Journal of Horticultural Science & Biotechnology, 2003
Summary A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidi... more Summary A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA3. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.
Natural herbicide is considered as a sustainable approach for weed management in agriculture. Her... more Natural herbicide is considered as a sustainable approach for weed management in agriculture. Here, allelopathic activities of Piper betle L. extract (BE) and known allelochemical eugenol (EU) were studied against rice and associated weeds in terms of germination and seedling growth. Five plant species including a rice crop (Oryza sativa L.); a dicot weed, false daisy (Eclipta prostrata (L.) L.); and three monocot weeds, barnyard grass (Echinochloa crus-galli (L.) P. Beauv.), swollen fingergrass (Chloris barbata Sw.), and weedy rice (Oryza sativa f. spontanea Roshev.) were studied. The paper-based results demonstrated that BE and EU had inhibitory effects on seed germination and seedling growth. The IC50 values of BE and EU for seed germination were ranked from swollen fingergrass, to false daisy, barnyard grass, rice, and weedy rice, respectively. The ratio of root to shoot length of the seedlings indicated that the roots were more affected by the treatments than the shoots. In add...
This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of th... more This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of the hybrid seedlings originated from Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume. Intra- and interspecific hybridization between Phaiusmishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume were examined to establish the primary hybrid, observe their cross ability and identify the F1 hybrids using sequence-characterized amplified region (SCAR) markers. Self-incompatibility and cross ability of P. mishmensis and P. tankervilliae were tested before starting the breeding program. Results showed that they were self-compatible orchids. The interspecific hybridization between P. mishmensis and P. tankervilliae was achieved with the highest pod setting (80%), seed germination percentage (94.8%) and the rate of protocorm development into mature seedlings (stage 6) (10.6%), but the smallest size of embryo with width 46.5 μm, length 67.3 μm w...
Jatropha podagrica Hook is one species of the genus Jatropha. The Roots and vegetative parts of J... more Jatropha podagrica Hook is one species of the genus Jatropha. The Roots and vegetative parts of J. podagrica contain biologically active compounds and its seeds comprise high oil content. There is urgent need to develop mass propagation of J. podagrica by in vitro cultures. The objective of this research was therefore to induce direct shoots from juvenile cotyledon. Seeds were grown in vitro for 12 days and juvenile cotyledons excised for direct shoot organogenesis. MS (Murashige & Skoog) solid medium supplemented with 2.22-13.32 μM BAP, 0.49 μM IBA and 2.27-6.81 μM TDZ enriched with 3% (w/v) sucrose and 0.8% agar were used. The best shoot induction (3.00 ± 0.89 shoots per explant) was achieved after 6 weeks at a combination of 2.22 μM BAP, 0.49 μM IBA and 4.54 μM TDZ. The elongated renewed shoots were transferred to root induction medium containing half strength MS salts supplemented with either indole 3-butyric acid (IBA) or indole acetic acid (IAA). Best rooting was achieved on t...
Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry ... more Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry for its oil. However, the cultivation of J. curcas L. is faced with numerous challenges unlike the cultivation of Ricinus communis L. (2n = 2× = 20), a closely related species. The generation of an intergeneric hybrid between J. curcas L. and R. communis L. was investigated. Intergeneric hybrids were produced by hand crossing. Immature embryos were rescued, in vitro, from the hybrid seeds and cultured on an enriched Murashige and Skoog (MS) medium for a month. The plantlets produced were grown in sterile peat moss in plastic pots and covered with polyethylene for 30 days, after which they were transferred into cement potted soil. The hybridity and the genuineness of the hybrids were successfully confirmed using randomly amplified polymorphic DNA (RAPD) markers. The number of branches, stem diameter, and leaf size of the F1 hybrids were similar to those of J. curcas L. while the plant hei...
In vitro shoot clumps of D rosera peltata were cultured on semi-solid half-strength MS medium wit... more In vitro shoot clumps of D rosera peltata were cultured on semi-solid half-strength MS medium with topping by liquid half-strength MS medium supplemented with various concentrations and types of biotic and abiotic elicitors (50-150 mg/L yeast extract, 50-150 mg/L chitosan, 50-200 µM methyl jasmonate, 1-4 mM cyclodextrin, 10-100 µM silver nitrate, 5-20 mg/L 2,4-D, 1-10 mM L-alanine and 50-150 µM salicylic acid for 3 days. Shoot clumps were investigated for plumbagin accumulation using HPLC. Highest plumbagin content (3.45 ± 0.90 mg/g dry wt . ) was detected when 5 mg/L 2,4-D was used as the elicitor, almost 3 times higher than the control treatment (1.22 ± 0.08 mg/g dry wt) while other elicitors also significantly stimulated higher plumbagin content than the control treatment. Results revealed that relatively high concentrations of 2,4-D , commonly used in tissue culture media, elicited high plumbagin contents from in vitro D. peltata shoot clumps
Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and sec... more Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and secondary metabolite production. Enhanced production of stilbene compounds such as trans-resveratrol, trans-arachidin-1 and trans-arachidin-3 was achieved using an elicitor treatment procedure. Two different hairy root inoculum densities were investigated and compared between shake flask and bioreactor cultures. The lowest growth index was observed using a 20 g/L inoculum size in the bioreactor, which differed significantly from bioreactor of 5 g/L. Increasing the hairy root inoculum size from 5 g/L to 20 g/L in both the shake flask and bioreactor significantly improve antioxidant activity, phenolic content and stilbene compound levels. The highest ABTS and FRAP antioxidant activity, and levels of total phenolic compounds, trans-arachidin-1 and trans-arachidin-3 in the crude extract were demonstrated in shake flask cultures with a 20 g/L inoculum after elicitation for 72 hr. The minimum inhibitory concentrations (MICs) of the crude extract to inhibit growth of foodborne microbes, S. aureus, S. typhimurium and E. coli, were 187.5, 250 and 500 μg/mL, respectively. This was due to the ability of the crude extract to disrupt the cell membrane, as observed by scanning electron microscopy (SEM) showing ruptured pores on the S. aureus and S. typhimurium cell surfaces. Moreover, the E. coli cell division process could be inhibited by the crude extract, which promoted an increase in cell size. A DNA nicking assay indicated that a 50 µg/mL concentration of the crude extract caused plasmid DNA damage that might be due to a genotoxic effect of the pro-oxidant activity of the crude extract.
The panda plant (Kalanchoe tomentosa Baker) is a popular ornamental succulent. The optimal method... more The panda plant (Kalanchoe tomentosa Baker) is a popular ornamental succulent. The optimal method for in vitro plantlet propagation was investigated. Effects of plant growth regulator on growth and development of leaf explants were determined using various concentrations of Thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.01, 0.1, 0.2 and 0.5 mg L-1. A concentration of 0.01 mg L-1 2,4-D gave the highest plantlet quality derived from direct organogenesis. Higher concentrations of 2,4-D promoted callus proliferation. The lowest concentration of 0.01 mg L-1 TDZ induced shorter shoots, while higher concentrations resulted in greater callogenesis and inhibit root production. After in vitro culture, plantlets from the optimal treatment were acclimatized by exposure to growth in sand, sand with coconut husk (2:1), sand with potting soil (2:1) and sand with perlite (2:1). Highest survival percentage (100%) was found in plantlets grown in sandy soil, the most well-drained...
Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidativ... more Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidative stress. The present work elucidated a schematic diagram of the proposed pathway of peanut hairy root tissue treated with different elicitors; paraquat (PQ), methyl jasmonate (MeJA), and cyclodextrin (CD). The different elicitation approaches could provoke intrinsic stress in plant cells and might activate a distinct response pathway, allowing plants to overcome the deleterious effects of oxidative stress. Among all strategies, hairy root culture pretreated with PQ followed by application of MeJA plus CD showed an extensive induction of antioxidant defense mechanisms. The expression of the antioxidant enzyme genes and stilbene-synthesized enzyme genes were up-regulated in accordance with the dramatic increase in the production of stilbene compounds. The non-enzymatic antioxidant substances exhibited a highly enhanced capability. The pathogenesis-related protein (PR) genes were also highly up-regulated. In summary, we demonstrated that the interplay among MeJA plus CD and PQ may activate a complex signaling network to regulate plant defense mechanisms involving the up-regulation of detoxifying enzymes, induction of free-radical scavengers and overexpression of genes associated with plant defense pathways.
In vitro propagation of a rare orchid, Bulbophyllum nipondhii was carried out. Five different med... more In vitro propagation of a rare orchid, Bulbophyllum nipondhii was carried out. Five different media were tested to find the suitable medium for seed germination and seedling development. The tangible results were obtained on VW medium. To assess the effect of pollination types on seed germination and seedlings development, seeds derived from different self?, cross? and open?pollination were examined. Open?pollinated seeds produced the best germination and the highest seedling development, followed by cross? and self? pollination. VW medium was supplemented with 0, 25, 50 or 75 g/l potato extract (PE) and 0, 50, 100, 150 or 200 ml/l coconut water (CW) to identify their most suitable concentration. PE (75 g/l) with 100 ml/l CW was found to be best combination.Plant Tissue Cult. & Biotech. 26(1): 37-46, 2016 (June)
Journal of Horticultural Science & Biotechnology, 2003
Summary A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidi... more Summary A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA3. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.
Natural herbicide is considered as a sustainable approach for weed management in agriculture. Her... more Natural herbicide is considered as a sustainable approach for weed management in agriculture. Here, allelopathic activities of Piper betle L. extract (BE) and known allelochemical eugenol (EU) were studied against rice and associated weeds in terms of germination and seedling growth. Five plant species including a rice crop (Oryza sativa L.); a dicot weed, false daisy (Eclipta prostrata (L.) L.); and three monocot weeds, barnyard grass (Echinochloa crus-galli (L.) P. Beauv.), swollen fingergrass (Chloris barbata Sw.), and weedy rice (Oryza sativa f. spontanea Roshev.) were studied. The paper-based results demonstrated that BE and EU had inhibitory effects on seed germination and seedling growth. The IC50 values of BE and EU for seed germination were ranked from swollen fingergrass, to false daisy, barnyard grass, rice, and weedy rice, respectively. The ratio of root to shoot length of the seedlings indicated that the roots were more affected by the treatments than the shoots. In add...
This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of th... more This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of the hybrid seedlings originated from Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume. Intra- and interspecific hybridization between Phaiusmishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume were examined to establish the primary hybrid, observe their cross ability and identify the F1 hybrids using sequence-characterized amplified region (SCAR) markers. Self-incompatibility and cross ability of P. mishmensis and P. tankervilliae were tested before starting the breeding program. Results showed that they were self-compatible orchids. The interspecific hybridization between P. mishmensis and P. tankervilliae was achieved with the highest pod setting (80%), seed germination percentage (94.8%) and the rate of protocorm development into mature seedlings (stage 6) (10.6%), but the smallest size of embryo with width 46.5 μm, length 67.3 μm w...
Jatropha podagrica Hook is one species of the genus Jatropha. The Roots and vegetative parts of J... more Jatropha podagrica Hook is one species of the genus Jatropha. The Roots and vegetative parts of J. podagrica contain biologically active compounds and its seeds comprise high oil content. There is urgent need to develop mass propagation of J. podagrica by in vitro cultures. The objective of this research was therefore to induce direct shoots from juvenile cotyledon. Seeds were grown in vitro for 12 days and juvenile cotyledons excised for direct shoot organogenesis. MS (Murashige & Skoog) solid medium supplemented with 2.22-13.32 μM BAP, 0.49 μM IBA and 2.27-6.81 μM TDZ enriched with 3% (w/v) sucrose and 0.8% agar were used. The best shoot induction (3.00 ± 0.89 shoots per explant) was achieved after 6 weeks at a combination of 2.22 μM BAP, 0.49 μM IBA and 4.54 μM TDZ. The elongated renewed shoots were transferred to root induction medium containing half strength MS salts supplemented with either indole 3-butyric acid (IBA) or indole acetic acid (IAA). Best rooting was achieved on t...
Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry ... more Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry for its oil. However, the cultivation of J. curcas L. is faced with numerous challenges unlike the cultivation of Ricinus communis L. (2n = 2× = 20), a closely related species. The generation of an intergeneric hybrid between J. curcas L. and R. communis L. was investigated. Intergeneric hybrids were produced by hand crossing. Immature embryos were rescued, in vitro, from the hybrid seeds and cultured on an enriched Murashige and Skoog (MS) medium for a month. The plantlets produced were grown in sterile peat moss in plastic pots and covered with polyethylene for 30 days, after which they were transferred into cement potted soil. The hybridity and the genuineness of the hybrids were successfully confirmed using randomly amplified polymorphic DNA (RAPD) markers. The number of branches, stem diameter, and leaf size of the F1 hybrids were similar to those of J. curcas L. while the plant hei...
In vitro shoot clumps of D rosera peltata were cultured on semi-solid half-strength MS medium wit... more In vitro shoot clumps of D rosera peltata were cultured on semi-solid half-strength MS medium with topping by liquid half-strength MS medium supplemented with various concentrations and types of biotic and abiotic elicitors (50-150 mg/L yeast extract, 50-150 mg/L chitosan, 50-200 µM methyl jasmonate, 1-4 mM cyclodextrin, 10-100 µM silver nitrate, 5-20 mg/L 2,4-D, 1-10 mM L-alanine and 50-150 µM salicylic acid for 3 days. Shoot clumps were investigated for plumbagin accumulation using HPLC. Highest plumbagin content (3.45 ± 0.90 mg/g dry wt . ) was detected when 5 mg/L 2,4-D was used as the elicitor, almost 3 times higher than the control treatment (1.22 ± 0.08 mg/g dry wt) while other elicitors also significantly stimulated higher plumbagin content than the control treatment. Results revealed that relatively high concentrations of 2,4-D , commonly used in tissue culture media, elicited high plumbagin contents from in vitro D. peltata shoot clumps
Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and sec... more Obtaining large-scale hairy root cultures is a major challenge to increasing root biomass and secondary metabolite production. Enhanced production of stilbene compounds such as trans-resveratrol, trans-arachidin-1 and trans-arachidin-3 was achieved using an elicitor treatment procedure. Two different hairy root inoculum densities were investigated and compared between shake flask and bioreactor cultures. The lowest growth index was observed using a 20 g/L inoculum size in the bioreactor, which differed significantly from bioreactor of 5 g/L. Increasing the hairy root inoculum size from 5 g/L to 20 g/L in both the shake flask and bioreactor significantly improve antioxidant activity, phenolic content and stilbene compound levels. The highest ABTS and FRAP antioxidant activity, and levels of total phenolic compounds, trans-arachidin-1 and trans-arachidin-3 in the crude extract were demonstrated in shake flask cultures with a 20 g/L inoculum after elicitation for 72 hr. The minimum inhibitory concentrations (MICs) of the crude extract to inhibit growth of foodborne microbes, S. aureus, S. typhimurium and E. coli, were 187.5, 250 and 500 μg/mL, respectively. This was due to the ability of the crude extract to disrupt the cell membrane, as observed by scanning electron microscopy (SEM) showing ruptured pores on the S. aureus and S. typhimurium cell surfaces. Moreover, the E. coli cell division process could be inhibited by the crude extract, which promoted an increase in cell size. A DNA nicking assay indicated that a 50 µg/mL concentration of the crude extract caused plasmid DNA damage that might be due to a genotoxic effect of the pro-oxidant activity of the crude extract.
The panda plant (Kalanchoe tomentosa Baker) is a popular ornamental succulent. The optimal method... more The panda plant (Kalanchoe tomentosa Baker) is a popular ornamental succulent. The optimal method for in vitro plantlet propagation was investigated. Effects of plant growth regulator on growth and development of leaf explants were determined using various concentrations of Thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.01, 0.1, 0.2 and 0.5 mg L-1. A concentration of 0.01 mg L-1 2,4-D gave the highest plantlet quality derived from direct organogenesis. Higher concentrations of 2,4-D promoted callus proliferation. The lowest concentration of 0.01 mg L-1 TDZ induced shorter shoots, while higher concentrations resulted in greater callogenesis and inhibit root production. After in vitro culture, plantlets from the optimal treatment were acclimatized by exposure to growth in sand, sand with coconut husk (2:1), sand with potting soil (2:1) and sand with perlite (2:1). Highest survival percentage (100%) was found in plantlets grown in sandy soil, the most well-drained...
Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidativ... more Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidative stress. The present work elucidated a schematic diagram of the proposed pathway of peanut hairy root tissue treated with different elicitors; paraquat (PQ), methyl jasmonate (MeJA), and cyclodextrin (CD). The different elicitation approaches could provoke intrinsic stress in plant cells and might activate a distinct response pathway, allowing plants to overcome the deleterious effects of oxidative stress. Among all strategies, hairy root culture pretreated with PQ followed by application of MeJA plus CD showed an extensive induction of antioxidant defense mechanisms. The expression of the antioxidant enzyme genes and stilbene-synthesized enzyme genes were up-regulated in accordance with the dramatic increase in the production of stilbene compounds. The non-enzymatic antioxidant substances exhibited a highly enhanced capability. The pathogenesis-related protein (PR) genes were also highly up-regulated. In summary, we demonstrated that the interplay among MeJA plus CD and PQ may activate a complex signaling network to regulate plant defense mechanisms involving the up-regulation of detoxifying enzymes, induction of free-radical scavengers and overexpression of genes associated with plant defense pathways.
In vitro propagation of a rare orchid, Bulbophyllum nipondhii was carried out. Five different med... more In vitro propagation of a rare orchid, Bulbophyllum nipondhii was carried out. Five different media were tested to find the suitable medium for seed germination and seedling development. The tangible results were obtained on VW medium. To assess the effect of pollination types on seed germination and seedlings development, seeds derived from different self?, cross? and open?pollination were examined. Open?pollinated seeds produced the best germination and the highest seedling development, followed by cross? and self? pollination. VW medium was supplemented with 0, 25, 50 or 75 g/l potato extract (PE) and 0, 50, 100, 150 or 200 ml/l coconut water (CW) to identify their most suitable concentration. PE (75 g/l) with 100 ml/l CW was found to be best combination.Plant Tissue Cult. & Biotech. 26(1): 37-46, 2016 (June)
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