Agrobacterium-Mediated Transformation Of Dendrobium Sonia-28

Effective propagation of protocorm-like bodies (PLBs) of Dendrobium sonia-28 was achieved by using micropropagation technique. The highest PLBs proliferation within 21 days was observed in semi-solid media over liquid culture. Half strength of Murashige and Skoog (MS) media produced the highest PLBs growth rate percentage compared to full and double strength MS media in semi-solid and liquid cultures. In sucrose treatment, the highest PLBs growth rate percentage was in 10 g.L-1 sucrose. In the conditions of absence of sucrose (0 g.L-1) or excessive of sucrose (30 g.L-1) had proven detrimental for the PLBs growth and development

Optimisation of transient green fluorescent protein (GFP) gene expression in Phalaenopsis Violacea Orchid mediated by Agrobacterium Tumefaciens-mediated transformation system

Numerous transformation factors were successfully optimised to develop a reliable and highly efficient Agrobacterium-mediated transformation into the protocorm-like bodies (PLBs) of Phalaenopsis violacea. The optimisation of factors influencing stable transformation efficiency in new species is very important as it can reduce the costs in labor and materials in the future. Hypervirulent Agrobacterium tumefaciens strains, EHA 101 and 105, harboring the pCAMBIA 1304 plasmid which contains gusA gene and gfp gene as the reporter markers, were used for transformation study. Transient gfp gene expression was used to evaluate the efficiency of T-DNA delivery in transformants due to its simple, non-destructive and cell autonomous procedure. Agrobacterium strain EHA 105 was proved to be better in transforming the targeted PLBs than EHA 101, based on the notably high transient expression of gfp gene in all the parameters tested. Different temperatures during cocultivation period, the concentration of L-cysteine, calcium (CaCl2) and silver nitrate (AgNO3) in cocultivation medium as well as pH and light and dark conditions during co-cultivation period were identified to be major factors in enhancing the percentage of transient gfp gene expression. Increased T-DNA delivery efficiencies were obtained when P. violacea PLBs were co-cultivated with Agrobacterium tumefaciens strain EHA 105 in half-strength MS medium supplemented with 5% of banana Mas extract containing 200 mg.L-1 L-cysteine, 60μM silver nitrate, without calcium, adjusted to pH 5.5 and incubated in the dark at 24°C. The results from transient transformation of PLBs suggested that Agrobacterium-mediated transfer of T-DNA to the naturally recalcitrant P. violacea is feasible and is highly efficient. Consequently, by combining the best treatments, an efficient and reproducible Agrobacterium-mediated transformation protocol could be continued to facilitate the insertion of any desirable traits for the production of transgenic Phalaenopsis violacea orchid

The Effect of Strain Virulence on Agrobacterium Rhizogenes Transformation Efficiency in Eurycoma Longifolia

Eurycoma longifolia, known locally as ‘Tongkat Ali' in Malaysia, is popularly sought out as herbal remedy in many parts of South East Asia. Consequently, this has resulted in the indiscriminate collection of the taproot from the wild, leading to fears of extinction among conservationists. Agrobacterium rhizogenes is a naturally occurring soil bacterium that infects injured plants and causes a massive proliferation of roots, known as hairy roots. The objective of this study is to determine the transformation efficiency of 2 different strains of Agrobacterium rhizogenes on somatic embryos of Eurycoma longifolia using transient GUS expression as an indicator of successful transformation. Somatic embryos cultured in vitro for 4 to 5 weeks were used as explants and were pre-cultured for 2 days in full strength MS medium containing 0.5 mg/L IBA added with 1% PVP and 2mg/L DTT. The explants were transformed using Agrobacterium rhizogenes strains AR12 and AR14. GUS assay was carried out 1 week after transformation and observed. Observations indicate that AR12 is more effective in transforming somatic embryos of Eurycoma longifolia compared to AR14. Therefore, Agrobacterium rhizogenes strain AR12 is a potential candidate for the successful transformation of Eurycoma longifolia somatic embryos, leading to the induction of sustainable hairy root culture

Liquid Biphasic Electric Partitioning System as a Novel Integration Process for Betacyanins Extraction From Red-Purple Pitaya and Antioxidant Properties Assessment

Nowadays, downstream bioprocessing industries inclines towards the development of a green and high efficient bioseparation technology. Betacyanins are presently gaining higher interest in the food science as driven by their high tinctorial strength and health promoting functional properties. In this study, a novel green integration process of liquid biphasic electric partitioning system (LBEPS) was proposed for betacyanins extraction from peel and flesh of red-purple pitaya. Initially, the betacyanins extraction using LBEPS with initial settings was compared with that of liquid biphasic partitioning system (LBPS), and the results revealed that both systems demonstrated a comparable betacyanins extraction. This was followed by further optimizing the LBEPS for better betacyanins extraction. Several operating parameters including operation time, voltage applied, and position of graphitic electrodes in the system were investigated. Moreover, comparison between optimized LBEPS and LBPS with optimized conditions of electric system (as post-treatment) as well as color characterization and antioxidant properties assessment were conducted. Overall, the betacyanins extraction employing the optimized LBEPS showed the significant highest values of betacyanins concentration in alcohol-rich top phase (Ct) and partition coefficient (K) of betacyanins from peel (99.256 ± 0.014% and 133.433 ± 2.566) and flesh (97.189 ± 0.172% and 34.665 ± 2.253) of red-purple pitaya. These results inferred that an optimal betacyanins extraction was successfully achieved by this approach. Also, the LBEPS with the peel and flesh showed phase volume ratio (Vr) values of 1.667 and 2.167, respectively, and this indicated that they have a clear biphasic separation. In addition, the peel and flesh extract obtained from the optimized LBEPS demonstrated different variations of red color as well as their antioxidant properties were well-retained. This article introduces a new, reliable, and effective bioseparation approach for the extraction of biomolecules, which is definitely worth to explore further as a bioseparation tool in the downstream bioprocessing

Crops For the Future (CFF): an overview of research efforts in the adoption of underutilised species

Main conclusion Crops For the Future (CFF), as an entity, has established a broad range of research activities to promote the improvement and adoption of currently underutilised crops. Abstract This paper summarises selected research activities at Crops For the Future (CFF) in pursuit of its mission 'to develop solutions for diversifying future agriculture using underutilised crops'. CFF is a research company focussed on the improvement of underutilised crops, so that they might be grown and consumed more widely with benefits to human food and nutritional security; its founding guarantors were the Government of Malaysia and the University of Nottingham. From its base in Malaysia, it engages in research around the world with a focus on species and system diversification. CFF has adopted a food system approach that adds value by delivering prototype food, feed and knowledge products. Bambara groundnut (Vigna subterranea) was adopted as an exemplar crop around which to develop CFF's food system approach with emphasis on the short-day photoperiod requirement for pod-filling and the hard-to-cook trait. Selective breeding has allowed the development of lines that are less susceptible to photoperiod but also provided a range of tools and approaches that are now being exploited in other crops such as winged bean (Psophocarpus tetragonolobus), amaranth (Amaranthus spp.), mor-inga (Moringa oleifera) and proso (Panicum miliaceum) and foxtail (Setaria italica) millets. CFF has developed and tested new food products and demonstrated that several crops can be used as feed for black soldier fly which can, in turn, be used to feed fish thereby reducing the need for fishmeal. Information about underutilised crops is widely dispersed; so, a major effort has been made to develop a knowledge base that can be interrogated and used to answer practical questions about potential exploitation of plant and nutritional characteristics. Future research will build on the success with Bambara groundnut and include topics such as urban agriculture, rural development and diversification, and the development of novel foods. CFF's remit and research objective

Vitrification and histological analyses of protocorm-like bodies of Vanda Kaseem's Delight orchid

Abstract This study was conducted to investigate the potential of cryopreserving protocorm-like bodies (PLBs) of Vanda Kaseem's Delight Orchid using plant vitrification solution 2 (PVS2). Measured parameters included the effect of PLB size (1-2 and 3-4mm), the effect of sucrose preculture concentrations and duration (0, 0.10, 0.25, 0.50 and 0.75 M, for 24 and 48 hours), the effect of dehydration duration (0, 10, 20, 30 and 40 minutes) and the effect of various unloading periods (0, 10, 20, 30 and 40 minutes) on survival of cryopreserved PLBs, as assessed using spectrophotometric 2,3,5-triphenyltetrazolium chloride (TTC) assay at 490nm. The PLBs were also subjected to a histological study to observe differences in both cryopreserved and non-cryopreserved PLBs of the orchid. The best results in the cryopreservation of PLBs of Vanda Kaseem's Delight was obtained when 3-4mm PLBs were precultured in VW medium supplemented with 0.1M sucrose for 24 hours, followed by a loading treatment, and 20 minutes of dehydration in PVS2 at 0°C, prior to cryostorage, and 30 minutes of unloading treatment after 90 seconds of thawing. Histological observations of cryopreserved PLBs indicated that most of the damages resulting from cryostorage occurred in the cell wall and nucleus of the cells

Nutritive bambara groundnut powdered drink mix: characterization and in-vivo assessment of the cholesterol-lowering effect

Drought has become more frequent due to climate change and its effects on the agricultural sector can be devastating. This increases the need for drought tolerant crops such as the Bambara groundnut (BGN) to be farmed in Malaysia. The development of BGN to a product suitable for consumers in this region has not been studied. Therefore, this research aimed to determine consumer acceptability of a nutritive BGN powdered drink mix using the Just-About-Right (JAR) method. BGN flour was produced by mixing soy powder with 0% BGN powder (0B100S), 10% BGN powder (10B90S), 20% BGN powder (20B80S) and 30% BGN powder (30B70S), respectively. The physicochemical properties of the powdered drink mix and its microbiological changes upon storage for six months were then determined. In addition, in-vivo assessment of the cholesterol-lowering effect of this product was conducted. Sample 10B90S was the most preferred sample among the sensory panellists (overall acceptability = 72%). Sample 10B90S remained stable after six months of ambient storage. Rats fed with sample 30B70S showed the most improvement in blood cholesterol levels. In conclusion, nutritive BGN powdered drink mix exhibited good physicochemical properties and could be useful for food applications

Application of liquid biphasic flotation for betacyanins extraction from peel and flesh of Hylocereus polyrhizus and antioxidant activity evaluation

This study investigated the use of liquid biphasic flotation (LBF) which composed of alcohol and salt for betacyanins extraction from peel and flesh of red-purple pitaya (Hylocereus polyrhizus). Betacyanins have received a great deal of research attention owing to their promising antioxidant activity. The operating parameters of LBF, including types/concentrations/volumes of alcohol and salt, addition of sodium chloride, flotation time and types/concentrations of crude extract, were adopted for the betacyanins extraction. Overall, the highest values of betacyanins concentration in alcohol-rich top phase (Ct), separation efficiency (E) and partition coefficient (K) of betacyanins that reveal a maximum betacyanins extraction from the peel (95.989%, 88.361% and 24.168, respectively) and flesh (95.488%, 94.886% and 21.195, respectively) were successfully achieved with LBF. In addition, both the peel and flesh extracts of red-purple pitaya provided different hues and intensities of red color, and their antioxidant properties were well retained

Preliminary analysis of cryopreservation of Dendrobium Bobby Messina orchid using an encapsulation-dehydration technique with Evans blue assay

In vitro grown protocorm-like bodies (PLBs) of Dendrobium Bobby Messina hybrid were cryopreserved in liquid nitrogen (LN) at -196°C by an encapsulation-dehydration technique. PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid MS media supplemented with six different concentrations of sucrose (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M). The PLBs were then encapsulated to form the beads in halfstrength liquid MS media supplemented with different concentrations of sodium alginate (2.5, 3.0 and 3.5%). The beads were placed in 2 ml cryovials and plunged into LN for 24 h. The beads were then thawed in a 40°C water bath for 90 s and were placed in recovery media composed of half strength semisolid MS media supplemented with 2% sucrose for four days under dark condition. After 12 days, the Evans blue dye assay was carried out to determine the viability of the PLBs. The highest viability was found in 1 to 2mm PLBs precultured in half strength semi-solid MS media supplemented with 1.0 M sucrose and encapsulated in 2.5% sodium alginate. Biochemical content analyses (chlorophyll, total soluble protein and peroxidase activities) were done to investigate the physiological responses of the PLBs after cryopreservation