Overexpression of Bt Cry2A gene conferred resistance to Chilo partellus in transgenic Sorghum plants (Sorghum bicolor)     

Overexpression of Bt Cry2A gene conferred resistance to Chilo partellus in transgenic Sorghum plants (Sorghum bicolor)     

1. Amali¹, M. Ramakrishnan^2,3,†*, Papolu pradeep kumar^{2, †}, Illimar Altosaar⁴, Savarimuthu Ignacimuthu³,

¹Department of Biotechnology, Sathyabama University, Chennai, India

²State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin’an, Hangzhou 311300, China

³Division of Plant Biotechnology, Entomology Research Institute, Loyola College, Chennai, India

⁴Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada.

† Thèse authors contributed equally to this article.

*Correspondence

Abstract

Stem borer (Chilo partellus) is a complex parasite with extensive host range and causes substantial yield losses in many economically important crops, including Sorghum (Sorghum bicolor). Which is an important cereal crop of tropical grasses, grown worldwide, where stemborer is a major yield constraint for the production fields. In the present study, transgenic sorghum expressing the Bt cry2A gene under the control of a double CaMV35S promoter has generated to evaluate the sorghum stem borer resistance. The shoot apical meristem-targeted to Agrobacterium-mediated plant transformation strategy coupled with stringent Hygromycin selection has used to develop sorghum transformants. Eight putative transformants selected through PCR and Southern hybridization assay revealed the T-DNA integration of the cry2A gene into transformed sorghum plants containing single and double copy insertions. Stable expression of cry2A transgene in all the events was confirmed using quantitative RT-PCR (qPCR) and histochemical GUS detection assay. Second instar larvae of C. partellus on transgenic sorghum shoots expressing cry2A gene showed a larval mortality rate between 95.5-100% with 10% shoot damage. It demonstrates that the effectiveness of cry2A gene expression in sorghum shoots upon challenges to the insect larvae to infect the plants. These reports suggest that cry2A toxins from Bt enhances pod borer resistance in sorghum bicolor, and it protects the plants against pod borer infestation and results in high crop yield.

Keywords: Sorghum bicolor, Insect resistance, Bt cry2A, Agrobacterium, Transgenic plants, Chilo partellus,  Bioassay.

Introduction

Sorghum [Sorghum bicolor (L.) Moench] is the fifth major cereal crop after rice, wheat, maize, and barley in terms of production (Rooney and Saldivar 2003). Sorghum improvement through molecular and genetic research has gained wide significance in recent years, due to greater adaptability of this cereal crop to extreme environmental conditions such as drought, salt, and heat. Further, its ability to develop and persist in arid tropical soils with minimal nutritional requirements has gained the farmer’s attention towards adoption for cultivation aiming at food, fodder, and bioethanol production (Mathur et al. 2017). Although its natural resistance has significantly shaped the productivity of sorghum to abiotic factors, considerable productivity loss and decline had encountered due to interference from insect pests (Laidlaw and Godwin 2009; Nwanze et al. 1995).

Farmers suffer extensive sorghum yield losses due to the attack of various diseases and pests, including stem borers, C. partellus (Swinhoe) (Lepidoptera: Pyralidae); sorghum midge, Stenodiplosis sorghicola (Coquillett) (Diptera: Cecidomyiidae); and the sorghum shoot fly, Atherigona soccata Rondani (Diptera: Muscidae) (Visarada et al. 2016; Visarada and Kishore 2007). Among them, sorghum stem borer is the major yield constraint for sorghum. However, the absolute control or management tool is still lacking for these pod borer species. The worldwide ban/restriction on using chemical pesticides has put forward the necessity to search for alternative strategies to get rid of the pod borer problems. In this critical juncture, the use of transgenic plants with pod borer resistance genes promises to be an effective strategy. Several studies involving overexpression of resistance genes, silencing of effector genes, expression of cry toxins, etc., in model plants as well as crop species, was found to effectively reduce the pathogenicity of different pod borers thereby improving the crop yield. Therefore, the transgenic approach can be a preferred alternative strategy for pod borer management.

Genetic transformation techniques have recognized as an alternate strategy to conventional approaches that precisely mobilize genes and confer desirable traits such as insecticidal property to host plants and thus GM crops as a source of resistance to insect pests (Vaeck et al. 1987). Ideally, transfer of various crystalline toxins (cry) genes from the soil bacterium, Bacillus thuringiensis (Bt) to plants has reported to be effective in the development of insect resistant transgenic plants such as transgenic Bt tomato, cotton, corn and soybean resistant to lepidopteran larvae (Fischhoff et al. 1987; Korth 2008). These techniques are relatively simple and repeatable in crops with stable in vitro regeneration systems (non-recalcitrant). Although recalcitrance in sorghum has identified as a major impediment in successful genetic transformation, great success has achieved in improving the transformation efficiency in recent years (Gao et al. 2017; Liu et al. 2014). There are several useable genes in vitro and in vivo of the plant kingdom, and those genes can impart desirable traits in crop cultivars such as insecticidal properties. Various insecticidal Cry proteins or δ-endotoxins from Bt have identified as effective candidates for genetic transformation especially against lepidopteran insects (Hofte and Whiteley 1989). Further, transgenic plants expressing Bt genes have shown a high level of resistance against stem borers in rice (Cheng et al. 1998; Khanna and Raina 2002), maize (Koziel et al. 1993) and sorghum (Girijashankar et al. 2005).

The initial attempts were employing electroporation and biolistic–mediated gene delivery systems to genetically transform sorghum met with slight success (Battraw and Hall 1991; Casas et al. 1993; Hagio et al. 1991). Conversely, the first report on the Agrobacterium-mediated transformation of sorghum (Zhao et al. 2000) revealed a higher transformation frequency (2.1%) than those obtained by the earlier biolistic gene delivery methods. Since then, there were several attempts to use Agrobacterium-mediated transformation in sorghum. Among the several explants used for plant transformation, immature embryos (Howe et al. 2006), immature inflorescences (Casas et al. 1997) and shoot meristem (Girijashankar et al. 2005) has found to be amenable to gene transfer protocols. However, several reports are specifying the use of immature embryo explants to be superior for the formation of transgenic sorghum lines (Nguyen et al. 2007; Zhao et al. 2000).

Fortunately, the molecular toolbox or protein portfolio to engineer insect resistance is considerably more advanced than tissue culture. There are 789 reported δ-endotoxin proteins based on 318 holotypes (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/) belonging to 74 cry gene families (Crickmore et al. 1998). The particular Cry toxins highly target specific and have a defined spectrum of insecticidal activity, usually restricted to a few species within a specific order of insects. The largest group of Cry proteins belongs to 3-domain toxins, a group containing 30 basic types with more than 100 subtypes, with unique specificities, as well as toxicity to multiple insect orders such as Lepidoptera, Diptera, Coleoptera and Hymenoptera (de Maagd et al. 2003). The holotoxins, cry1A, cry1B, cry2A,  belong to this group.

The initial step towards the introduction of Cry endotoxins for imparting insect resistance in sorghum has attempted by directing cry1Ab and cry1B genes under the control of CaMV35S promoter into meristematic shoot explants by particle bombardment (Gray et al. 2004). Similarly, efforts to express the cry1Ac gene under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) in shoot tip explants proved to be promising with the net resultant transgenic sorghum resistant to the stem borer, C. partellus (Girijashankar et al. 2005).

A cuboidal crystal protein of approximately 65-kDa, encoded by the cry2A gene, is toxic to both dipteran and lepidopteran insects (Widner and Whiteley 1989; Yamamoto and McLaughlin 1981). However, little information is available about the insecticidal property of Cry2A type of endotoxins in insect pests of sorghum. Further, to the best of our knowledge, there are no reports available on the introgression of the cry2A gene in sorghum through any genetic transformation route and its efficacy against sorghum pests. The present investigation reports for the first time the successful transfer and integration of the cry2A gene into the sorghum genome, and validation of its insecticidal properties by conferring resistance to stem borer, C.partellus in a bioassay system. In the present study, we have used shoot meristem based Agrobacterium-mediated plant transformation for generating the transgenic plants.

Materials and methods

Plant material and tissue culture                                 

Seeds of Sorghum bicolor M35-1 have obtained from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, India, and were grown in vitro for excising the shoot apex explants. Before germinating, the seeds have washed in tap water for 10 min, followed by washing with 0.2 % Tween-20 for five minutes. Then, the seeds have washed with distilled water, and surface sterilized using 70 % ethanol for 30 sec followed by washed with distilled water twice. Seeds were sterilized again by soaking in 0.1 % HgCl₂ for five minutes and washed finally with sterilized distilled water twice and sown for germination in Murashige-Skoog (MS) medium (Murashige and Skoog 1962) in Petri plates. The shoot apices were excised from 5-days old in vitro grown seedlings and inoculated in the callus induction medium (CIM) prepared by supplementing MS salts with 2.5 mg/L 2,4-D(2,4-Dichlorophenoxyacetic acid), 0.5 mg/L kinetin and 500 mg/L casein hydrolysate with pH of 5.8 adjusted before autoclaving. The compact, healthy calli obtained after two weeks of culture in the dark at 25±2ºC were selected and used for agrobacterium infection (Amali et al. 2014).

Expression vector used for plant transformation

The binary vector pCAMBIA1305.1 containing the expression cassette of the cry2A gene under the control of 2x CaMV35S promoter (Kay et al. 1987) and NOS-terminator (Figure1), was mobilized into competent Agrobacterium tumefaciens strain LBA4404 by freeze and thaw method (Hoekema et al. 1983). The PCR positive colonies were selected and maintained in yeast extract agar medium with the selection of antibiotic kanamycin (50μg ml−1) and rifampicin (10μg ml−1).

In planta transformation of cry2A toxins from Bt using sorghum

Callus explants from matured sorghum seed were used for Agrobacterium mediated transformation. Explants were kept in pre-cultivation medium for 3 days (MS+ 0.1mg L-1 NAA + 2 mg L-1 BAP). Subsequently, they were infected with Agrobacterium suspension harbouring pCAMBIA1305.1 construct for 30 min with continuous. Later, the leaf explants were blotted dry on sterile tissue paper and co-cultivated for 2 days on cocultivation media (MS+ 200 µM acetosyringone). Subsequently, they were incubated on selection medium (MS+ 500mg L-1 cefotaxime + 4 mg L-1 BAP + 100 mg kanamycin+ 250 mg L-1 cefotaxime). After 30 days, shoots produced from the explants were subcultured at 10-15 days interval into fresh selection medium. The elongated shoots were further transferred to rooting media (1/2 MS + 100 mg L-1 kanamycin+ 250 mg L-1 cefotaxime). The resulting plants with well-established roots were hardened and transferred to green house for acclimatization, further growth and production of T0 seeds. Plants transformed with A.tumefaciens harboring empty vector, hence underwent without Hygromycin selection treated as control.

Hygromycin sensitivity test

Hygromycin B was used in the selection medium to select for transformed calli using hpt  gene. The effective lethal concentration of hygromycin B for the selection of transformed calli was determined by culturing the callus explants in CIM supplemented with hygromycin B at five different concentrations ranging from 1-5 mg/L. A positive control in CIM without hygromycin B was also maintained. Three replications were maintained at each level of concentration. The percentage of survival of callus was calculated after two weeks of culture in the dark at 25±2°C. Based on the results, 5 mg/L was identified as optimum concentration for constituting the selection medium.

GUS histochemical assay

The embryonic calluses and regenerated shoots of T0, T1, or T2 lines harbouring pCAMBIA1305.1 construct were selected via screening against antibiotic (hygromycin) resistance followed by  histochemical staining for GUS expression analysis. The expression of GUS was assayed with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) as the substrate (Jefferson et al. 1987). The explant tissues were incubated at 37°C in sodium phosphate buffer (50 mM Na₃PO₄, pH 6.8) that contained 1% Triton-X-100 for 1 hour. Subsequently, incubated at overnight in a solution containing 1.0 mM X-Gluc, 10 mM ETDA, 100 mM NaH₂PO₄, 0.1X Triton-X-100 and 50% methanol (pH 5.8), followed by two times washings with 99% methanol for two hours to remove chlorophyll pigment. The stained tissues were examined for blue spots under visible light and photographed under the microscope without damaging the tissue.

Molecular analysis of transformants using PCR, qRT-PCR and Southern hybridization

Genomic DNA was extracted from the fresh plants using modified cetyl trimethylammonium bromide (CTAB) method (Murray and Thompson 1980). For PCR-based detection of the presence of the gene, gene-specific primers (cry2A forward 5′- TGG ACA ACA ACG TGC TCA AC -3′, cry2A reverse 5′- GGA TCC TTA GTA GAG CGG CG -3′)  and primers specific for kanamycin resistance gene (hpt forward 5′-CAA TCG GCT GCT CTC ATG CCG-3′ and hpt reverse 5′-AGG CGA TAG AAG GCG ATG CGC-3′) were used.

Total RNA was isolated from the roots of fresh plants using Nucleospin Plant II RNA extraction kit (Macherey-Nagel, Germany) following manufacturer’s instructions. Approximately 500 ng of the purified RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Superscript VILO, Invitrogen). Quantitative RT-PCR (qPCR) amplification was performed to assess the level of  expression in sorghum tissue  using specific primers (cry2A RT- forward 5′- AAT GGC TAC TCA ACG AAG GG -3′ and cry2A RT-reverse 5′- GCA TAG TAG TAC AAG CGG AGA C -3′) with the PCR condition as follows: a hot start of 95°C for 5 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. In order to ascertain the specificity of amplification, a melt curve analysis or dissociation program was run as 95°C for 15 s; 60°C for 15 s followed by a slow ramp from 60-95°C. qPCR was performed in a realplex² thermal cycler (Eppendorf, Germany) using SYBR Green PCR master-mix kit (Eurogentec) and gene expression was normalized using Eukaryotic Initiation Factor 4A-1 (EIF4a) (Sudhakar et al. 2016) as a reference gene. Relative differences in expression were analysed through   2^-ΔΔCT method (Livak and Schmittgen 2001) after incorporating the data from three independent experiments, i.e. three separate plants from the same transgenic line.

In order to determine the integration pattern and copy number of cry2A transgene in the T0 sorghum plants, Southern hybridization was performed in the PCR-confirmed events. Approximately 12 µg of genomic DNA of each event was digested with BamHI enzyme (New England Biolabs), electrophoresed in a 0.8% agarose gel and transferred to nitrocellulose membrane (BioRad Zeta Probe). For probing, a 450 bp fragment of the cry2A gene was used. Probe labelling, hybridisation and blot development were performed using Dig luminescent detection kit (Roche, Germany) according to manufacturer’s instructions.

Resistance evaluation of transgenic sorghum against C. partellus

The second instar larvae of sorghum stem borer, C. partellus were collected from the fields at Paiyur, Krishnagiri District, Tamil Nadu, India, authenticated and maintained at the insect collection unit in the Entomology Research Institute, Loyola College, Chennai. Homozygous T1, and T2  lines harbouring the pCAMBIA1305.1 construct of cry2A were subjected to bioefficacy studies against C. partellus .The shoots of  8-10 cm²  cut from 30 days old plants were weighed and placed in petri dishes lined with moist cotton. The larvae were released on the transgenic and control plants were incubated for 72 hours at 28±1°C , under 70% RH, 16h light and 8h dark. The larvae were starved for five hours prior to inoculation. The incubated petri dishes were examined after every 24 hours for feeding damage on shoots and change in larval characteristics. The percentage of larval mortality and shoot damage were calculated after 72 hours (Cheng et al. 1998). These were compared with the wild type plants which were also inoculated and grown under similar conditions. Six replicates were used in the study. The data were made for all the replicates separately and subjected to subjected to one-way ANOVA (analysis of variance) test followed by Tukey’s multiple-comparison test with significance level at P < 0.05 using SAS software (version9.3).

Results

Hygromycin sensitivity test for the selection of transgenic plants

The hpt gene encoding hygromycin phosphotransferase that conferred resistance to hygromycin B used in the selection medium showed differential effect on the callus with different concentrations of hygromycin B ranging from 1-5 mg/L. In the CIM, complete death of cells were obtained with hygromycin concentration of 5 mg/L after two weeks of inoculation (Table 1), indicating this dose to be the lethal one for selective screening.  The toxicity level was high beyond 2 mg/L concentration as indicated by the sudden decline in the survival percentage of calli. The hygromycin concentration of 5 mg/L was used to constitute a selection medium for selecting the transgenic calli in the transformation programme.

Effect of Hygromycin concentration on callus induction

Hygromycin B is widely used aminoglycoside antibody and the resistance to different plant tissue is quite variable (Rosellini 2012). In order to assess the suitable concentration of hygromycin for transgenic sorghum bicolor call selection, shoot apex derived from meristematic tissues were cultured on petri plates with section medium containing 2, 5, 10 and 15 mg L^-1 Hygromycin for 30 days in dark at 25ºC. The shoot apex callus was maintained on similar medium without hygromycin selection were treated as control. The callus differentiation proportion were defined as the percentage of shoot apex tissue that showed viable transformed cells, was used in analysing the hygromycin inhibition during calli formation. As shown in figure, nearly all cells of the shoot apex (89.5 %) produced calli without hygromycin (Supplementary Figure S3). Correspondingly, when hygromycin was added to selection medium the callus differentiation proportion decline in growth rate, showed a negative correlation on selected hygromycin concentration. When 5 and 10 mg L^-1 was added to regeneration medium, only 9.8 % and 50.5 % shoot apex callus were generated (Table2). This suggest that the growth of calli was greatly inhibited when the concentration of hygromycin was increased. After 30 days, only immature embryo’s less than 0.5 mm³ could be converted into plantlets when 10 mg L^-1 hygromycin was added to differentiation medium. While almost invisible calli were obtained on medium with 5 mg L^-1 hygromycin (Supplementary Figure S3). Demonstrates the differentiation and multiplication of untransformed calli from shoot apex were strongly inhibited. Hence 5 mg L hygromycin was used for the selection of putative transgenic calli from sorghum shoot apex.

GUS expression activity in transgenic sorghum callus

GUS analysis was targeted to four distinct organs, such as callus, embryogenic calli, somatic embryo and regenerated shoots. After PCR assay of chimeric transgene in genomic DNA of genetically modified sorghum, the reporter gene expression was quantified by using GUS fluorometric assay. Sorghum tissue expressing the pCAMBIA1305.1: GUS construct were produced a significantly highest expression in embryogenic calli (Figure 2). Notably, transient GUS expression was absent in untransformed callus. Signifying that cry2A gene was highly expressed in different plant tissues. Suggesting that the role of promoter gene in reducing parasitic success

Molecular characterization of transgenic sorghum harbouring pCAMBIA Construct of cry2A

A total of 15 independent transgenic events (T0) were generated based on the hygromycin selection (Supplementary Figure S3). Each event was characterized for the presence of hpt and cry2A genes by PCR with different sets of primers amplifying the gene specific, as well as the selectable marker region. The amplified PCR products were gel electrophoresed and analysed on 1% agarose gel. The resolved gel showed single fragments with the desired size of cry2A (1.9 kb) and hpt (750 bp) were in 15 of 22 events (Supplementary Figure S1). Untransformed wild-type plants did not differ in morphological characteristics with transgenic plants (data not shown). Overall 8 T0 lines (4, 5, 6, 7, 8, 9, 10 and 11) were subjected to Southern blotting to analyse the integration patterns of cry2A. The T-DNA single copy insertion was reported for the event numbers 4, 8 and 11, and double copy insertion was reported for the event numbers 5, 9, and 10; the rest (1,2 6 and 7) not showed the desired copy insertions. (Figure 3).

T1 progeny plants were produced by self-pollination of the selected T0 lines in the green house. T1 lines were genotype through PCR using gene specific and hpt regions (Supplementary Figure S1). At least eight plants per event were tested, all plants showed expected fragment length of the specific gene. Homozygous T1 lines were self-pollinated to generate T2 plants. Correspondingly selected T2 plants were genotyped through PCR analysis (Supplementary Figure S1), shown the expected fragment amplification in all progeny plants (Data not shown). Using southern blotting assay, single copy integration of cry2A was confirmed in progeny plants of the selected T2 lines, namely 4.2,4.3,4.4, 4.5, 4.6, 4.7, 4.8,4.9 and 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9,11.10 (Supplementary Figure S2).This indicates the stable integration of Bt, cry2A, transgene and inherited in the progeny plants. The homozygous single and double copy plants were used for subsequent studies.

Expression of cry2A in transgenic lines of Sorghum

Using a qPCR assay, amplified transcripts of cry2A were observed in all the T0 and T2 transgenic events. On the contrary, no transcripts corresponding to the cry2A were detected in the RNA isolated from non-transgenic control plants. The data for the transgenic lines is therefore simply presented relative to the normalizer gene. Since the mean Ct of cry2A was greater than the mean Ct of the normalizer gene, EIF4a for each transgenic line. However, there was significant variation amongst the expression of transgenic plants. The average expression was higher is single copy plants (4.2 And 11.1) than the double copy ones (5.2,9.2 and 10.2) (Figure 4). However, EIF4a expression was found undetectable in empty vector control plants. Transformation frequency

Evaluation of sorghum transformants for resistance against C. partellus

To assess whether the in-planta expression of B. thuringiensis gene conferring resistance to             C. partellus, eight independent T2 sorghum lines (4.2, 5.2, 8.2, 9.2, and 11.2) harbouring pCAMBIA1305.1 construct of cry2A was evaluated in terms of larval mortality and shoot damage in the host plant shoots. No apparent phenotypic variation was observed in the shoot mass between wild-type plants grown on non-selective medium and T2 plants grown on selective medium containing Hygromycin (Figure 5), indicating that neither the antibiotic marker nor the cry2A endotoxin expression affected the shoot growth of transgenic plants. Following the          C.partellus larvae invasion, the damage was induced in shoots of wild-type and transgenic plants (Figure 4). At 72 hrs, the average larval damage was significantly (P < 0.05) reduced by 95.5 – 99.3% in sorghum transgenic plants compared to control lines harbouring empty vector (Table 2). Accordingly, marked reduction (3.2-9.2%) in the shoot damage was recorded in all transgenic lines compared to control plants (Table2). As the adult moth of C. partellus female produces its progeny in single egg mass, the number of egg masses indicates the similar number of reproducing females. Therefore B. thuringiensis cry2A toxin resulted in reduced shoot damage, apparently because attenuated development of C. partellus moth to females in transgenic lines. Our results suggest that B. thuringiensis corresponding to cry2A gene were ingested by              C. partellus during early parasitic process.

Discussion

Genetic transformation of sorghum for insect resistance and other traits such as nutritional superiority has been researched for two decades (Girijashankar et al. 2005; Ignacimuthu and Premkumar 2014; Zhao et al. 2000). The introduction of insect resistance, however, took a prime seat in these endeavours because of the poor availability of natural resistance and due to the magnitude of crop loss occurring annually though pest damages. Although few reports are available pertaining to the transfer of insecticidal Bt cry genes in sorghum, earlier genetic transformation works for insect resistance were targeted towards transfer of genes such as cry1Ab (Girijashankar et al. 2005; Zhang et al. 2009), cry1Ac (Girijashankar et al. 2005) and cry1C (Ignacimuthu and Premkumar 2014). While insect resistance in transgenic sorghum has profoundly been marked by successful reports using cry1Ac, cry1Ab, cry1Aa, cry1B and (Visarada et al. 2009; Visarada et al. 2016), cry1C (Ignacimuthu and Premkumar 2014), insects have developed resistance against some of these toxins. There are reports on insects that have developed resistance against cry1Ac expressed in Bt-cotton (Akhurst et al. 2003). Alternatively, Cry2A-type toxins have been deployed to overcome such impediments towards the generation of Bt crops. The cry2A genes encode proteins that differ in their structure and insecticidal mechanism from Cry1- type proteins, proving to be promising candidates for management of resistance development in insects (Kumar et al. 2004). Transgenic rice expressing cry2A was demonstrated to effectively control yellow stem borer and rice leaf folder. Further, cry2A gene in combination with other insecticidal genes can be used for pyramiding resistance against insect pests (Maqbool et al. 1998; Riazet al. 2006). There are no reports available on the transfer and effect of cry2 genes in sorghum. We formulated this study to introduce cry2A into sorghum by Agrobacterium-mediated gene transfer method. Furthermore, very little is known about the individual effect of cry2A type toxins in conferring an insecticidal property in this crop. Therefore, our prime objective was to successfully integrate this gene in sorghum, and to evaluate its potential in combating the attack by C. partellus in transgenic sorghum plants.

Sorghum is the least successful crop for manipulation by tissue culture and Agrobacterium-mediated transformation, due to its recalcitrant nature that is attributed to its high genotypic dependency, release of phenolics and lack of regeneration during long term in vitro cultures (Visarada and Kishore 2007). Further, the transformation frequency in sorghum is influenced by the Agrobacterium-host plant interaction and this interaction differs between genotypes, type of explants used and Agrobacterium strain. Very few sorghum genotypes have proven to be successful in transgenic experiments. Majority of transformation studies in sorghum with reports of higher transformation efficiency using either Agrobacterium or particle bombardment had focused on the use of elite genotypes such as P898012 (Gurel et al. 2009), C401 (Gao et al. 2005) and Tx430 (Liu and Godwin 2012). These were validated for their high efficiency in tissue culture and transformation. In addition, the transformation protocols were confined to the optimal transfer and expression of selectable marker genes in transgenic genotypes (Liu and Godwin 2012). In the present study, an agronomically desirable sorghum genotype M35-1 of Maldandi landrace, a traditional Indian cultivar of post-rainy season, popular among farmers for its grain and stover qualities (Kholová et al. 2013) was used.  Although reported to be less responsive in tissue culture (Jogeswar et al. 2007), we have found in sorghum  and organised to achieve successful transformation mediated by Agrobacterium (Amali et al 2014).

While most genetic transformation reports in sorghum have claimed immature embryos to be the most beneficial explants (Gao et al. 2005; Gurel et al. 2009; Howe et al. 2006), our study has shown the use of shoot apices with meristematic tissues as promising explants that respond better in transformation protocols (Amali et al 2014). Shoot apices proved to be better explants for achieving somatic embryogenesis in sorghum, via embryogenic calli formation and regeneration yielding a total of 22 hygromycin resistant plants with a transformation frequency of 10.5 % (Hiei, Ohta et al. 1994). Few reports are available till  date pertaining to the use of shoot apex explants for Agrobacterium-mediated transformation of sorghum with transformation frequencies reported in the range of 1.2-3.9% (Devi et al. 2004; Gray et al. 2004; Tadesse et al. 2003). Shoot apex explants derived from meristematic tissues at the early stages of development responded better in tissue culture conditions and were most desirable for genetic transformation to reduce somaclonal variations (Girijashankar and Swathisree 2009). Shoot apex has also been successfully used in Agrobacterium-mediated transformation of other grasses especially in finger millet (Ceasar and Ignacimuthu 2011, Satish, Ceasar et al. 2017) and  foxtail millet (Ceasar, Baker et al. 2017), etc.

The first successful report on production of transgenic sorghum plants through Agrobacterium-mediated transformation with a transformation efficiency of 2.1% was achieved using immature embryos of sorghum (Zhao et al. 2000). Subsequently several works pertaining to the application of Agrobacterium for sorghum transformation have been reported, with a progressive increase in the transformation frequency up to 7.7% (Gurel et al. 2009). The use of Agrobacterium for gene delivery enabled the transfer and integration of the cry2A gene along with the marker genes in the sorghum genome, resulting in stable transformation with insertion of single copy insertions.

The current investigation developed transgenic sorghum plants that expressed a codon optimized cry2A gene under the control of double 35S CaMV promoter, through Agrobacterium-mediated transformation. The first insecticidal genes used for plant transformation were indeed cry genes (Vaeck et al. 1987). Earlier study of Girijashankar et al. (2005) had reported the generation of transgenic sorghum genotype BTx623 expressing cry1Ac using the bombardment of shoot apices, yields a low transformation frequency of 1.5 %. Similarly, a very low transformation frequency in the range of 0.1-0.3 % was reported for generating transgenic sorghum lines expressing cry1Aa and cry1B by bombardment of immature embryos (Visarada et al. 2009). In this aspect, the results of this investigation with 3.8 % transformation frequency would serve as a benchmark to spur further improvements in transformation-based protocols, for the efficient application of insecticidal transgenics in sorghum.

By analysing various callus tissue of transgene under the constitutive expression of the promoter 2XCaMV35S, four major regulating regions (callus, embryogenic calli, somatic embryos and regenerated shoots) were detected by difference in GUS expression. 35S promoter is a widely used promoter that exhibits a high level of transcriptional activity in a variety of plant species (Benfey and Chua 1990). Although the 35S promoter is considered a constitutive promoter, the tissue specific expression levels appear to be developmentally and spatially controlled within a plant. Our results are consistent with previous reports that plants carrying the 35S promoter had higher GUS activity in somatic embryos and regenerated shoots, and lower expression in the roots (Casas, Kononowicz et al. 1993, Omirulleh, Ábrahám et al. 1993). A substantial resistance of was achieved against Colorado potato beetle larvae while expressing cry3A gene in potato plants under the control of CaMV35S promoter (Adang, Brody et al. 1993).Transgenic canola plants that expressing the cry1Ac from the shoots under the control of constitutive CaMV35S promoter resulted in reduced establishment of Diamondback moth (Plutella xylostella L.), cabbage looper (Trichoplusia ni Hübner), and corn earworm (Helicoverpa zea Boddie) (Stewart Jr, Adang et al. 1996). Similar attempts of 35S promoter in rice and maize transgenic plants expressing Cry9C and Cry6A proteins exhibited delayed larval development and increased mortality rates of yellow stem borer (Scirpophaga incertulas), striped stem borer (Chilo suppressalis) and Indian meal moth, Plodia interpunctella (Wünn, Klöti et al. 1996, Giles, Hellmich et al. 2000). In tobacco transgenic lines, over expression of cry3A and cry1Ab genes under the control of CaMV 35S promoter gene had significantly reduced shoot damage due to tobacco hornworm (Manduca sexta), tobacco budworm (Heliothis virescens), and potato beetle larvae (Carozzi, Warren et al. 1992, Sutton, Havstad et al. 1992) respectively. Further the in-planta expression of Cry2Aa and cry1Ac driven by the dual promoter of CaMV 35S in transgenic pigeon pea and Loblolly pine (Pinus taeda L.) had increased resistance to   gram pod borer, Helicoverpa armigera (Singh, Kumar et al. 2018), Crypyothelea formosicola (Staud) and Dendrolimus punctatus (Walker) (Tang and Tian 2003). This exemplifies the potential of GUS activity obtained in differentiated tissue of stably transformed sorghum plants indicates the value of the CaMV35S promoter and gene activity in transgenic monocots and dicots.

Using shoot apex targeted agrobacterium medium genetic transformation, transgenic sorghum plants expressing cry2A gene were generated. After, sorghum plants were successfully transformed with pCAMBIA1305.1 construct of cry2A gene, in order to evaluate the effect on   C. partellus larvae infectivity and transcription pattern of B. thuringiensis cry2A gene. Eight T0 (4, 5, 6, 7, 8, 9, 10 and 11) transgenic lines, containing single and double copy insertions of cry2A gene, were selected for further analysis. Only the single-copy plants harbouring the cry2A toxin were used for subsequent studies. Selected eight homozygous T0 events were self-pollinated to generate T1 lines, followed by selfing of subsequent homozygous lines, T2 progeny plants were generated. The stable integration and inheritance of the cry2A transgene was determined in all the selected transgenic lines. Interestingly, qRT-PCR assay revealed significantly greater expression of cry2A transgene in single copy plants than the double copy lines. Supporting this hypothesis, single copy T1 and T2 plants conferred significantly greater pod borer resistance, in terms of reduction in C. partellus larval mortality and shoot damage than the double copy lines of cry2A gene. This result suggests that B. thuringiensis cry2A gene was stably integrated at transcriptionally active in the sorghum genome.

Resistance screening was performed on shoot tissue of the six selected T2 progeny plants. Larval mortality was recorded in the progeny plants after 72 hr of incubation. Mortality of the challenged larvae, which determines the parasitic success of pod borer in host plants, was reduced by 97.2 – 99.3% in single copy lines and 95.5- 98.1 in double copy ones compared to non-transgenic plants. Our results are consistent with the earlier reports of transgenic sorghum expressing cry1C with pod borer C. partellus resistance (Ignacimuthu and Premkumar 2014). Rice plants expressing 2 % of cry1B protein displayed 100 % resistance to second-instar larvae of striped stem borer, C. suppressalis (Breitler et al. 2001). Low level expression of cry1Aa and cry1B in sorghum, decreased the productive success of C. partellus by 20-30% (Visarada et al. 2014). Among, transgenic pigeon pea expressing chimeric Cry1Aabc and Cry2Aa confers resistance to gram pod borer Helicoverpa armigera (Das, Datta et al. 2016, Singh, Kumar et al. 2018). In order to increase the resistance level of Bt toxin genes, cryIAb, cryI E-C, cryIA, cryIAcF have been reported earlier, demonstrating a significant reduction in pod borer infestation (Surekha, Beena et al. 2005, Sharma, Lavanya et al. 2006, Ramu, Rohini et al. 2012, Koul 2020). The lepidopteran specific δ-endotoxins of Bt, Cry1A is the most effective and specific toxin against gram pod borer Helicoverpa armigera (Chakrabarti, Mandaokar et al. 1998). It is important to mention that the best transgenic event of cry2A (4.2) in this study conferred about 99.3% resistance to  C. partellus in terms of the reduced larval mortality and shoot damage. Our data demonstrate that Cry2A overexpression in sorghum deliberates substantial resistance to C. partellus by inducing deleterious effects on pod borer survival and development. Moreover, this is the first report signifying the utility of Cry2A in sorghum, except for a single report (visharada), molecular characterization and bioassays proved the stability in the expression of Cry2A and denotes the potential of such strategy for the development of insect resistant transgenic plants.

Acknowledgements: We acknowledge the help rendered by ICRISAT, India, by providing sorghum germplasm. We also thank the Entomology Research Institute, Loyola College, Chennai for the insect rearing facilities.

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References

Adang, M. J., M. S. Brody, G. Cardineau, N. Eagan, R. T. Roush, C. K. Shewmaker, A. Jones, J. V. Oakes and K. E. McBride (1993). “The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants.” Plant molecular biology 21(6): 1131-1145.

Akhurst RJ, James W, Bird LJ, Beard C (2003) Resistance to the Cry1Ac δ-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J Eco Entomol 96(4):1290-1299

Amali P, Kingsley SJ, Ignacimuthu S (2014) Factors influencing Agrobacterium-mediated transformation of shoot apex derived calli explants of Sorghum. International J of Current Agri Research 3(9):85-88

Bale J, Van Lenteren J, Bigler F (2008) Biological control and sustainable food production. Philos Trans R Soc Lond B Biol Sci 363(1492):761-776

Battraw M, Hall T (1991) Stable transformation of Sorghum bicolor protoplasts with chimeric neomycin phosphotransferase II and β-glucuronidase genes. Theor. Appl. Genet 82(2):161-168

Benfey, P. N. and N.-H. Chua (1990). “The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants.” Science 250(4983): 959-966.

Breitler JC, Cordero MJ, Royer M, Meynard D, San Segundo B, Guiderdoni E (2001) The− 689/+ 197 region of the maize protease inhibitor gene directs high level, wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack. Molecular Breeding 7(4):259-274

Carozzi, N. B., G. W. Warren, N. Desai, S. M. Jayne, R. Lotstein, D. A. Rice, S. Evola and M. G. Koziel (1992). “Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco.” Plant molecular biology 20(3): 539-548.

Carvalho CH (1999) Agrobacterium-mediated transformation of sorghum and analysis of putative transposable element-induced mutants in sorghum. Ph.D. thesis, Purdue University

Carvalho CHS, Zehr UB, Gunaratna N, Anderson J, Kononowicz HH, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet. Mol. Biol 27(2):259-269

Carvalho CHS, Zehr UB, Gunaratna N, Anderson J, Kononowicz HH, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet. Mol. Biol 27(2):259-269

Casas AM, Kononowicz AK, Haan TG, Zhang L, Tomes DT, Bressan RA, Hasegawa PM (1997) Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences. In Vitro Cell.Dev.Biol.-Plant 33(2):92-100

Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM (1993) Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci U S A 90(23):11212-11216

Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM (1993) Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci U S A 90(23):11212-11216

Ceasar, S. A. and S. Ignacimuthu (2011). “Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.” Plant cell reports 30(9): 1759-1770.

Ceasar, S. A., A. Baker and S. Ignacimuthu (2017). “Functional characterization of the PHT1 family transporters of foxtail millet with development of a novel Agrobacterium-mediated transformation procedure.” Scientific reports 7(1): 1-16.

Chakrabarti, S., A. Mandaokar, P. Ananda Kumar and R. Sharma (1998). “Efficacy of lepidopteran specific δ-endotoxins of Bacillus thuringiensis against Helicoverpa armigera.” Journal of invertebrate pathology (Print) 72(3): 336-337.

Cheng X, Sardana R, Kaplan H, Altosaar I (1998) Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer. Proc Natl Acad Sci U S A 95(6):2767-2772

Crickmore N, Zeigler D, Feitelson J, Schnepf E, Van Rie J, Lereclus D, Baum J, Dean D (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):807-813

Das, A., S. Datta, G. Sujayanand, M. Kumar, A. K. Singh, A. Shukla, J. Ansari, L. Faruqui, S. Thakur and P. Kumar (2016). “Expression of chimeric Bt gene, Cry1Aabc in transgenic pigeonpea (cv. Asha) confers resistance to gram pod borer (Helicoverpa armigera Hubner.).” Plant Cell, Tissue and Organ Culture (PCTOC) 127(3): 705-715.

de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 37(1):409-433

de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 37(1):409-433

Devi P, Zhong H, Sticklen M (2004) Production of transgenic sorghum plants with related HVA1 gene. In: Seetharama N, Godwin ID (Eds.), Sorghum tissue culture and transformation, Oxford Publishrs, New Delhi, India 75-79

Dong J-Z, Yang M-Z, Jia S-R, Chua N-H (1991) Transformation of melon (Cucumis melo L.) and expression from the cauliflower mosaic virus 35S promoter in transgenic melon plants. Nature Biotechnol 9(9):858-863

Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCormick SM, Niedermeyer JG, Dean DA, Kusano-Kretzmer K, Mayer EJ, Rochester DE, Rogers SG, Fraley RT (1987) Insect tolerant transgenic tomato plants. Nature Biotechnol. 5:807–813

Gao L, Lihua W, Yanlong L, Yanyang L, Xiaocui Y, Qiuwen Z, Jiachen Z, Jieqin L (2017) Construction of an efficient tissue culture system for sorghum using mature embryos. Pak. J. Bot 49(3): 995-1000

Gao Z, Xie X, Ling Y, Muthukrishnan S, Liang GH (2005) Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotechnol. J 3(6):591-599 doi:10.1111/j.1467-7652.2005.00150.x

Giles, K. L., R. L. Hellmich, C. T. Iverson and L. C. Lewis (2000). “Effects of transgenic Bacillus thuringiensis maize grain on B. thuringiensis-susceptible Plodia interpunctella (Lepidoptera: Pyralidae).” Journal of economic entomology 93(3): 1011-1016.

Girijashankar V, Sharma H, Sharma KK, Swathisree V, Prasad LS, Bhat B, Royer M, San Secundo B, Narasu ML, Altosaar I (2005) Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus). Plant Cell Rep 24(9):513-522

Girijashankar V, Swathisree V (2009) Genetic transformation of Sorghum bicolor. Physiol. Mol. Biol. Plants 15(4):287-302

Girijashankar V, Swathisree V (2009) Genetic transformation of Sorghum bicolor. Physiol. Mol. Biol. Plants 15(4):287-302

Gray S, Zhang S, Rathus C, Lemaux P, Godwin I (2004) Development of sorghum transformation: organogenic regeneration and gene transfer methods. In: Seetharama N, Godwin ID (Eds.), Sorghum tissue culture and transformation, Oxford Publishers, New Delhi, India 35-43

Gurel S, Gurel E, Kaur R, Wong J, Meng L, Tan H-Q, Lemaux PG (2009) Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Rep 28(3):429-444

Guttikonda SK, Marri P, Mammadov J, Ye L, Soe K, Richey K, Cruse J, Zhuang M, Gao Z, Evans C, Rounsley S, Kumpatla SP (2016) Molecular characterization of transgenic events using next generation sequencing approach. PLoS One 11(2):e0149515 doi:10.1371/journal.pone.0149515

Hagio T, Blowers AD, Earle ED (1991) Stable transformation of sorghum cell cultures after bombardment with DNA-coated microprojectiles. Plant Cell Rep 10(5):260-264

Hiei, Y., S. Ohta, T. Komari and T. Kumashiro (1994). “Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T‐DNA.” The Plant Journal 6(2): 271-282.

Hoekema A, Hirsch P, Hooykaas P, Schilperoort R (1983) A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179-180

Hofte H, Whiteley H (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev 53(2):242-255

Howe A, Sato S, Dweikat I, Fromm M, Clemente T (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant Cell Rep 25(8):784-791

Ignacimuthu S, Premkumar A (2014) Development of transgenic Sorghum bicolor (L.) Moench resistant to the Chilo partellus (Swinhoe) through Agrobacterium-mediated transformation. Mol Biol Genet Eng 2:1 doi:10.7243/2053-5767-2-1

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901-3907

Jogeswar G, Ranadheer D, Anjaiah V, Kishor PK (2007) High frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from immature inflorescence explants. In Vitro Cell.Dev.Biol.-Plant 43(2):159-166

Kay R, Chan A, Daly M, Mcpherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236(4806):1299-1302 doi:10.1126/science.236.4806.1299

Khanna HK, Raina SK (2002) Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas). Transgenic Res 11(4):411-423

Kholová J, McLean G, Vadez V, Craufurd P, Hammer GL (2013) Drought stress characterization of post-rainy season (rabi) sorghum in India. Field Crops Research 141:38-46 doi:http://dx.doi.org/10.1016/j.fcr.2012.10.020

Korth KL (2008) Genes and traits of interest for transgenic plants.  In: Stewart, C.N. (Ed.), Plant Biotechnology and Genetics: Principles, Techniques and Applications. John Wiley & Sons Inc., Hoboken, NJ, USA

Koul, B. (2020). Genetically Modified (GM) Crops Harbouring Bacillus thuringiensis (BT) Gene (S) to Combat Biotic Stress Caused by Insect Pests. Phyto-Microbiome in Stress Regulation, Springer: 21-61.

Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Nature Biotechnol 11(2):194-200

Kumar KK, Poovannan K, Nandakumar R, Thamilarasi K, Geetha C, Jayashree N, Kokiladevi E, Raja JA, Samiyappan R, Sudhakar D (2003) A high throughput functional expression assay system for a defence gene conferring transgenic resistance on rice against the sheath blight pathogen, Rhizoctonia solani. Plant Sci 165(5):969-976

Kumar S, Udayasuriyan V, Sangeetha P, Bharathi M (2004) Analysis of Cry2A proteins encoded by genes cloned from indigenous isolates of Bacillus thuringiensis for toxicity against Helicoverpa armigera. Curr. Sci. 86(4):566-570

Laidlaw HK, Godwin ID (2009) Sorghum. Compendium of Transgenic Crop Plants. 1(6):157-176

Leifert C, Camotta H, Waites W (1992) Effect of combinations of antibiotics on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia. Plant Cell Tiss Organ Cult 29(2):153-160

Liu G, Campbell BC, Godwin ID (2014) Sorghum genetic transformation by particle bombardment. In: Henry RJ, Furtado A (Eds.), Cereal Genomics. Methods in Molecular Biology (Methods and Protocols), Humana Press, Totowa, NJ 1099:219-234

Liu G, Godwin ID (2012) Highly efficient sorghum transformation. Plant Cell Rep 31(6):999-1007 doi:10.1007/s00299-011-1218-4

Livak, K. J. and T. D. Schmittgen (2001). “Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method.” methods 25(4): 402-408.

Maqbool SB, Husnain T, Riazuddin S, Masson L, Christou P (1998) Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene. Molecular Breeding 4(6):501-507

Mathur S, Umakanth AV, Tonapi VA, Sharma R, Sharma MK (2017) Sweet sorghum as biofuel feedstock: recent advances and available resources. Biotechnol Biofuels 10:146

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3):473-497

Murray M, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321-4326

Nguyen T-V, Thu TT, Claeys M, Angenon G (2007) Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant Cell Tiss Organ Cult 91(2):155-164

Nwanze K, Seetharama N, Sharma H, Stenhouse J (1995) Biotechnology in pest management: improving resistance in sorghum to insect pests. African Crop Science Journal 3(2):
209-215

Omirulleh, S., M. Ábrahám, M. Golovkin, I. Stefanov, M. K. Karabev, L. Mustárdy, S. Mórocz and D. Dudits (1993). “Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize.” Plant molecular biology 21(3): 415-428.

Pipatpanukul T, Bunnag S, Theerakulpisut P, Kosittrakul M (2004) Transformation of indica rice (Oryza sativa L.) cv. RD6 mediated by Agrobacterium tumefaciens. Songklanakarin J. Sci. Technol 26(1): 1-13

Ramu, S., S. Rohini, G. Keshavareddy, M. Gowri Neelima, N. Shanmugam, A. Kumar, S. Sarangi, P. Ananda Kumar and M. Udayakumar (2012). “Expression of a synthetic cry1AcF gene in transgenic Pigeon pea confers resistance to Helicoverpa armigera.” Journal of applied entomology 136(9): 675-687.

Riaz N, Husnain T, Fatima T, Makhdoom R, Bashir K, Masson L, Altosaar I, Riazuddin S (2006) Development of Indica Basmati rice harboring two insecticidal genes for sustainable resistance against lepidopteran insects. South African Journal of Botany 72(2): 217-223

Rooney LW, Saldivar SOS (2003) Sorghum. In Encyclopedia of Food Sciences and Nutrition (Second Edition), Academic Press 5370-5375

Rosellini, D. (2012). “Selectable markers and reporter genes: a well furnished toolbox for plant science and genetic engineering.” Critical reviews in plant sciences 31(5): 401-453.

Satish, L., S. A. Ceasar and M. Ramesh (2017). “Improved Agrobacterium-mediated transformation and direct plant regeneration in four cultivars of finger millet (Eleusine coracana (L.) Gaertn.).” Plant Cell, Tissue and Organ Culture (PCTOC) 131(3): 547-565.

Schledzewski K, Mendel RR (1994) Quantitative transient gene expression: comparison of the promoters for maize polyubiquitin1, rice actin1, maize- derived Emu and CaMV 35S in cells of barley, maize and tobacco. Transgenic Res 3(4):249-255

Seltzer J (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44(6):789-798

Sharma HC (2012) Conventional and biotechnological approaches for pest management: potential and limitations. In Environmental Safety of Biotech and Conventional IPM Technologies. Studium Press LLC, Texas, USA, pp. 3-25

Sharma, K. K., M. Lavanya and V. Anjaiah (2006). “Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L. Millsp.) expressing the synthetic Bt cry1Ab gene.” In Vitro Cellular & Developmental Biology-Plant 42(2): 165-173.

Shrawat AK, Lörz H (2006) Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers. Plant Biotechnology Journal 4(6):575-603

Silva Jd, Fukai S (2001) The impact of carbenicillin, cefotaxime and vancomycin on chrysanthemum and tobacco TCL morphogenesis and Agrobacterium growth. J. Appl. Hort 3(1):3-12

Singh, S., N. R. Kumar, R. Maniraj, R. Lakshmikanth, K. Rao, N. Muralimohan, T. Arulprakash, K. Karthik, N. Shashibhushan and T. Vinutha (2018). “Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.” Scientific reports 8(1): 1-12.

Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503-517

Sridevi G, Parameswari C, Rajamuni P, Veluthambi K (2006) Identification of hemizygous and homozygous transgenic rice plants in T1 generation by DNA blot analysis. Plant Biotechnology 23(5):531-534

Stewart Jr, C. N., M. J. Adang, J. N. All, P. L. Raymer, S. Ramachandran and W. A. Parrott (1996). “Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis cryIAc gene.” Plant physiology 112(1): 115-120.

Sudhakar Reddy, P., D. Srinivas Reddy, K. Sivasakthi, P. Bhatnagar-Mathur, V. Vadez and K. K. Sharma (2016). “Evaluation of sorghum [Sorghum bicolor (L.)] reference genes in various tissues and under abiotic stress conditions for quantitative real-time PCR data normalization.” Frontiers in plant science 7: 529.

Tadesse Y, Sagi L, Swennen R, Jacobs M (2003) Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Plant Cell Tiss Organ Cult 75(1):1-18

Vaeck M, Reynaerts A, Höfte H, Jansens S, De Beuckeleer M, Dean C, Zabeau M, Montagu MV, Leemans J (1987) Transgenic plants protected from insect attack. Nature 328(6125):33-37

Veluthambi K, Gupta AK, Sharma A (2003) The current status of plant transformation technologies. Curr. Sci 84(3):368-380

Visarada K, Kishore NS (2007) Improvement of sorghum through transgenic technology. ISB News Report 1-3

Visarada K, Padmaja P, Saikishore N, Pashupatinath E, Kanti Meena, Rao S, Seetharama N (2009) Genetic transformation of sorghum for resistance to Stem borer. In: International conference on current trends in biotechnology and its implications in agriculture and technology, Meerut, India

Visarada K, Padmaja P, Saikishore N, Pashupatinath E, Royer M, Seetharama N, Patil J (2014) Production and evaluation of transgenic sorghum for resistance to stem borer. In Vitro Cell.Dev.Biol.-Plant 50(2):176-189

Visarada K, Sarma N (2004) Transformation of indica rice through particle bombardment: factors influencing transient expression and selection. Biologia Plantarum 48(1):25-31

Visarada KBRS, Prasad GS, Royer M (2016) Genetic transformation and evaluation of two sweet sorghum genotypes for resistance to spotted stem borer, Chilo partellus (Swinhoe). Plant Biotechnol Rep 10(5): 277-289

Widner WR, Whiteley H (1989) Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol 171(2):965-974

Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho M-J, Zhao Z-Y (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. In Vitro Cell.Dev.Biol.-Plant 50(1):9-18

Yamamoto T, McLaughlin RE (1981) Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var, kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem. Biophys. Res. Commun 103(2):414-421

Zaidi MA, Mohammadi M, Postel S, Masson L, Altosaar I (2005) The Bt gene cry2Aa2 driven by a tissue specific ST-LS1 promoter from potato effectively controls Heliothis virescens. Transgenic Res. 14:289-298.

.

Zhang M, Tang Q, Chen Z, Liu J, Cui H, Shu Q, Xia Y, Altosaar I (2009) Genetic transformation of Bt gene into sorghum (Sorghum bicolor L.) mediated by Agrobacterium tumefaciens. Sheng wu gong cheng xue bao, Chinese journal of biotechnology 25(3):418-423

Zhao Z, Tomes D (2003) Sorghum transformation. In: Jackson JF, Linskens HF, Inman RB (Eds.), Genetic Transformation of Plants, Verlag Berlin Heidelberg, Springer 23:91-102

Zhao Z-Y, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44(6):789-798