Analysis of embryogenic callus induction and regeneration of indica rice variety of Malaysia

Rice is the main food-crop for more than half of the global population and its demand is increasing due to population growth. Different abiotic and biotic stresses are among the major reasons that lower the yield. Development of new rice varieties through in vitro somatic embryogenesis not only contributes to enhance the yield but also improves the quality of rice. However, exact timing of maintenance of embryogenic competent callus was yet to be established for indica rice, which is considered as a main barrier in genetic modification. Somatic embryogenesis receptor kinase (SERK1) gene is extensively used as an embryogenic marker in many plant species, which is expressed specifically in embryogenic callus. To obtain high callus induction, effect of plant growth regulators (i.e. 2,4-D and NAA), carbon sources (i.e. sucrose, maltose and sorbitol), basal media (i.e. MS, N6 and LS), and pre-heat treatments (i.e. 35°C, 40°C, 45°C and 50°C) with different imbibition periods (3 days, 5 days and 7 days) were investigated for four Malaysian indica rice varieties (i.e. MR220, MR220-CL2, MR232 and Bario). SERK1 gene was quantified by real time PCR in differential stages of callus (14 days, 21 days, 28 days, 35 days and 42 days), different PGR (2,4-D, NAA, NAA+ 2,4-D), and pre-heat treatment. Plant regeneration was also optimised by using different concentrations of plant growth regulators. After regeneration, agronomic studies was carried out between control plant and treatment plant for all varieties. In the present study, highest percentage of callus induction was obtained for MR220 (96%), MR220-CL2 (100%) MR232 (100%) and Bario (95.7%) on MS media with 3 mg/L 2, 4-D and 3% maltose after 21 days of culture of pre-heat treated seed at 45°C for 3 days. The characteristics of embryogenic callus were found to be embryogenic from SEM and histology. Amplification of SERK1 cDNA was referred as detection of the gene of aged 21-days was successfully amplified in all four varieties. The phylogenetic tree analysis showed that SERK1 gene of all varieties were similar to the SERK1 of Oryza sativa Japonica. The Real Time PCR analysis revealed that SERK1 transcript was significantly higher at 21 days old callus on MS media with 2,4-D at 45°C pre- heat treated callus for all four varieties. Further, regeneration was tested for 21 days old callus, where the regeneration frequency were found to be 72%, 89%, 71% and 50% in MR220, MR220-CL2, MR232 and Bario respectively in optimised regeneration media (2mg/L BAP+ 2mg/L Kinetin+0.5mg/L NAA). Regenerated plants grew easily in the glasshouse with 90 –95% survival rate. Agronomic studies did not show any morphological variation but grain weight of in vitro raised plant was significantly higher than control plant in all tested varieties. These findings establish a suitable protocol for in vitro regeneration system to be used in genetic modification studies in indica rice in future

Analysis of embryogenic callus induction and regeneration of indica rice variety of Malaysia

Rice is the main food-crop for more than half of the global population and its demand is increasing due to population growth. Different abiotic and biotic stresses are among the major reasons that lower the yield. Development of new rice varieties through in vitro somatic embryogenesis not only contributes to enhance the yield but also improves the quality of rice. However, exact timing of maintenance of embryogenic competent callus was yet to be established for indica rice, which is considered as a main barrier in genetic modification. Somatic embryogenesis receptor kinase (SERK1) gene is extensively used as an embryogenic marker in many plant species, which is expressed specifically in embryogenic callus. To obtain high callus induction, effect of plant growth regulators (i.e. 2,4-D and NAA), carbon sources (i.e. sucrose, maltose and sorbitol), basal media (i.e. MS, N6 and LS), and pre-heat treatments (i.e. 35°C, 40°C, 45°C and 50°C) with different imbibition periods (3 days, 5 days and 7 days) were investigated for four Malaysian indica rice varieties (i.e. MR220, MR220-CL2, MR232 and Bario). SERK1 gene was quantified by real time PCR in differential stages of callus (14 days, 21 days, 28 days, 35 days and 42 days), different PGR (2,4-D, NAA, NAA+ 2,4-D), and pre-heat treatment. Plant regeneration was also optimised by using different concentrations of plant growth regulators. After regeneration, agronomic studies was carried out between control plant and treatment plant for all varieties. In the present study, highest percentage of callus induction was obtained for MR220 (96%), MR220-CL2 (100%) MR232 (100%) and Bario (95.7%) on MS media with 3 mg/L 2, 4-D and 3% maltose after 21 days of culture of pre-heat treated seed at 45°C for 3 days. The characteristics of embryogenic callus were found to be embryogenic from SEM and histology. Amplification of SERK1 cDNA was referred as detection of the gene of aged 21-days was successfully amplified in all four varieties. The phylogenetic tree analysis showed that SERK1 gene of all varieties were similar to the SERK1 of Oryza sativa Japonica. The Real Time PCR analysis revealed that SERK1 transcript was significantly higher at 21 days old callus on MS media with 2,4-D at 45°C pre- heat treated callus for all four varieties. Further, regeneration was tested for 21 days old callus, where the regeneration frequency were found to be 72%, 89%, 71% and 50% in MR220, MR220-CL2, MR232 and Bario respectively in optimised regeneration media (2mg/L BAP+ 2mg/L Kinetin+0.5mg/L NAA). Regenerated plants grew easily in the glasshouse with 90 –95% survival rate. Agronomic studies did not show any morphological variation but grain weight of in vitro raised plant was significantly higher than control plant in all tested varieties. These findings establish a suitable protocol for in vitro regeneration system to be used in genetic modification studies in indica rice in future

Utilization of modified wood shavings as growing media for selected horticultural crops

Plant production in soilless systems is attracting increased interest day by day. The major reason for these interest is the fact that soilless medium can help to eliminate soil-borne diseases and give an understanding of plant nutritional requirements. The aim of this thesis is to evaluate the suitability of chemically and physically modified wood shavings as growing media for horticultural crop production. Five different wood shavings derived from Scots pine trees, namely untreated wood, heat treated wood, organic acid treated wood, Q-Treat, Q-Treat and organic acid treated wood, along with mixtures of peat and Q-Treat as 50/50 v/v (P50Q50), 25/75 (v/v) (P25Q75) and peat (control ) were used. In order to assess the characteristics of the growing media phytotoxicity, pH, water holding capacity, N immobilization, EC, water content of the substrates were analysed. Plant performances on the substrates was evaluated by observing the vegetative and generative growth of Hosta ‘Golden Tiara’ and strawberry ‘Elsanta’ plants. Q-Treat, organic acid treated wood and P50Q50 showed a high water holding capacity. No nitrogen immobilization was observed in Q-Treat. At the same time, EC and water content of the substrates were favourable for both Hosta ‘Golden Tiara’ and strawberry production. Hosta ‘Golden Tiara’ plants grew satisfactorily on all the substrates but the visual quality of the plants was unacceptable on untreated wood. For strawberry, vegetative growth was strong on peat and P50Q50. Least runners were formed on P25Q75 and all of the wood substrates. However, the yield from strawberries was highest on peat and P25Q75. The quality of strawberry fruits on wood substrates was equal to that on peat. In conclusion, based on the results obtained in this experiment, 50% of peat may be replaced by 50% Q-Treat in soilless cultivation for Hosta ‘Golden Tiara’ and 75% of peat may be replaced by Q-Treat in soilless strawberry production

The effects of temperature on callus induction and regeneration in selected Malaysian rice cultivar Indica

The development of an efficient tissue culture protocol for somatic embryo would facilitate the genetic modification in breeding program. The present study describes the reproducible protocols for three wetland Malaysian rice cultivars (MR232, MR220 and MR220-CL2) and upland rice (Bario) via somatic embryogenesis. In the present study, four pre-heat treatments (35, 40, 45 and 50°C) were applied to mature seeds with different imbibition periods (3, 5 and 7 days) prior to culture on MS media with 3 mg/L 2,4-D. The results showed that the cultivars exhibited the highest callus induction percentage from 45°C pre-heated seeds and 3 days imbibition (100%, 96%, 100% and 95% for MR232, MR220, MR220-CL2 and Bario, respectively). Callus was induced early ranging from 3 to 12 days compared to without pre-heat treatment. The regeneration efficiency for MR220 and MR220-CL2 cultivars was significantly higher compared to the control treatment. However, both 45°C and 25°C (control) treatments produced higher plantlet regeneration for MR232 and Bario. This study observed that pre-heat treated seeds prior to callus induction did promote callusing and hence regeneration. These findings can be used to establish a suitable protocol for the in vitro regeneration system for several genetic improvements in the numerous stress tolerances of Malaysian rice

Integrated approach for species identification and quality analysis for Labisia pumila using DNA barcoding and HPLC

Labisia pumila is a precious herb in Southeast Asia that is traditionally used as a health supplement and has been extensively commercialized due to its claimed therapeutic properties in boosting a healthy female reproductive system. Indigenous people used these plants by boiling the leaves; however, in recent years it has been marketed as powdered or capsuled products. Accordingly, accuracy in determination of the authenticity of these modern herbal products has faced great challenges. Lack of authenticity is a public health risk because incorrectly used herbal species can cause adverse effects. Hence, any measures that may aid product authentication would be beneficial. Given the widespread use of Labisia herbal products, the current study focuses on authenticity testing via an integral approach of DNA barcoding and qualitative analysis using HPLC. This study successfully generated DNA reference barcodes (ITS2 and rbcL) for L.pumila var. alata and pumila. The DNA barcode that was generated was then used to identify species of Labisia pumila in herbal medicinal products, while HPLC was utilized to determine their quality. The findings through the synergistic approach (DNA barcode and HPLC) implemented in this study indicate the importance of both methods in providing the strong evidence required for the identification of true species and to examine the authenticity of such herbal medicinal products

Combination of Plant Growth Regulators, Maltose, and Partial Desiccation Treatment Enhance Somatic Embryogenesis in Selected Malaysian Rice Cultivar

The development of efficient tissue culture protocol for somatic embryo would facilitate the genetic modification breeding program. The callus induction and regeneration were studied by using different parameters i.e., auxins, cytokinins, and desiccation treatment. Scanning electron microscopy and histological analysis were performed to identify the embryogenic callus for regeneration. The callus percentage results showed that MS (Murashige and Skoog) basal medium supplemented with 3 mg/L 2, 4-D and 30g/L maltose were the optimal callus induction medium for MR220 (80%) and MR220-CL2 (95%). The morphology of the embryogenic callus was confirmed by the SEM (Scanning Electron Microscopy) (presence of extracellular matrix surface network) and later by histological analysis. Finally, MS media supplemented with 0.5 mg/L NAA (Naphthalene Acetic Acid), 2 mg/L kin, and 1 mg/L BAP were selected as the optimum regeneration media treatment while callus desiccated for 48 h was proved to produce more plantlets in MR220 (60%) and MR220-CL2 (73.33%) compared to control treatment (without desiccation). The protocol presented here showed the necessity for the inclusion of partial desiccation as an important step in the tissue culture protocol of Malaysian indica rice genotypes in order to enhance their regeneration potential