Production of monoterpenoids and aroma compounds from cell suspension cultures of <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Cell suspension cultures of Camellia sinensis were established in 250 ml shake flasks. Flasks contained 50 ml liquid medium of either Murashige and Skoog (MS), N/5 MS or Heller medium containing different levels of 6-benzyladenine (BA) (0.05–2 mg l⁻¹), 2,4-dichlorophenoxyacetic acid (2,4-D) (1–10 mg l⁻¹), and sucrose (10–50 g l⁻¹). Moreover, the pH of the medium was varied from 5.2–6.2. In addition, cultures were subjected to light irradiation as well as to complete darkness. Following optimization of aroma and terpenoid extraction methods, cell cultures were analyzed for the volatile compounds using GC/MS. A total of 43 compounds were identified using the micro SDE apparatus. Among the major monoterpenoids obtained were α-terpineol and nerol. Moreover, other high aroma-value compounds, including 2-ethyl hexanol, benzyl alcohol, benzene acetaldehyde, nonanal and phenylethylalcohol were also detected. The highest levels of these compounds were obtained from cell suspension cultures grown in MS medium containing 5 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA and 30 g l⁻¹ sucrose at pH of 5.8 with incubation in complete darkness.     

Biotransformation of menthol and geraniol by hairy root cultures of <Emphasis Type="Italic">Anethum graveolens</Emphasis>: effect on growth and volatile components

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l⁻¹), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC–MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17–52%), apiole (11–24%), palmitic acid (7–16%), linoleic acid (4–9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, α-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Production of saponions and polysaccharide in the presence of lactoalbumin hydrolysate in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

In this article, ginsenosides and polysaccharide contents in suspension cells and native roots of Panax quinquefolium L. were studied. In order to enhance the contents of ginsenosides and polysaccharide in P. quinquefolium suspension cells, we tested the effects of lactoalbumin hydrolysate on the growth of P. quinquefolium suspension cell, synthesis of ginsenosides and polysaccharide in flask and bioreactor. In flask culture, cells growth ratio was significantly enhanced by the addition of lower concentration of lactoalbumin hydrolysate. Addition of 100 mg L⁻¹ lactoalbumin hydrolysate significantly enhanced the contents of total saponins (5.44 mg g⁻¹ DW) and the contents were 3.89-fold over the control group. Addition of lactoalbumin hydrolysate significantly promoted the accumulation of polysaccharide, except 200 mg L⁻¹ lactoalbumin hydrolysate. The highest total saponins yield (36.72 mg L⁻¹ DW) and polysaccharide yield (0.83 g L⁻¹ DW) were obtained at 100 mg L⁻¹ lactoalbumin hydrolysate. In a 5-L stirred tank bioreactor, the highest contents of total saponins and TRb group ginsenosides were achieved on day 26, while the effect of lactoalbumin hydrolysate on the contents of TRg group ginsenosides were insignificant. This result suggests that lactoalbumin hydrolysate might have triggered the enzyme activities for the synthesis of TRb group ginsenosides. Overall, the highest total saponins yield (31.37 mg L⁻¹ DW) and polysaccharide yield (1.618 g L⁻¹ DW) were obtained on day 26 and day 24 respectively and the polysaccharide yield was 1.95-fold higher than the shake flask culture (0.83 g L⁻¹ DW). These results provided theoretical reference for two-stage culture in suspension cells of P. quinquefolium in bioreactor.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

Combination effect of lactoalbumin hydrolysate and methyl jasmonate on ginsenoside and polysaccharide production in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

Lactoalbumin hydrolysate (LH) at 100 mg L⁻¹ with methyl jasmonate (MJ) at 2 mg L⁻¹ synergistically stimulated ginsenoside accumulation in Panax quinquefolium cells compared with 100 mg L⁻¹ LH. Combination elicitors led to higher ginsenoside productivity (45.93 mg L⁻¹) than single treatment of 100 mg L⁻¹ LH (31.37 mg L⁻¹). This present result will be helpful in providing a tool for enhancing the productivity of ginsenoside by Panax quinquefolium cell cultures on a commercial scale.     

Micropropagation and plant regeneration from embryogenic callus of Miscanthus sinensis

Miscanthus sinensis (Poaceae) is a typical perennial giant grass of East Asia. Due to its high photosynthetic efficiency, low input requirements, and high biomass production, M. sinensis shows outstanding potential as a biofuel feedstock. However, the lack of an efficient tissue culture system may limit its utilization potential. Different explants of M. sinensis were evaluated to develop an efficient tissue culture system. Shoot apices from in vitro-germinated seedling explants were tested for adventitious bud proliferation. The highest level of proliferation (multiplication coefficient 6.69) was obtained when shoot apices were cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 6-benzyladenine (BA), 2.0 mg L⁻¹ kinetin, 0.05 mg L⁻¹ α-naphthalene acetic acid (NAA), 3% sucrose, and 0.8% agar. The highest rooting percentage (95.4%) was obtained when adventitious buds were cultured on half-strength MS medium supplemented with 0.2 mg L⁻¹ NAA, 3% sucrose, and 0.8% agar. Significant differences were found in the formation of embryogenic callus among different explant types. The embryogenic callus derived from epicotyls had the highest regeneration capacity when cultured on a medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, 0.5 mg L⁻¹ BA, and 0.1 mg L⁻¹ thiamine. Under these conditions, the callus induction percentage was 82%.     

Decalactone Production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under increased O<Subscript>2</Subscript> Transfer Rates

Yarrowia lipolytica converts methyl ricinoleate to γ-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-γ-decalactone produced by the yeast (263 mg l^-1) occurred by increasing the k_La up to 120 h⁻¹ at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O₂ solubility, but, although growth was accelerated, γ-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec−2-en-4-olide and dec-3-en-4-olide (70 mg l⁻¹).     

Decolorization of Acid red 151 by Aspergillus niger SA1 under different physicochemical conditions

The fungal strain A. niger SA1 isolated from textile wastewater pond proved to be an important source of remediation (decolorization/degradation) for textile dye, AR 151 (Reactive diazo dye) under different physicochemical conditions. Decolorization assays of AR 151 were carried out in Simulated textile effluent under shake flask condition for 8 days. Decolorization (at 20 mg l⁻¹ of dye) and related biomass production overall decreased with increase in pH from 5 to 9, at 30°C. It was maximum (95.71%) at pH 5 with highest amount of three residual products (36.91 (α-naphthol = 5.72) (sulfanilic acid = 24.81) (aniline = 6.38)) besides 2.05 mg ml⁻¹ of biomass production at an optimum concentration 6 and 0.1 mg l⁻¹ of glucose and urea respectively. The formation of the three products followed a quite different pattern at different pH values, however, it was considerably low (Total = 2.81 mg l⁻¹) compared to the amount of decolorization (67.26%) at pH 8. Decolorization (95–97%) was most favored under mesophilic temperature (25–45°C). It increased i.e., 90–98% with subsequent increase in dye from 10 to 100 mg l⁻¹, kept ≥50% below 400 mg l⁻¹ and drastically declined to 17% at 500 mg l⁻¹ of dye. Apparently, decolorization is found to be associated with fungal growth and hyphal uptake mechanism (Biosorption/Bioadsorption), however, mineralization of AR 151 and related products under different operational conditions also suggested a metabolically mediated decolorization/degradation.     

Microbial degradation of pyridine and α-picoline using a strain of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Nocardia</Emphasis> sp.

Biodegradation of pyridine and α-picoline (2-methyl pyridine) by Pseudomonas pseudoalcaligenes-KPN and Nocardia sp. isolated from garden soil were investigated in batch culture experiments. Pyridine and α-picoline (50–200 mg L⁻¹) were used as sole source of carbon and energy in the investigation. The kinetic constants were evaluated for pyridine and α-picoline degradation under optimized nutritional (C, N, P) and environmental (pH, temperature) conditions. The values of bio-kinetic constant obtained in the present investigation indicate the usefulness of both the cultures for treatment of waste containing pyridine and its derivatives.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).     

Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. cv. Poinsett76) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L⁻¹ benzyladenine (BA), 20 mg L⁻¹ l-glutamine and 2 mg L⁻¹ phosphinothricin (PPT) or 100 mg L⁻¹ kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L⁻¹) containing MS media with 0.5 mg L⁻¹ gibberellic acid (GA₃) and 0.6 mg L⁻¹ indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T₀ and the progenies. T₁ segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants.     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants.     

In vitro propagation of four threatened <Emphasis Type="Italic">Paphiopedilum</Emphasis> species (Orchidaceae)

The effects of seed maturity, media type, carbon source, and organic nutrient additives on seed germination, protocorm development, and plant growth of Paphiopedilum villosum var. densissimum Z. J. Liu et S. C. Chen were investigated. Micropropagation frequency was enhanced through the use of 200-day-old seed, Knudson C (KC) medium, and the presence of both glucose and coconut milk in the medium. The effects of various plant growth regulators on the frequency of shoot organogenesis in four Paphiopedilum species were also investigated. Explants of P. villosum var. densissimum and P. insigne (Lindl.) Stein incubated in the presence of 5 mg l⁻¹ 6-benzyladenine (BA) with 0.5 mg l⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, showed a twofold increase in the frequency of shoot organogenesis. For explants of P. bellatulum (Rchb. f.) Stein and P. armeniacum S. C. Chen et F. Y. Liu, the combination of 5.5 mg l⁻¹ BA with 0.5 mg l⁻¹ NAA and 4 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, resulted in the highest frequencies of shoot organogenesis.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Elicitation of guggulsterone production in cell cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis> by plant gums

Plant gum as an elicitor for guggulsterone production in cell cultures of Commiphora wightii is reported for the first time. Guggulsterone production increased 2.4 fold in the cell cultures by gum Arabic (100 mg l⁻¹), while mesquite gum elicited 2 fold. The cells treated with gum Arabic at 7th and 9th day accumulated enhanced guggulsterones within 24 h, which increased further up to 48 h and then declined. The cells treated at 9th day accumulated higher amount (218 μg l⁻¹) of guggulsterones after 48 h of elicitation as compared to cells treated at 7th day (164 μg l⁻¹). The optimized elicitation conditions were used in vessels of varying capacity where maximum yield of 285 μg l⁻¹ of guggulsterones was recorded in 3 l shake flasks. These experiments enabled highest guggulsterones yield in a short duration of 11 days in cell cultures of C. wightii.     

Micropropagation of <Emphasis Type="Italic">Pinus massoniana</Emphasis> and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l⁻¹ BA and 0. 2 mg l⁻¹ NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l⁻¹ BA and 0.2 mg l⁻¹ NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l⁻¹ NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.     

Growth,secondary metabolite production and antioxidant enzyme response of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> adventitious root as affected by auxin and cytokinin

Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l⁻¹ indole butyric acid (IBA) and at 7 and 9 mg l⁻¹ naphthalene acetic acid (NAA). On the other hand, 9 mg l⁻¹ NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l⁻¹ IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l⁻¹) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l⁻¹) in combination with 5 mg l⁻¹ IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l⁻¹ IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.     

A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L⁻¹ Kinetin (Kn), 0.5 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.5 mg L⁻¹ 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl₂ solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS₂ solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m⁻² s⁻¹ provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.