Genetic engineering technique offers myriads of applications in improvement of horticultural crops for biotic and abiotic stress tolerance, and produce quality enhancement. During last two decades, a large number of transgenic horticultural crops has been developed and more are underway. A number of genes including natural and synthetic Cry genes, protease inhibitors, trypsin inhibitors and cystatin genes have been used to incorporate insect and nematode resistance. For providing protection against fungal and bacterial diseases, various genes like chitinase, glucanase, osmotin, defensin and pathogenesis-related genes are being transferred to many horticultural crops world over. RNAi technique has been found quite successful in inducing virus resistance in horticultural crops in addition to coat protein genes. Abiotic stresses such as drought, heat and salinity adversely affect production and productivity of horticultural crops and a number of genes encoding for biosynthesis of stress protecting compounds including mannitol, glycine betaine and heat shock proteins have been employed for abiotic stress tolerance besides various transcription factors like DREB1, MAPK, WRKY, etc. Antisense gene and RNAi technologies have revolutionized the pace of improvement of horticultural crops, particularly ornamentals for color modification, increasing shelf-life and reducing post-harvest losses. Precise genome editing tools, particularly CRISPR/Cas9, have been efficiently applied in tomato, petunia, citrus, grape, potato and apple for gene mutation, repression, activation and epigenome editing. This review provides comprehensive overview to draw the attention of researchers for better understanding of genetic engineering advancements in imparting biotic and abiotic stress tolerance as well as on improving various traits related to quality, texture, plant architecture modification, increasing shelf-life, etc. in different horticultural crops.

Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review

Despite the importance of okra, as one of the important vegetable crop, very little attention has been paid to its genetic improvement using advanced biotechnological tools. The exploitation of marker assisted breeding in okra is often limited due to the availability of a few molecular markers, the absence of molecular genetic-map(s) and other molecular tools. Chromosome linkage-groups were not yet constructed for this crop and reports on marker development are very scanty and mostly hovering around cultivar characterization. Besides, very little progress has been observed for transgenic development. However, high throughput biotechnological tools like chromosome engineering, RNA interference (RNAi), marker-assisted recurrent selection (MARS), genome-wide selection (GWS), targeted gene replacement, next generation sequencing (NGS) and nanobiotechnology can provide a rapid way for okra improvement. Further, the etiology of many deadly viral diseases like the yellow vein mosaic virus (YVMV) and okra enation leaf curl virus (OELCV) in okra is broadly indistinct and has been shown to be caused by various begomovirus species. These diseases cause systemic infections and have a very effective mode of transmission; thus, preventing their spread has been very complicated. Biotechnological interventions have the potential to enhance okra production even under different viral-stress conditions. In this background, this review deals with the biotechnological advancements in okra per se along with the begomoviruses infecting okra, and special emphasis has been laid on the exploitation of advanced genomic tools for the development of resistant varieties.

/pdf/biotechnological-advancements-and-begomovirus-management-in-lwwyyuqtf5.pdf

Biotechnological Advancements and Begomovirus Management in Okra (Abelmoschus esculentus L.): Status and Perspectives.

Recent trends and advances of RNA interference (RNAi) to improve agricultural crops and enhance their resilience to biotic and abiotic stresses.

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

Assessment of factors influencing the tissue culture-independent Agrobacterium -mediated in planta genetic transformation of okra [ Abelmoschus esculentus (L.) Moench]

RNA interference (RNAi) is a method of gene silencing where dsRNA is digested into small interfering RNA (siRNA) in the presence of enzymes. These siRNAs then target homologous mRNA sequences aided by the RNA-induced silencing complex (RISC). The mechanism of dsRNA uptake has been well studied and established across many living organisms including insects. In insects, RNAi is a novel and potential tool to develop future pest management means targeting various classes of insects including dipterans, coleopterans, hemipterans, lepidopterans, hymenopterans and isopterans. However, the extent of RNAi in individual class varies due to underlying mechanisms. The present review focuses on three major insect classes viz hemipterans, lepidopterans and coleopterans and the rationale behind this lies in the fact that studies pertaining to RNAi has been extensively performed in these groups. Additionally, these classes harbour major agriculturally important pest species which require due attention. Interestingly, all the three classes exhibit varying levels of RNAi efficiencies with the coleopterans exhibiting maximum response, while hemipterans are relatively inefficient. Lepidopterans on the other hand, show minimum response to RNAi. This has been attributed to many facts and few important being endosomal escape, high activity dsRNA-specific nucleases, and highly alkaline gut environment which renders the dsRNA unstable. Various methods have been established to ensure safe delivery of dsRNA into the biological system of the insect. The most common method for dsRNA administration is supplementing the diet of insects via spraying onto leaves and other commonly eaten parts of the plant. This method is environment-friendly and superior to the hazardous effects of pesticides. Another method involves submergence of root systems in dsRNA solutions and subsequent uptake by the phloem. Additionally, more recent techniques are nanoparticle- and Agrobacterium-mediated delivery systems. However, due to the novelty of these biotechnological methods and recalcitrant nature of certain crops, further optimization is required. This review emphasizes on RNAi developments in agriculturally important insect species and the major hurdles for efficient RNAi in these groups. The review also discusses in detail the development of new techniques to enhance RNAi efficiency using liposomes and nanoparticles, transplastomics, microbial-mediated delivery and chemical methods.

/pdf/next-generation-dsrna-based-insect-control-success-so-far-34r9z4k6l5.pdf

Next Generation dsRNA-Based Insect Control: Success So Far and Challenges.

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic
 Bt
 plants showed resistance to the target pest, okra shoot, and fruit borer (
 Earias vittella
 ).
 Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

Protecting crops against insect pests is a major focus area in crop protection. Over the past two decades, biotechnological interventions, especially Bt proteins, have been successfully implemented across the world and have had major impacts on reducing chemical pesticide applications. As insects continue to adapt to insecticides, both chemical and protein-based, new methods, molecules, and modes of action are necessary to provide sustainable solutions. RNA interference (RNAi) has emerged as a significant tool to knock down or alter gene expression profiles in a species-specific manner. In the past decade, there has been intense research on RNAi applications in crop protection. This chapter looks at the current state of knowledge in the field and outlines the methodology, delivery methods, and precautions required in designing targets. Assessing the targeting of specific gene expression is also an important part of a successful RNAi strategy. The current literature on the use of RNAi in major orders of insect pests is reviewed, along with a perspective on the regulatory aspects of the approach. Risk assessment of RNAi would focus on molecular characterization, food/feed risk assessment, and environmental risk assessment. As more RNAi-based products come through regulatory systems, either via direct application or plant expression based, the impact of this approach on crop protection will become clearer.

Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit

Field experiments were conducted during Kharif-2009 and 2010 to evaluate the efficacy of chlorantraniliprole 18.50% SC (Coragen), flubendamide 39.35% SC (Fame), indoxacarb 14.50% SC (Avaunt), chlorfenapyr 10% SC (Intrepid) and spinosad 45% SC (Spintor) against brinjal fruit and shoot borer, Leucinodes orbonalis (Guenee). Chlorantraniliprole 18.50% SC and flubendamide 39.35% SC proved their superiority over other insecticides in reducing infestation of L. orbonalis and resulted in higher yields.

Newer Insecticides for the Management of Brinjal Fruit and Shoot Borer, Leucinodes orbonalis

A major pest of chickpea, Helicoverpa armigera, can be controlled by expressing genes from the bacterium Bacillus thuringiensis as an environmentally compatible option.
 Here we show that transgenic chickpeas containing a cry1Ac gene conferred a high degree of resistance to H. armigera. The Agrobacterium binary vector contained the nptII gene as the selectable marker and cry1Ac gene driven by the Arabidopsis rubisco small subunit gene (ats1A) promoter.
 We generated 54 and 47 independent transgenic lines using truncated (trcry1Ac) and full-length versions of the cry1Ac (flcry1Ac) gene, respectively. Of these lines, twelve transmitted the trcry1Ac transgene to the next generation at a 3:1 ratio, while only 8 flcry1Ac lines segregated in a 3:1 ratio.
 Five lines expressed trCry1Ac protein > 50 μg/g fresh weight, however, only one line accumulated about 30 μg/g flCry1Ac protein. Such high levels of trCry1Ac protein have not been reported before in chickpea. When trCry1Ac lines were challenged to whole plant bioassays in the greenhouse, lowest pod damage was observed in BS100B (1.4%) followed by BS81P (4.4%), and BS100E (6.2%) compared to the parental line (49.9%). The phenotypes of the lines expressing high levels of Cry1Ac protein were indistinguishable from their null segregants and controls. Thus, trCry1Ac lines could be suitable for crossing with our existing Cry2Aa lines for generation of a pyramided Bt chickpea for enhanced insect resistance management in the field.

Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera

Production of marker-free insect resistant transgenic brinjal (solanum melongena l.) carrying cry2ab gene

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