Showing papers on "Murashige and Skoog medium published in 2012"
TL;DR: It is found that melatonin plays an important role in regulating both seminal root length and root growth after germination in monocotyledonous rice plants, and treatment with 0.5 and 1 μm melatonin promoted seminal root growth of the wild type under continuous light.
Abstract: The effect of melatonin on root growth after germination was examined in transgenic rice seedlings expressing sheep serotonin N-acetyltransferase (NAT). Enhanced melatonin levels were found in T(3) homozygous seedlings because of the ectopic overexpression of sheep NAT, which is believed to be the rate-limiting enzyme in melatonin biosynthesis in animals. Compared with wild-type rice seeds, the transgenic rice seeds showed enhanced seminal root growth and an analogous number of adventitious roots 4 and 10 days after seeding on half-strength Murashige and Skoog medium. The enhanced initial seminal root growth in the transgenic seedlings matched their increased root biomass well. We also found that treatment with 0.5 and 1 μM melatonin promoted seminal root growth of the wild type under continuous light. These results indicate that melatonin plays an important role in regulating both seminal root length and root growth after germination in monocotyledonous rice plants. This is the first report on the effects of melatonin on root growth in gain-of-function mutant plants that produce high levels of melatonin.
160 citations
TL;DR: The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant.
138 citations
TL;DR: In this article, a callus-derived adventitious root culture was used for the enhanced production of withanolides upon chitosan elicitation in a half-strength MS medium.
115 citations
TL;DR: In this paper, an in vitro method was developed for the production of phenolic compounds from callus suspension cultures of Habenaria edgeworthii Hook, a rare Himalayan medicinal orchid, induced within eight weeks when seeds were cultured on Murashige and Skoog (MS) or ½ MS medium supplemented with 1μM α-naphthaleneacetic acid (NAA).
105 citations
TL;DR: High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA, and these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis.
Abstract: Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.
70 citations
TL;DR: An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn.
Abstract: An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn., an important medicinal climber species. Optimum embryogenic callus (75 %) was induced on Murashige and Skoog (MS) medium supplemented with 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D). On subculturing the callus on MS medium supplemented with 2 mg/l 6-benzyladenine (BA) and 0.5 mg/l α-naphthalene acetic acid (NAA), 61 % of cultures responded with a mean number of 22 somatic embryos per gram callus at different stages of development after 45 days of culture. The addition of higher concentrations of sucrose and abscisic acid (ABA) significantly increased the embryogenic response. MS medium supplemented with 2 mg/l BA, 0.2 mg/l NAA and 4 % of sucrose resulted in 76 % of cultures responding with a mean number of 28 embryos per one gram callus. The highest embryogenic response, frequency of 83 % and mean number of 37 embryos per gram callus, was observed when the MS medium was supplemented with 2 mg/l BA, 0.2 mg/l NAA, and 3 mg/l ABA. Synthetic seeds were produced by encapsulating embryos in calcium alginate gel. The gel contained MS medium with 3 % of sucrose, 1.0 mg/l BA and 0.2 mg/l NAA. The synthetic seeds germinated on MS medium supplemented with BA (1.0-4.0 mg/l) alone or in combination with NAA (0.1–0.7 mg/l) or indole-3-butyric acid (IBA; 0.1–0.7 mg/l). The highest synthetic seed germination (92 %) was observed on MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA. The synthetic seeds were stored at 4 °C and lab conditions (25 ± 2 °C) up to 5 months. The synthetic seeds kept at 4 °C showed 86 % viability even after 5 months of storage. Both somatic embryos and synthetic seeds germinated and were transferred to soil successfully.
65 citations
TL;DR: It is necessary to do more complementary works to identify mechanisms involved for the increased calli size and embryogenesis of explants in darkness, as there is no doubt that TiO2 nanoparticles could dramatically increase callugenesis and the size of calli.
Abstract: In the last decades, extensive research on the effects of nano-TiO2 on plant systems and different microorganisms has confirmed its photocatalytic and antimicrobial activity. However, there is no report on its application in plant cell and tissue culture as well as its role in eliminating contaminating microorganisms in tissue culture. In this work, barley mature embryos were cultured in Murashige and Skoog medium with four concentrations (0, 10, 30, 60 μg/ml) of TiO2 suspension in four repetitions. Quantitative and qualitative characteristics of calli were analyzed after each subculture. Data analysis for calli number in the first culture and callus size in all three cultures showed that the effect of treatment was significant at p > 0.95. As a result, quantitative features such as callus color, shape, embryogenesis, etc. were completely similar in both control and TiO2 nanoparticle treatments; there is no doubt that TiO2 nanoparticles could dramatically increase callugenesis and the size of calli. As well, TiO2 nanoparticles are effective bactericides with an aseptic effect, causing no negative change in the quality of the callus. It is necessary to do more complementary works to identify mechanisms involved for the increased calli size and embryogenesis of explants in darkness.
63 citations
TL;DR: This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids and the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.
Abstract: The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures (max 22.60 and 21.17 mg/100 g d.w., respectively), while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.
61 citations
TL;DR: Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds in plantation eucalypts and exhibited almost 100% survival in the nursery without mist irrigation.
Abstract: A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.
59 citations
TL;DR: An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments, which successfully acclimatized and established in field where they grew well without any detectable variation.
Abstract: An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl2·2H2O). A gelling matrix of 4 % Na-alginate and 100 mM CaCl2·2H2O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00 ± 2.09 %) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 30.0 μM adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5 μM α-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.
59 citations
TL;DR: Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.
Abstract: An efficient and improved in vitro propagation method has been developed for Terminalia bellirica, a medicinally important tree from nodal explants of 10-year-old mature tree. Shoot multiplication was influenced not only by cytokinin types, their concentrations and their interaction with auxin but also by successive transfer of mother explants for different passages, subculture of excised shoots on fresh medium and different medium composition. MS medium containing 2.22 μM BAP was found to be the best for shoot multiplication in a single step. After excision of newly formed shoots, mother explants successively transferred to the same medium produced maximum shoots per explant after IV passage. Further enhancement in morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on MS medium supplemented with 2.22 μM BAP, 1.16 μM Kn and 0.57 μM IAA. Half-strength MS medium supplemented with 24.60 μM IBA and 100 mg l−1 AC was most effective for rooting of the shoots. To reduce labor, cost and time, an experiment on ex vitro rooting was also carried out and it was observed that highest percent shoots rooted ex vitro when treated with 2,460 μM IBA for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. In comparison to plantlets developed from in vitro rooted, percent survival of plants those rooted ex vitro was significantly higher. Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.
TL;DR: An efficient and reproducible method for plant regeneration through direct shoot bud induction from cotyledonary petiole explants of elite genotypes of J. curcas was developed and could be established in soil with more than 90% survival rate.
TL;DR: The present study develops a protocol for rapid in vitro micropropagation of a critically endangered and floriculturally most important epiphytic orchid, Dendrobium primulinum Lindl.
Abstract: The present study develops a protocol for rapid in vitro micropropagation of a critically endangered and floriculturally most important epiphytic orchid, Dendrobium primulinum Lindl. through the culture of small shoot tip explants (0.3 to 0.5mm) derived from in vitro grown seedlings. The shoot tip explants cultured on solidified Murashige and Skoog (MS) basal medium and MS medium alone or supplemented with combination of various concentrations of growth regulators; α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP), produced shoots and multiple shoots. The maximum numbers of rootless healthy shoots were observed on MS medium fortified with BAP 1.5 mgl -1 with an average value of 4.5 shoots per culture where shoot multiplication was initiated after 5 weeks of culture of shoot tip. Among the different tested combination, MS medium with BAP (1.5 mg l -1 ) and NAA (0.5 mg l -1 ) were most effective for the shoot multiplication. MS medium supplemented with various concentrations of rooting hormones viz. NAA, IAA and IBA showed positive response in development of roots, except NAA 0.5 mgl -1 . The rooting was observed after 3 weeks of culture of shoot tip. The various concentrations of IAA and IBA were found to be effective hormone for rooting of D. primulinum in comparison to NAA. The best rooting response was observed on MS medium with exogenous supply of IAA 0.5 mgl -1 . The well developed in vitro rooted plantlets were hardened successfully in the potting mixture containing cocopeat and sphagnum moss in the ratio of 2:1. Nearly 70% of plantlets survived.
TL;DR: The results obtained confirm the therapeutic potency of Cichorium pumilum used in the traditional medicine and verify that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds.
Abstract: Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb An efficient protocol for callus formation and whole plant propagation has been developed Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 15 mg l−1 6-Benzyladenine (BA) and 05 mg l−1 Naphthalene acetic acid (NAA) Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 15 mg l−1 BA or Kinetin (Kin) The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid In the present study, the antibacterial activity of C pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C pumilum
TL;DR: An improved and efficient in vitro regeneration system has been developed for Eclipta alba, a medicinally important plant, through transverse thin cell layer culture (tTCL) and RAPD profile analysis indicated that micropropagated plants were genetically similar to mother plant.
TL;DR: Investigation of the effect of thidiazuron (TDZ) in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia found it to enhance multiplication, proliferation, and elongation of induced shoots.
Abstract: The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 μM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0 μM BA and 0.5 μM NAA. The highest shoot regeneration frequency (90 %) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2 cm) was recorded on MS medium fortified with 5.0 μM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 μM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.
TL;DR: The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers and confirmed the genetic stability among the clones.
Abstract: An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg’s B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 μM BA and 1.0 μM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 μM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.
TL;DR: This is the first report for in vitro propagation and tuberization of E. nuda and the protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species.
TL;DR: A two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.
Abstract: An efficient and reproducible two-step in vitro propagation system for tomato (Lycopersicon esculentum) was developed by using the combinations of seaweed biostimulant (Gracilaria edulis and Sargassum wightii) extracts and plant growth regulators (PGRs). Double cotyledonary nodal (DCN) explants of Co-3 cultivar were initially cultured on Murashige and Skoog (MS) and Gamborg’s medium (B5) containing thidiazuron (TDZ) and 6-benzylaminopurine (BA); the best responding cytokinin was tested in combinations with different auxins (NAA, IAA and IBA), and seaweed extracts (G. edulis and S. wightii) of about basal MS medium +10–70% was used for shoot proliferation. The best organogenic culture response was obtained on MS medium fortified with 1.5 mg L−1 TDZ and 1.5 mg L−1 IBA. Up to 24 shoots per explants were formed at an optimal duration of exposure to 35 days. Mini shoots of about 3–4 cm were transferred to medium supplemented with MS + iP, MS + zeatin, MS + G. edulis and MS + S. wightii at different concentrations. High frequency of shoot elongation was observed in the medium supplemented with 30% G. edulis (15.2 cm), and profuse rooting was observed in the medium supplemented with 50% S. wightii of about 16.1 cm. Shoot elongation and rooting were observed in the medium supplemented with seaweed extracts. The plantlets were transferred to the plant growth chamber (70% of relative humidity and 9 light cycles) and maintained in it for a week, and then they were transferred to a greenhouse condition. The plant growth chamber to green house transferred plantlets showed an increase in the survival rate from 70 to 85%. Thus a two-step regeneration protocol was developed in this study with a combination of seaweed extracts and PGRs, which provides a basis for the production of transgenics with high frequency and survivability of tomato plants.
TL;DR: High fidelity micro-propagated system for efficient and rapid micro- Propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability is provided.
Abstract: An efficient plant propagation system through nodal explants was established in Ocimum gratissimum L, a medicinally important herbaceous perennial herb belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzyladenine (BA) (0.5 - 3.0 mg/l), Kinetin (KN) (0.5 - 3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5 - 3.0 mg/l). Maximum numbers of shoots (5.17 ± 0.04) with average length (2.50 ± 0.07) were induced on medium containing 1.0 mg/l BA. Shoot multiplication was maintained by repeated subculturing the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. Histological study shows that the organogenesis occurs directly, without callus formation on epidermal and sub epidermal layer of the explants. Rooting of shoots was achieved on half strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed complete plantlets were transferred to plastic pots containing a mixture of (1:1) soil and vermiculite showed 82.5 % survival rate. Genetic fidelity was assessed by chromosome analysis and DNA fingerprinting using random amplified polymorphic DNA (RAPD) of in vitro and in vivo plants. Nine arbitrary decamers displayed same banding profile showed no genomic alterations, indicating homogeneity among the tissue culture regenerates and genetic uniformity with that of donor plants. The present study provides high fidelity micro-propagated system for efficient and rapid micro-propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability.
TL;DR: The regeneration protocol developed in this study provides a basis for germplasm conservation and harnessing the total phenol and phenolic compounds of H. edgeworthii and can open avenues for application in other Orchidaceous plants of the Indian Himalayan region.
Abstract: An efficient in vitro propagation protocol for Habenaria edgeworthii Hook. f. ex. Collett using seed-derived callus was established. The maximum seed germination was observed in Murashige and Skoog (MS) medium supplemented with 1.0 μM α-naphthalene acetic acid (NAA). Induction of callus was achieved on full and ½-strength MS medium supplemented with 1.0 μM NAA. The highest number of shoot (11.9 shoots/explant) was achieved in MS medium supplemented with 0.1 μM 6-benzyladenine (BA) and 0.01 μM NAA. Further, elongated shoots when transferred to ½-strength MS rooting medium with different auxin concentrations induced roots (41.6–83.3%) and tubers (0–20.8%); however, a maximum of 87.5% rooting was achieved in a plant growth regulator (PGR)-free MS medium. Rooted shoots (plantlets) when transferred to a mixture of soil:sand:perlite (1:1:1 ratio) resulted in 68% survival. Inter-simple sequence repeats (ISSR) markers confirmed the genetic stability among regenerated plants. The phytochemical analysis of tissue culture-raised tubers showed higher phenolic content than wild tuber. The regeneration protocol developed in this study provides a basis for germplasm conservation and harnessing the total phenol and phenolic compounds of H. edgeworthii. Further, the methods can open avenues for application in other Orchidaceous plants of the Indian Himalayan region.
TL;DR: The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh, Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivar.
Abstract: An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l−1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l−1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.
TL;DR: A simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation and has successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.
Abstract: Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.
TL;DR: The present investigation was undertaken to mass propagate Cymbidium mastersii, an ornamental orchid of Northeast India by in vitro propagation method, which could help for the conservation as well as commercialization of C. mastersii and other threatened and Ornamental orchids.
Abstract: Background and aims Cymbidium mastersii is an epiphytic orchid distributed mainly in Northeast India. Owing to its high commercial value in the floricultural industry, natural populations are under threat from over-exploitation. Mass propagation provides an alternative means of satisfying the demand. Unfortunately, conventional propagation is slow and difficult, suggesting in vitro methods for mass multiplication may be more appropriate. The objective of this study was to develop an efficient protocol. Methodology and principal results Four nutrient media were evaluated for seed germination and early protocorm development: Murashige and Skoog (MS), half-strength MS, Knudson ‘C’ (KC), and Vacin and Went (VW). In addition, the effects of plant growth regulators 6-benzylaminopurine (BAP), kinetin (KN), a-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were studied alone and in combination. The maximum percentage seed germination (93.58+ 0.56) was obtained in MS basal medium after 8 –9 weeks of culture. Secondary protocorms (protocorm-like bodies) were developed from primary protocorms on MS medium fortified with different concentrations and combinations of cytokinins (BAP and KN) and auxins (NAA and IBA). The highest numbers of secondary protocorms (20.55+ 0.62)/primary protocorms were obtained in MS medium supplemented with 5.0 mM BAP and 2.5 mM NAA. The most effective auxin source promoting root production (7.46+ 0.09 per shoot) was 10.0 mM IBA. The plants were acclimatized effectively (survival percentage 88 %) in a greenhouse using a rooting medium of crushed sterile brick and charcoal (1 : 1 v/v) and vermicompost (leaf litter + cow dung, 1 : 1 v/v). Conclusions An efficient protocol was established for in vitro propagation of C. mastersii using seed as the starting material. The percentage seed germination varied with the composition of the nutrient media and was highest in full-strength MS basal medium. The number of secondary protocorms that developed from primary protocorms was increased by the addition of 5.0 mM BAP and 2.5 mM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.
TL;DR: An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L. using shoot tip and nodal explants, excised from 15 days old aseptic seedlings with 95 % survival rate.
Abstract: An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L using shoot tip and nodal explants, excised from 15 days old aseptic seedlings A range of cytokinins were investigated for multiple shoot regeneration Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at an optimal concentration of 25 μM was most effective in proliferating apical and axillary buds The highest regeneration frequency (95 %) and number of shoots (361 ± 033) were obtained on MS medium fortified with BA (25 μM) and NAA (05 μM) from nodal segments High frequency of rooting (100 %) was obtained in in vitro raised shoots when transferred to half-strength MS medium supplemented with NAA (05 μM) Histological sections revealed that additional shoot bud primordia were differentiated within the explants just underneath the suberized cells which appeared to be arrested in their development The presence of additional bud primordia within the explants is thereby helpful to maximize the potential of this system The regenerated plantlets with well developed shoots and roots were hardened successfully, established in earthen pots containing garden soil and maintained in greenhouse with 95 % survival rate
TL;DR: The observed changes reflect the plant’s capacity to develop antioxidant mechanisms during acclimatization which determine the ability to survive in oxidative stress and should eliminate the dependence on natural stands of plants for pharmaceutical purposes, and will also serve as a means of conservation as the species is highly overexploited.
Abstract: The aim of the study was to develop an improved and rapid regeneration system via axillary shoot proliferation for Tecomella undulata, an important endangered medicinal tree in India. The Murashige and Skoog (MS) medium augmented with different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 2.5, 5.0, 7.5 and 10.0 μM) of Thidiazuron (TDZ) was tested for the ability to induce axillary shoot development from cotyledonary node explants excised from 7-day-old sterile seedlings. Among the tried concentrations of TDZ, 0.7 μM showed optimum response in inducing maximum number (25.00 ± 2.30) of shoots and shoot length (4.06 ± 0.58 cm) after 3 weeks of incubation. The regenerated shoots when subcultured on MS medium lacking TDZ gave twofold shoot multiplication rate with maximum number (43.00 ± 2.86) of shoots per explant and longer shoot length (7.40 ± 0.34 cm) during the fourth subculture passage. Attempts were also made to study the morphogenetic effect of medium pH and sucrose concentration on axillary shoot induction and proliferation. The highest efficiency of shoot regeneration was recorded in MS medium supplemented with 0.7 μM TDZ and 3% sucrose at pH 5.8 after 3 weeks of culture. Different concentrations of Indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with IBA (200 μM) pulse treatment given to the basal end of the microshoots for 30 min followed by their transfer in plastic cups containing soilrite and eventually established in natural soil with 80% survival rate. During acclimatization of plants, catalase (CAT) activity increased reaching maximum at 28th day after transplantation, while superoxide dismutase (SOD) activity increased reaching a maximum in the 21 days. Likewise, changes in the glutathione reductase (GR) and Ascorbate peroxidase (APX) were also detected. The observed changes reflect the plant’s capacity to develop antioxidant mechanisms during acclimatization which determine the ability to survive in oxidative stress. The standardized protocol for mass propagation of Tecomella undulata should eliminate the dependence on natural stands of plants for pharmaceutical purposes, and will also serve as a means of conservation as the species is highly overexploited.
TL;DR: A method for cloning selected genotypes of E. benthamii using a micropropagation technique, enabling the formation of a clonal micro-garden, and normal development of adventitious roots showing a vascular connection with the vascular cambium is shown.
Abstract: Eucalyptus benthamii is an important component of forestry plantations in cold regions, but it is difficult to obtain clonal plants of this species, especially by low rooting. In this study, we developed a method for cloning selected genotypes of E. benthamii using a micropropagation technique, enabling the formation of a clonal micro-garden. Nodal segments from sprouts of mini-stumps in the clonal mini-garden were used as explants. After in vitro establishment of the explants, we tested two selected clones (BP101 and BP118), three culture media (Wood Plant Medium (WPM), Correia and colleagues JADS medium, and Murashige and Skoog medium), and two plant growth regulators (6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA)) for the multiplication of adventitious buds. Additionally, combinations of two other plant growth regulators (BAP and gibberellic acid (GA3)) were tested for the elongation of shoots. The in vitro and ex vitro rooting of micro-plantlets prior to acclimatization were compared. The in vitro bud multiplication of E. benthamii depended on the clone, culture medium, and concentration of plant growth regulators. The best results were obtained with WPM supplemented with 0.5 mg L−1 BAP and 0.05 mg L−1 NAA. The elongation of shoots depended on the clone and plant growth regulator, and the best results were obtained with nutrient medium free of GA3 and BAP. Histological analysis showed that both in vitro and ex vitro rooting were successful, resulting in normal development of adventitious roots showing a vascular connection with the vascular cambium. The new protocol is efficient for micro-plantlet production of E. benthamii and can be used for the formation of a clonal micro-garden for other Eucalyptus or tree species.
TL;DR: A gradual decline was observed in the number of shoots regenerated from irradiated petiole explants compared to control, and growth responses of callus were strongly influenced by the radiation dose.
Abstract: The present work was carried out to study the effects of gamma irradiation on in vitro growth of explants, callus and the formation of shoots and plantlets. Irradiation is known to exhibit or inhibit the differentiation of cells and growth of plants in vitro, which helps in producing new plant varieties. Gamma irradiation is one of the physical mutagens that are widely used for mutation breeding. A gradual decline was observed in the number of shoots regenerated from irradiated petiole explants compared to control. Numbers of shoots regenerated from irradiated petiole explant cultured on Murashige & Skoog medium supplemented with 2.0 mg L-1 BAP and 0.5 mg L-1 NAA was reduced to 6.6±0.9 from 7.5±0.4 (control) when explants were exposed to 20 Gray of irradiation dose. Similar observation was reported on effects of gamma irradiation on in vitro propagated plantlets. Gradual decline was observed based on plant height as the dose of gamma irradiation increased. A significant decline was observed in the fresh weight of irradiated callus compared to control. In this case, growth responses of callus were strongly influenced by the radiation dose. The fresh weight of callus was reduced to 76.4±2.2% compared to 89.7±0.5% of control when callus tissues were exposed to 20 Gy.
TL;DR: More than 25,000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.
Abstract: Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February–April) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS + 15 μM BAP. For shoot multiplication, MS medium supplemented with 10 μM BAP and 75 μM Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ½ strength MS medium supplemented with 5 μM each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6 months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.
TL;DR: An important contribution has been made to the conservation of the endangered species T. lotocephalus with the development of this micropropagation protocol using seedlings as explants, using MS medium for proliferation studies.
Abstract: The aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in ¼MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l−1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.