Adding value to banana farming: Antibody production in post-harvest leaves.

Banana, a globally popular fruit, is widely cultivated in tropical and sub-tropical regions. After fruit harvest, remaining banana plant materials are low-value byproducts, mostly composted or used as fibre or for food packaging. As an aim to potentially increase farmer income, this study explored underutilised banana biomass as a novel plant tissue for production of a high-value product. Protein scFvTG130 used in this study, is an anti-toxoplasma single chain variable fragment antibody that can be used in diagnostics and neutralising the Toxoplasma gondii pathogen. Using detached banana leaves, we investigated the factors influencing the efficacy of a transient expression system using reporter genes and recombinant protein, scFvTG130. Transient expression was optimal at 2 days after detached banana leaves were vacuum infiltrated at 0.08 MPa vacuum pressure for a duration of 3 min with 0.01% (v/v) Tween20 using Agrobacterium strain GV3101 harbouring disarmed virus-based vector pIR-GFPscFvTG130. The highest concentration of anti-toxoplasma scFvTG130 antibody obtained using detached banana leaves was 22.8 µg/g fresh leaf tissue. This first study using detached banana leaf tissue for the transient expression of a recombinant protein, successfully demonstrated anti-toxoplasma scFvTG130 antibody expression, supporting the potential application for other related proteins using an underutilised detached banana leaf tissue.

First Report of Pectobacterium carotovorum and Pectobacterium aroidearum Causing Bacterial Soft Rot on Curly Dwarf Pak Choy (Brassica rapa var. Chinensis) in Malaysia.

Brassica rapa var. Chinensis (curly dwarf pak choy) is commonly grown in large-scale vertical farming aquaponic systems. In October 2022, soft rot symptoms and dark brown lesions were observed on B. rapa grown in a commercial aquaponic farm located in Perak, Malaysia. The infected stem appeared brown and water soaked. Severely infected plants produced creamy white ooze on the surface before collapsing entirely (Fig. 1A and B). Infected leaves displayed yellow-brown symptoms and eventually rotted (Fig. 1C); the healthy plants were symptomless (Fig. 1D). About 20 % of the 20,000 B. rapa plants on the farm exhibited symptoms. Ten randomly selected symptomatic plants, five with infected stems and five with infected leaves, were surface sterilized. Each tissue (1.0 cm2) was homogenized and suspended in a saline solution. The suspensions were then serially diluted and plated separately on Luria-Bertani agar. After a 16-h incubation period, stem tissue yielded 12 isolated colonies, while leaf tissue produced 8 colonies. These isolates were subjected to dereplication using RAPD-PCR (Krzewinski et al., 2001), revealing two distinct RAPD patterns. The cultures, named Pathogen Stem 2 (PS2, obtained from the stem) and Pathogen Leaf 2 (PL2, obtained from the leaf), were initially identified as Pectobacterium sp. through 16S rRNA sequence analysis (Frank et al., 2008) on the EzBioCloud 16S database (Yoon et al., 2017). Further identification of the Pectobacterium species was conducted using multilocus sequence analysis (MLSA) of the icdA, mdh, proA, and mltD genes (Ma et al., 2007). The sequences were deposited in GenBank (OQ660180, OQ660181, and OR206482-OR206489). Based on MLSA phylogeny, PS2 and PL2 were identified as Pectobacterium carotovorum and Pectobacterium aroidearum, respectively (Fig. 2A). Anaerobic assays confirmed their facultative anaerobic nature, while Gram staining revealed Gram-negative, rod-shaped morphology consistent with Pectobacterium (Fig. 2B and C). For the re-inoculation study, one-month-old healthy B. rapa plants were used. PS2 was inoculated into petioles, while PL2 was inoculated into leaves separately (3 biological replicates × 3 leaves for each replicate) using the prick inoculation method (Wei et al., 2019). Sterile needles were used to prick the plant tissues, and 10 µL of bacterial suspensions (2.40×109 CFU/mL) in saline were inoculated onto the pricked spots. Negative control using sterile saline was included. The inoculated plants were maintained in a controlled growth chamber (25 ± 1°C, relative humidity 80 ± 5%). After 48 hpi, the petiole tissue inoculated with PS2 showed bacterial soft rot symptoms (Fig. 1F) and leaves inoculated with PL2 appeared dark brown around the wound (Fig. 1G), similar to the symptoms observed in the commercial farm (Fig. 1B, C); while control plants remained asymptomatic (Fig. 1E). Bacteria were re-isolated from the inoculated petiole and leaf tissue and their identities were confirmed by RAPD-PCR. The RAPD profiles of the bacteria reisolated from the petiole and leaf tissues were the same as those of PS2 and PL2 respectively (Fig. 1H). The pathogenicity of PS2 and PL2 was thus confirmed. To our knowledge, this is the first report of bacterial soft rot on B. rapa in aquaponic systems caused by P. carotovorum and P. aroidearum in Malaysia. The identification of these pathogens is crucial for the prevention of disease outbreaks and to develop an effective disease management strategy.

A review of Hydrocotyle bonariensis, a promising functional food and source of health-related phytochemicals.

Hydrocotyle bonariensis is an edible herb, that is also used for traditional medical purposes. It is high in antioxidants, phenols, and flavonoids. However, there is limited information on the nutritional composition and the mechanisms by which nutritional and functional constituents of H. bonariensis affect human metabolism. With an aim to identify gaps in evidence to support the mainstream use of H. bonariensis for health and as a functional food, this review summarises current knowledge of the taxonomy, habitat characteristics, nutritional value and health-related benefits of H. bonariensis and its extracts. Ethno-medical practices for the plant are supported by pharmacological studies, yet animal model studies, clinical trials and food safety assessments are needed to support the promotion of H. bonariensis and its derivatives as superfoods and for use in the modern pharmaceutical industry.

Arbuscular Mycorrhizal Symbiosis: A Strategy for Mitigating the Impacts of Climate Change on Tropical Legume Crops.

Climate change is likely to have severe impacts on food security in the topics as these regions of the world have both the highest human populations and narrower climatic niches, which reduce the diversity of suitable crops. Legume crops are of particular importance to food security, supplying dietary protein for humans both directly and in their use for feed and forage. Other than the rhizobia associated with legumes, soil microbes, in particular arbuscular mycorrhizal fungi (AMF), can mitigate the effects of biotic and abiotic stresses, offering an important complementary measure to protect crop yields. This review presents current knowledge on AMF, highlights their beneficial role, and explores the potential for application of AMF in mitigating abiotic and biotic challenges for tropical legumes. Due to the relatively little study on tropical legume species compared to their temperate growing counterparts, much further research is needed to determine how similar AMF-plant interactions are in tropical legumes, which AMF species are optimal for agricultural deployment and especially to identify anaerobic AMF species that could be used to mitigate flood stress in tropical legume crop farming. These opportunities for research also require international cooperation and support, to realize the promise of tropical legume crops to contribute to future food security.

Halotolerant rhizobacteria isolated from a mangrove forest alleviate saline stress in Musa acuminata cv. Berangan.

Saline soils resulting from anthropogenic activity and climate change present a challenge to future food security. Towards addressing this, we isolated and characterized halotolerant bacteria from a Malaysian mangrove forest, and explored their effect on morpho-physiological and biochemical parameters of banana plantlets under salt stress. A total of 88 rhizobacterial and 16 endophytic bacterial isolates collected from the roots and rhizosphere of Rhizophora apiculata, Avicennia alba and Sonneratia alba, were found to tolerate up to 400 mM of sea salt. Based on best performance in multiple plant growth traits, three rhizobacterial strains RB1, RB3 and RB4 and three endophytic bacterial strains EB1, EB2 and EB3 were used for further analysis. The rhizobacterial strains were identified as Bacillus sp. and endophytic bacteria as Pseudomonas sp. based on 16 S rRNA gene sequence. SEM observation confirmed colonization of each strain on banana plantlet roots. When colonized plantlets were subjected to 90 mM salt and compared to uninoculated (control) and mock inoculated plants, improved plant growth was observed with each of the strains, especially with bacterial strains EB3 and RB3. Biochemical analysis of plantlets revealed that root colonization with EB3 and RB3 enhanced levels of plant chlorophyll (> 5-fold), carotenoid (> 2.85-fold) and proline (2.6-fold and 2.3-fold), while plantlets also showed reduced MDA content (0.45-fold and 0.51-fold), significantly reduced generation of ROS (0.23-fold and 0.47-fold) and lower levels of electrolyte leakage (0.77 and 0.51-fold). Antioxidant enzymes also showed enhanced activity with EB3 and RB3. Our results indicate that these halotolerant Bacillus and Pseudomonas strains from the mangrove have multifunctional plant growth promoting activity and can reduce salt stress in bananas. This data provides a reference for exploring halotolerant microbes from hypersaline environments to overcome salt stress in plants.

DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium hybrid.

BACKGROUND: Flower pigment and shape are determined by the coordinated expression of a set of structural genes during flower development. R2R3-MYB transcription factors are known regulators of structural gene expression. The current study focused on two members of this large family of transcription factors that were predicted to have roles in pigment biosynthesis and organ shape development in orchids. METHODS: Phylogenetic analysis was used to identify candidate Dendrobium catenatum R2R3-MYB (DcaMYB) sequences associated with pigment and cell shape development. Gene silencing of candidate DhMYBs in Dendrobium hybrid by direct application of dsRNA to developing flowers was followed by observation of gene expression level and flower phenotypes. Silencing of the structural gene chalcone synthase was used as a comparative control. KEY RESULTS: Ten candidate flower-associated DcaMYBs were identified. Flowers treated with dsRNA of DhMYB22 and DhMYB60 sequences were less pigmented and had relatively low expression of anthocyanin biosynthetic genes (F3'H and DFR), lower total anthocyanin concentration and markedly lower levels of cyanidin-3-glucoside and cyanidin-3-rutinoside. Petals of DhMYB22-treated flowers and sepals of DhMYB60-treated flowers showed the greatest colour difference relative to the same organs in untreated flowers. DhMYB22-treated flowers had relatively narrow and constricted lips, while DhMYB60-treated flowers had narrow and constricted sepals. No significant difference in shape was observed for DhCHS-treated or untreated flowers. CONCLUSIONS: Our results demonstrate that DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium. This is a first report of MYB regulation of floral organ shape in orchids.

Drought tolerance improvement in Solanum lycopersicum: an insight into "OMICS" approaches and genome editing.

Solanum lycopersicum (tomato) is an internationally acclaimed vegetable crop that is grown worldwide. However, drought stress is one of the most critical challenges for tomato production, and it is a crucial task for agricultural biotechnology to produce drought-resistant cultivars. Although breeders have done a lot of work on the tomato to boost quality and quantity of production and enhance resistance to biotic and abiotic stresses, conventional tomato breeding approaches have been limited to improving drought tolerance because of the intricacy of drought traits. Many efforts have been made to better understand the mechanisms involved in adaptation and tolerance to drought stress in tomatoes throughout the years. "Omics" techniques, such as genomics, transcriptomics, proteomics, and metabolomics in combination with modern sequencing technologies, have tremendously aided the discovery of drought-responsive genes. In addition, the availability of biotechnological tools, such as plant transformation and the recently developed genome editing system for tomatoes, has opened up wider opportunities for validating the function of drought-responsive genes and the generation of drought-tolerant varieties. This review highlighted the recent progresses for tomatoes improvement against drought stress through "omics" and "multi-omics" technologies including genetic engineering. We have also discussed the roles of non-coding RNAs and genome editing techniques for drought stress tolerance improvement in tomatoes.

Late blight in tomato: insights into the pathogenesis of the aggressive pathogen Phytophthora infestans and future research priorities.

MAIN CONCLUSION: This review provides insights into the molecular interactions between Phytophthora infestans and tomato and highlights research gaps that need further attention. Late blight in tomato is caused by the oomycota hemibiotroph Phytophthora infestans, and this disease represents a global threat to tomato farming. The pathogen is cumbersome to control because of its fast-evolving nature, ability to overcome host resistance and inefficient natural resistance obtained from the available tomato germplasm. To achieve successful control over this pathogen, the molecular pathogenicity of P. infestans and key points of vulnerability in the host plant immune system must be understood. This review primarily focuses on efforts to better understand the molecular interaction between host pathogens from both perspectives, as well as the resistance genes, metabolomic changes, quantitative trait loci with potential for improvement in disease resistance and host genome manipulation via transgenic approaches, and it further identifies research gaps and provides suggestions for future research priorities.

High-quality RNA isolation from pigment-rich Dendrobium flowers.

Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.

Sheath blight of rice: a review and identification of priorities for future research.

MAIN CONCLUSION: Rice sheath blight research should prioritise optimising biological control approaches, identification of resistance gene mechanisms and application in genetic improvement and smart farming for early disease detection. Rice sheath blight, caused by Rhizoctonia solani AG1-1A, is one of the most devasting diseases of the crop. To move forward with effective crop protection against sheath blight, it is important to review the published information related to pathogenicity and disease management and to determine areas of research that require deeper study. While progress has been made in the identification of pathogenesis-related genes both in rice and in the pathogen, the mechanisms remain unclear. Research related to disease management practices has addressed the use of agronomic practices, chemical control, biological control and genetic improvement: Optimising nitrogen fertiliser use in conjunction with plant spacing can reduce spread of infection while smart agriculture technologies such as crop monitoring with Unmanned Aerial Systems assist in early detection and management of sheath blight disease. Replacing older fungicides with natural fungicides and use of biological agents can provide effective sheath blight control, also minimising environmental impact. Genetic approaches that show promise for the control of sheath blight include treatment with exogenous dsRNA to silence pathogen gene expression, genome editing to develop rice lines with lower susceptibility to sheath blight and development of transgenic rice lines overexpressing or silencing pathogenesis related genes. The main challenges that were identified for effective crop protection against sheath blight are the adaptive flexibility of the pathogen, lack of resistant rice varieties, abscence of single resistance genes for use in breeding and low access of farmers to awareness programmes for optimal management practices.

Impact of sea-salt on morpho-physiological and biochemical responses in banana (Musa acuminata cv. Berangan).

Banana is often grown in coastal-regions, and while known for its sensitivity towards seawater, little is documented on the effect of sea-salt on the growth, physiology and metal homeostasis. Here we report that banana plantlets exposed to sea-salt at extreme (average seawater concentration; 52.7 dS m-1), severe (28.5 dS m-1) or moderate (10.2 dS m-1) salinity levels had reduced root length (2.0-6.0-fold), plant height (1.2-1.6-fold), leaf number (2.0-2.3-fold) and leaf area (3.3-4.0-fold) compared to control plantlets. Degradation of pigments (total chlorophyll: 1.3-12.3-fold, chlorophyll a: 1.3-9.2-fold; chlorophyll b: 1.3-6.9-fold lower and carotenoids: 1.4-3.7-fold lower) reflected vulnerability of photosystems to salt stress. Relative water content showed a maximum decrease of 1.5-fold in salt stress. MDA analysis showed sea-salt exposure triggers 2.3-3.5-fold higher lipid peroxidation. Metal content analysis showed a 73-fold higher Na value from roots exposed to extreme salinity compared to control plantlets. While phenotype was clearly affected, moderate salinity showed no significant alteration of macro (N, P, K and Ca) and micro (Fe, Mn and Cu) metal content. The antioxidant enzymes: SOD (3.2-fold), CAT (1.7-fold) and GR (6-fold) showed higher activity at moderate salinity level compared to control plantlets but lower activity at severe (SOD: 1.3-fold; CAT: 1.5-fold; GR: 2-fold lower) and extreme seawater salinity (SOD: 1.5; CAT: 1.9; GR: 1.3-fold lower). Mild changes in growth and physiology at sea-salt levels equivalent to moderate seawater flooding, indicate that banana will survive such flooding, while extreme seawater inundation will be lethal. This data provides a reference for future salinity-mediated work in banana.

Ectopic expression of a Musa acuminata root hair defective 3 (MaRHD3) in Arabidopsis enhances drought tolerance.

Genetic improvement is an important approach for crop improvement towards yield stability in stress-prone areas. Functional analysis of candidate stress response genes can provide key information to allow the selection and modification of improved crop varieties. In this study, the constitutive expression of a banana cDNA, MaRHD3 in Arabidopsis improved the ability of transgenic lines to adapt to drought conditions. Transgenic Arabidopsis plants expressing MaRHD3 had roots with enhanced branching and more root hairs when challenged with drought stress. The MaRHD3 plants had higher biomass accumulation, higher relative water content, higher chlorophyll content and an increase in activity of reactive oxygen species (ROS) scavenging enzymes; SOD, CAT, GR, POD and APX with reduced water loss rates compared to control plants. The analysis of oxidative damage indicated lower cell membrane damage in transgenic lines compared to control plants. These findings, together with data from higher expression of ABF-3 and higher ABA content of drought-stressed transgenic MaRHD3 expressing plants, support the involvement of the ABA signal pathway and ROS scavenging enzyme systems in MaRHD3 mediated drought tolerance.

Codon usage and codon pair patterns in non-grass monocot genomes.

BACKGROUND AND AIMS: Studies on codon usage in monocots have focused on grasses, and observed patterns of this taxon were generalized to all monocot species. Here, non-grass monocot species were analysed to investigate the differences between grass and non-grass monocots. METHODS: First, studies of codon usage in monocots were reviewed. The current information was then extended regarding codon usage, as well as codon-pair context bias, using four completely sequenced non-grass monocot genomes (Musa acuminata, Musa balbisiana, Phoenix dactylifera and Spirodela polyrhiza) for which comparable transcriptome datasets are available. Measurements were taken regarding relative synonymous codon usage, effective number of codons, derived optimal codon and GC content and then the relationships investigated to infer the underlying evolutionary forces. KEY RESULTS: The research identified optimal codons, rare codons and preferred codon-pair context in the non-grass monocot species studied. In contrast to the bimodal distribution of GC3 (GC content in third codon position) in grasses, non-grass monocots showed a unimodal distribution. Disproportionate use of G and C (and of A and T) in two- and four-codon amino acids detected in the analysis rules out the mutational bias hypothesis as an explanation of genomic variation in GC content. There was found to be a positive relationship between CAI (codon adaptation index; predicts the level of expression of a gene) and GC3. In addition, a strong correlation was observed between coding and genomic GC content and negative correlation of GC3 with gene length, indicating a strong impact of GC-biased gene conversion (gBGC) in shaping codon usage and nucleotide composition in non-grass monocots. CONCLUSION: Optimal codons in these non-grass monocots show a preference for G/C in the third codon position. These results support the concept that codon usage and nucleotide composition in non-grass monocots are mainly driven by gBGC.

Jatropha (Jatropha curcas L.).

The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance. Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by histochemical GUS assay, polymerase chain reaction, and Southern hybridization.

Excess copper induced oxidative stress and response of antioxidants in rice.

To investigate the effects of copper (Cu), rice plant (Oryza sativa. L. var. MSE-9) was treated with different Cu concentrations (0, 10, 50 and 100 µM) for 5 days in hydroponic condition. Gradual decrease in shoot and root growth was observed with the increase of Cu concentration and duration of treatment where maximum inhibition was recorded in root growth. Cu was readily absorbed by the plant though the maximum accumulation was found in root than shoot. Hydrogen peroxide (H(2)O(2)) production and lipid peroxidation were found increased with the elevated Cu concentration indicating excess Cu induced oxidative stress. Antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) and glutathione reductase (GR) were effectively generated at the elevated concentrations of Cu though catalase (CAT) did not show significant variation with respect to control. Ascorbate (ASH), glutathione (GSH) and proline contents were also increased in all the Cu treated plants compared with the control. SOD isoenzyme was greatly affected by higher concentration of Cu and it was consistent with the changes of the activity assayed in solution. The present study confirmed that excess Cu inhibits growth, induced oxidative stress by inducing ROS formation while the stimulated antioxidative system appears adaptive response of rice plant against Cu induced oxidative stress. Moreover proline accumulation in Cu stress plant seems to provide additional defense against the oxidative stress.