Development of salt-tolerant lines of Malaysian indica rice (Oryza sativa L. cv. MR219) using tissue culture approach

Rice is one of the most important staple foods for human. However, millions hectares of rice land in the South and Southeast Asia were left uncultivated and/or grown with very low yields due to salinity. Due to this, development and production of salt-tolerant rice seed as apart of integrated management practices is needed. By using tissue culture approach, salt-tolerant lines of Malaysia indica rice cv. MR219 were produced. This work started with production and propagation of MR219 callus. Then, callus was sub-cultured separately on MS media supplemented with 2 mg/L 2,4-D and different concentration of NaCl (0, 50, 100, 200, and 300 mM NaCl) to produce salt-tolerant MR219 callus. Screening and selection of salt-tolerant MR219 callus were conducted using morphological and biochemical markers which are total proline content, total soluble sugar, lipid peroxidase and the activity of ascorbate peroxidase and catalase. At regeneration of salt-tolerant plantlets, selected salt-tolerant callus was sub-cultured on MS media supplemented with 2 mg/L kinetin and 1 mg/L BAP for shoot induction, followed by sub-cultured in MS media supplemented with 0.5 mg/L BAP, 1 mg/L kinetin, 1 mg/L IBA and 0.5 mg/L NAA for root formation. At acclimatization stage, MR219 plantlets from 50 mM NaCl found survived and transferred to pots containing paddy soil. These plantlets is called First generation (F1) salt-tolerant MR219. After 70 days, seeds of F1-salt-tolerant MR219 lines was successfully obtained. The grain characteristics of mother plant and F1-salt-tolerant MR219 lines were compared. Germination capability of F1-salt-tolerant MR219 seed in saline showed that seeds of F1-salt-tolerant MR219 able to germinate and growth in 50 -100 mM NaCl. As conclusion, salt-tolerant MR219 rice was produced in vitro and have potential to be commercialized. The protocol to produce salt-tolerant rice can be used to produce other salt-tolerant plant

Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase

Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants

Isolation, similarity and subcellular localisation of transaldolase from sugarcane (Saccharum officinarum)

This study focused on isolation, cloning of TAL from sugarcane. Transaldolase is one of the enzymes of the Pentose Phosphate Pathway (PPP). Transaldolase in non-oxidative phase of OPPP transfer a three carbon dihydroxyacetone moiety from sedoheptulose-7-phosphate and glyceraldehyde-3-phophate to produce Erythrose-4-Phophate (E4P) and fructose-6-phophate. E4P is the precursor for many secondary metabolic pathways including aromatic amino acids, lignin and flavonoid synthesis. Earlier studies revealed that OPPP is incomplete in the cytosol of plants as no genes encoding for a cytosolic TAL. Moreover, there is no study about the TAL genes from sugarcane until to date. Thus, the objective of this study is to isolate TAL gene from sugarcane, to compare its similarity with other plants, to determine its subcellular localization. A total of 1601 bp of TAL has been isolated by PCR. Similarity, studies by ClustalW revealed that TAL show highest similarity (75%) with Zea mays. Analysis of subcellular localization by using Target 1.1 revealed that of TAL from sugarcane was not located in the plastidic

Isolation, subcellular localization and phylogenetic of transaldolase genes from Zea mays cv sweet corn bi-color

The Pentose Phosphate Pathway (PPP) also is known as 6-phosphogluconate pathway which occurs in the cytosol of the plant cell. There are two main arms in PPP; the oxidative and non-oxidative arms. The main functions of this pathway is to generate reducing equivalents in the form of NADPH, for reductive biosynthesis reactions within cells in the biosynthesis of fatty acids and steroid. This pathway also provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. Transaldolase (TAL) is an enzyme which plays an important role in the non-oxidative portion of the pentose phosphate pathway. The actual reaction is between glyceraldehydes 3-phosphate and sedoheptulose 7-phosphate, resulting the formation of C4 product erythrose 4-phosphate and fructose 6-phosphate. In this study, transaldolase (TAL) has been successfully isolated and identified from Zea mays cv Sweet corn bi-color. The objectives of the study were achieved where the specific primer designed has function effectively in isolated TAL with 773 bp of nucleotide sequences. Analysis of homology through multiple sequence alignment revealed that TAL from Z. mays cv Sweet corn bi-color is highly similar with the plant species compared to animal and bacteria. ClustalW analysis found that TAL from Z. mays hit the score of 85.0 as they are close related in terms of taxonomy level which they are from the family of Poaceae. Other plants such as Solanum lycopersicum and Solanum tuberosum are from family Solanaceae, Dimorcarpus longan is from family Sapindaceae whereas Hyacinthus orientalis is from Hyacinthus family. However, it is discovered that TAL from Z. mays cv Sweet corn bi-color with TAL in Oryza sativa subsp. Indica only possess score of 64.0

Isolation, cloning, and sub-cellular localization of transketolase from Amaranthus tricolor L.

Transketolase (TK) is one of the key enzymes that involved in the Oxidative Pentose Phosphate Pathway (OPPP), and produce erythrose-4-phosphate which is the precursor for many secondary metabolites such as aromatic amino acids, lignin and flavonoid. The OPPP is composed of two functionally-connected phases, the oxidative and non-oxidative phase. The complete OPPP is localized in cytosol of animal and prokaryotic. However, in plant, the first phase is localised in cytosol but the sub-cellular localization of the second phase of OPPP is still under debate. There is no study available on transketolase in Amaranthus tricolor till to day? Therefore, the objectives of study are to isolate TK gene from Amaranthus tricolor, to compare its identity with other plant species and to determine its sub-cellular localization. The full length of 2021bp nucleotide sequence of TK had been isolated from A. tricolor by RT-PCR. ClustalW revealed that A. tricolor TK sequences showed high similarity (more than 81 %) within plants’ other species. Subcellular localization by using TargetP 1.1 and ChloroP revealed that of A. tricolor TK was located in the plastid. Thus it can be concluded that the OPPP is incomplete in the cytosol

Effect of four different salts on seed germination and morphological characteristics of Oryza sativa L. cv. MR219

The response of Oryza sativa L. cv. MR219 to NaCl, KCl, MgCl2 and MgSO4 at different salinity levels (0, 50, 100, 150, 200 and 250 mM) was studied with emphasis on seed germination and early seedling stage. High salinity delayed mean germination time of seeds and increased biomass, relative injury rate and seedling height reduction. Seeds are more tolerant to NaCl among four salts even at the highest salinity. Results showed that 50mM KCl enhanced the root growth with more roots developed at this salinity. Abnormal seed germination was found in MgCl2 and MgSO4 due to inhibition of root growth. This study proposes that degree of tolerance of MR219 to salts from morphological results is NaCl>KCl>MgCl2>MgSO4. This study might be useful for further research of salinity effect on growth and physiological processes at advanced stage of MR 219 growth

Hydro priming stimulates seedling growth and establishment of Malaysian Indica rice (MR219) under drought stress

Drought stress severely effects on seed germination and seedling establishment as critical stages in crops lifetime. Seed hydro priming is useful process for improving crops tolerance to drought stress. The present investigation was designed to evaluate the effect of hydro priming on adaptation strategies of Malaysian Indica rice (MR219) under drought conditions. Rice aseptic seeds were soaked at 20°C for 8h distilled water. Primed and non-primed seeds were subjected to polyethylene glycol (0, - 0.4, - 0.8 and -1.2) MPa treatments. Results showed that germination percentage, germination index, the fresh and dry weight, shoots and roots lengths decreased with increasing polyethylene glycol concentrations. We observed that polyethylene glycol tolerance of primed seeds is higher than non-primed seeds at all polyethylene glycol levels. Mean germination time and relative polyethylene glycol injury are in-creased in non-primed seeds as compare to primed seeds under drought stress. Proline content positively correlated increased with the increasing polyethylene glycol concentrations. The results indicated that hydro priming of Malaysian Indica rice (MR219) seeds is associated with the accumulation of proline and modulating the activity of ascorbate peroxidase and catalase under drought stress. This study suggests the hydro priming as an effective technique on rice seeds to withstand under drought condition which could be a step forward to commercialization

Selection, characterizations and somatic embryogenesis of Malaysian salt-tolerant rice (Oryza sativa cv. MR219) through callogenesis

Salt-tolerant lines of MR219 were produced through somatic embryogenesis from salt-tolerant callus of Oryza sativa L. cv.MR219 using in vitro selection procedure. Callus was developed aseptically from seeds on MS media supplemented with 2 mg L -1 2, 4-D. Then, callus directly was sub-cultured on MS media with different concentrations of NaCl (0, 50, 100, 200, and 300 mM) to produce salt-tolerant callus. Based on the callus characteristics which are morphological, physiological and biochemical cascades such as proline content, total protein, total soluble sugar, lipid peroxidation, activity of ascorbate peroxidase and catalase, salt-tolerant callus was screened and selected. After 4 months, callus cultured in 50 and 100 mM NaCl showed yellow color, soft, friable and nodular proliferating. However, callus cultured in 200 and 300 mM NaCl turn blackish-brown and stiff and acutely-necrotic. The selected salt-tolerant callus was sub-cultured on MS media for somatic embryogenesis. The salt-tolerant plantlets were transferred into pots individually for acclimatization purpose. Salt stress caused significant reduction in water content, fresh and dry weight of callus. The level of total soluble sugar, proline, lipid peroxidation and ascorbate peroxidase significantly increased under salt stress. Salt-tolerant callus indicated high activity of catalase that determined more protection against production of reactive oxygen species. According to growth performance and antioxidant capacity, the plantlets from 50 and 100 mM NaCl, selected as salt-tolerant line. This study suggests the methodology to produce salt-tolerant cultivar of rice which could be a step forward to commercialization

Factors affecting shoot and root apical meristem tissue culture of Thai supersweet corn (Zea mays var. rugosa)

During last recent years, in vitro propagation technic is widely used to produce plants with desirable traits. This experiment was conducted to produce an ideal protocol for in vitro propagation of Thai supersweet corn by using shoot apical meristem (SAM) and root apical meristem (RAM) as explants. Four-day-old germinating seedlings were used as the experimental materials on culture media supplemented with a range of auxin, kinetin, and carbohydrates. The primary establishment for SAM showed the highest percentage of survival (80%) while RAM showed the highest survival (67%) and in Murashige and Skoog (MS) media supplemented. Upon acclimatization, regenerated plantlets from shoot showed the highest survival rate (12%) with the production of 21 plantlets; however, the survival rate of plantlets from root was only 20% with the production of 9 plantlets. The efficient and economic protocol that is produced in this study can be applied as an alternative to conventional propagation method for the large-scale production of Thai supersweet corn throughout the year