Pod Borer Resistance Research Articles

Breeding for Pod Borer and Wilt Resistance in Chickpea (Cicer arietinum L.)

Breeding for Pod Borer and Wilt Resistance in Chickpea (Cicer arietinum L.)

Genetic resources and genes/QTLs for gram pod borer (Helicoverpa armigera Hübner) resistance in chickpea from the Western Himalayas.

Helicoverpa armigera (also known as gram pod borer) is a serious threat to chickpea production in the world. A set of 173 chickpea genotypes were evaluated for H. armigera resistance, including mean larval population (MLP), percentage pod damage (PPD), and pest resistance (PR) for 2 consecutive years (year 2020 and 2021). The same core set was also genotyped with 50K AxiomCicerSNP Array. The trait data and 50,000 single nucleotide polymorphism genotypic data were used together to work out marker-trait associations (MTAs) using different genome-wide association studies models. For MLP, a total of 53 MTAs were identified, including 25 MTAs in year 2020 and 28 MTAs in year 2021. A set of three MTAs was found common in both environments. For PPD, two MTAs in year 2020 and five MTAs in year 2021 were identified. A set of two MTAs were common in both environments. Similarly, for PR, only two MTAs common in both environments were identified. Interestingly, a common MTA (Affx_123255526) on chromosome 2 (Ca2) was found to be associated with all the three component traits (MLP, PPD, and PR) of pod borer resistance in chickpea. Further, we report key genes that encode SCAMPs (that facilitates the secretion of defense-related molecules), quinone oxidoreductase (enables the production of reactive oxygen species that promotes diapause of gram pod borer), and NB-LRR proteins that have been implicated in plant defense against H. armigera. The resistant chickpea genotypes, MTAs, and key genes reported in the present study may prove useful in the future for developing pod borer-resistant chickpea varieties.

Field screening and identification of biochemical indices of pod borer (Helicoverpa armigera) resistance in chickpea mutants.

Chickpea pod borer (CPB) (Helicoverpa armigera) is one of the major pests, causing significant yield losses. The objectives were to screen chickpea mutants for pod borer resistance/tolerance under field conditions and identification of biochemical markers of tolerance. Chickpea mutant CM216-A/15 had highest leaf (25 trichomes/mm2) and stem trichome density (17 trichomes/mm2) with least pod damage at Kallur Kot and highest pod weight per plant (22.8 ± 2.6g) at AZRI. Higher total phenolic contents (TPCs) and antioxidant capacity were detected in tolerant mutants, i.e., CM216-A/15 and CM664/15. TPC was positively associated with pod yield and had negative correlation with pod damage. Mutants CM216-A/15, CM664/15, and CM766/15 depicted the highest resilience to CPB, owing to higher hairiness, better antioxidant defense response, and lower levels of hydrolytic enzymes and sugars. Identified biochemical markers like TPC, total oxidant status, superoxide dismutase, and pigments can be used for screening of CPB-tolerant/resistant mutants.

Identifying Soybean Pod Borer (Leguminivora glycinivorella) Resistance QTLs and the Mechanism of Induced Defense Using Linkage Mapping and RNA-Seq Analysis.

The soybean pod borer (Leguminivora glycinivorella) (SPB) is a major cause of soybean (Glycine max L.) yield losses in northeast Asia, thus it is desirable to elucidate the resistance mechanisms involved in soybean response to the SPB. However, few studies have mapped SPB-resistant quantitative trait loci (QTLs) and deciphered the response mechanism in soybean. Here, we selected two soybean varieties, JY93 (SPB-resistant) and K6 (SPB-sensitive), to construct F2 and F2:3 populations for QTL mapping and collected pod shells before and after SPB larvae chewed on the two parents to perform RNA-Seq, which can identify stable QTLs and explore the response mechanism of soybean to the SPB. The results show that four QTLs underlying SPB damage to seeds were detected on chromosomes 4, 9, 13, and 15. Among them, qESP-9-1 was scanned in all environments, hence it can be considered a stable QTL. All QTLs explained 0.79 to 6.09% of the phenotypic variation. Meanwhile, 2298 and 3509 DEGs were identified for JY93 and K6, respectively, after the SPB attack, and most of these genes were upregulated. Gene Ontology enrichment results indicated that the SPB-induced and differently expressed genes in both parents are involved in biological processes such as wound response, signal transduction, immune response, and phytohormone pathways. Interestingly, secondary metabolic processes such as flavonoid synthesis were only significantly enriched in the upregulated genes of JY93 after SPB chewing compared with K6. Finally, we identified 18 candidate genes related to soybean pod borer resistance through the integration of QTL mapping and RNA-Seq analysis. Seven of these genes had similar expression patterns to the mapping parents in four additional soybean germplasm after feeding by the SPB. These results provide additional knowledge of the early response and induced defense mechanisms against the SPB in soybean, which could help in breeding SPB-resistant soybean accessions.

Deciphering of Pod Borer [Helicoverpa armigera (Hübner)] Resistance in Cajanus platycarpus (Benth.) Offers Novel Insights on the Reprogramming and Role of Flavonoid Biosynthesis Pathway

Management of pod borer, Helicoverpa armigera in pigeonpea (Cajanus cajan L.), an important legume crop, has been a pertinent endeavor globally. As with other crops, wild relatives of pigeonpea are bestowed with various resistance traits that include the ability to deter the H. armigera. Understanding the molecular basis of pod borer resistance could provide useful leads for the management of this notorious herbivore. Earlier studies by our group in deciphering the resistance response to herbivory through multiomics approaches in the pigeonpea wild relative, Cajanus platycarpus, divulged the involvement of the flavonoid biosynthesis pathway, speculating an active chemical response of the wild relative to herbivory. The present study is a deeper understanding of the chemical basis of pod borer (H. armigera) resistance in, C. platycarpus, with focus on the flavonoid biosynthesis pathway. To substantiate, quantification of transcripts in H. armigera-challenged C. platycarpus (8 h, 24 h, 48 h, 96 h) showed dynamic upregulation (up to 11-fold) of pivotal pathway genes such as chalcone synthase, dihydroflavonol-4-reductase, flavonoid-3′5′-hydroxylase, flavonol synthase, leucoanthocyanidin reductase, and anthocyanidin synthase. Targeted LC-MS analyses demonstrated a concomitant increase (up to 4-fold) in naringenin, kaempferol, quercetin, delphinidin, cyanidin, epigallocatechin, and epicatechin-3-gallate. Interestingly, H. armigera diet overlaid with the over-produced flavonoids (100 ppm) showed deleterious effects on growth leading to a prolonged larval period demonstrating noteworthy coherence between over-accumulation of pathway transcripts/metabolites. The study depicts novel evidence for the directed metabolic reprogramming of the flavonoid biosynthesis pathway in the wild relative to pod borer; plant metabolic potential is worth exploiting for pest management.

Relevance of methionine sulfoxide reductase(s) (MSR) as candidate proteins in redox homeostasis-mediated resistance response to Helicoverpa armigera (Hübner) in the pigeonpea wild relative Cajanus platycarpus (Benth.) Maesen

Pod borer, Helicoverpa armigera, a polyphagus herbivore causes extensive economic losses to crops, including pigeonpea. Exploitation of pod borer resistance in wild relatives is pertinent due to the absence of resistance sources in cultivated pigeonpea and crossing-incompatibility with the resistant wild relatives. We present leads obtained in deeper understanding of pod borer resistance mechanism in Cajanus platycarpus, a pigeonpea wild relative. Surge in cellular ROS during herbivory leads to redox-PTMs (post translational modifications) of methionine-rich proteins including antioxidant enzymes, causing oxidative damage. Plants then officiate methionine sulfoxide reductases (MSRs), that maintain the redox status of methionine and hence homeostasis. We demonstrate functionality of MSRs (MSRA and MSRB) in the resistance response of the wild relative to pod borer. Among 5 MSRA and 3 MSRB genes, CpMSRA2 and CpMSRB1 were herbivore-responsive based on expression during herbivory. Clues about the stress-responsiveness were obtained upon analyses of cis-elements and co-expressing genes. Apparently, the wild relative followed a non-canonical mode of redox management, as divulged by antioxidant enzymes and the scavenging capacity. Differential lipid peroxidation as an early response provided evidences for an effective redox management in the wild relative. This is the first report signifying redox homeostasis in the resistance response towards herbivory.

Identification of dynamic microRNA associated with systemic defence against Helicoverpa armigera infestation in Cajanus scarabaeoides

Helicoverpa armigera is a major insect pest of several crop plants, including pigeonpea. Resistant gene sources are not available in the cultivated gene pool, but resistance has been observed in its crop wild relative, Cajanus scarabaeoides. Gene regulatory mechanisms governing the systemic immune response of this plant to pod borer infestation have not yet been deciphered. MicroRNA (miRNA) profiles of H. armigera-infested and undamaged adjacent leaves of C.scarabaeoides were compared to gain an insight into the plant-insect interactions and to identify dynamic miRNA molecules potentially acting as mediators of systemic defence responses. A total of 211 conserved, temporally dynamic miRNA were identified in the unfed adjacent leaves, out of which 98 were found to be differentially expressed in comparison to control leaves. On further analysis, most of the miRNA detected in the adjacent leaves was found to target genes involved in the defence pathways and plant immune response. An overlap of the differentially expressing miRNAs was observed between insect-fed and adjacent unfed leaves, indicating the transmission of signal from the site of infestation to the undamaged parts of the plant, indicative of induction of a systemic defence response. The miRNA response in the unfed leaves had the signatures of induced changes in metabolism and signal transduction for induction of defence pathway genes. This study reveals the participation of miRNAs in imparting pod borer resistance and mounting a systemic defence response against pod borer infestation in C. scarabaeoides. © 2022 Society of Chemical Industry.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg−1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Comparative analysis of herbivory responsive miRNAs to delineate pod borer (Helicoverpa armigera) resistance mechanisms in Cajanus cajan and its wild relative Cajanus scarabaeoides.

Comparative analysis of herbivory responsive miRNAs between pod borer susceptible C. cajan and its resistant Crop Wild Relative (CWR) C. scarabaeoides revealed miRNA-based regulation of defense genes and plant-insect interactions. Gram pod borer (Helicoverpa armigera) is one of most devastating pests of pigeon pea (Cajanus cajan) worldwide, responsible for huge losses in crop productivity. The lack of genes conferring resistance to pod borer in pigeon pea has proven to be a bottleneck for its improvement. One of its CWR, C. scarabaeoides has demonstrated resistance to this pest and can be exploited for developing pest resistant crop varieties. Differences in expression patterns of herbivory responsive microRNAs in the susceptible C. cajan and resistant C. scarabaeoides after different time duration of pod borer infestation (2h, 8h and 18h) were identified, characterized and functionally validated to understand their role in insect defense response. A total of 462 conserved and 449 novel miRNAs and 273 conserved and 185 novel miRNAs, were identified in C. cajan and C. scarabaeoides, respectively. Among the identified miRNAs, 65, 68 and 65 miRNAs were found to be differentially expressing between the C. scarabaeoides and C. cajan libraries 2h, 8h and 18h post infestation, respectively. These miRNAs were found to target genes involved in a number of pathways contributing to defense and acquired resistance in C. scarabaeoides against pod borer, indicating miRNA-based regulation of defense pathways. Expression patterns of eight of these miRNAs were validated by qRT-PCR. This study provides novel insights into the miRNA-mediated plant-insect interactions and the mechanisms of regulatory pathways involved in insect defense. These findings can be utilized for further exploring the mechanism of herbivore defense in plant systems.

Reaping the Potential of Wild Cajanus Species through Pre-Breeding for Improving Resistance to Pod Borer, Helicoverpa armigera, in Cultivated Pigeonpea (Cajanus cajan (L.) Millsp.).

Simple SummaryPigeonpea is an important legume crop that is severely affected by various insect pests, especially pod borer. Because there are low resistance levels to pod borers in the cultivated pigeonpea gene pool, it is necessary to introduce resistance-related traits from its wild relatives. In this study, we conducted a series of crosses to introduce traits related to pod borer resistance from two wild relatives of pigeonpea into two popular cultivated varieties. We generated populations from four different crosses and screened these populations for traits related to pod borer resistance: i.e., low levels of insect damage, high concentrations of insect-deterring compounds in the seeds, and the presence of trichomes on the leaves. The most promising lines were tested across seasons and locations. Ultimately, we identified 21 lines with excellent traits related to pod borer resistance. These lines will be useful for breeding new insect-resistant pigeonpea cultivars. The availability of such cultivars will reduce the use of pesticides to control pests on pigeonpea crop.Pod borer (Helicoverpa armigera) causes the highest yield losses in pigeonpea, followed by pod fly (Melanagromyza obtusa). High levels of resistance to pod borer are not available in the cultivated genepool. Several accessions of wild Cajanus species with strong resistance, and different resistance mechanisms (antixenosis and antibiosis) to pod borer have been identified. These accessions can be utilized to improve the pod borer resistance of cultivated pigeonpea. Using pod borer resistant Cajanus scarabaeoides and Cajanus acutifolius as pollen donors and popular pigeonpea varieties as recipients, pre-breeding populations were developed following simple- and complex-cross approaches. Preliminary evaluation of four backcross populations consisting of >2300 introgression lines (ILs) under un-sprayed field conditions resulted in identifying 156 ILs with low visual damage rating scores (5.0–6.0) and low pod borer damage (<50%). Precise re-screening of these ILs over different locations and years resulted in the identification of 21 ILs having improved resistance to pod borer. Because these ILs were derived from wild Cajanus species, they may contain different alleles for different resistance components to pod borer. Hence, these ILs are ready-to-use novel and diverse sources of pod borer resistance that can be utilized for improving the pod borer resistance of cultivated pigeonpea.

Secondary genepool of Australian Cajanus species contains sources of resistance to Helicoverpa armigera (Hübner)

AbstractA narrow genetic base in cultivated pigeonpea (Cajanus cajan (L.) Millsp.) limits the host plant resistance (HPR) to the devastating pest Helicoverpa armigera (Hübner). Earlier research on pod borer resistance in wild Cajanus relatives has mainly explored the tertiary genepool, making breeding for resistance difficult because of linkage drag. Previous investigations on a wide range of Australian native germplasm from the secondary and tertiary gene pools revealed resistance in the secondary gene pool, associated with total phenolic content (TPC). This study reports the application of the extreme phenotype genome‐wide association (XP‐GWAS) to associate single nucleotide polymorphisms (SNPs) with resistance traits. Mapping of 19 accessions, including 12 wild accessions to a reference pigeonpea genome have identified a total of 16,489 and 16,227 nonsynonymous SNPs for the resistance and TPC traits, respectively. These SNPs were subjected to functional analysis, and KEGG pathway analysis revealed the presence of candidate genes coding for the crucial enzymes in five defence‐related pathways that could be contributing to resistance. These pathways involve ‘Jasmonic acid synthesis’ (palmitoyl‐CoA hydrolase, phospholipase A1 and phospholipase A2), ‘insect hormone biosynthesis’ (aldehyde dehydrogenase) ‘Steroid biosynthesis’ (squalene monooxygenase, farnesyl transferase and lanosterol synthase), ‘Cutin, suberine and wax synthesis’ (O‐acyltransferase) and ‘Brassinosteroid biosynthesis’ (Det2). Analysis of SNPs associated with the TPC trait led to the identification of a divergent pathway of ‘secondary metabolite synthesis’ (flavonoid 3′ hydroxylase, l‐rhamnosyltransferase) in wild Cajanus species consistent with a high concentration of isoorientin and the possible presence of its derivative maysin; a strong insecticidal compound in leaves.

Transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein imparts resistance to Maruca vitrata legume pod borer.

Fertile independent transgenic cowpea lines expressing the BtCry2Aa toxin with increased resistance to the most devastating lepidopteran insect pest, Maruca pod borer has been developed for the first time. Cowpea is a staple legume important for food and nutritional security in sub-Saharan Africa and Asia, where its production is limited by the key pest, legume pod borer (Maruca vitrata). Cowpea varieties resistant to M. vitrata are not known, hence, development of Maruca pod borer resistance cowpea through genetic engineering is a promising approach to improve its production. In the present study, transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein were developed for the first time using Agrobacterium tumefaciens-mediated transformation of cotyledonary explants. T0 plants recovered from Agrobacterium cocultured explants on medium containing 120 mgl-1 of kanamycin were identified on the basis of the presence of transgenes by PCR, their integration into genome by Southern hybridization and expression of their transcripts by semi quantitative PCR (sqRT-PCR) and quantitative Real-time-PCR (qRT-PCR) and protein by Western blot analysis. The transformation efficiency obtained was 3.47% with 11 independent T0 transgenic lines. The bioefficacy of Cry2Aa protein expressed in randomly selected four T0 plant's leaves and pods was evaluated by feeding Maruca pod borer demonstrated a significant lower damage and a high level of Maruca mortality (more than 90%) for all these Bt lines. The inheritance of transgenes from T0 to T1 progeny plants was demonstrated by PCR analysis. The transgenic plants generated in this study can be used in cowpea breeding program for durable and sustainable legume pod borer resistance.

Optimization of kanamycin concentration for screening of primary transformants in pigeonpea

Pigeonpea is an important pulse crop which is widely cultivated throughout the tropical and sub-tropical regions. It is the major source of protein for the people with vegetarian diet. Major constraint in pigeonpea is the attack by pod borer. Conventional plant breeding techniques have limitations to develop resistance to the pest due to non-availability of resistance in the available germplasm. Therefore genetic engineering tools can be employed to transfer foreign genes into pigeonpea to impart pod borer resistance. Development of a reliable regeneration system coupled with efficient transformation has been a major limitation in developing transgenic pigeonpea with useful genes. In planta is an alternative approach to obtain transformed plants in short duration without involving tissue culture regeneration. However major limitations with this approach is screen more number of seeds to identify putative transformants which is time consuming and expensive. Therefore the present investigation was carried out to optimize the concentration of kanamycin for quick identification of putative transformants carrying cry1Ac and npt II using sand based assay. Seeds of Asha and BSMR736 were soaked in different concentrations of kanamycin (10-100, 200, 300, 400 and 500 ppm) for 5hrs and later sown in sand based medium. There was no effect of kanamycin on seedling growth up to 60 ppm and decrease in survival percentage in both the genotypes beyond this concentration. There was significant differences in growth of the seedlings in terms of shoot and root length at 100 ppm and beyond. Therefore this concentration was used to screen seeds of 32 primary transformants carrying cry1Ac and npt II and subjected to gene specific PCR analysis to confirm the usability of simple screening on kanamycin based sand medium for the identification of true transgenic plants. However some discrepancies between the growth inhibition and PCR results were observed.

Unraveling the proteomic changes involved in the resistance response of Cajanus platycarpus to herbivory by Helicoverpa armigera.

The pigeonpea wild relative Cajanus platycarpus is resistant to Helicoverpa armigera, one of the major pests responsible for yield losses in Cajanus cajan. Deciphering the molecular mechanism underlying host plant resistance is pertinent to identify proteins that aid in the mitigation of the insect pest. The present study adopted comparative proteomics as a tool to interpret the resistance mechanism(s) in C. platycarpus vis-à-vis C. cajan during continued herbivory (up to 96 h). Over-representation analysis of the differentially expressed proteins implicated a multi-dimensional resistance response accomplished by both physical and chemical barriers in C. platycarpus. While the chemical basis for resistance was depicted by the upregulation of proteins playing a rate limiting role in the phenylpropanoid pathway, the physical basis was provided by the regulation of proteins involved in microtubule assembly and synthesis of lignins. Upregulation of proteins in the polyamine pathway indicated the role of metabolite conjugates to be negatively affecting herbivore growth. Reallocation of resources and diversion of metabolic flux to support the production of secondary metabolites could be the probable approach in the wild relative against herbivory. Our study provided deeper insights into the pod borer resistance mechanism in C. platycarpus for utility in crop improvement. KEY POINTS: • Pod borer resistance in Cajanus platycarpus is multi-dimensional. • Pod borer resistance has been arbitrated to cell wall rigidity and secondary metabolites. • Phenylpropanoid pathway derivatives apparently shaped the plant chemical defense against pod borer.

Evaluation of morpho-molecular diversity and pod borer resistance in country bean

Evaluation of morpho-molecular diversity and pod borer resistance in country bean

Variability in Morpho-biochemical Traits Associated with Pod Borer (Helicoverpa armigera) Resistance in Pigeonpea Pods

Pigeonpea contributes to food and nutrition security among poor households in urban and rural areas. Globally it is characterized by stagnant and unstable yield due to its susceptibility to various stresses including the pod borer (Helicoverpa armigera) which causes substantial damage to the crop and may result in absolute economic yield loss. The existing cultivated pigeonpea are susceptible to pod borer with only a few genotypes reported to be tolerant based on pod and seed damage. Limited information is available on morphological and biochemical traits associated with pod borer resistance among the existing genotypes. This study was therefore carried out to identify diversified sources of resistance against pod borer damage. The study was set up to assess traits that may contribute to pod borer resistance among 12 selected elite pigeonpea genotypes in three replicates and means were separated based on LSD test using Genstat software. The field study was carried out in Kerio Valley during the long rains of April-September of 2017. The genotypes varied significantly for all the parameters measured at P≤0.05 with a mean of 608.33 g/100 g (crude protein), 175.61 mg/100 g (total phenol), 19.85 mg/100 g (total flavonoid), 0.448 mm (trichome length), 210.6 / 4 mm2(trichome density) and 0.353 mm(depth of locules). Significant negative correlation was also observed between total phenol, total flavonoid, depth of locules, trichome length and trichome density with pod damage. However, a positive correlation was recorded between crude proteins with pod damage. These results reveal that, host plant resistance is an association of several morphological and biochemical traits. Therefore, these genotypes with elevated levels can be selected and utilized in breeding towards improving resistance to pod borer in pigeonpea.

Assessment of phenotypic and genetic variation against pod borer among a subset of elite pigeonpea (Cajanus cajan) genotypes in Kenya

Molecular marker information supported by quality morphological data facilitates the choice of suitable parents for applied breeding. The main aim of this study was to assess genetic diversity among 55 cultivated yield elite pigeonpea lines using 21 simple sequence repeat (SSR) markers that are well distributed across the genome. Among the 55 pigeonpea genotypes, 16 medium duration were selected and evaluated in the field for response to pod borer resistance in varied agro-ecological zones of Kenya during long rains of April-October cropping season Twenty one primer pairs detected 80 alleles with a mean of 3.9 alleles per locus and polymorphism information content (PIC) ranging from 0.09 to 0.75 averaging to 0.39 suggesting a low genetic diversity. However, marker CcM1820 revealed the highest number of alleles (9) with a PIC value of 0.75. The genotype response to pod borer attack was significant (P<=0.05) with three genotypes (ICEAPs 01541, 01154-2 and 00902) revealing tolerance to pod borer. The markers based on Neighbor Joining, grouped the 55 genotypes into three main clusters based on parentage selection. Most genotypes developed from ICEAP 00068 as the maternal parent were grouped in Cluster I while cluster II comprised of improved genotypes and cluster III comprised genotypes developed from ICPL 87091 as maternal parent. The resistant genotypes identified in the field experiment were grouped in cluster I except ICEAP 00902 which grouped in cluster II. Future studies should focus on broadening genetic base by including more landraces and wild relatives to maximize selection and improve breeding work.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80–100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25–80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Protease inhibitors of Cajanus conferring resistance to pod borer of pigeonpea (Cajanus cajan L. Millsp.)

Pigeonpea is susceptible to pod borer damage with resistance lacking in its primary gene pool. Many Cajanus species harbor high levels of resistance. Host plant resistance can play an important role in minimizing the extent of losses due to insects and pests as well as the use of insecticides/pesticides and thus protect the environment. A major initiative was undertaken to tap the defence genes from wild relatives of secondary and tertiary gene pool through wide hybridization and thereby introgress resistance to pod borer. A range of interspecific derivatives derived from C. lanceolatus, C. cajanifolius, C. volubilis and C. platycarpus along with their parents were screened for the pod borer resistance under unprotected field conditions at ICRISAT, Patancheru, India. Biochemical basis of resistance was also identified by studying the levels of defence proteins active against bovine pancreatic trypsin, chymotrypsin and trypsin-like enzymes of H. armigera mid-gut proteases. Protease inhibitor profiles of parents and interspecific derivatives differed in terms of activity units, number and intensities of activity bands visualized on gelatin-PAGE. As the protease inhibitors are anti-nutritional factors, parents and interspecific derivatives, which resulted in high levels of Helicoverpa gut protease inhibitor (HGPI) units were screened for Human pancreatic trypsin inhibitor (HPTI) activity levels. Samples with high ratio of HGPI/HPTI represent less or no effect on human pancreatic trypsin and high effect on insect gut proteases.