Bimal Kumar Ghimire

Direct somatic embryogenesis from pericycle cells of broccoli (Brassica oleracea L. var. italica) root explants

Cotyledon, hypocotyl or root explants of 7-day-old broccoli seedlings were cultured on Murashige and Skoog (MS) agar or liquid medium supplemented with 1.0xa0mgxa0l−1 2,4-dichlorophenoxyacetic acid (2,4-D). The frequency of direct somatic embryo formation was 100% when root explants were cultured in liquid medium. Histological analysis indicated that somatic embryos were initiated directly from the pericycle cell layers of root explants as early as 1xa0day after liquid culture. Genotype did not affect the frequency of somatic embryo formation or the number of somatic embryos per explant. All broccoli genotypes examined had 100% somatic embryo induction efficiency, and the number of somatic embryos per 0.8xa0mm root segment ranged from 22.9 in ‘Luhui’ to 26.0 in ‘Haizi’. The number of normally developed somatic embryos in culture increased with increasing 2,4-D concentration. Plantlet regeneration frequency was the highest (73.3%) when germinated plantlets were transferred to 1/2 strength MS agar medium containing 1.0xa0mgxa0l−1 6-benzyladenine (BA). When regenerated plantlets were transferred to a greenhouse, approximately 75% survived and there were no morphological differences between regenerated plants and seed-derived controls. The protocols established in this study will benefit large-scale vegetative propagation and transformation-based genetic improvement of broccoli.

High-frequency direct shoot regeneration from Drymaria cordata Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65xa0±xa01.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1xa0mg/l NAA and 1.0xa0mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1xa0mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67xa0±xa03.68 roots per leaf segment, was obtained in the presence of 1xa0mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.

Direct shoot organogenesis from petiole and leaf discs of Withania somnifera (L.) Dunal

An efficient and reproducible procedure is described for direct shoot regeneration using petiole and leaf explants of Withania somnifera (L.). The shoots were mainly induced from the distal end of the petiole, whereas in leaf explants, shoot regeneration was initiated from the basal part and wounded tissue. The regeneration medium that induced the highest numbers of shoots in the petiole and leaf explants was Murashige and Skoog (MS) medium supplemented with 2 mg/l N 6 -benzyladenine (BA) alone or with 0.1 mg/l a-naphthalene acetic acid (NAA). The frequency of shoot regeneration was greatly influenced by the type of explant, the carbon source, the orientation of the explant, and the basal medium used in the regeneration medium. Explants produced shoot buds and adventitious shoots within four weeks. Histological analysis of the regenerating shoots showed that the shoot buds emerged from sub epidermal parenchymal cells, with no intermediate callus formation. Plantlets were rooted on MS alone or MS containing different concentrations of 3-indolebutyric acid (IBA). The addition of 1 mg/l IBA to the medium was most effective in inducing root formation. The regenerated plantlets were acclimatized in the greenhouse and successfully transferred to the field, with a 90% survival rate. The acclimatized plants showed normal flowering and were not morphologically different from the seed-derived mother plants.

Improving antioxidant activity in transgenic Codonopsis lanceolata plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene

Codonopsis lanceolata Trautv (Companulaceae) is a folk medicine in Korea. To shift the content of tocopherol and enhance its antioxidant properties, we overexpressed the γ-tocopherol methyltransferase (γ-tmt) gene in C. lanceolata. The antioxidant activity of methanolic crude extracts of the transgenic plants was compared to that of control plants using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, with α-tocopherol and butylated hydroxy toluene as standard antioxidants. The antioxidant activity of the leaf and root extracts of transgenic plants was higher (IC 50 12–17.33 and 408–524xa0μg/ml, respectively) than that of control plant leaf and root extracts (18 and 529xa0μg/ml, respectively). High-performance liquid chromatography analysis of phenolic compounds confirmed an increase in the levels of 12 major phenolic acids and flavonoids in the leaf and root extracts of transgenic plants compared to control plants. We also found that the rate of photosynthesis was 48% higher in transgenic plants than in control plants. Based on these results, we suggest that increases in the α-tocopherol level in transgenic C. lanceolata plants may result in increases in the photosynthetic performance and antioxidant metabolism of these plants.

Overexpression of the γ-TMT gene in Codonopsis lanceolata.

A cDNA-encoding γ-tocopherol methyltransferase (γ-TMT) from Arabidopsis thaliana was overexpressed in deoduck (Codonopsis lanceolata L.) to improve the tocopherol composition. Deoduck (T2) containing the γ-TMT transgene was produced by Agrobacterium-mediated transformation. Transgene expression was confirmed by polymerase chain reaction and RNA gel blot analysis. The transgenic plants produced more leaves than control plants. In addition, the transgenic plants showed higher levels of the CSOD, CTRX, CAPX, CNADP+-IDCH, and CSO transcripts and higher SOD-like activity compared with the control plants.

Ginseng authenticity testing by measuring carbon, nitrogen, and sulfur stable isotope compositions that differ based on cultivation land and organic fertilizer type

Background The natural ratios of carbon (C), nitrogen (N), and sulfur (S) stable isotopes can be varied in some specific living organisms owing to various isotopic fractionation processes in nature. Therefore, the analysis of C, N, and S stable isotope ratios in ginseng can provide a feasible method for determining ginseng authenticity depending on the cultivation land and type of fertilizer. Methods C, N, and S stable isotope composition in 6-yr-old ginseng roots (Jagyeongjong variety) was measured by isotope ratio mass spectrometry. Results The type of cultivation land and organic fertilizers affected the C, N, and S stable isotope ratio in ginseng (p < 0.05). The δ15NAIR and δ34SVCDT values in ginseng roots more significantly discriminated the cultivation land and type of organic fertilizers in ginseng cultivation than the δ13CVPDB value. The combination of δ13CVPDB, δ15NAIR, or δ34SVCDT in ginseng, except the combination δ13CVPDB–34SVCDT, showed a better discrimination depending on soil type or fertilizer type. Conclusion This case study provides preliminary results about the variation of C, N, and S isotope composition in ginseng according to the cultivation soil type and organic fertilizer type. Hence, our findings are potentially applicable to evaluate ginseng authenticity depending on cultivation conditions.

Improved antioxidant activity in transgenic Perilla frutescens plants via overexpression of the γ-tocopherol methyltransferase (γ-tmt) gene.

The main goal of this study was to generate transgenic Perilla frutescens with enhanced antioxidant properties by overexpressing the γ-tocopherol methyltransferase (γ-tmt) gene. In this study, the antioxidant activity of methanolic crude extracts of transgenic and non-transgenic control plants was investigated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. Free radical scavenging activity was evaluated using α-tocopherol and butylated hydroxyl toluene as standard antioxidants. In general, the ethyl acetate fraction of transgenic P. frutescens showed stronger DPPH radical scavenging activity than the ethyl acetate fraction from non-transgenic control plants (IC50 2.00u2009±u20090.10 and 5.53u2009±u20090.40xa0μg∙ml−1, respectively). High-performance liquid chromatography analysis of phenolic acids in leaf extracts confirmed increased levels of 16 individual phenolic compounds in two transgenic lines (pf47-5 and pf47-8) compared with control plants. Changes in the phenolic compound profile and α-tocopherol content were correlated with the antioxidant properties of transgenic plants, indicating that the introduction of transgene γ-tmt influenced the metabolism of phenolic compounds and subsequently produced biochemical changes in the transformants. There were no significant differences in photosynthetic rate in the transgenic plants as compared to the non-transgenic control plants, suggesting that the alteration of phenolic compounds and tocopherol composition had little impact on photosynthesis.

Karyotype and nucleic acid content in Zantedeschia aethiopica Spr. and Zantedeschia elliottiana Engl.

Analysis of karyotype, nucleic deoxyribonucleic acid (DNA) content and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed in Zantedeschia aethiopica and Zantedeschia elliottiana. Mitotic metaphase in both species showed 2n=32. The chromosomes of both species were quite similar with medium length ranging from 1.55 ± 0.04 to 3.85 ± 0.12 µM in Z. aethiopica and 2.15 ± 0.04 to 3.90 ± 0.12 µM in Z. elliottiana. However, some differences were found in morphology and centromeric position among the chromosomes. Identification of individual chromosomes was carried out using chromosomes length, and centromeric positions. The karyotype of Z. aethiopica was determined to be 2n = 32 = 14 m + 18 sm and of Z. elliottiana to be 2n = 32 = 10 m + 22 sm. The 2C nuclear DNA content was found to be 3.72 ± 0.10 picograms (equivalent to 3638.16 mega base pairs) for Z. aethiopica and 1144.26 ± 0.05 picograms (equivalent to 1144.26 mega base pairs) for Z. elliottiana. Leaf protein analysis showed 11 and 9 bands for Z. aethiopica and Z. elliottiana, respectively, among which some were species specific. These results may provide useful information regarding Zantedeschia for the study of taxonomic relationships, genetics and breeding.

In vitro regeneration of Melastoma malabatricum Linn. through organogenesis and assessment of clonal and biochemical fidelity using RAPD and HPLC

Melastoma malabatricum Linn. is an important medicinal plant used in folk medicine for the treatment of various infectious diseases. We developed an in vitro protocol for direct adventitious shoot regeneration from leaf explants of M. malabatricum. Murashige and Skoog (MS) was the most efficient basal medium for shoot regeneration. Multiple adventitious shoot formation was higher in medium supplemented with 3xa0% sucrose than maltose or fructose. Leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA) and thidiazuron (TDZ) showed the highest shoot regeneration (78.00xa0±xa00.58xa0%) and the largest number of shoots per explant (11.67xa0±xa03.05). MS supplemented with gibberellic acid (GA3) was the most effective for shoot elongation. The highest number of roots per explant (10.67xa0±xa03.51) occurred on MS supplemented with indole-3-butyric acid (IBA), indicating the IBA was more effective to induce rooting than indole acetic acid. Explants with intact petioles and lamina were more responsive and produced a larger number of shoots per explant (14.67xa0±xa02.52) than explants with lamina alone (8.00xa0±xa02.00). Histology and scanning electron microscopy of regenerated shoots confirmed the occurrence of direct organogenesis. Random amplified polymorphic DNA analysis confirmed that in vitro regenerated plants were genetically similar to their mother plant. High-performance liquid chromatography of phenolic acids in leaf extracts of regenerants revealed no significant differences in the phenolic compound profile compared with mother plants. Our in vitro regeneration protocol represents a valuable tool for germplasm conservation and genetic transformation of M. malabatricum.

Assessment of the phenolic profile, antimicrobial activity and oxidative stability of transgenic Perilla frutescens L.overexpressing tocopherol methyltransferase (γ-tmt) gene

This study evaluated the effects of enhanced concentrations of α-tocopherol and phenolic compounds on the resistance and stability of Perilla oil in transgenic Perilla frutescens plants against various tested pathogenic bacteria by over-expressing the γ-tmt gene. The concentration of phenolic compounds in the non-transgenic samples was 9313.198xa0±xa018.887xa0μgxa0g-1 dry weight (DW), whereas the total concentration of the transgenic samples ranged from 9118.015xa0±xa018.822 to 10527.612xa0±xa020.411xa0μgxa0g-1 DW. The largest increases in phenolic compounds in the transgenic plants in comparison with the control plants were observed in gallic acid, pyrogallol, 5-sulfosalicylic acid, catechin, chlorogenic acid, vanillin, syringic acid, naringenin, salicylic acid, quercetin, o-coumaric acid, kaempferol, and hesperetin. o-coumaric and benzoic acid acid were the most abundant phenolic acids found in the transgenic plants. Gram-negative bacteria (Salmonella typhimurium) were the most susceptible microorganism against transgenic ethyl acetate extracts with lower measurement of minimum inhibitory concentration (MICs) (0.25xa0±xa00.03xa0mg/ml) at an extract concentration of 2xa0mg/ml in dried plant material. The same extracts were more effective against gram-positive bacteria (Bacillus subtilis) when compared to control plants with MICs values of 0.52xa0±xa00.02xa0mg/ml. The suplementation of 20xa0μg of α-tocopherol (1000xa0ppm) in combination with ethyl acetate extracts enhanced the antimicrobial activity against S.xa0typhimurium and B.xa0subtilis, compared to the non-transgenic plants. The acid value of transgenic Perilla oil improved by 91.2% and 35.54% relative to the non-transgenic control oil and commercial Perilla oil, respectively. The low acid value suggests that the oil will be less susceptible to lipase action, and more economically viable and thus, may also improve the oil quality for industrial purposes. In addition, extracts obtained from transgenic plants could be a potential source of antimicrobial agents for the treatment of bacterial infections.