Application of TILLING for Orphan Crop Improvement

People in developing countries mostly depend for their diet on special staple crops, so called orphan crops. These crops play a key role in food security since they are grown by many resource-poor farmers and consumed locally. Despite their huge importance in the economy and livelihood of the developing world, orphan crops have received little attention in terms of scientific improvement. Although conventional breeding is widely implemented to improve crop plants, alternative methods such as marker-assisted breeding and reverse genetics approaches have proved to be efficient in developing crop cultivars. In this review, we present detailed description of a non-transgenic and reverse genetics technique called TILLING (Targeting Induced Local Lesion IN Genomes). The method was originally optimized in the model plant Arabidposis thaliana and subsequently applied to crops such as maize, wheat, and rice. We also present detailed procedures for several TILLING strategies and discuss their benefits and drawbacks. The application of the technique for orphan crop improvement is also discussed based on several TILLING platforms currently carried-out on these understudied crops of the world.

Dynamics of Sodium‐22 and Cesium‐134 in Young Wheat Plants

The dynamics of the two alkali metals sodium and cesium in crop plants are relevant in an ecological context. Redistribution processes for these elements in young wheat plants were investigated in the work reported here. Two days old wheat plants (Triticum aestivum L. cv. Arina) were fed for 24 h with sodium‐22 (22Na) and cesium‐134 (134Cs) via the main root and incubated afterwards in a culture room. Cesium‐134 accumulated in newly formed parts of the main root and in the expanding leaves during the first 20 days after labeling, while 22Na accumulated transiently in these plant parts, reached a peak and declined after a few days. A high percentage of 22Na was released from the roots to the medium. Total Na in leaves also accumulated transiently, but its highest accumulation appeared later than the peak of 22Na. Therefore, the distribution and retranslocation processes differ considerably for sodium and cesium in wheat plants. Such differences must be considered for the evaluation of environmental effects (e.g., release of pollutants into the environment) on the quality of harvested cereal products.

Toward a successful multinational crop plant genome initiative

Plant genome research is needed as the foundation for an entirely new level of efficiency and success in the application of genetics and breeding to crop plants and products from crop plants. Genetic improvements in crop plants beyond current capabilities are needed to meet the growing world demand not only for more food, but also a greater diversity of food, higher-quality food, and safer food, produced on less land, while conserving soil, water, and genetic resources. Plant biology research, which is poised for dramatic advances, also depends fundamentally on plant genome research. The current Arabidopsis Genome Project has proved of immediate value to plant biology research, but a much greater effort is needed to ensure the full benefits of plant biology and especially plant genome research to agriculture. International cooperation is critical, both because genome projects are too large for any one country and the information forthcoming is of benefit to the world and not just the countries that do the work. Recent research on grass genomes has revealed that, because of extensive senteny and colinearity within linkage groups that make up the chromosomes, new information on the genome of one grass can be used to understand the genomes and predict the location of genes on chromosomes of the other grasses. Genome research applied to grasses as a group thereby can increase the efficiency and effectiveness of breeding for improvement of each member of this group, which includes wheat, corn, and rice, the world’s three most important sources of food.

Mexican papita viroid: putative ancestor of crop viroids.

The potato spindle tuber disease was first observed early in the 20th century in the northeastern United States and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (PSTVd). No wild-plant PSTVd reservoirs have been identified; thus, the initial source of PSTVd infecting potatoes has remained a mystery. Several variants of a novel viroid, designated Mexican papita viroid (MPVd), have now been isolated from Solanum cardiophyllum Lindl. (papita güera, cimantli) plants growing wild in the Mexican state of Aguascalientes. MPVd's nucleotide sequence is most closely related to those of the tomato planta macho viroid (TPMVd) and PSTVd. From TPMVd, MPVd may be distinguished on the basis of biological properties, such as replication and symptom formation in certain differential hosts. Phylogenetic and ecological data indicate that MPVd and certain viroids now affecting crop plants, such as TPMVd, PSTVd, and possibly others, have a common ancestor. We hypothesize that commercial potatoes grown in the United States have become viroid-infected by chance transfer of MPVd or a similar viroid from endemically infected wild solanaceous plants imported from Mexico as germplasm, conceivably from plants known to have been introduced from Mexico to the United States late in the 19th century in efforts to identify genetic resistance to the potato late blight fungus, Phytophthora infestans.

Plants useful to man /

Includes bibliographical references (p. 421-422)

Boron nutrition and chilling tolerance of warm climate crop species

Background Field observations and glasshouse studies have suggested links between boron (B)-deficiency and leaf damage induced by low temperature in crop plants, but causal relationships between these two stresses at physiological, biochemical and molecular levels have yet to be explored. Limited evidence at the whole-plant level suggests that chilling temperature in the root zone restricts B uptake capacity and/or B distribution/utilization efficiency in the shoot, but the nature of this interaction depends on chilling tolerance of species concerned, the mode of low temperature treatment (abrupt versus gradual temperature decline) and growth conditions (e.g. photon flux density and relative humidity) that may exacerbate chilling stress. Scope This review explores roles of B nutrition in chilling tolerance of continual root or transient shoot chills in crop species adapted to warm season conditions. It reviews current research on combined effects of chilling temperature (ranging from > 0 to 20 degrees C) and B deficiency on growth and B nutrition responses in crop species differing in chilling tolerance. Conclusion For subtropical/tropical species (e.g. cucumber, cassava, sunflower), root chilling at 10-17 degrees C decreases B uptake efficiency and B utilization in the shoot and increases the shoot : root ratio, but chilling-tolerant temperate species (e.g. oilseed rape, wheat) require much lower root chill temperatures (2-5 degrees C) to achieve the same responses. Boron deficiency exacerbates chilling injuries in leaf tissues, particularly under high photon flux density. Suggested mechanisms for B x chilling interactions in plants are: (a) chilling-induced reduction in plasmalemma hydraulic conductivity, membrane fluidity, water channel activity and root pressure, which contribute to the decrease in root hydraulic conductance, water uptake and associated B uptake; (b) chilling-induced stomatal dysfunction affecting B transport from root to shoot and B partitioning in the shoot; and (c) B deficiency induced sensitivity to photo-oxidative damage in leaf cells. However, specific evidence for each of the mechanisms is still lacking. Impacts of B status on chilling tolerance in crop species have important implications for the management of B supply during sensitive stages of growth, such as early growth after planting and early reproductive development, both of which can coincide with the occurrence of chilling temperatures in the field.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Isolation and functional characterisation of banana phytoene synthase genes as potential cisgenes

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy-transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Bananas are a major commercial crop and serve as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrate that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

Making mangoes move

A landscape of mangoes most likely brings to mind a place in a tropical location. By the end of the nineteenth century that place could have been located on any continent in the world. Mangoes were found in geographic locations; in scientific institutions; as crop plants; and as a backyard trees. Here I trace the movement of mangoes Mangifera indica Linn., focusing on the transnational links formed through colonial botanic gardens in Australia. Botanic gardens were largely understood through their work in establishing economically successful plantation crops, such as sugar and tea. Mangoes were not a success crop of the age of botanic imperialism. Instead, mangoes were simply one species among the millions of plants that botanic gardens moved in addition to these well known commercial crops. Colonial science moved plants for a myriad of other types of reasons, for ornament, for curiosity, for lesser commercial purposes and for pure science. In each site the mango emerged, the discourses and technologies that traveled with it changed according to local needs. Indeed, rather than finding mangoes located in one place, tracing their movement demonstrates that this was an extended landscape connecting these things across time and space...

Fate of potassium fertilisers applied to clay soils under rainfed grain cropping in south-east Queensland, Australia

Negative potassium (K) balances in all broadacre grain cropping systems in northern Australia are resulting in a decline in the plant-available reserves of K and necessitating a closer examination of strategies to detect and respond to developing K deficiency in clay soils. Grain growers on the Red Ferrosol soils have increasingly encountered K deficiency over the last 10 years due to lower available K reserves in these soils in their native condition. However, the problem is now increasingly evident on the medium-heavy clay soils (Black and Grey Vertosols) and is made more complicated by the widespread adoption of direct drill cropping systems and the resulting strong strati. cation of available K reserves in the top 0.05-0.1 m of the soil pro. le. This paper reports glasshouse studies examining the fate of applied K fertiliser in key cropping soils of the inland Burnett region of south-east Queensland, and uses the resultant understanding of K dynamics to interpret results of field trials assessing the effectiveness of K application strategies in terms of K availability to crop plants. At similar concentrations of exchangeable K (K-exch), soil solution K concentrations and activity of K in the soil solution (AR(K)) varied by 6-7-fold between soil types. When K-exch arising from different rates of fertiliser application was expressed as a percentage of the effective cation exchange capacity (i.e. K saturation), there was evidence of greater selective adsorption of K on the exchange complex of Red Ferrosols than Black and Grey Vertosols or Brown Dermosols. Both soil solution K and AR(K) were much less responsive to increasing K-exch in the Black Vertosols; this is indicative of these soils having a high K buffer capacity (KBC). These contrasting properties have implications for the rate of diffusive supply of K to plant roots and the likely impact of K application strategies (banding v. broadcast and incorporation) on plant K uptake. Field studies investigating K application strategies (banding v. broadcasting) and the interaction with the degree of soil disturbance/mixing of different soil types are discussed in relation to K dynamics derived from glasshouse studies. Greater propensity to accumulate luxury K in crop biomass was observed in a Brown Ferrosol with a KBC lower than that of a Black Vertosol, consistent with more efficient diffusive supply to plant roots in the Ferrosol. This luxury K uptake, when combined with crops exhibiting low proportional removal of K in the harvested product (i.e. low K harvest index coarse grains and winter cereals) and residue retention, can lead to rapid re-development of stratified K profiles. There was clear evidence that some incorporation of K fertiliser into soil was required to facilitate root access and crop uptake, although there was no evidence of a need to incorporate K fertiliser any deeper than achieved by conventional disc tillage (i.e. 0.1-0.15 m). Recovery of fertiliser K applied in deep (0.25-0.3 m) bands in combination with N and P to facilitate root proliferation was quite poor in Red Ferrosols and Grey or Black Vertosols with moderate effective cation exchange capacity (ECEC, 25-35 cmol(+)/kg), was reasonable but not enough to overcome K deficiency in a Brown Dermosol (ECEC 11 cmol(+)/kg), but was quite good on a Black Vertosol (ECEC 50-60 cmol(+)/kg). Collectively, results suggest that frequent small applications of K fertiliser, preferably with some soil mixing, is an effective fertiliser application strategy on lighter clay soils with low KBC and an effective diffusive supply mechanism. Alternately, concentrated K bands and enhanced root proliferation around them may be a more effective strategy in Vertosol soils with high KBC and limited diffusive supply. Further studies to assess this hypothesis are needed.

Size optimization of a cantilever beam under deformation-dependent loads with application to wheat stalks

In this paper, we consider the optimization of the cross-section profile of a cantilever beam under deformation-dependent loads. Such loads are encountered in plants and trees, cereal crop plants such as wheat and corn in particular. The wind loads acting on the grain-bearing spike of a wheat stalk vary with the orientation of the spike as the stalk bends; this bending and the ensuing change in orientation depend on the deformation of the plant under the same load.The uprooting of the wheat stalks under wind loads is an unresolved problem in genetically modified dwarf wheat stalks. Although it was thought that the dwarf varieties would acquire increased resistance to uprooting, it was found that the dwarf wheat plants selectively decreased the Young's modulus in order to be compliant. The motivation of this study is to investigate why wheat plants prefer compliant stems. We analyze this by seeking an optimal shape of the wheat plant's stem, which is modeled as a cantilever beam, by taking the large deflection of the stem into account with the help of co-rotational finite element beam modeling. The criteria considered here include minimum moment at the fixed ground support, adequate stiffness and strength, and the volume of material. The result reported here is an example of flexibility, rather than stiffness, leading to increased strength.

Isohydric and anisohydric characterisation of vegetable crops. The classification of vegetables by their physiological responses to water stress

Research on the physiological response of crop plants to drying soils and subsequent water stress has grouped plant behaviours as isohydric and anisohydric. Drying soil conditions, and hence declining soil and root water potentials, cause chemical signals—the most studied being abscisic acid (ABA)—and hydraulic signals to be transmitted to the leaf via xylem pathways. Researchers have attempted to allocate crops as isohydric or anisohydric. However, different cultivars within crops, and even the same cultivars grown in different environments/climates, can exhibit both response types. Nevertheless, understanding which behaviours predominate in which crops and circumstances may be beneficial. This paper describes different physiological water stress responses, attempts to classify vegetable crops according to reported water stress responses, and also discusses implications for irrigation decision-making.

Allelopathy for weed control in agricultural systems

Weeds are a hidden foe for crop plants, interfering with their functions and suppressing their growth and development. Yield losses of ∼34 are caused by weeds among the major crops, which are grown worldwide. These yield losses are higher than the losses caused by other pests in the crops. Sustainable weed management is needed in the wake of a huge decline in crop outputs due to weed pressure. A diversity in weed management tools ensures sustainable weed control and reduces chances of herbicide resistance development in weeds. Allelopathy as a tool, can be importantly used to combat the challenges of environmental pollution and herbicide resistance development. This review article provides a recent update regarding the practical application of allelopathy for weed control in agricultural systems. Several studies elaborate on the significance of allelopathy for weed management. Rye, sorghum, rice, sunflower, rape seed, and wheat have been documented as important allelopathic crops. These crops express their allelopathic potential by releasing allelochemicals which not only suppress weeds, but also promote underground microbial activities. Crop cultivars with allelopathic potentials can be grown to suppress weeds under field conditions. Further, several types of allelopathic plants can be intercropped with other crops to smother weeds. The use of allelopathic cover crops and mulches can reduce weed pressure in field crops. Rotating a routine crop with an allelopathic crop for one season is another method of allelopathic weed control. Importantly, plant breeding can be explored to improve the allelopathic potential of crop cultivars. In conclusion, allelopathy can be utilized for suppressing weeds in field crops. Allelopathy has a pertinent significance for ecological, sustainable, and integrated weed management systems.

Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.