Transient Transformation of Oncidium Taka by Particle Bombardment

Oncidium Taka was used in this study for its importance as cut flower in Malaysia floriculture industry. Protocorm likes bodies (PLBs) that proved to have vigorous propagation and regeneration capacity were induced from shoot tips and flower buds in MS hormone free medium. Subsequently, callus induction by using different concentrations of auxins (2,4-D, dicamba, picloram, IAA and NAA) was investigated in dark condition. Picloram in a concentration of 40 μM was found to be the most effective for inducing callus from PLBs. However, the initiated callus was maintained and proliferated in medium consisted of combination hormones of 50 μM picloram and 20 μM kinetin. Both PLBs and callus were used as target tissues in the genetic transformation study.Regeneration study of these calli was carried out in half strength MS medium under light condition. The calli regenerated by intervening PLB stage and the shoot primordial was developed as early as 44 days after culture. Besides that, media composition and different concentrations of BAP were examined to enhance plant regeneration and plant growth from PLBs. Half strength MS medium supplemented with full strength B5 vitamin, 3 % sucrose and 20 μM BAP was found to be efficient for plant regeneration from PLBs. Apparently, better plant growth can be promoted in full strength MS medium supplemented with full strength B5 vitamin, 2 % sucrose and 20 μM BAP. The effectiveness of hygromycin as selective agents to inhibit growth of PLBs and callus was evaluated. Toxicity effect of hygromycin was found rapid and effective to kill the untransformed PLBs and callus in low concentration at 25 mg/L and 10 mg/L, respectively. GFP as a non-destructive, early and rapid detection reporter system was used in this transformation studies. The p35S-sgfp-TYG-nos GFP plasmid was determined to give the highest transient expression in both target tissues among five different plasmids evaluated. The fluorescent signals expressed with elevated level of green fluorescent on nuclei were observed using Leica Stereo-fluorescence System MZ FLIII with GFP 2 filter set that masked out chlorophyll. The highest numbers of transient expression achieved on day two postbombardment for both target tissues. The physical and biological parameters affecting DNA delivery, based on the highest scorable transient GFP expression and minimal tissue dislocation upon impact, were optimised. The physical parameters optimised in helium pressure, distance from stopping plate to target tissue, vacuum pressure, size of microcarrier, quantity of DNA and effect of CaCl2 and Spermidine were found no different between PLBs and callus. As for biological parameters tested, PLBs in sizes of 5 – 6 mm had significantly higher transient expression compared to calli. Seven days old calli with one day pre-culture period and fourteen days old PLBs that pre-culture on the same day gave the highest transient expression. The quantity of DNA per bombardment used was 1.5 μg for both target tissues to achieve the highest transient expression. The bombarded tissues were subsequently transferred to hygromycin containing medium for a minimum of six months selection. The survived and proliferated putative transformed tissues were subjected to molecular analysis. Insertion of the transgenes (gfp, gus A, hpt II and chs) into host genome was confirmed using PCR and Southern vi Blot analysis. Transformation efficiency was determined at 1.5 % to 1.67 % and 1.67 % to 2.5 % when using PLBs and callus, respectively as target tissue. In addition, PCR analysis showed co-transformation frequency of the un-linked genes from different plasmids was 40 % to 67 %

Antioxidant-mediated response of a susceptible papaya cultivar to a compatible strain of Erwinia mallotivora

Dieback disease caused by Erwinia mallotivora is the most devastating disease of papaya in Malaysia. Most papaya cultivars here are susceptible to the disease, indicating a compatible relationship between the cultivars and the pathogenic strains of the bacteria. In this study, we evaluated early responses of a susceptible cultivar to a virulent strain of the bacteria by monitoring the disease symptoms, measuring the expression patterns and activities of the oxidative enzymes superoxide dismutase (SOD) and peroxidase (POD), and profiling the leaf proteome. Water-soaked lesions and necrotic spots were evidenced on the inoculated leaves at 6 days post-inoculation (dpi) which were deteriorated rapidly with death at 12 dpi. The two oxidative enzymes were responsive to the infection as shown by their induced enzymatic activities and gene expressions in the leaves of the infected plants. The 2D-PAGE profile of the leaf proteome exhibited a significant increase in the intensity of the SOD spot, confirming its involvement in the response. In addition, differential expressions of the protein spots known to be related to stress response, energy production and photosynthesis provided insights into the plant's global response to the infection. Overall, this study suggested that the early response to E. mallotivora inoculation in the susceptible papaya cultivar was likely to be mediated by the antioxidant defense system but the effectiveness of the defense could have been compromised with the down-regulation of important proteins required for normal plant growth and development. This study provides the basis for studying the defect in the defense mechanism that turned this cultivar susceptible to the infection

RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit

The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants

Iron Biofortification of Rice: Progress and Prospects

Biofortification is the process of improving the bioavailability of essential nutrients in food crops either through conventional breeding or modern biotechnology techniques. Rice is one of the most demanding staple foods worldwide. Most global population live on a diet based on rice as the main carbohydrate source that serve as suitable target for biofortification. In general, polished grain or white rice contains nutritionally insufficient concentration of iron (Fe) to meet the daily requirements in diets. Therefore, iron biofortification in rice offers an inexpensive and sustainable solution to mitigate iron deficiency. However, understanding on the mechanism and genes involved in iron uptake in rice is a prerequisite for successful iron biofortification. In this chapter, the overview of iron uptake strategies in plants and as well as different iron-biofortified approaches used in rice will be outlined. Then, the challenges and future prospects of rice iron biofortification to improve global human health will also be discussed

A quick protocol to facilitate the selection of putative delayed ripening transgenic papaya lines for field evaluation

A quick protocol is needed to determine transgene, ACC oxidase 2 expression in putative transgenic Eksotika papaya lines targeting at selecting potential lines for field evaluation. In this study, three housekeeping genes, 18S ribosomal RNA, 40S ribosomal protein and actin, were used for normalisation of the ACC oxidase 2 gene expression. Comparison with a non-transformed seedling-derived plants revealed that 42 putative transgenic lines exhibited between 2- and 5-fold reduction of ACC oxidase 2 expression level. Based on the gene expression data of the in vitro putative transgenic papaya plants obtained, 24 independent potential lines were selected for field evaluation. Gene expression analysis on field grown papaya plants showed similar profile of ACC oxidase 2 expression levels as compared to in vitro papaya plants. Increase in storage shelf life was also examined in the transgenic lines grown in the field with the most potential transgenic line was 27-3, which required 14 days to achieve full yellow colour index. Overall, the findings in this study revealed that reduction of ethylene was successful in the Eksotika papaya by manipulating the ACC oxidase 2 using the antisense technique. This reflects that future production of longer shelf life Eksotika papaya fruits is possible through antisense technique and it will help boost the papaya industry further by opening up new export markets in distant destinations

Effects of lignosulfonates on callus proliferation and shoot induction of recalcitrant indica rice

In vitro culture of recalcitrant indica rice cultivar through intervening callus is difficult due to long regeneration period. Therefore, this study was undertaken to evaluate the growth promoting effects of lignosulfonate (LS) on callus proliferation and shoot induction of Malaysian recalcitrant indica rice cv. MR219. LS is a by-product of wood industry, commonly used as a plant growth enhancer. Seed derived calli were proliferated on Murashige and Skoog (MS) medium supplemented with different ion-chelated LS (calcium LS: CaLS and sodium LS: NaLS) at 50, 100, 150, and 200 mg/L. MS supplemented with 100 mg/L CaLS significantly increased the callus proliferation rate and adventitious root formation. In shoot induction study, both LSs did not enhance the shoot induction efficiency as compared to the control. However, the formation of albino shoot increased in MS fortified with 100 mg/L CaLS. Further chlorophyll and molecular analyses showed that, albino shoots induced from 100 mg/L CaLS had severe reduction in total chlorophyll content and expression of both chlorophyll-associated genes, chlorophyll a/b-binding protein 1 (OsCAB1R) and young seedling albino (OsYSA). Taken together, LS improves callus proliferation rate and modulate different physiological responses during plant growth of recalcitrant indica rice

Effects of lignosulfonates on callus proliferation and shoot induction of recalcitrant Indica rice

In vitro culture of recalcitrant indica rice cultivar through intervening callus is difficult due to long regeneration period. Therefore, this study was undertaken to evaluate the growth promoting effects of lignosulfonate (LS) on callus proliferation and shoot induction of Malaysian recalcitrant indica rice cv. MR219. LS is a by-product of wood industry, commonly used as a plant growth enhancer. Seed derived calli were proliferated on Murashige and Skoog (MS) medium supplemented with different ion-chelated LS (calcium LS: CaLS and sodium LS: NaLS) at 50, 100, 150, and 200 mg/L. MS supplemented with 100 mg/L CaLS significantly increased the callus proliferation rate and adventitious root formation. In shoot induction study, both LSs did not enhance the shoot induction efficiency as compared to the control. However, the formation of albino shoot increased in MS fortified with 100 mg/L CaLS. Further chlorophyll and molecular analyses showed that, albino shoots induced from 100 mg/L CaLS had severe reduction in total chlorophyll content and expression of both chlorophyll-associated genes, chlorophyll a/b-binding protein 1 (OsCAB1R) and young seedling albino (OsYSA). Taken together, LS improves callus proliferation rate and modulate different physiological responses during plant growth of recalcitrant indica rice

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

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

First Draft Genome Assembly of the Malaysian Stingless Bee, Heterotrigona itama (Apidae, Meliponinae)

The Malaysian stingless bee industry is hugely dependent on wild colonies. Nevertheless, the availability of new queens to establish new colonies is insufficient to meet the growing demand for hives in the industry. Heterotrigona itama is primarily utilized for honey production in the region and the major source of stingless bee colonies comes from the wild. To propagate new colonies domestically, a fundamental understanding of the biology of queen development, especially from the genomics aspect, is necessary. The whole genome was sequenced using a paired-end 150 strategy on the Illumina HiSeq X platform. The shotgun sequencing generated approximately 89 million raw pair-end reads with a total output of 13.37 Gb and a GC content of 37.31%. The genome size of the species was estimated to be approximately 272 Mb. Phylogenetic analysis showed H. itama are much more closely related to the bumble bee (Bombus spp.) than they are to the modern honey bee (Apis spp.). The genome data provided here are expected to contribute to a better understanding of the genetic aspect of queen differentiation as well as of important molecular pathways which are crucial for stingless bee biology, management and conservation