Optimization of Agrobacterium-Mediated Transformation Parameters for Melastomatacea Spp. Using Green Fluorescent Protein(GFP) as A Reporter

Agrobacterium-mediated transformation for both Melastoma malabathricum and Tibouchinu semidecanda were optimized using green fluorescent protein (GFP) as a reporter. The binary vector pCAMBIA1304 harboring the modified green fluorescent protein (mgfp) gene driven by the CuMV 35S promoter was used. Parameters optimized were bacterial strain, bacterial concentration, pre-culture period, co-cultivation period,immersion time, acetosyring concentration and wounding type. Results obtained obtained were based on the percentage of (GFP expression which was observed 3 days post-transformation. Agrobacterium tumefaciens starin LBA4404 and EHA105 at concentration 1 X 107 cfu ml -1 (OD 600mm 0.8) showed the highest virulence on M. malabathricum and T.semidecandra, respectively. Four days of pre-culture and 2 days of co-cultivation were optimum for M.malabathricum transformation, while 3 days of pre-culture and co-cultivation fot T, semidecandra, . result also showed that 60 min of immersion and addition of 200 p.M acetosyringone gave the highest percentage of positive transformants for both M. malabathricum and T.semidecandra. Mild wounding also significantly increased the efficiency 0f M.malabathricum transformation

Agrobacterium-mediated transformation of melastoma malabathricum and tibouchina semidecandra with sense and antisense dihydroflavonol-4-reductase(DFR)genes.

Genetic engineering of a wide variety of plant species has led to the improvement of plant traits. In this study, the genetic transformation of two potentially important flowering ornamentals, Melastoma malabathricum and Tibouchina semidecandra, with sense and antisense dihydroflavonol-4-reductase (DFR) genes using the Agrobacterium mediated method was carried out. Plasmids pBETD10 and pBETD11, each harbouring the DFR gene at different orientations (sense and antisense) and selectable marker npt II for kanamycin resistance, were used to transform M. malabathricum and T. semidecandra under the optimized transformation protocol. Putative transformants were selected in the presence of kanamycin with their respective optimized concentration. The results indicated that approximately 4.0% of shoots and 6.7% of nodes for M. malabathricum regenerated after transforming with pBETD10, whereas only 3.7% (shoots) and 5.3%(nodes) regenerated with pBETD11 transformation. For the selection of T. semidecandra, 5.3% of shoots and 9.3% of nodes regenerated with pBETD10 transformation, while only 4.7% (shoots) and 8.3% (nodes) regenerated after being transformed with pBETD11. The presence and integration of the sense and antisense DFR genes into the genome of M. malabathricum and T. semidecandra were verified by polymerase chain reaction (PCR) and nucleotide sequence alignment and confirmed by southern analysis. The regenerated putative transformants were acclimatized to glasshouse conditions. Approximately 31.0% pBETD10-ransformed and 23.1% pBETD11-transformed M. malabathricum survived in the glasshouse, whereas 69.4% pBETD10-transformed and 57.4% pBETD11-transformed T. semidecandra survived. The colour changes caused by transformation were observed at the budding stage of putative T. semidecandra transformants where greenish buds were produced by both T. semidecandra harbouring the sense and antisense DFR transgenes. Besides that, the production of four-petal flowers also indicated another morphological difference of putative T. semidecandra transformants from the wild type plants which produce five-petal flowers

Mass spectrometry-based metabolomics approach for metabolite profiling and biomarker discovery of Acanthaceae medicinal plants and tissue cultures

Clinacanthus nutans (Sabah Snake Grass) and Strobilanthes crispus {daun pecah beling) from the family of Acanthaceae, are gaining research interest as the plants have been reported to possess various bioactivities. The present study was aimed to profile and identify the biomarker compounds of C. nutans and S. crispus plants and its tissue cultures using GCMS-based metabolomics approach. Using this high-throughput instrument coupled with advanced chemoinformatics softwares, the plants were found to rich with squalene, pentacyclic triterpenoids such as lupeol, betulin, phytosterols such as stigmasterol, campesterol and others such as alpha­tocopherol, didecan-2-yl phthalate, beta-tocopherol. As the metabolomes of C. nutans and S. crispus unfold, it is believed that such information will play critical role in the standardization and development of herbal drugs derived from the plants in the future

Genetic Identification and Mass Propagation of Economically Important Seaweeds

Seaweeds are a primary source of hydrocolloids, which can be processed into various food additives, cosmetics, and pharmaceuticals. The inability of current commercial seaweed farming projects to meet industrial demands is underscored by a plethora of challenges, which include the lack of high-quality germplasm with the desired cultural characteristics. This chapter describes the current trends in commercial seaweed production and the potential technological advances in production methods and genetic selection strategies, which can be applied to raise the productivity of seaweed farms. Molecular markers have become increasingly relevant to the selection of a diverse range of wild varieties for domestication, and this augurs well for strain identification. The development of high-density linkage maps based on molecular markers offers an avenue for the implementation of molecular breeding strategies based on quantitative trait loci (QTLs). Concurrently, productivity of existing varieties can be enhanced by the analysis of exogenous factors known to affect the growth and survival of tissue-cultured seedlings. The application of photobioreactors for tissue culture is another important development, which will be digressed upon. In addition to this, quality control which focuses on the comparison of chemical and physical qualities of the tissue-cultured and conventional cultivated seaweeds will become increasingly relevant to the development of industry standards for sustainable seaweed production to fulfill the increasing demands of seaweed-related industries

Quantitative real-time PCR for determination of Transgene in Callus of Jatropha curcas

Jatropha curcas is an important plant belonging to the family Euphorbiaceae which is a potential candidate for biofuel production. Genetic transformation protocol for J. curcas callus mediated by Agrobacterium tumefaciens were optimized using a pCAMBIA1303 plasmid which carries green fluorescent protein (GFP) gene as a reporter. Results obtained were based on the highest percentage of GFP expression which was observed three days post-transformation. Immersion of callus into 1×105 cfu ml-1 (OD600nm 0.6) of A. tumefaciens LBA4404 with addition of 300 µM of acetosyringone for 45 min, two days of pre-culture and three days of co-cultivation periods were determined to be ideal for J. curcas callus transformation. Putative transformants were selected in the presence of 25 mg/l hygromycin. Surviving calli were transferred into proliferation media (MS with 1 mg/l NAA and 1 mg/l BAP) to proliferate the callus for further molecular analyses and to confirm the presence of the target GFP transgene in the putative transformants. Polymerase chain reaction (PCR) was carried out using a 35S specific primer pair confirmed the presence of the 454 bp of 35S promoter region from the transformed callus. Quantitative real-time PCR (qRT-PCR) was carried out to demonstrate the integration and copy number of the 35S promoter in the putative tranformants. The 35S promoter gene (178 bp) as a target gene and J. curcas actin gene (179 bp) which functions as reference gene was designed to detect the positive transformants and control sample in real-time PCR reaction analysis. The results indicated that the actin specific PCR product was present in both the control and transformed calli, however the 35S PCR product was found only in the positive transformants. The similarity in CT values confirmed that both the genes were present as single copy thus confirming a single integration event

Enhancers of agrobacterium-mediated transformation of Tibouchina semidecandra selected on the basis of GFP expression

Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers (e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride and ascorbic acid) to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using GFP as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM aluminium chloride in the presence of 15 mg/L ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM aluminium chloride in the presence of 15 mg/L ascorbic acid was optimum for T. semidecandra nodes. The presence of the hptII transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers

Morphological and molecular studies of undescribed Kappaphycus species

Morphological and molecular studies were carried on an undescribed species of Kappaphycus (AS 12) from Sabah, Malaysia. The species displays unique and different physical characters from Kappaphycus alvarezii, K. striatus, K. malesianus and Eucheuma denticulatum in terms of branching patterns, thalli texture and colors. The phylogenetic position of this plant was inferred by cox2-3 spacer, intergenic transcribed spacer (ITS) region and RuBisCo spacer. Based on the results from this study, it is clear that the specimen AS 12 pointed to significant differences from K. alvarezii, K. striatus, K. malesianus and E. denticulatum as supported by both morphological and molecular analyses

A glance at molecular Identification of bamboo (poaceae: bambusoideae)

Conservation of plant species plays a vital role in preventing the loss of valuable plant resources. The success of conservation depends on the correct identification and characterization of plant species. Bamboo is one of the most important plants with multiple uses that have contributed to the economy and socio-economy of many people in rural areas. It is under the subfamily of Bambusoideae that includes both woody and herbaceous bamboo. Conventionally, like other plants, bamboo has been classified dependently based on morphological characteristics. However, morphological identification leads to difficulties and misclassification of bamboo species due to their infrequent flowering behaviour and peculiar reproductive biology. Since then, molecular markers have been introduced to overcome the problems associated with bamboo taxonomy and phylogeny. This paper provides an overview of the diverse, predominantly molecular techniques used to assess and determine the genetic diversity of bamboo species

Carrageenan from Kappaphycus alvarezii (Rhodophyta, Solieriaceae): Metabolism, structure, production, and application

Carrageenan is a polysaccharide derived from red algae (seaweed) with enormous economic potential in a wide range of industries, including pharmaceuticals, food, cosmetics, printing, and textiles. Carrageenan is primarily produced through aquaculture-based seaweed farming, with Eucheuma and Kappaphycus species accounting for more than 90% of global output. There are three major types of carrageenan found in red algae: kappa (κ)-, iota (ι)-, and lambda (λ)-carrageenan. Kappaphycus alvarezii is the most common kappa-carrageenan source, and it is primarily farmed in Asian countries such as Indonesia, the Philippines, Vietnam, and Malaysia. Carrageenan extracted from K. alvarezii has recently received a lot of attention due to its economic potential in a wide range of applications. This review will discuss K. alvarezii carrageenan in terms of metabolic and physicochemical structure, extraction methods and factors affecting production yield, as well as current and future applications