Genetic stability and phytochemical profiling of the in vitro regenerated plants of <Emphasis Type="Italic">Angelica glauca</Emphasis> Edgew.: an endangered medicinal plant of Himalaya

The present study describes the first successful report on in vitro propagation through direct organogenesis for multiple shoot induction of Angelica glauca. Rhizomes were used as explant, and maximum shoot multiplication was observed on MS medium supplemented with 6-Benzylaminopurine 8.0 µM and Indole-3-acetic acid 0.1 µM. Roots were observed within 14 days in the MS medium enriched with 0.5 µM IAA and 0.1 µM Naphthalene acetic acid (NAA) with an average production of 4.2 roots per shoot. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in field, with a recorded survival rate of 72% after 45 days. The total phenolic content showed significant difference (p?<?0.05) between in vitro raised plants (5.87 mM AAE/ g DW) and control (2.36 mM AAE/ g DW). Antioxidant activity, calculated through two in vitro assays, i.e. 1,1-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays revealed higher antioxidant activity in in vitro grown plants in comparison to control plants. Essential oil constituent’s analysis was also carried out in control and in vitro raised plants. Thirty-one compounds were identified in the oil samples through Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analysis also identified 31 compounds in the essential oil, representing 98.1–98.7% of total oil compositions. The major components of the essential oils were (Z)-ligustilide (51.1–51.5%), (Z)-butylidene phthalide (31.2–31.6%), (E)-butylidene phthalide (2.6–2.9%) and (E)-ligustilide (2.1–1.8%). Genetic stability of in vitro raised plants, evaluated using 20 Inter Simple Sequence Repeats primers, proved true to typeness of in vitro raised plants.     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.     

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg^?1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l^?1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml^?1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.     

In vitro propagation,genetic and phytochemical assessment of <Emphasis Type="Italic">Habenaria edgeworthii</Emphasis>: an important Astavarga plant

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.     

In vitro and ex vitro evaluation of long-term micropropagated turmeric as analyzed through cytophotometry,phytoconstituents, biochemical and molecular markers

Turmeric (Curcuma longa L.), a high valued medicinal plant, was micropropagated through induction of multiple shoots using latent axillary buds of rhizome. Cytophotometric and random amplified polymorphic DNA (RAPD) as well as inter simple sequence repeats (ISSR) analysis were used to periodically monitor the genetic stability of micropropagated clones of Curcuma longa conserved in vitro up to 7 years at every 6 months interval. A total of eighteen RAPD and eight ISSR primers gave 45,537 distinct and reproducible bands, monomorphic across all 353 plants analyzed. Micropropagated turmeric after being conserved for 7 years in vitro was transplanted into soil in field. Drug yielding potential of tissue culture derived plants was evaluated in field through estimation of phytoconstituents like curcumin and essential oil contents. The result of 2 years of field trial showed that micropropagated turmeric retained stability in all the characteristics examined when compared with the field performance of conventionally propagated plants. Thus long term conservation of an elite genotype of turmeric with epigenetic and genetic stability is significant for stable supply of drug i.e., curcumin and essential oil to the market.     

Evaluation of phytomedicinal yield potential and molecular profiling of micropropagated and conventionally grown turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Drug yielding potential of turmeric (Curcuma longa L.) is due to the presence of important phytoconstituents such as curcumin, oleoresin and essential oil. Slow multiplication rate, high susceptibility to rhizome rot and leaf spot disease and restricted availability of elite genotype necessitated application of tissue culture technique to alleviate the problems. A protocol has been developed for in vitro micropropagation of an elite genotype (cv. suroma) using latent axillary bud explants from unsprouted rhizome, available throughout the year. MS media containing 3 mg/l 6-Benzyladenine (BA) and 1 mg/l Indole Acetic acid (IAA) was found optimum for regeneration, multiplication and in vitro conservation of plantlets. After 3 years of in vitro conservation regenerants were transplanted to field and assessed for drug yielding potential through evaluation of curcumin, oleoresin and essential oil contents of rhizomes and leaves. One year of field grown tissue culture derived turmeric were found uniform in all the characteristics examined, when compared with those grown conventionally. Micropropagated turmeric showing stable drug yielding potential also proved to have genetic basis of stability as revealed by RAPD based molecular profiling.     

Optimization of in vitro and ex vitro regeneration and micromorphological studies in <Emphasis Type="Italic">Basella alba</Emphasis> L.

The optimum concentrations of the plant hormones for in vitro regeneration and subsequent effect of auxins on rooting (in vitro and ex vitro) of shoots of Basella alba L. have been investigated in present study. Nodal shoot segments were used as explants to initiate the cultures. The bud breaking from explants was observed within 1 week of incubation on agar gelled Murashige and Skoog’s (MS) medium. Multiple axillary shoots (7.30 ± 0.56 shoots per explant) were induced on MS medium supplemented with 2.0 mg/L 6-benzylaminopurine (BAP). The shoots were multiplied (maximum 17.10 ± 0.44 shoots per explant) on the same medium fortified with 0.5 mg/L each of BAP and Kin (Kinetin) +0.1 mg/L IAA. These shoots were excised and rooted in vitro (10.73 ± 0.92 roots per shoot) on half-strength MS medium augmented with 2.0 mg/L indole-3 butyric acid (IBA). Hundred percentage success rates have been achieved by ex vitro rooting of the in vitro regenerated shoots with IBA at 300 mg/L. The in vitro and ex vitro rooted shoots were acclimatized in greenhouse and subsequently transferred to the natural field conditions where 100 % survival rate was reported. The ex vitro rooting method was found more advantageous than in vitro rooting in terms of time, energy and survival percentage of B. alba. A comparative foliar micromorphological study of B. alba was conducted to understand the micromorphological changes in plants while shifting from in vitro to the in vivo conditions in terms of variations in stomatal index, venation pattern and vein density, and the arrangement of crystals. The study could help in understanding the response of in vitro raised plants towards in vivo environment.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Glandular trichomes and essential oil characteristics of in vitro propagated <Emphasis Type="Italic">Micromeria pulegium</Emphasis> (Rochel) Benth. (Lamiaceae)

Main conclusion

In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N⁶–benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 μM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.

     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

We developed a micropropagation protocol for Cleome gynandra, a C₄ model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l^?1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l^?1 BA and 0.1 mg l^?1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l^?1 NH₄NO₃ and 950 mg l^?1 KNO₃) and 100 mg l^?1 (NH₄)₂SO₄. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l^?1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) + 100 mg l^?1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l^?1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.     

Potential of thidiazuron in improved micropropagation of Picrorhiza kurroa– an endangered medicinal herb of alpine Himalaya

The potential of thidiazuron (1-phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea (TDZ) in the micropropagation of Picrorhiza kurroa, a western Himalayan herb was studied. The roots and rhizomes of this plant are rich in medicinally important glycosides i.e., picroside I, picroside II and kutkoside. Nodal segments (2.0–2.5 cm) from plants were pretreated with 0, 0.25, 0.50, 0.75, 1.0 μM TDZ for 7, 15 and 30 days. Maximum shoot multiplication was recorded after 60 days, provided, the nodal segments pretreated with 0.5 μM TDZ for 15 days were transferred to 0.8% agar gelled MS medium containing 3.0% sucrose. The shoots also showed profuse rooting having maximum length. When these plantlets were incubated at 15°C for 10 days and transferred to sand under polyhouse conditions, 100% survival was recorded after one month. In contrast, when the plantlets were not incubated at 15°C, only 86% survival was recorded. While the stomata and chlorophyll content of the tissue culture-raised plantlets treated at 15°C were comparable to that of plants growing in the field, the ones that were not treated at 15°C showed lower number of stomata and chlorophyll content.     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.     

Selenium Modifies the Effect of Short-Term Chilling Stress on Cucumber Plants

The objective of this study was to investigate the effect of selenium (Se) supply (0, control; 2.5, 5, 10, or 20 μM) on cucumber (Cucumis sativus L.) cv. Polan F1 plants grown under short-term low temperature stress. About 14–16 day-old seedlings, grown at an optimal temperature (25/20°C; day/night), were exposed to short-term chilling stress with a day/night temperature of 10°C/5°C for 24 h, for a further 24 h at 20°C/15°C, and then transferred to 25/20°C (re-warming) for 7 days. Se did not affect the fresh weight (FW) of plants at a concentration of 2.5–10 μM, but in the presence of 20 μM Se, the biomass of shoots significantly decreased. The contents of chlorophylls and carotenoids witnessed no significant change after Se supplementation. Compared with the control, the Se-treated plants showed an increase of proline content in leaves, once after chilling and again after 7 days of re-warming. However, proline levels were much higher immediately after chilling than after re-warming. The malondialdehyde (MDA) content in the root of plants treated with 2.5–10 μM Se decreased directly after stress. This was in comparison with the plants grown without Se, whereas it increased in roots and leaves of plants exposed to 20 μM Se. Seven days later, the MDA level in the root of plants grown in the presence of Se was still lower than those of plants not treated with Se and generally witnessed no significant change in leaves. Although Se at concentrations of 2.5–10 μM modified the physiological response of cucumber to short-term chilling stress, causing an increase in proline content in leaves and diminishing lipid peroxidation in roots, the resistance of plants to low temperature was not clearly enhanced, as concluded on the basis of FW and photosynthetic pigments accumulation.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

5-Azacytidine as a tool to induce somaclonal variants with useful traits in sugarcane (Saccharum spp.)

A possible strategy to produce variant sugarcane plants with beneficial traits was tested by promoting somaclonal variation in vitro through the action of the hypomethylation and mutagenic agent 5-Azacytidine (Azac). Treatment of calli in liquid medium caused high levels of necrosis. Consequently, 6- to 8-week-old calli of cultivar NCo376 were exposed to 50 and 100 μM Azac in semi-solid callus induction medium (CIM) (MS salts and vitamins, sucrose, casein hydrolysate, agar, with or without 3 mg l^?1 2,4-D) for 1 week. They were then transferred to fresh CIM with 2,4-D and to CIM without 2,4-D, for 2 and 8–10 weeks, respectively. The highest callus necrosis (>60 %) and reduced recovery (<40 %) were recorded for calli treated with 100 μM Azac without 2,4-D, which also resulted in lower plant yield (12 plantlets/0.2 g calli) than the control (18 plantlets/0.2 g calli). From methylation-sensitive amplified fragment length polymorphism analyses, the highest polymorphisms (4.2 %) were also obtained from plants derived from the 100 μM Azac treatment without 2,4-D. After 9 months of field growth, Azac-derived plants exhibited phenotypic differences compared with the controls. Ex vitro screening resulted in the identification of one plant from the 100 μM Azac with 2,4-D treatment putatively tolerant to smut, and three plants from the 100 μM Azac with 2,4-D and one from the 50 μM Azac with 2,4-D treatments, potentially tolerant to the herbicide imazapyr.