Schenk And Hildebrandt Medium Research Articles

Adventitious root culture of Lessertia frutescens for the production of triterpenoid saponins and polysaccharides

Lessertia frutescens is a perennial shrub of commercial importance in South Africa, but the scarcity of plant resources has limited current product production. In this study, to provide an alternative approach for obtaining L. frutescens material, adventitious roots (ARs) were induced from sterilized seedlings and cultured in a suspension culture system. During this process, selection tests were conducted to find a suitable auxin and its concentration for AR induction and a suitable basal medium for AR growth and metabolite accumulation; a kinetic study was then performed to constructure kinetic models. The results showed that compared to other auxins and concentrations, indole-3-butyric acid at 3 mg/L was suitable for increasing the number and length of ARs during AR induction. In AR suspension culture, Schenk and Hildebrandt (SH) was better than other basal media, and the maximum AR fresh (86.9 g/L) or dry weight (5.5 g/L), total triterpenoid saponin (92.6 mg/g DW), and polysaccharide (114.7 mg/g DW) contents were determined in the 1.5×SH medium. In addition, AR biomass and metabolite contents reached the maximum on day 42. The kinetic models for AR growth and triterpenoid and polysaccharide production were constructed, providing the basis for further optimization of culture conditions and large-scale culture.

Shoot Cultures of Vitis vinifera (Vine Grape) Different Cultivars as a Promising Innovative Cosmetic Raw Material-Phytochemical Profiling, Antioxidant Potential, and Whitening Activity.

The primary purpose of this work was the initiation and optimization of shoot cultures of different Vitis vinifera L. cultivars: cv. Chardonnay, cv. Hibernal, cv. Riesling, cv. Johanniter, cv. Solaris, cv. Cabernet Cortis, and cv. Regent. Cultures were maintained on 30-day growth cycles using two media, Murashige and Skoog (MS) and Schenk and Hildebrandt (SH), with various concentrations of plant growth regulators. Tested media ('W1'-'W4') contained varying concentrations of 6-benzylaminopurine (BA) in addition to indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA). High performance liquid chromatography coupled with mass spectrometry (UPLC-MS) was used for metabolomic profiling. In all tested extracts, 45 compounds were identified (6 amino acids, 4 phenolic acids, 13 flavan-3-ols, 3 flavonols, and 19 stilbenoids). Principal component analysis (PCA) was performed to assess the influence of the genotype and medium on metabolic content. PCA showed that metabolic content was mainly influenced by genotype and to a lesser extent by medium composition. MS media variants induced the amino acid, procyanidin, and flavan-3-ol production. In addition, the antioxidant potential and anti-tyrosinase activity was measured spectrophotometrically. The studies on antioxidant activity clearly reveal very high efficiency in reducing free radicals in the tested extracts. The strongest tyrosinase inhibition capacity was proved for shoots cv. Hibernal cultured in SH medium and supplemented with NAA, with an inhibition of 17.50%. These studies show that in vitro cultures of V. vinifera cvs. can be proposed as an alternative source of plant material that can be potentially used in cosmetic industry.

Induction and submerged cultivation of Valeriana jatamansi adventitious root cultures for production of valerenic acids and its derivatives

In vitro adventitious roots were induced from leaves of Valeriana jatamansi to assess their potential as a sustainable alternative to extract pharmaceutically important phytoconstituents. Among the different media used, a significantly (p ≤ 0.05) high root induction (90%) was achieved on Schenk and Hildebrandt (SH) medium fortified with 9.84 µM indole-3-butyric acid (IBA). In addition, various process parameters i.e. IBA concentration, sucrose and medium strength were also optimized under submerged cultivation. The maximum fresh root biomass (144.09 ± 11.36 g/L) with a high relative growth rate (2.01 ± 0.04) and growth index (13.41) was achieved in half-strength SH medium having 2% sucrose and 4.92 µM IBA. Further, a significantly high yield of total valerenic acid derivatives [1525.14 µg/g dry weight (DW)] was recorded in adventitious roots as compared to donor plant parts. Individually, valerenic acid (506.27 µg/g DW) was accumulated higher in plant rhizomes, while acetoxyvalerenic (534.91 µg/g DW) and hydroxyl valerenic acid (919.57 µg/g DW) in adventitious roots. Interestingly, hydroxy valerenic acid was unmeasurable in donor plant parts. The phenolic compounds were also found maximum in adventitious roots (451.85 µg/g DW) with the dominance of pharmaceutically important kaempferol and rutin. A substantial increase in phytochemicals was evident at subsequent culture stages with shortened in vitro cultivation cycle (2 months) than field-grown plants (24 months). Moreover, adventitious roots also accumulated 0.059% essential oil with patchouli alcohol (24%) as a key constituent. Conclusively, an enriched metabolic profile and substantially shorter growth cycle under submerged cultivation undoubtedly demonstrated the potential of induced V. jatamansi adventitious roots as a feasible source of phytoconstituents. In vitro adventitious roots induced leaf of Valeriana jatamansi showed improved metabolic profile and shorter cultivation cycle, thereby exhibiting potential as a sustainable alternative for extraction of industrially important phytoconstituents.

In Vitro Propagation, Huperzine A Content and Antioxidant Activity of Three Genotypic Huperzia serrata.

Huperzia serrata is a traditional herb and endangered Chinese medicinal material, which has attracted much attention due to its production of Huperzine A (HupA). In vitro propagation of H. serrata is considered a new way to relieve the resource pressure of H. serrata. In this study, three different genotypic wild H. serrata were used for in vitro propagation. Then, the antioxidant activity and the content of HupA in the regenerated H. serrata were investigated. The results showed the survival rate of the explant was increased to 25.37% when using multiple sterilization processes. The best induction medium for H. serrata was the Schenk and Hildebrandt (SH) medium supplemented with 0.5 mg·L−1 Naphthalene acetic acid (NAA) and 0.1 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), where the regeneration rate of the explant was to 57.04%. The best proliferation medium was the SH medium with NAA (1.0 mg·L−1), as the biomass of in vitro tissue increased 164.17 ± 0.41 times. High-performance liquid chromatography analysis showed that the in vitro culture of three genotypes could produce HupA and the content of HupA was 53.90–87.17 µg·g−1. The antioxidant experiment showed that the methanol extract of in vitro H. serrata had higher antioxidant activity than that of wild H. serrata. This study provides a reliable in vitro H. serrata culture protocol and laid an important foundation for the antioxidant capacity of the thallus and the content of HupA.

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA4, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g−1 (DW)) in hairy root extracts.

Effects of shoot tip removal, wounding manipulation, and plant growth regulators on shoot regeneration and plantlet development in Paphiopedilum species

The micropropagation procedure of three endangered Paphiopedilum species (Paphiopedilum callosum, Paphiopedilum gratrixianum, and Paphiopedilum delenatii) by removing shoot apex and wounding method is reported in this study. The results showed that shoot regeneration of P. callosum, P. gratrixianum, and P. delenatii was at high frequency (5.61; 5.48; and 6.00 shoot/explant, respectively) when decapitated shoot apex was cultured on liquid Schenk and Hildebrandt (SH) medium added with thidiazuron (TDZ, 0.4–0.6 mg L−1). Multiple shoots were also induced in vitro shoots by wounding method, when cultured on medium containing TDZ (0.4–0.6 mg L−1) which were 4.48; 5.37; and 5.31 shoots/explant, respectively in each species. The shoots regenerated via both shoot apex decapitation and wounding method were well-developed to plantlets on SH medium supplement with 1.0 mg L−1 Naphthaleneacetic acid (NAA) after 90 days of culture. High-quality plants of P. callosum, P. gratrixianum, and P. delenatii. were observed when the plantlets were transferred onto peat moss with 100% survival rate after 24 months in greenhouse.

Establishment of a high-frequency plant regeneration system from rhizome-derived embryogenic cell-suspension cultures of Curcuma longa L.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

Enhancing Astragaloside Production in Astragalus membranaceus through Growth Media and Auxins

The secondary metabolites of triterpenoid saponins, isoflavonoids and polysaccharides are present in Astragalus membranaceus those are biologically active. Astragalosides are a major group of identified compounds that have various pharmacological properties. The present study was considered to enhance astragaloside biosynthesis in hairy root cultures of A. membranaceus by using different growth media and concentrations of different auxins. Our findings revealed that Schenk and Hildebrandt (SH) medium responded greatly for the production of the highest dry weight. However, the highest astragaloside production was detected while using SH medium of its half strength. The growth of hairy roots and accumulation of astragaloside in response to different auxinlike Indole-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA) and 1-naphthaleneacetic acid (NAA) were different. Hairy root growth rate was increased by the treatment of IBA and NAA, but was unaffected by IAA. Accumulation of Astragaloside was enhanced in response to the treatment of IBA and NAA. In most cases, treatment with the 0.5 mg L-1 NAA accumulated the highest level of stragaloside in the hairy root of A. membranaceus, which might, therefore, be utilized as an ideal mass-scale production system for astragalosides.

High frequency somatic embryogenesis and plant regeneration of interspecific ginseng hybrid between Panax ginseng and Panax quinquefolius

BackgroundInterspecific ginseng hybrid, Panax ginseng × Panax quenquifolius (Pgq) has vigorous growth and produces larger roots than its parents. However, F1 progenies are complete male sterile. Plant tissue culture technology can circumvent the issue and propagate the hybrid. MethodsMurashige and Skoog (MS) medium with different concentrations (0, 2, 4, and 6 mg/L) of 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and somatic embryogenesis (SE). The embryos, after culturing on GA3 supplemented medium, were transferred to hormone free ½ Schenk and Hildebrandt (SH) medium. The developed taproots with dormant buds were treated with GA3 to break the bud dormancy, and transferred to soil. Hybrid Pgq plants were verified by random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses and by LC-IT-TOF-MS. ResultsWe conducted a comparative study of somatic embryogenesis (SE) in Pgq and its parents, and attempted to establish the soil transfer of in vitro propagated Pgq tap roots. The Pgq explants showed higher rate of embryogenesis (~56% at 2 mg/L 2,4-D concentration) as well as higher number of embryos per explants (~7 at the same 2,4-D concentration) compared to its either parents. The germinated embryos, after culturing on GA3 supplemented medium, were transferred to hormone free ½ SH medium to support the continued growth and kept until nutrient depletion induced senescence (NuDIS) of leaf defoliation occurred (4 months). By that time, thickened tap roots with well-developed lateral roots and dormant buds were obtained. All Pgq tap roots pretreated with 20 mg/L GA3 for at least a week produced new shoots after soil transfer. We selected the discriminatory RAPD and ISSR markers to find the interspecific ginseng hybrid among its parents. The F1 hybrid (Pgq) contained species specific 2 ginsenosides (ginsenoside Rf in P. ginseng and pseudoginsenosides F11 in P. quinquefolius), and higher amount of other ginsenosides than its parents. ConclusionMicropropagation of interspecific hybrid ginseng can give an opportunity for continuous production of plants.

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

ABSTRACTThe biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89 mg g−1 DW; equivalent to 63.3 mg L−1), taxodione (1.15 mg g−1 DW; equivalent to 17.4 mg L−1), 15-deoxy-fuerstione (2.15 mg g−1 DW; equivalent to 32.5 mg L−1), and 7-(2′-oxohexyl)-taxodione (0.076 mg g−1 DW; equivalent to 1.1 mg L−1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC50 = 0.05 µM with high selectivity, selectivity index (SI) = 38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1 µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC50 values ranging from 0.62 to 194.7 µM) and lower selectivity (SI value ranged from 0.4 to 5.0).

Improved in vitro propagation, genetic stability and analysis of corosolic acid synthesis in regenerants of Lagerstroemia speciosa (L.) Pers. by HPLC and gene expression profiles

A rapid in vitro propagation protocol for large-scale cultivation of Lagerstroemia speciosa L., an important medicinal and ornamental tree was established using nodal explants in Schenk and Hildebrandt (SH) medium optimized with various concentration and combinations of auxins and cytokinins. The study revealed, for the first time, accumulation of corosolic acid (CRA), an anti-diabetic and anti-obese pentacyclic triterpene in the in vitro plantlets. Multiple shoot induction was maximum in SH medium supplemented with 4.44 µM Benzyl Amino Purine (BAP) and shoot initials, when transferred to SH medium supplemented with 2.22 µM BAP, resulted in shoot multiplication and elongation. Rooted plantlets, after 2 weeks of hardening in mist-house and 3 months of rearing under shade-net, transferred to forest segments showed successful establishment (100 %). In a short duration of 24 weeks, ~2,450 hardened plantlets were obtained from a single nodal explant. The ISSR banding profiles of the regenerants and the mother plant were highly monomorphic and similarity matrix (0.96–0.99) confirmed genetic fidelity of the clones. Chemical analysis using HPLC showed that CRA content is more in mature (0.012–0.062 % DW) than young leaves (0.004–0.007 % DW) of the in vitro derived plantlets. The differential synthesis of CRA in micropropagated and mother plant was studied using gene expression profiles. The expression of upstream rate limiting genes in terpenoid biosynthesis was relatively high in young leaves while downstream Cytochrome P450 hydroxylase (CYP450H), catalyzing the final step(s) in CRA synthesis, was more in mature leaf, suggesting a strong correlation between observed CRA variation and gene expression. The study signifies the role of in vitro methodology for true-to-type regeneration and its possible utility for biosynthesis of CRA, throughout the year.

English

An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures. Embryogenic callus was induced from leaf explants on Schenk and Hildebrandt (SH) medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 to 3.0 mg/l) along with Kinetin (Kn) (0.5 mg/l). High frequency of somatic embryogenesis was effective on SH medium with 2, 4-D (2.5 mg/l) + Kn (0.5 mg/l) from leaf explants. Secondary somatic embryogenesis was also observed when primary somatic embryos were subculture on the same somatic embryo induction medium. Well developed cotyledonary shaped embryos regenerate 80% of shoots on media containing 2,4-D 0.5 mg/l + 2.0 mg/l BA. The regenerated shoots transferred to rooting media containing Indole- 3- butyric acid (IBA). Efficient rooting of 90% was noted on SH media with 1.0 mg/l IBA. Finally, these in vitro regenerated plantlets were hardened, acclimatized and successfully transferred to the field. The post transplantation survival rate of these regenerated plants was 65 to 70%. The in vitro regenerated plants and flowers were similar to mother plants. This protocol will be useful for genetic transformation experiments in R. vesicarius L. Keywords: Rumex vesicarius L, 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin (Kn), Benzyl adenine (BA), Indole- 3- butyric acid (IBA). African Journal of Biotechnology , Vol 13(45) 4268-4274

Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

The conditions for the regeneration of plants through organogenesis from callus tissues of <em>Menyanthes trifoliata</em> are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M) containing indole-3-acetic acid (IAA 0,5 mg/l) and 6-benzyladenine (BA 1 mg/l) or zeatin (2 mg/l). Under these conditions ca 7 shoots (mostly 1 cm or more in length) per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages) there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

Micropropagation of Catalpa bignonioides Walt. through tissue cultures

The conditions used in the micropropagation of <em>Catalpa bignonioides </em>Walt. <em>(Bignoniaceae) </em>from callus tissue are described. The multiplication of shoots was best on Schenk and Hildebrandt (SH) solid medium supplemented with 0.5mg dm<sup>-3</sup> indolil-3-acetic acid (IAA) and 2.0 mg dm<sup>-3</sup> benzylaminopurine (BAP). Under these conditions, 11 shoots per culture could be developed within 4 weeks. To develop roots, the shoots were then transferred to basal SH medium without growth regulators. Rooted plantlets were obtained within 2-8 weeks. Regenerated plants were fully capable of further development in soil. Generally, ca 10 plants could be obtained from a fragment of callus within 10-12 weeks.

Shoot organogenesis and plant regeneration in Agave hybrid, No. 11648

The effects of explant type and the combination and concentration of plant growth regulators on callus induction and shoot regeneration in Agave hybrid No. 11648 were examined. Shoot tip and immature leaf segments were cultured on Murashige and Skoog (MS) medium supplied with different concentrations of benzyladenine (BA), at 0.2–3.0mgl−1, and α-naphthalene acetic acid (NAA), at 0.1–1.0mgl−1, or 2,4-dichlorophenoxyacetic acid (2,4-D), at 0.2–2.0mgl−1 for callus induction. The highest rate of light yellow-green, compact and nodular-like calli was formed on the MS medium supplemented with 2.0mgl−1 BA in combination with 0.2mgl−1 NAA, with 93.2% of shoot tips producing a compact, light yellow-green and nodular-like callus. The calli derived from the different explants were subcultured on Schenk and Hildebrandt (SH) medium (300mgl−1 ammonium nitrate, 2267mgl−1 potassium nitrate, 353mgl−1 potassium dihydrogen phosphate, 3mgl−1 glycine, 1.0mgl−1 thiamine hydrochloride (B1), 1.0mgl−1 pyridoxine hydrochloride (B6), 1.0mgl−1 niacin, 200mgl−1 inositol) containing different concentrations of BA, NAA and indole-3-butyric acid (IBA) for shoot induction. Shoot organogenesis was achieved when the callus tissue derived from shoot tips was transferred to SH medium. Maximum organogenesis was obtained with 5.0mgl−1 BA in combination with 0.1mgl−1 NAA and 0.1mgl−1 IBA, yielding 98.0% shoot induction frequencies. Regenerated shoots were rooted on growth regulator-free MS medium, with an average number of 5.39 roots per shoot, and a length of 8.44cm per root within 30 days, and acclimatized in a greenhouse, with 98.57±1.43% survival. This is the first report of plant regeneration from Agave hybrid No. 11648 shoot tips.

Production of somatic embryo and transgenic plants derived from breeding lines of Rosa hybrida L.

This study was conducted to obtain intron-GUS gene-transferred plants by using somatic embryos (including embryogenic calluses) derived from 2 in vitro root explants of breeding lines (KR056002 and KR056006) bred by crossing between Rosa hybrida ‘Tineke’ and ‘Mirinae Gold’. Calluses were induced from their root explants cultured in Schenk and Hildebrandt (SH) medium, which was supplemented with 5 or 11 mg·L−1 of 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos were generated from the calluses, which were cultured in SH medium supplemented with 3 mg·L−1 of 2,4-D. The ratio of callus formation from in vitro root explants was dependent on the concentration of 2,4-D supplement in the SH medium. Somatic embryos were generated from both KR056002 and KR056006, and embryogenesis was observed from calluses around the somatic embryo. The regenerative capacity of the embryo was maintained longer in calluses derived from in vitro root explants cultured on SH medium supplemented with 11 mg·L−1 of 2,4-D than with 5 mg·L−1 of 2,4-D. Six pseudo-intron-GUS transgenic lines were obtained. The expression rate of the intron-GUS gene in multi-shoots was 100%. After the formation of healthy roots, 3 transgenic lines were transferred to the greenhouse. All 3 lines were reconfirmed as intron-GUS gene transgenic plants by PCR and Southern analyses.

Conservation through in vitro propagation and restoration of Mahonia leschenaultii, an endemic tree of the Western Ghats

Conservation of Mahonia leschenaultii was achieved through multiple axillary shoot formation in single node cultures, in vitro and ex vitro rooting of the shoots, and successful reintroduction and establishment of the rooted plants in the forests of Palani hills in Southern India. A preliminary experiment on the culture of young nodal explants (1.0-1.5 cm) from field-grown plants in different basal nutrient media revealed high frequency bud beak in Schenk and Hildebrandt (SH) medium followed by Woody Plant medium and Murashige Skoog medium in 4 weeks. Subsequent culture of both shoot tips (0.5-0.8 cm) and nodes for 6 weeks in SH medium supplemented with various concentration of cytokinins resulted in optimal formation of 3.6 shoots in 80% of the nodes against 1.35 shoots in 75% of the shoot tips in the presence of 2.0 and 1.0 mg/l benzyl adenine (BA), respectively. Invariably, the resident apical and axillary meristems grew into a single shoot (1.0-1.8 cm) in 1-2 weeks followed by the characteristic differentiation of additional buds from its base. A synergistic combination of 1.0 mg/l BA and 0.02 mg/l indole-3-acetic acid (IAA) induced the maximum number (5.9) of axillary shoot formation which were relatively high (75%) when the explant was collected during May-June and the fifth nodes from the top of the growing shoots were used. Repeated subculture of the nodes from shoot cultures at 5-6 week intervals in medium supplemented with reduced concentrations of the growth regulators (0.5 mg/l BA, 0.01 mg/l IAA) through at least 10 passages enabled consistent production of 6-7 shoots per node at 92% success rate without loss of vigour, growth and morphological abnormalities. Shoots of 3-6 cm were rooted in vitro in the presence of 1.0 mg/l indole-3-butyric acid (IBA) and hardening in the mist house at 76-78% and these rooted plants established in a potting medium of river sand and top soil (1:1) under constant mist irrigation. The plants reared in the nursery for 5-8 weeks were successfully reintroduced into the natural forest segment of the Institute's campus (MSL 200 m) at Palode and Vattakanal shola forests of Palani hills (MSL 2500 m) with establishment frequencies of 78.8 and 90.6, respectively, after 18 months. Growth characteristics of the plants (height, number of leaves and branches) reintroduced into native habitat at Vattakanal were better than those at Palode. Genetic fidelity of the 12-month old plants established in the nursery/field was confirmed by analysis of amplified products of genomic DNA obtained with random primers.

Shoot regeneration and micropropagation of Panax vietnamensis Ha et Grushv. from ex vitro leaf-derived callus

The methods for leaf-derived callus induction, callus proliferation, adventitious shoot induction and plant regeneration of Vietnamese ginseng ( Panax vietnamensis Ha et Grushv.) were examined. In this study, callus induction was formed on both medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with thidiazuron (TDZ). The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2,4-D and 0.2 mg/l TDZ. The best callus proliferation medium was Schenk and Hildebrandt (SH) supplemented with 0.2 mg/l TDZ and 1.0 mg/l 2,4-D. The maximum callus-derived shoot number (8.2) was obtained on SH medium supplemented with 50 g/l sucrose in combination with 2.0 mg/l 6-benzylaminopurine (BA). The most successful rooting of regenerated adventitious shoots was obtained on SH medium with 1.0 mg/l α-naphthalene acetic acid (NAA). Plantlets were successfully acclimatized without growth chamber facility on Ngoc Linh mountain with a survival rate of 85% after two months. On the other hand, substantial increase of root length was observed. This study describes an efficient method for in vitro regeneration of P. vietnamensis , which could be considered for large-scale multiplication and propagation of this important medicinal plant. Key words : Acclimatization, callus, Panax vietnamensis, regeneration, root, shoot.

멸종위기 종 솔나리의 기내 대량생산을 통한 보존 연구

This study was conducted to establish the optimal condition for conservation of genetic resources and micropropagation of Lilium cernum. Induction of bulbet of L. cernum was highly effective (9.2 bulb/explant) on 1/2 SH (Schenk and Hildebrandt) medium supplemented with 1.0 mg/L TDZ (Thidiazuron) and 0.1 mg/L NAA (Naphthaleneacetic acid). The treatment of 0.1 mg/L NAA increased root development (6.4 root/explant) under the in vitro condition. In addition, treatments of AC (Activated Charcoal) and ventilation were enhanced to develop number of shoots and to elongate length of leaf, bulb and root. Futhermore, the process of short-term soil acclimatization was promoted to strengthen the plantlets induced under the in vitro condition.

Micropropagation of Oleander (Nerium oleander L.)

Successful propagation of Nerium oleander L. (oleander) was achieved by in vitro methods. Shoot cultures were initiated from seedlings of wild-growing plants and from shoot apices of adult plants belonging to the commercial cultivars Splendens Giganteum, Revanche, and Alsace. Axillary shoot breaking from shoot tips excised from these cultures required the presence of either 6-benzylaminopurine (BA) or thidiazuron (TDZ). The higher number of axillary shoots from juvenile material was obtained by culturing shoot tips from BA-pretreated material derived from seedlings on a modified Schenk and Hildebrandt medium (SHM) supplemented with BA or TDZ (average of 3.9 shoots per explant with a mean length of 10.4 mm) and when the media were supplemented with 8.8 μM TDZ (average of 3.5 shoots per explant with a mean length of 7.3 mm) or 4.4 μM BA (average of 3.3 shoots per explant with a mean length of 12.3 mm). Among cultivars, cv. Revanche showed best shoot proliferation rates, especially when explants were cultured on Woody Plant Medium (average of 3.2 shoots per explant with a mean length of 10.2 mm). Adventitious bud differentiation from oleander leaves is also reported. Leaves excised from seedling-derived shoot cultures responded better than those from adult plant-derived shoot cultures (40% versus 5%, respectively). Bud differentiation required the presence of TDZ in the SHM medium, although shoot development was only achieved on transference of explants to media without TDZ but supplemented with BA and indoleacetic acid (IAA) or BA, kinetin, and IAA. Axillary and adventitious shoots were easily rooted (99%) and successfully (95% to 100%) transferred to soil.