴﭙﻴﻜﻲ ﻧﺸﺎن دادﻧﺪ ﺑﺮاي ﻧﺴﻞ ﻫﺎي ﺑﻌﺪي اداﻣﻪ داده ﺷﺪﻧﺪ. ﻧﺘﻴﺠﻪ ﭘﮋوﻫﺶ ﻧﺸﺎن داد ﻛﻪ ﻛﺎر ﺑﺮد ژن Bt در ﮔﻮﺟﻪ ﻓﺮﻧﮕﻲ ﻣﻮﻓﻖ ﺑﻮد و اﻳﻦ ﮔﻴﺎه ﻧﺴﺒﺖ ﺑﻪ ﻻرو ﭘﻮره اي ( helicoverpa armigera (neonate در ﺷﺮاﻳﻂ آزﻣﺎﻳﺸﮕﺎﻫﻲ ﻣﻘﺎوﻣﺖ ﻧﺸﺎن داد. اﻳﻦ ﻧﺘﺎﻳﺞ ﻣﻮﻳﺪ آن اﺳﺖ ﻛﻪ ﻻﻳﻦ ﻫﺎي ﺗﺮارﻳﺨﺖ ﺑﻴﺎن ﻣﻘﺪار ﻗﺎﺑﻞ ﺗﻮﺟﻬﻲ از ﭘﺮوﺗﺌﻴﻦ Bt Cry2A را داﺷﺘﻨﺪ ﻛﻪ در ﻣﺪﻳﺮﻳﺖ Helicoverpa armigera ﻣﻮﺛﺮ ﺑﻮد.ﻏﺮﺑﺎل ﻛﺮدن دﻗﻴﻖ رﮔﻪ ﻫﺎي (ﻻﻳﻦ ﻫﺎي) ﺗﺮارﻳﺨﺖ T1 ﺑﺮاي ﺑﻪ دﺳﺖ آوردن ﺗﻚ ﻧﺴﺨﻪ اي ﮔﻴﺎﻫﺎن ﺑﺴﻴﺎر ﻣﻬﻢ اﺳﺖ زﻳﺮا ﺑﻴﺎن ﭘﺮوﺗﺌﻴﻦ Bt در ﻧﺴﻞ ﻫﺎي ﭘﻲ در ﭘﻲ، ﻣﻨﺠﺮ ﺑﻪ ارﺗﻘﺎي ﻣﺪﻳﺮﻳﺖ ﻣﻮﺛﺮ اﻳﻦ آﻓﺖ در آﻳﻨﺪه ﻣﻲ ﺷﻮد.

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