Over-expression of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) in transgenic pigeonpea confers resistance to the Helicoverpa armigera

Pigeonpea is an important food legume crop cultivated in tropical and sub-tropical regions around the world wherein the Indian subcontinent accounting for over 90 % of global production. It is a rich source of protein and is an important component of a well-balanced diet for the majority of Indians. Among the other insect pests, pigeonpea productivity is mostly affected by Helicoverpa armigera which is causing severe yield loss. Non-availability of resistant genes in germplasm and constraints with traditional breeding induce the application of a genetic engineering approach to generate insect resistance in pigeonpea. Expression of plant defensins in various crops provided enhanced resistance towards a variety of pests and pathogens. In the current study, two defensins Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) integrated by a linker peptide was transferred into pigeonpea as a fusion gene by Agrobacterium mediated transformation. Putative transgenic lines were confirmed through PCR and the promising lines were identified in the following generations based upon integration, expression and bioefficiency of the fusion gene. Leaf bioassay conducted against H. armigera larvae showed increased levels of insect resistance compared to the control, where six T2 plants were identified as superior lines showing less than 25 % of leaf damage. Our findings illustrates that Tfgd2–RsAFP2 fusion protein is efficient in imparting protection against the insect pest and the transgenic lines developed in this study could be used for further pigeonpea improvement projects. Over-expression of Tfgd2-RsAFP2 fusion gene conferred enhanced insect resistance against Helicoverpa armigera in transgenic pigeonpea plants.


Introduction
Pigeonpea (Cajanus cajan (L.) Millisp.) is one of the significant versatile grain legume crop that serves as a backbone of poor farmers in tropical and sub-tropical regions of Asia, Africa and Latin America. Pigeonpea has social, economic and medicinal significance in developed countries (Ramdas etal. 2015). It is the second-largest pulse crop in terms of area and yield, following chickpea and is cultivated in 6.97 million hectares around the world, with yield and production of 724 kg per hectare and 5.05 million tonnes respectively (FAO STAT, 2016). Pigeonpea is cultivated in 60.96 lakh hectares around the world, with a yield of 50.12 lakh tonnes and productivity of 822.2 kg/ha (FAO STAT, 2020). In the Communicated by Sergio J. Ochatt.
* Vasavirama Karri vasavi8@gmail.com year 2020-21, globally India ranks first in pigeonpea production with 42.8 lakh tonnes grown under 48.24 lakh hectares with the yield of 887 kg/hectare (agricoop.nic.in). It can be cultivated in different climatic conditions all over the year owing to its adaptive nature. This adaptability nature reduces the cost of cultivation, resulting in higher profits for marginal farmers (FAOSTAT, 2014). High protein content of pigeonpea makes it as a potential constituent of diet mainly among the Indian vegetarian population. Its seed contains 20-22 % protein, where sulphur containing amino acids such as methionine and cysteine are present in threefold times higher than cereals (Srivastava, 2013). Additionally, seeds also consists of other important substances like crude fiber (1.2-8.1 %), lipids (0.6-3.8 %) and carbohydrates (57.3-58.7 %), etc. (Sinha, 1977). Global yield speculates the pigeonpea yield in India where its yield is declined over 0.70 tonnes/ha over the past few years. As it is primarily a rain fed crop, poor rainfall causes moisture loss leading to lesser yield. Irrespective of its major requirement, pigeonpea production has increased by only 1 % during the previous years. This resulted in a severe shortage of this pulse, primarily in India (Jhoshi et al. 2001). The primary reasons for their low yield are due to various abiotic and biotic stresses, as well as absence of proper crop management practices. Helicoverpa armigera, a lepidopteran pest, is amongst the most severe biotic stresses in pigeonpea cultivation (Ghosh et al. 2017;Choudhary et al. 2013). It is difficult to control this pest due to its high reproductive potential and potent migratory nature giving rise to yield loss of nearly 85 %. Its larvae attack green colored parts like pods, flowers and leaves of the plants causing significant loss of around 40-50 % and yearly yield loss of 400 million US $ globally (Kaur et al. 2016). The insect's broad host range, higher level of migration, random application of pesticides by farmers and insect's innate immunity in developing resistance against pesticides have attained this insect as a major pest (Tripathi et al. 2001;Vishwadhar et al. 2008). To control this, prime insect resistant pigeonpea varieties can be developed through traditional breeding methods, however it's not been successful because of the inconsistency with wild species and confined genetic diversity in the cultivated germplasm (Nene and Sheila 1990). Moreover, evaluating over 14,000 cultivated pigeonpea accessions has illustrated an average or low rates of resistance against this pest (Reed and Lateef 1990;Rana et al. 2017). So, introducing pod borer resistant trait is very important in pigeonpea crop development program. In recent times, plant genetic engineering techniques have shown promising results in overcoming this type of challenges.
In various plants, insect pest resistant genes have been successfully incorporated (Dunwell, 2000;Vasavirama K and Kirti PB 2013) to develop resistance against various pests. As a result, transgenic techniques for the generation of insect resistant varieties have become a viable option to the integrated pest management programme (Ranga Rao and Shanower 1999;Sharma et al. 2006). To incorporate the novel traits and to generate successful transgenic pigeonpea plants effective regeneration & transformation mechanisms are required (Nirmala Nalluri and Vasavi Rama Karri 2019; Sarkar et al. 2019;Yadav et al. 2016;Srivastava J 2013;Rao SK et al. 2008;Singh ND et al. 2002). Pigeonpea is leguminous crop that is not easily regenerated in vitro by tissue culture. However, this limitation was effectively resolved by following the protocol for multiple shoot bud induction from leaf petiole explants reported by Nirmala Nalluri and Vasavi Rama (2019). There has already been some progress in the generation of pod borer tolerant pigeonpea and chickpea varieties expressing the insecticidal genes like cry1Ab, cry1E-C, cry1Ac and chimeric cry1AcF (Kaur et al. 2016;Ramu et al. 2012;Singh et al. 2018). But, there is insufficient evidence in substantiating the transgenic pigeonpea activities in respect of stability in gene expression and insect mortality rates (Ghosh et al. 2014). Apart from these early achievements, more promising events involving effectual toxic gene expression resulting in major effect on pod borer tolerant plants under natural conditions are needed.
Genetically modified crops were usually produced by using a single gene with enhanced insect, viral and fungal resistance (Shin et al. 2002;Khanna and Raina 2002;Horvath et al. 2003;Lentini et al. 2003;Zhu et al. 2012;Ghag et al. 2012). On the other hand, long-lasting antifungal resistance could be obtained by integrated generation of antifungal proteins with various ways of action (Jayaraj and Punja 2007;Chen et al. 2009), which even needs the assimilation of different transgenes into the plant genome and their symphonious expression in transformed plants. There are several methods for developing genetically modified plants expressing different transgenes. One of the routinely preferred alternatives is to incorporate each transgene separately through different transformation activities and to cross the individual single transgene expressing lines (Bizly et al. 2000). One of the drawbacks of this method is that different transgenes in the succeeding lines were integrated at separate loci constraining the successive breeding. Additionally, this approach is not suitable to many ornamental plants and fruit trees which are cultivated vegetatively. Francois et al. (2004) stated that polyprotein made of two specific proteins, viz DmAMP1 isolated from Dahlia merckii seeds (Osborn et al. 1995) and RsAFP2 isolated from seeds of Raphanus sativus fused with a linker peptide of IbAMP polyprotein precursor from Impatiens balsamina (Tailor et al. 1997) has been transferred into plants with good level of gene expression. Both Tfgd2 and RsAFP2 are efficient anti-fungal proteins belonging to the antimicrobial peptide family. Tfgd2 was proved to show in vitro anti-fungal activity against plant pathogens like Rhizoctonia solani and Fusarium moniliformae (Olli et al. 2007). Likewise, RsAFP2 was also proved as a potent anti-fungal protein and its constitutive expression showed increased resistance to Altenaria longiceps in tobacco (Terras et al. 1995) against Alternaria solani in tomato (Parashina et al. 2000) and antagonistic to fungi like Rhizoctonia cerealis and Fusarium graminearum in wheat (Li et al. 2011). Further, Vasavirama and Kirti (2013a, b) expressed fusion gene made up of two defensins Tfgd2 and RsAFP2 in transgenic tobacco which showed disease and insect resistance against Phytophthora parasitica var. nicotianae & Rhizoctonia solani fungi and Spodoptera litura larvae. In view of these reports, an effort has been made to generate an insect resistant transgenic pigeonpea expressing the fusion gene made up of the two defensins Tfgd2 and RsAFP2 associated together with a linker peptide isolated from Impatiens balsamina seeds to acquire resistance against the pod borer Helicoverpa armigera.

Preparation of Agrobacterium tumefaciens strain with fusion gene construct
The fusion gene (GenBank accession number: KF498667) (Vasavirama and Kirti 2013a, b; Karri and Pulugurtha Bharadwaja 2013) consisting of Tfgd2 and RsAFP2 genes associated with each other by linker peptide sequence was cloned by directional cloning of 1.2 kb synthetic gene cassette at HindIII position of pBI121 vector. This fusion gene was controlled by CaMV35S promoter and nos terminator. pBI121 binary vector bearing synthetic gene construct was transferred to Agrobacterium tumefaciens (EHA 105 strain) through freeze thaw method. The disarmed rifampicin and kanamycin resistant Agrobacterium strain was cultured at 200 rpm till the OD reaches 0.6-0.8 and was further used for co-cultivation.

Generation of transgenic pigeonpea plants harboring the Tfgd2-RsAFP2 fusion gene
The sterilized seeds of pigeonpea ICP 8863 cultivar were germinated on MS basal media (Murashige and Skoog 1962) and maintained at 28 ± 1°C for 16 h of light. Afterwards 7-day-old leaf petioles were collected and utilized as explants for Agrobacterium mediated transformation and regeneration was done following the protocol of Nirmala Nalluri and Vasavi Rama (2019). The transformed regenerated plants were selected on MS media consisting of 50 mg/l kanamycin as a selective agent. Further, the rooted plantlets were transferred to 1:1 ratio of soil and vermiculate amalgam and were acclimatized in culture room and subsequently transferred to the green house. Afterwards, transgenes integration and expression in putative transgenic pigeonpea plants and segregation analysis of T 0 progenies was performed.

Segregation analysis of putative progenies harbouring Tfgd2-RsAFP2 fusion gene
It is important to identify the inheritance pattern of the fusion gene in the T 0 progenies to find out the stability of the transgenes integrated. Based on the analysis, PCR positive putative transgenic lines were selected to analyze the segregation motif of the fusion gene by kanamycin sensitivity test. Selection was performed by inoculating the overnight imbibed seeds for 5 h in MS media consisting of 50 mg/l kanamycin and was later sown on autoclaved soilrite. After three weeks, germination response was noted in both sensitive and resistant T 1 seeds, where healthy seedlings were considered as kanamycin resistant (Kan R ) whereas the non germinated seedlings were considered as kanamycin sensitive (Kan S ). Segregation analysis for T 1 plantlets was done by χ2 test and further these plants were analyzed for transgenes integration, expression and insect resistance.

Molecular analysis of T 1 and T 2 transgenic lines
PCR screening was done for kanamycin resistant T 1 transformants in order to find out the inheritance motif of the fusion gene integrated. For this study, genomic DNA from the tender leaves of kanamycin resistant (T 1 ) and control plants were isolated through CTAB method (Murray and Thompson 1980) and PCR analysis was conducted to check the amplification of the fusion gene using fusion gene specific primers under appropriate PCR conditions.

Southern analysis and RT-PCR
To analyze the copy number by southern hybridization, 5-10 μg of genomic DNA was isolated from PCR positive T 1 , T 2 and non-transgenic pigeonpea plants and were overnight digested with HindIII restriction enzyme. These restriction digested DNA fragments were separated on 1 % agarose gel electrophoresis and were transferred onto Hybond-N + nylon membrane through capillary blotting. Further, the PCR amplified nptII fragment was radiolabeled employing α-32P-dATP probe. Further, the membrane was rinsed with 0.1 % SDS, 1 X SSC for 15 min and consequently with 0.1 % SDS, 0.1 X SSC at 65 °C for 10 min twice. The radioactivity count was calculated by the means of GM counter. Further, RT-PCR was conducted to evaluate the fusion gene expression. To carry out this experiment, total RNA was isolated from one month old leaves of PCR positive and untransformed plants utilizing TRI reagent 1 3 (Bangalore Genei) as per the manufacturer's guidelines. For RT-PCR, RNAase free water was utilized to prevent the contamination of RNase. The first cDNA strand was produced from 5 μg of total RNA utilizing cDNA synthesis kit (Bangalore Genei). About 1/20th volume of the primary strand cDNA reaction was utilized to carry out the PCR amplification reactions for fusion gene.

Insect bioassay of transgenic pigeonpea plants
For in vitro bioassay H. armigera egg masses were acquired from NBAIR, Bangalore and were hatched on castor leaves by placing them in well aerated boxes. Completely expanded trifoliate leaves of 50-60 days old transgenic and control plants were taken and placed in petri dishes containing one layer of wet cotton and double layer of wet tissue paper to maintain moisture. On each trifoliate leaf, five number of second instar larvae were released. The experiment was repeated thrice and the mean and standard error of the intensity of the leaf damage and the larval mortality was calculated after 72 h of larval release. The petri dishes were placed in an incubator at 25 ± 2 °C temperature and 70 % relative humidity. After 72 h of feeding the damage rate was scored on a 1 to 9 scale (1 = < 10 % leaf area damaged, 2 = 11 to 20 %, 3 = 21 to 30 %, 4 = 31 to 40 %, 5 = 41 to 50 %, 6 = 51 to 60 %, 7 = 61 to 70 %, 8 = 71 to 80 % and 9 = > 80 % leaf area damaged). The transgenic lines that showed the damage rate score of 1 to 3 were selected to conduct RT-PCR assay. Further, the T 1 progenies of the RT-PCR positive lines were subjected to segregation analysis. The data of in vitro leaf bioassay was subjected to student's t test, where P-value of < 0.05 was considered as significant. In addition to leaf bioassay, detached pod assay was also conducted for the pods collected from the T 2 transgenic plants, where two weeks old pods were taken for evaluating the efficacy against third instar H. armigera larvae by releasing single larvae on each pod.

Protein isolation from pigeonpea leaves and Western blotting
Total protein was isolated from 50 mg of leave tissue of both T 2 transgenic and control plants by homogenizing with 1 ml of extraction buffer 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 10 mM Phenyl Methyl Sulphonylfluoride, 10 mM MgCl 2 , 150 mM NaCl, 10 % Glycerol and protease inhibitors: 50 g/ml TLCK, 50 mg/ml TPCK and 10 mM PMSF). Total protein content was quantified by Bradford's test and was separated by PAGE and was transferred to nitrocellulose membrane utilizing transfer buffer (7.2 g glycine, 1.5 g Tris and 100 ml methanol made up to 500 ml with distilled water). Further the membrane was blocked with TBST in 5 % skimmed milk powder (w/v) at room temperature for 1 h to avoid non specific binding. After blocking the membrane was probed with primary antibody that is rabbit anti-Tfgd antibody (IMGENEX, Bhubaneswar, India) and rabbit anti-RsAFP2 antibody and was further washed in TBST. This was again probed with alkaline phosphatase-conjugated antirabbit/IgG secondary antibody. Finally, the bands were observed by reacting with BCIP/NBT (5-bromo-4-chloro-3indolyl phosphate /nitro blue tetrazolium tablets) substrate.

Stastical analysis
The experiment was conducted in a Completely Randomized Design (CRD) and the experimental results in both the generations were graphically represented using sigma plot 14.5 software. Chi-square (χ2) test was done in both T 1 and T 2 generations to evaluate the segregation ratio (3:1) of the fusion gene depending on the performance in kanamycin sensitivity test.

Results
To acquire long-term pest resistance against devastating insect pest Helicoverpa armigera, Tfgd2-RsAFP2 fusion gene was expressed in pigeonpea ICP 8863 cultivar through genetic transformation. Under laboratory conditions, the fusion protein was expressed and evidenced its insecticidal efficiency towards H. armigera. The findings were illustrated in the below section.

Generation of transgenic pigeonpea lines and transformation efficacy of putative transformants
To produce transgenic pigeonpea, multiple shoots were initiated from co-cultivated leaf petiole explants of ICP 8863 pigeonpea variety using the standardized protocol. Out of 90 explants cultured in three batches, 81 shoots were identified to be kanamycin positive and among them 35 plants were established well in the green house. PCR analysis performed for these plants showed 500 bp band with fusion gene primers ( Supplementary Fig. 1) and 700-bp band with nptII-specific primers ( Supplementary Fig. 2) respectively in 12 plants confirming the presence of fusion gene. The transformation efficiency obtained was 16.66 %, which is calculated as the percentage of total number of rooted shoots versus fusion gene and nptII positive plants.

Analysis of T 0 and T 1 progenies for the inheritance of the T-DNA
Among the 12 PCR positive T 0 transgenic lines, six transgenic lines (T-2, T-8, T-9, T-14, T-28, T-32) which were fertile and produced seeds were selected for segregation analysis. After kanamycin sensitivity test, the seeds were further transferred to the soilrite for germination. In this experiment, plantlets generated from T 0 plants seeds showed normal development with apparent cotyledonary leaves. Subsequently, these seedlings were developed into matured plants with normal roots, height and leaf pattern without any abnormalities. On the other hand, null segregant seeds were unable to form proper cotyledonary leaves and were apparently dwarfed and impeded in stature with distorted primitive roots and were unable to achieve appropriate height. Control kanamycin untreated seeds were germinated normally and grown to normal height in about three weeks whereas, kanamycin treated negative control seeds responded identically to null segregants, which were not able to germinate normally. Segregation analysis done by the chisquare test for all the six T 0 plants seeds showed 3:1 ratio (p ≥ 0.05) ( Table 1) and were further subjected to PCR analysis. In the same way, the RT-PCR positive and insect resistant positive T 1 progeny seeds were subjected to segregation analysis (Table 2).

Molecular analysis of the T 1 transgenic plants for stable integration and inheritance of fusion gene
PCR screening was performed to 48 well established T 1 plants out of 71 kanamycin resistant plants and among them 16 showed strong PCR amplification (Supplementary Fig. 3) and were further subjected to southern analysis. Amongst the 16 PCR positive plants, 13 plants were southern positive, where 10 plants showed single copy insertion and 3 plants showed two copy insertions (Fig. 1). These southern positive plants were maintained in the green house (Fig. 2) and the seeds collected from them were used to generate T 2 generation for further analysis.

Improved resistance of T 1 transgenic pigeonpea plants expressing the fusion gene antagonistic towards Helicoverpa armigera
Insect leaf bioassay was conducted in ten southern positive T 1 transgenic plants against second instar H. armigera larvae which exhibited significant variance in degree of leaf damage and larval mortality. The immensity of leaf damage was evaluated after 72 h of leaf feeding in both transgenic and control plants. The plants that exhibited high mortality showed lesser leaf damage and the larval mortality varied between 20 and 86 % (Table 4, Fig. 3). It was observed that the area of leaf damage in untransformed after 72 h was noticed as 24.0 cm 2 , while in transgenic plants it varied from 3.6 to 8.06 cm 2 respectively (Table 3, Fig. 1). In the current investigation, it was noticed that, more than 80 % of the T 1 transgenic plants showed less than 25 % damage. Larvae fed on the transgenic pigeonpea plants were stunted in growth with darkening and shrinking of body color, whereas larvae fed on the control plants showed weight gain after 72 h of leaf feeding (Fig. 4). In this generation according to 1 to 9 scale rating leaf damage and percentage of mortality, eight lines showing the leaf damage rate of 1 to 3 (Table 3) and mortality percentage ranging from 86 to 60 % (Table 4) were selected as superior lines (9-5, 2-4, 2-1, 14-10, 8-14, 14-1, 8-3, 9-8) for further analysis.

RT-PCR assay of T 1 transgenic plants for the expression of fusion gene
Eight single copies transgenic lines of T 1 generation that are identified as superior in leaf assay were selected to analyze the level of fusion gene expression through RT-PCR analysis including control. This revealed the expression of fusion gene in all the eight transgenic lines while no expression was noticed in the control (Fig. 1). RT-PCR analysis signified that the fusion gene was easily visible in the transgenic plants. In addition, it represented that the level of expression was different in the transgenic resistant lines and it was higher in pigeonpea transgenics compared to the control, which was related with the enhanced pod borer resistance in these transgenic plants. Further, these selected plants were progressed to T 2 generation for analyzing the stable integration of T-DNA and their stable potency.

Improved insect resistance of T 2 transgenic pigeonpea plants against H. armigera
T 2 transgenic lines were subjected to in vitro leaf bioassay to determine their efficiency against the insect pest H. armigera, where they exhibited improved efficiency which is correlated to high larval mortality and less leaf damage. Variance in larval mortality rate was noticed in transgenic plants (40 to 86 %) ( Table 6); furthermore the plants that displayed high mortality rate showed lesser leaf damage. The leaf damage rate was identified as 25.0 cm 2 in control and in case of transgenic plants it varied between 5.1 to 6.96 cm 2 respectively (Table 5, Fig. 5). The bioassay finally stated that the transformants selected not only have the fusion gene stably integrated in their genome but also showed improved ability in resistance against H. armigera as represented in the histogram indicating the percentage of leaf damage and larval mortality rate (Fig. 6). Moreover, it was noticed that, 100 % of the T 2 transgenic plants displayed < 25 % of leaf damage. After 72 h of larval feeding, increase in larval body weight was observed in case of control which is 40.6 mg, where as larval weight was decreased in case of larvae fed on transgenic plants leaves (7.36 and 8.33 mg) ( Table 6, Fig. 7). In the current study, difference in larval physiology was also observed where, larvae fed on the transgenic leaves showed stunted in growth compared to the larvae fed on the untransformed  control leaves (Fig. 5). So, it was visible that the weight and size of the larvae fed on the transgenic leaves was significantly less than the larvae fed on the control leaves.

Detached pod assay
Two week old pods of single copy transgenic lines were analyzed for efficiency towards third instar larvae of Helicoverpa armigera. Decrease in pod consumption was noticed in transgenic plants (0.21 g) compared to the control (0.92 g) and further complete pod was damaged after 24 h of feeding in the case of control (Fig. 5, Table 7). In the present study, it was clearly implied that the functionality of the transgenic lines displayed a clear stability in efficiency of the transgenic lines to resist larval attack with decreased damage. 22.36 ± 0.10 20.00 ± 14.14 32-3 19.2 ± 0.14 40.00 ± 14.14 9-8 14.53 ± 0.04 66.66 ± 8.16 14-1 14.33 ± 0.10 60 ± 0.00 Control 43.33 ± 0.08 0 ± 0.00

Western blot analysis
Protein isolated from the leaves of T 2 transgenic lines was subjected to western blot analysis to study Tfgd2 and RsAFP2 expression utilizing their corresponding antibodies. The protein bands of anticipated size (8 kDa for Tfgd2 and 9 kDa for RsAFP2) (Fig. 8) were seen in these transgenic lines, while no bands of similar size were observed in the control plants.

Discussion
The loss of agricultural produce owing to insect pest damage is a serious concern all over the world, which has already taken a lot of time and effort to resolve. Immense use of synthetic pesticides to combat these losses not only endangers the environment but also costs a lot of money.
Marginal farmers in developing countries are frequently unable to afford this cost. These diseases not only lead to  Fig. 6 A histogram representing the variance in the performance of T 2 transgenic pigeonpea plants and control in the leaf bio-assay depicting the percentage of larval mortality and percentage of leaf damage. Values represented were Mean ± SE productivity loss but even decrease the crop quality. Many scientific ways to achieve long-term disease resistance in pigeonpea cultivars have been developed, concentrating on the objective of stacking numerous R-genes or introducing broad-spectrum insect resistance genes into cultivated lines. Application of transgenic technologies is advantageous and one amongst the crucial components in integrated pest management (IPM) program to generate wide range of insect and disease tolerance in plants (Meiyalaghan et al. 2011). Concurrently, it reduces the necessity of insecticides and is effective than traditional breeding methods. Despite the controversy surrounding GM or Bt crops, biotechnologically generated plants have gained importance over the varieties developed through traditional crop improvement methods like breeding, particularly for the development of insect resistant crops (Tamiru et al. 2015). As a result, compared to normal traditional breeding techniques, transgenic technology which takes less time would be the ideal choice to address the rising issue of food scarcity. Among the effective techniques to generate insect resistant plants, generation of transgenic plants expressing the insecticidal protein of Bacillus thuringiensis is the important one in providing insect pest resistance. Employing the Bt toxin gene, more than 30 insect resistant crops have been produced so far (Sarkar et al. 2021). Previous work stated that, defensins are the essential elements of the innate immune system in plants (Lacerda et al. 2014). These defensins existing in majority of plant parts display wide array of in vitro antimicrobial function and currently, there are various records depicting the generation  Gomes 2009, 2011). Thomma et al. (2002) reported that, over 80 defensins genes were sequenced from various plant species. Primarily, Terras et al. (1992a) identified two anti-fungal defensins from radish seeds, that are RsAFP1 and RsAFP2 and their activity was assessed towards various fungi such as, Phomabetae, Cercospora beticola and Pyricularia oryza and concluded that these two defensins constrained their growth (Terras et al. 1992b). Rs-AFP1, Rs-AFP2and Rs-AFP3/4 were the most extensively investigated defensins isolated from Raphanus sativus seeds (Carvalho and Gomes 2009). Genetically modified peanut plants expressing SniOLP and RsAFP2 genes displayed increased disease tolerance to P. personata (Vasavirama and Kirti 2012). Further, transgenic apple plants with RsAFP2 gene displayed enhanced resistance to F. culmorum (De Bondt et al. 1999). Constitutive expression of RsAFP2 gene in GM tomato plants displayed resistance to many fungal phytopathogens like A. tenuis, R. solani, A. solan and P. Infestans (Parashina et al. 2000). Vijayan et al. (2013) has stated that the GM plants expressing TvD1 defensin isolated from the Tephrosia villosa, a weedy legume enhances both insect and disease resistance in transgenic tobacco plants. Previously, it was also reported that, synchronous use of two defensins may lead to enhanced insecticidal and antimicrobial activity than using a single gene (Vasavirama and Kirti 2013a, b;Bezirganoglu et al. 2013;Guler et al. 2014). So, we chose a polyprotein type of gene expression with an aim that Tfgd2-RsAFP2 fusion gene linked by a linker peptide could impart enhanced insect resistance in transgenic pigeonpea. In accordance with this, genetic transformation in pigeonpea was performed to analyze the insect resistance in fusion gene expressing pigeonpea plants.
The Agrobacterium mediated transformation of the Tfgd2-RsAFP2 fusion genes resulted in the generation of 35 putative transgenic plants (T 0 ) and among them 12 plants were observed to be fusion gene and nptII positive by PCR analysis. The transformation efficiency obtained was 16.66 %, which is measured as the frequency of fusion gene and nptII positive plants versus total number of rooted shoots. In other transgenic pigeonpea studies, 15 % PCR positive T 0 plants were attained by GV2260 Agrobacterium strain harboring pPK202 vector (Surekha et al. 2005) and with C58 Agrobacterium strain harboring pHS723 vector 1-50 % transformation frequency was obtained (Sharma etal. 2006). Whereas Krishna et al. (2011) reported transformation frequency of 44.61 % with A. tumefaciens strain GV3101 carrying the pPZP211 binary vector and Dayal et al. (2003) reported transformation potency of 50 % using leaf explants by biolistic methods. The variance in the efficiency of transformation may be due to explant and genotypic variations in pigeonpea, co-cultivation method and the genetic backdrop of the Agrobacterium strains (Surekha et al. 2005;Surekha et al. 2007). The survival of T 0 progenies in kanamycin containing media exhibited the segregation of fusion gene according to the 3:1 Mendelian ratio and was transmitted to the following generation. In this study, fusion gene integration in the T 1 and T 2 progenies was confirmed through PCR and southern blotting and the expression of the fusion gene was evaluated by RT-PCR assay. The southern positive plants in the T 1 and T 2 generations were treated as stably integrated transgenic lines. Finally, an in vitro leaf bioassay was carried out with H. armigera second instar larvae, which displayed that Tfgd2-RsAFP2 fusion gene present in the transgenic pigeonpea leaves provided protection against the insect pest damage compared to the control untransformed lines. There was a significant relation between the expression level of the fusion gene and the level of resistance against H. armigera in the transgenic plants. In this experiment, high percentage of mortality and lesser leaf Fig. 8 a Represents Western blot analysis for the identification of Tfgd2 protein in the six T 2 transgenic pigeonpea lines. Lane NT represents total protein extract from the non transgenic plant; Lanes 1, 2, 3, 4, 5 & 6 represents 9-5-2, 2-1-1, 2-4-9, 9-5-5, 14-1-8 & 14-10-1 transgenic plants. b Represents Western blot analysis for the identification of RsAFP2 protein in transgenic pigeonpea plants. First lane represents negative control; Lanes 1, 2, 3, 4, 5 & 6 represents 9-5-2, 2-1-1, 2-4-9, 9-5-5, 14-1-8 & 14-10-1 transgenic plants damage showed by the transgenic lines represents that all the transformants could confront the insect damage to certain degree. Small variations in the percentage of larval mortality and area of leaf damage in all the transformants may be featured due to the variations in the expression levels of the fusion gene. The T 2 transgenic plants 9-5-5, 9-5-2 and 2-4-9 showed highest percentage of larval mortality ranging from 80 to 86 % within three days of incubation. Gosh et al. (2017) reported 80-100 % mortality towards second instar H. armigera larvae in transgenic plants transformed with Cry1Ac and Cry2Aa insecticidal genes. In acceptance to the earlier findings, the difference in the percentage of larval mortality in our study could be attributed due to the type of gene employed and the period of data collection (Kranthi and Kranthi 2004;Lacey and Kaya 2000). The current insect bioassay experiment displayed a significant decrease in the larval weight resulted by increased percentage of mortality related to the control plants, representing the expression of sufficient amount of the protein. Detached pod assay against third instar larvae of H. armigera conducted for the pods collected from the six transgenic lines of T 2 generation showed decreased pod consumption compared to the control. This was supported by the findings of pod assay done by Das et al. (2016) against 7-day-old H. armigera which showed 90 to 100 % larval mortality in T 4 and T 5 generations. In addition, Kaur et al. (2016) reported 97.78 % mortality of third instar larvae fed on the pods of T 2 transgenic lines after 48 h and Singh et al. (2018) reported less than 5-10 % of pod damage by the H. armigera second instar larvae. Based on these findings, it was observed that the fusion gene was effective in imparting insect resistance against H. armigera larvae in the gene expressing pigeonpea plants.

Conclusion
In conclusion, superior lines with high expression and efficiency of fusion gene against the pod borer were identified through molecular analysis and insect bioassay. The method used and the promising lines developed in T 2 generation could be a significant contribution to the community trying to minimise the damaging pest through gene transfer. Overall, the present results conclude that over-expression of Tfgd2-RsAFP2 fusion gene imparts resistance to H. armigera in pigeonpea plants carrying the fusion gene.