Highly efficient proliferation and regeneration of protocorm-like bodies (PLBS) of the threatened orchid, phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production

Highly efficient proliferation and regeneration of protocorm-like bodies (PLBS) of the threatened orchid, phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production

The CRISPR/Cas9 System for Crop Improvement: Progress and Prospects

The global demand for high-quality crops is continuously growing with time. Crop improvement techniques have a long history and they had been applied since the beginning of domestication of the first agricultural plants. Since then, various new techniques have and are being developed to further increase the commercial value and yield of crops. The latest crop improvement technique known as genome editing is a technique that enables precise modification of the plant genome via knocking out undesirable genes or enabling genes to gain new function. The variants generated from the genome editing are indistinguishable from naturally occurring variation. It is also less time-consuming and more readily accepted in the market commercially. The usage of genome editing has proven to be advantages and plays a promising role in future crop improvement efforts. Therefore, in this chapter, we aim to highlight the progress and application of genome editing techniques, in particular, the CRISPR/Cas9 system as a powerful genome editing tool for crop improvement. In addition, the challenges and future prospects of this technology for crop improvement will also be discussed

Development of an efficient particle bombardment transformation system for the endemic orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous orchid with high commercial value. In this study, we established an efficient particle bombardment transformation system for P. bellina using the protocorm-like-bodies (PLBs) as target tissues. Leaf derived PLBs were proliferated on ½ strength Murashige and Skoog (MS) medium supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2, 4-D). Both physical and biological parameters affecting the transformation system were optimised using the green-fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter systems. Optimal bombardment conditions with 6 cm target tissues distance, 1100 psi acceleration pressure, 1.0 μm gold particle size, 27 mmHg chamber vacuum pressure, single bombardment time, spermidine as DNA precipitation agent, 72 h post bombardment incubation time, 2 μg plasmid DNA in 0.15:0.12 pmol ratio (pSMCHS:p35SGFP) were successfully determined. Surviving PLBs transformants were successfully recovered from the hygromycin selection medium and verified using genomic PCR analysis. The established system is not only useful for a simple and reliable transient gene analysis but as well as generating stable transformants for selective traits improvement in orchids

Growth promoting effects of Pluronic F-68 on callus proliferation of recalcitrant rice cultivar

This study was undertaken to evaluate growth-promoting effects of Pluronic F-68 (PF-68) on recalcitrant MR 219 rice callus. Our study shows that calli grown on Murashige and Skoog medium supplemented with 0.04% PF-68 significantly increased callus proliferation by 58.80% (fresh weight) and 23.98% (dry weight) while root formation from callus was enhanced by 28.57%. Enhanced callus proliferation was supported by biochemical analysis, whereby highest amount of soluble sugar (1.77 mg/mL) and protein (0.17 mg/mL) contents were recorded in calli grown on 0.04% PF-68. Furthermore, enhanced expression of sucrose synthase (2.65-folds) and NADH-dependent glutamate synthase (1.86-folds) genes in calli grown on 0.04% PF-68 also correlates with enhanced callus proliferation. In contrast, high concentration of PF-68 (0.10%) recorded highest amount of phenolic (0.74 mg/mL), flavonoid (0.08 mg/mL), and hydrogen peroxide content (0.06 mg/mL) as compared to other treatment groups indicates activation of plant defence mechanism towards stress. Similarly, high expression of 4-coumarate:CoA ligase 3 (1.28-folds), chalcone-flavonone isomerase (1.65-folds) and ascorbate peroxidase (1.61-folds) genes were observed in calli grown on 0.10% PF-68 further supports increasing stress caused by the high concentration of PF-68. Taken together, our study revealed that optimum concentration of PF-68 could improve recalcitrant rice callus proliferation via enhanced sugar metabolism and amino acid biosynthesis which are crucial towards plant growth and development. However, at high concentration, PF-68 induces stress in plant which enhance the production of secondary metabolite to maintain cellular homeostasis

Characterization of vacuolar processing enzyme gene family and its expression in response to Fusarium oxysporum infection in Musa acuminata colla

Panama disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) has been the major threat for global banana production. Countless mitigation approaches have been implemented to avert Foc infections previously which include utilising of chemical fungicide, improvement of cultural practices and eradication through physical measures. However, most of the attempts failed to provide a satisfactory outcome in field trials. Cultivation of resistant cultivars has been shown to be the most effective approaches in counteracting the Panama disease which could be generated via genetic modification or conventional breeding. Hence, understanding the molecular events during compatible and incompatible interaction of Foc with banana could provide valuable insight into the development of genetically modified resistant banana which is crucial for protection strategies against Panama disease. This research was undertaken to study the molecular characteristic of Musa acuminata vacuolar processing enzyme (MaVPE) – a cysteine proteinase that mediates programmed cell death during FocTR4 infection. A total of seven MaVPE genes (designated as MaVPE1 through MaVPE7) were successfully identified through systemic in silico analysis of DH-Pahang (AA group) banana genome. Phylogenetic study showed that MaVPEs could be divided into seed type or vegetative type. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) further revealed that most of MaVPE genes expressions were induced in susceptible M. acuminata cv. Berangan after FocTR4 infection, specifically at 1 and 2 days post inoculation (DPI). However, in resistant M. acuminata cv. Jari Buaya, MaVPEs expression remained at low level at all time points after inoculation with FocTR4. The enzymatic caspase-1 activity of MaVPE also corroborated with the gene expression analysis. Interestingly, comparative proteomic profiling analysis revealed an increase in the abundance of cysteine proteinase in inoculated susceptible M. acuminata cv. Berangan as opposed to cysteine proteinase inhibitors in resistant M. acuminata cv. Jari Buaya. Consistently, inhibition of MaVPE activity through caspase-1 inhibitor reduced vacuolar membrane disintegration and decreased lesion formation of FocTR4 infected banana root. Further functional analysis using an Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infections and decreased cell death incidence. Taken together, the findings suggest that VPE acts as a key molecule in modulating susceptibility response in FocTR4-infected plant. This study could be the stepping stone for the development of silenced MaVPE banana via exploitation of gene editing technology. This would be useful for the production of FocTR4-resistant banana cultivar in future

Functional characterization of a new terpene synthase from Plectranthus amboinicus

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60–70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems

Vacuolar processing enzymes modulate susceptibility response to Fusarium oxysporum f. sp. cubense tropical race 4 infections in banana

Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4) is a destructive necrotrophic fungal pathogen afflicting global banana production. Infection process involves the activation of programmed cell death (PCD). In this study, seven Musa acuminata vacuolar processing enzyme (MaVPE1–MaVPE7) genes associated with PCD were successfully identified. Phylogenetic analysis and tissue-specific expression categorized these MaVPEs into the seed and vegetative types. FocTR4 infection induced the majority of MaVPE expressions in the susceptible cultivar “Berangan” as compared to the resistant cultivar “Jari Buaya.” Consistently, upon FocTR4 infection, high caspase-1 activity was detected in the susceptible cultivar, while low level of caspase-1 activity was recorded in the resistant cultivar. Furthermore, inhibition of MaVPE activities via caspase-1 inhibitor in the susceptible cultivar reduced tonoplast rupture, decreased lesion formation, and enhanced stress tolerance against FocTR4 infection. Additionally, the Arabidopsis VPE-null mutant exhibited higher tolerance to FocTR4 infection, indicated by reduced sporulation rate, low levels of H2O2 content, and high levels of cell viability. Comparative proteomic profiling analysis revealed increase in the abundance of cysteine proteinase in the inoculated susceptible cultivar, as opposed to cysteine proteinase inhibitors in the resistant cultivar. In conclusion, the increase in vacuolar processing enzyme (VPE)-mediated PCD played a crucial role in modulating susceptibility response during compatible interaction, which facilitated FocTR4 colonization in the host

Carrageenan promotes plant growth in banana via enhancement of cellular metabolism, nutrient uptake, and cellular homeostasis

Banana (Musa acuminata) is an important fruit crop and source of income for various countries, including Malaysia. To date, current agrochemical practice has become a disputable issue due to its detrimental effect on the environment. λ-carrageenan, a natural polysaccharide extracted from edible red seaweed, has been claimed to be a potential plant growth stimulator. Hence, the present study investigates the effects of λ-carrageenan on plant growth using Musa acuminata cv. Berangan (AAA). Vegetative growth such as plant height, root length, pseudostem diameter, and fresh weight was improved significantly in λ-carrageenan-treated banana plants at an optimum concentration of 750 ppm. Enhancement of root structure was also observed in optimum λ-carrageenan treatment, facilitating nutrients uptake in banana plants. Further biochemical assays and gene expression analysis revealed that the increment in growth performance was consistent with the increase of chlorophyll content, protein content, and phenolic content, suggesting that λ-carrageenan increases photosynthesis rate, protein biosynthesis, and secondary metabolites biosynthesis which eventually stimulate growth. Besides, λ-carrageenan at optimum concentration also increased catalase and peroxidase activities, which led to a significant reduction in hydrogen peroxide and malondialdehyde, maintaining cellular homeostasis in banana plants. Altogether, λ-carrageenan at optimum concentration improves the growth of banana plants via inducing metabolic processes, enhancing nutrient uptake, and regulation of cell homeostasis. Further investigations are needed to evaluate the effectiveness of λ-carrageenan on banana plants under field conditions

Effect of lignosulphonates on Vanilla planifolia shoot multiplication, regeneration and metabolism

Vanilla planifolia (V. planifolia) is a valuable orchidaceous plant, commonly grown for its pods that are used to produce the flavouring vanilla extract. Here, we evaluated the effect of calcium lignosulphonate (Ca-LIGN) and sodium lignosulphonate (Na-LIGN) on multiplication and regeneration of V. planifolia shoot tip culture. In 150 mg L−1 Ca-LIGN medium, the most number of shoots per explant (5.78 ± 0.63) was successfully obtained. Besides, Ca-LIGN also enhanced the shoot bud and primordial formation rate, as seen under scanning electron microscopy. In contrast, medium containing 150 mg L−1 Na-LIGN recorded the highest average of shoot length (4.72 ± 0.30 cm). Meanwhile, the best growth of root length (1.8 ± 0.32 cm) and root induction (96.67 ± 5.16%) were recorded on the explants treated with 150 mg L−1 Na-LIGN rooting medium. All rooted plantlets successfully acclimatized in the greenhouse (100.00% survival rate). Further biochemical analysis revealed that Ca-LIGN increased the total protein, chlorophyll, sugar, flavonoid and phenolic contents of V. planifolia. Notably, expression of both ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) genes were also elevated under the treatment of Ca-LIGN, implying a positive role in the photosynthetic process. Taken together, LIGN being an environmental friendly product could be used to enhance the growth and micropropagation of V. planifolia