Development Of In Vitro Regeneration System For Capsicum Annuum And Transformation With Cucumber Mosaic Virus Coat Protein Gene

Chilli is one of the most important crops grown worldwide. It ranks as the most popular fruit vegetable and occupies the highest hectarage among the fruit vegetables in Malaysia. However, like any other crop of economic importance, chilli production is hampered by various virus diseases, especially Cucumber Mosaic Virus (CMV) disease. Genetic engineering seems to be the most important technique for the development of novel chilli cultivars with virus resistant property, since other conventional controls were highly ineffective. This study was carried out in three stages: first, to establish an efficient in vitro regeneration system for a local chilli cultivar Cilibangi-4 (CB4); second, to obtain a transforming vector containing Cucumber Mosaic Virus (CMV) Coat Protein (CP) gene; and third, development of transgenic chilli plants with CMV resistant. The investigation was initiated to study the in vitro regenerative ability of seven local chilli cultivars (Capsicum annum cv. CB2, CB3, CB4, CB6, MC11, MC12 and Kulai) and to select the most responsive cultivar for subsequent experiments. Explants (hypocotyls and cotyledonary leaves) were collected from 10-12 day-old seedlings and subjected to differentiation medium (DM). Genotypic differences for the in vitro regeneration ability were observed in this study. Of all the genotypes tested, cultivar CB4 was found to be the most responsive for both hypocotyls and cotyledonary leaves tested. Hence, subsequent experiments were carried out by using CB4. BA and IAA concentrations have been optimised for DM. Five concentrations of both PGRs were tested, 5 mg/l(w/v) BA and 0.5 mg/l(w/v) IAA were only found to be the most suitable for bud induction. Up to 87.5% of the cultured hypocotyls formed buds in the induction medium and cotyledonary leaves with a lower percentage of 65%. Effects of other cytokinins and auxins were investigated as well for bud induction in DM. Kinetin and zeatin were found to be less effective on bud induction compared to BA. While, Thidiazuron (TDZ) showed an extremely high percentage of bud formation, yet the buds induced were mostly stunted buds due to its high cytokinin activity. For auxins, Phenylacetic acid (PAA) was tested. PAA has shown some encouraging results among the concentrations tested. However, the buds seemed likely to form a rosette of distorted leaves and refused to develop further. Elongation of shoot buds was examined. Treatment DMM (Buds were induced in DM and elongated on MS with 3% sucrose) was found to be the best among all the treatments applied. The leaves formed expanded as normal, shoots elongated well and the roots developed vigorously at the basal part of the explants. Shoots elongated were excised and allowed to root in PGR-free MS medium. A construct of a plant expression cassette with CMV CP gene has been successfully cloned. The cloned CMV CP fragment was 655 bp and exhibited more than 90% similarity to those published CMV CP gene sequences. The effectiveness of kanamycin in selecting transformed tissue has been investigated based on the minimal kanamycin concentration was that capable to thoroughly inhibit and/or kill all the non-transformed tissues. The minimal inhibitory concentrations of kanamycin were 100 and 200 mg/l(w/v) for cotyledonary leaves and hypocotyls, respectively. Agrobacterium tumefaciens strain EHA105 harbouring the CMV CP construct was used to transform the chilli hypocotyls and cotyledonary leaf explants. The putative transformants were screened by subjecting to polymerase chain reaction (PCR) with specific primers. Unfortunately, no positive result was obtained

Waterlogging stress induces antioxidant defense responses, aerenchyma formation and alters metabolisms of banana plants

Flooding caused or exacerbated by climate change has threatened plant growth and food production worldwide. The lack of knowledge on how crops respond and adapt to flooding stress imposes a major barrier to enhancing their productivity. Hence, understanding the flooding-responsive mechanisms of crops is indispensable for developing new flooding-tolerant varieties. Here, we examined the banana (Musa acuminata cv. Berangan) responses to soil waterlogging for 1, 3, 5, 7, 14, and 24 days. After waterlogging stress, banana root samples were analyzed for their molecular and biochemical changes. We found that waterlogging treatment induced the formation of adventitious roots and aerenchyma with conspicuous gas spaces. In addition, the antioxidant activities, hydrogen peroxide, and malondialdehyde contents of the waterlogged bananas increased in response to waterlogging stress. To assess the initial response of bananas toward waterlogging stress, we analyzed the transcriptome changes of banana roots. A total of 3508 unigenes were differentially expressed under 1-day waterlogging conditions. These unigenes comprise abiotic stress-related transcription factors, such as ethylene response factors, basic helix-loop-helix, myeloblastosis, plant signal transduction, and carbohydrate metabolisms. The findings of the study provide insight into the complex molecular events of bananas in response to waterlogging stress, which could later help develop waterlogging resilient crops for the future climate

Plant Nitric Oxide Signaling under Drought Stress

Water deficit caused by drought is a significant threat to crop growth and production. Nitric oxide (NO), a water- and lipid-soluble free radical, plays an important role in cytoprotection. Apart from a few studies supporting the role of NO in drought responses, little is known about this pivotal molecular amendment in the regulation of abiotic stress signaling. In this review, we highlight the knowledge gaps in NO roles under drought stress and the technical challenges underlying NO detection and measurements, and we provide recommendations regarding potential avenues for future investigation. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool with which plant fitness can be improved under adverse growth conditions

Green Revolution to Gene Revolution: Technological Advances in Agriculture to Feed the World

Technological applications in agriculture have evolved substantially to increase crop yields and quality to meet global food demand. Conventional techniques, such as seed saving, selective breeding, and mutation breeding (variation breeding), have dramatically increased crop production, especially during the ‘Green Revolution’ in the 1990s. However, newer issues, such as limited arable lands, climate change, and ever-increasing food demand, pose challenges to agricultural production and threaten food security. In the following ‘Gene Revolution’ era, rapid innovations in the biotechnology field provide alternative strategies to further improve crop yield, quality, and resilience towards biotic and abiotic stresses. These innovations include the introduction of DNA recombinant technology and applications of genome editing techniques, such as transcription activator-like effector (TALEN), zinc-finger nucleases (ZFN), and clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas) systems. However, the acceptance and future of these modern tools rely on the regulatory frameworks governing their development and production in various countries. Herein, we examine the evolution of technological applications in agriculture, focusing on the motivations for their introduction, technical challenges, possible benefits and concerns, and regulatory frameworks governing genetically engineered product development and production

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance