Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Indirect somatic embryogenesis of purple coneflower (Echinacea purpurea (L.) Moench): a medicinal-ornamental plant: evaluation of antioxidant enzymes activity and histological study.

Purple coneflower (Echinacea purpurea (L.) Moench) is a widely used medicinal and ornamental plant. In the present study, the callus embryogenesis was examined using benzyl adenine (BA) at three levels (3, 4, 5 mg L-1), 1-Naphthalene acetic acid (NAA) at three levels (0.1, 0.2 and 0.5 mg L-1) with or without activated charcoal (1 g L-1), coconut milk (50 ml L-1) and casein hydrolysate (50 mg L-1) in the MS (Murashige and Skoog 1962) medium. The embryogenesis indirectly occurred with the production of callus. The calli were observed in three forms: undifferentiated, embryogenic and organogenic. The embryogenic calli were dark green and coherent with a faster growth rate. The highest embryogenesis (100%) and embryonic regeneration (plantlet production) were obtained in the combined BA + NAA treatments with the activated charcoal, coconut milk and casein hydrolysate. However, the combined treatments of growth regulators failed to produce somatic embryos without the use of coconut milk and casein hydrolysate. The maximum amount of protein, peroxidase and catalase activity of embryogenic calli (2.02, 1.79 and 6.62ΔOD/Min/mg.protein, respectively), and highest percentage of acclimatization success (29.3% of plants) were obtained in the combined treatment of 5 mg L-1 BA + 0.5 mg L-1 NAA + activated charcoal + coconut milk + casein hydrolysate. The highest amount of chlorophyll content (33.3 SPAD value) and growth characteristics of acclimatized plantlets were observed in the media containing 3 mg L-1 BA + 0.1 and 0.2 mg L-1 NAA + 1 g. L-1 combined activated charcoal, coconut milk, casein hydrolysate. The histological studies confirmed the somatic embryogenesis in purple coneflower. Generally, it was found that the somatic embryogenesis of E. purpurea occurs at high levels of BA and low levels of NAA with the addition of coconut milk and casein hydrolysate.

In Vitro Micropropagation of Industrially and Medicinally Useful Plant Aloe trichosantha Berger Using Offshoot Cuttings.

This study was aimed to develop in vitro micropropagation protocol of Aloe trichosantha Berger using offshoots as explants. MS media supplemented with plant growth regulators helped explants develop shoots within about 14 to 17 days. The mean number of days to shooting has decreased from 16.8 ± 0.8 with 0.5/0.5 mg/L BAP/NAA supplement to 15.5 ± 0.5 with 2.0/0.5 mg/L BAP/NAA. While the mean shoot number has increased with increasing the concentration of BAP supplements, the reverse was true with mean shoot lengths, whereas supplement of 2.0/0.5 mg/L BAP/NAA has generated significantly more shoots (17 ± 3.8), and longer shoots were produced with the addition of 0.5/0.5 and 1.0/0.5 mg/L BAP/NAA. In regard to rooting, though higher concentrations of NAA have resulted in quick rooting, the rooting performance in terms of mean number and length of roots was better with low concentrations. All the plantlets subjected to greenhouse acclimatization in cocopeat have survived. Secondary acclimatization in composted and manured soil media has also resulted in 93 to 95% survival rate. Lighting conditions (nursery shade or direct sunlight) of secondary acclimatization did not lead to any difference in the survival rate of the plantlets.

Auxin treatment of grapevine (Vitis vinifera L.) berries delays ripening onset by inhibiting cell expansion.

KEY MESSAGE: Auxin treatment of grape (Vitis vinifera L.) berries delays ripening by inducing changes in gene expression and cell wall metabolism and could combat some deleterious climate change effects. Auxins are inhibitors of grape berry ripening and their application may be useful to delay harvest to counter effects of climate change. However, little is known about how this delay occurs. The expression of 1892 genes was significantly changed compared to the control during a 48 h time-course where the auxin 1-naphthaleneacetic acid (NAA) was applied to pre-veraison grape berries. Principal component analysis showed that the control and auxin-treated samples were most different at 3 h post-treatment when approximately three times more genes were induced than repressed by NAA. There was considerable cross-talk between hormone pathways, particularly between those of auxin and ethylene. Decreased expression of genes encoding putative cell wall catabolic enzymes (including those involved with pectin) and increased expression of putative cellulose synthases indicated that auxins may preserve cell wall structure. This was confirmed by immunochemical labelling of berry sections using antibodies that detect homogalacturonan (LM19) and methyl-esterified homogalacturonan (LM20) and by labelling with the CMB3a cellulose-binding module. Comparison of the auxin-induced changes in gene expression with the pattern of these genes during berry ripening showed that the effect on transcription is a mix of changes that may specifically alter the progress of berry development in a targeted manner and others that could be considered as non-specific changes. Several lines of evidence suggest that cell wall changes and associated berry softening are the first steps in ripening and that delaying cell expansion can delay ripening providing a possible mechanism for the observed auxin effects.

NAA and 6-BA promote accumulation of oleanolic acid by JA regulation in Achyranthes bidentata Bl.

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.

Phenolic compounds, antioxidant capacity and antimutagenic activity in different growth stages of in vitro raised plants of Origanum vulgare L.

Efficient micropropagation procedure was developed for Origanum vulgare, a high-value culinary herb, and the phytochemicals, phenolic content, antioxidant and antimutagenic activity of leaf and stem, derived from different growing stages were analyzed. The agar solidified Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine and α-naphthaleneacetic acid was optimized as best shoot-multiplication-medium. Shoots were rooted best on 1/2 strength MS medium supplemented with 50 µM indole-3-butyric acid (IBA). The plantlets were successfully acclimatized ex vitro in a soil, sand and farmyard manure mixture (2:1:1 v/v/v) with 100% survival rate in greenhouse. The total anthocyanin and total phenolic content were observed significantly higher in leaves of in vitro-raised plants. However, total tannin, flavonoid and antioxidant activity remained higher in leaves of mother plant maintained under ployhouse condition. All the plant extracts have shown significant antimutagenic activity except in vitro-growing plants. A total of 13 polyphenolic compounds were detected in different extracts using high performance liquid chromatography. Among these, catechin was detected maximum in in vitro-growing cultures and chlorogenic acid in leaves of mother plant. These findings will help the farmers, medicinal plant growers, and industries for mass multiplication and effective extraction of phytochemicals from O. vulgare.

Establishment and Phytochemical Analysis of a Callus Culture from Ageratina pichinchensis (Asteraceae) and Its Anti-Inflammatory Activity.

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L-1 NAA with 0.1 mg L-1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 µM), IL-6 (IC50 = 73.71 ± 3.21 µM), and TNF-α (IC50 = 73.20 ± 5.99 µM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 µM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.

Direct Organogenesis from Immature Female Inflorescence of Date Palm by Gradual Reduction of 2,4-D Concentration.

Inflorescences represent an alternative explant source for superior date palm trees, especially those that do not produce offshoots. They provide large numbers of explants free of fungal and bacterial contamination for successful tissue culture initiation. Furthermore, they are characterized by the capacity of plant regeneration within a short time as compared to other explant types. This chapter focuses on the procedures employed for plant regeneration by direct organogenesis using immature female inflorescence explants, including initiation of adventitious buds, differentiation, multiplication, shoot elongation, rooting, and acclimatization. Adding 5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) into the initiation medium and gradually reducing it to 1 and then to 0.5 mg/L in the subsequent 2 subcultures, respectively, are determining factors in direct adventitious bud formation from the inflorescence. Bud differentiation is obtained on MS medium containing 0.25 mg/L kinetin (Kin), 0.25 mg/L benzyladenine (BA), 0.25 mg/L abscisic acid (ABA), 0.1 mg/L naphthaleneacetic acid (NAA), and 0.2 g/L activated charcoal (AC). Regenerated shoots exhibit sufficient root formation on MS medium supplemented with 2 mg/L indole butyric acid (IBA) and 1 mg/L NAA and subsequent survival in the greenhouse.

Schisandra lignans production regulated by different bioreactor type.

Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA® and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA® bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively).

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

Elicitation of Medicinally Important Antioxidant Secondary Metabolites with Silver and Gold Nanoparticles in Callus Cultures of Prunella vulgaris L.

Prunella vulgaris L. (P. vulgaris) is an important medicinal plant with a wide range of antiviral properties. Traditionally, it is known as self-heal because of its faster effects on wound healing. It is commonly known as a natural antiseptic due to the presence of various polyphenols. There is lack of research efforts on its propagation and production of bioactive compounds under field and in vitro conditions. In this study, the effects of different ratios (1:2, 1:3, 2:1, and 3:1) of silver (Ag) and gold (Au) nanoparticles (NPs) alone or in combination with naphthalene acetic acid (NAA) were investigated for callus culture development and production of secondary metabolites. The Ag (30 µg l-1), AgAu (1:2), and AgAu (2:1) NPs in combination with NAA (2.0 mg l-1) enhanced callus proliferation (100 %) as compared to the control (95 %). Among the different NPs tested, AuNPs with or without NAA produced higher biomass in log phases (35-42 days) of growth kinetics. Furthermore, AgAu (1:3) and AuNPs alone enhanced total protein content (855 µg-BSAE/mg-fresh weight (FW)), superoxide dismutase (0.54 nM/min/mg-FW), and peroxidase (0.39 nM/min/mg-FW) enzymes in callus cultures. The AgAuNPs (1:3) in combination with NAA induced maximum accumulation of phenolics (TPC 9.57 mg/g-dry weight (DW)) and flavonoid (6.71 mg/g-DW) content. Moreover, AgAuNPs (3:1) without NAA enhanced antioxidant activity (87.85 %). This study provides the first evidence of NP effect on callus culture development and production of natural antioxidants in P. vulgaris.

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant.

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L(-1) BA and 0.2 mg L(-1) NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L(-1) BA and 0.5 mg L(-1) NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot-root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.

Shiraz wines made from grape berries (Vitis vinifera) delayed in ripening by plant growth regulator treatment have elevated rotundone concentrations and "pepper" flavor and aroma.

Preveraison treatment of Shiraz berries with either 1-naphthaleneacetic acid (NAA) or Ethrel delayed the onset of ripening and harvest. NAA was more effective than Ethrel, delaying harvest by 23 days, compared to 6 days for Ethrel. Sensory analysis of wines from NAA-treated fruit showed significant differences in 10 attributes, including higher "pepper" flavor and aroma compared to those of the control wines. A nontargeted analysis of headspace volatiles revealed modest differences between wines made from control and NAA- or Ethrel-treated berries. However, the concentration of rotundone, the metabolite responsible for the pepper character, was below the level of detection by solid phase microextraction-gas chromatography-mass spectrometry in control wines, low in Ethrel wines (2 ng/L), and much higher in NAA wines (29 ng/L). Thus, NAA, and to a lesser extent Ethrel, treatment of grapes during the preveraison period can delay ripening and enhance rotundone concentrations in Shiraz fruit, thereby enhancing wine "peppery" attributes.

[Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis].

To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.

Effect of 1-naphthaleneacetic acid on organic acid exudation by the roots of white lupin plants grown under phosphorus-deficient conditions.

The effect of NAA (1-naphthaleneacetic acid) on organic acid exudation in white lupin plants grown under phosphorus deficiency was investigated. Plants were sampled periodically for collecting of organic acids (citrate, malate, succinate), and also were used to study the effect on proton extrusion and release of Na(+), K(+), Ca(2+) and Mg(2+). The tissues were later processed to quantify the organic acids in tissues, the phosphorus content and the effects on plant biomass. The exogenous addition of NAA led to an increase in organic acid exudation, but this response was not proportional to the concentration of the dose applied, noticing the largest increments with NAA 10(-8)M. In contrast the increase in root weight was proportional to the dose applied, which shows that with higher doses the roots produced are not of proteoid type. Proton extrusion and the release of cations were related to the NAA dose, the first was proportional to the dose applied and the second inversely proportional. Regarding the analysis of tissues, the results of citrate and phosphorus content in shoots show that the overall status of these parts are the main responsible of the organic acids exuded. NAA served as an enhancer of the organic acid exudation that occurs under phosphorus deficient conditions, with a response that depends on the dose applied, not only in its magnitude, but also in the mechanism of action of the plant hormone.

Combination of the auxins NAA, IBA, and IAA with GA3 improves the commercial seed-tuber production of potato (Solanum tuberosum L.) under in vitro conditions.

The study compared the effects of 1.0 × MS medium containing various concentrations of α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), alone or in combination with gibberellic acid (GA3) in micropropagation of three potato (Solanum tuberosum L.) cultivars Pasinler, Granola, and Caspar using binodal stem cuttings. The results testified improved regeneration on 1.0 × MS medium containing variants of NAA, IAA, and IBA plus GA3 on all cultivars. The minimum days to shoot induction on three cultivars ranged 4.25-5 d on 1.0 × MS medium containing 0.25 mg L(-1) GA3 + 1 mg L(-1) NAA. The longest shoots (11.8 cm), maximum number of nodes (13.50), and maximum number of leaves (11.00) were recorded on cv. Caspar on 1.0 × MS medium containing 1 mg L(-1) NAA + 0.25 mg L(-1) GA3. The minimum time to root induction (12.25 d) was noted on cv. Pasinler on the same medium. All of the regenerated shoots could be easily rooted. The results showed that the combined effect of various concentrations of NAA, IAA, and IBA plus GA3 was more pronounced compared to the auxins used alone. The results of this research are of significant importance for potato breeders.

Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

Effect of various growth hormone concentration and combination on callus induction, nature of callus and callogenic response of Nerium odorum.

Nerium odorum, Linn. (Apocynaceae) is an important evergreen shrub. It is heat, salinity and drought tolerant. Plants with milky sap have medicinal value, mainly cardenolides, flavonoids and terpenes. It is used for wastewater purification and for restoration of riparian woodlands. In view of these facts, the study was conducted for micropropagation of N. odorum. Murashige and Skoog (MS) media supplemented with different concentrations (0.5-10.0 mg/l) of 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and kinetin (Kin) were used singly and in combinations. Among all the growth hormones, 2,4-D was the best for callus induction (75% in stem and 79% in leaf) and in combination 2,4-D and BAP (78% in stem and 81% in leaf). The day of callus induction started from the 19th to the 37th day. This variation is due to the differences in culture conditions and the age of explants. The fresh and dry weight and moisture content showed good growth of callus, which is used in further studies of alkaloid production. Micropropagation of this plant allows the production of clones at a fast rate and in continuous manner. This work can lead to the development of an efficient protocol for callus induction and other issues.

Induction and analysis of the alkaloid mitragynine content of a Mitragyna speciosa suspension culture system upon elicitation and precursor feeding.

This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin, 6-benzylaminopurine (BAP), and 1-naphthaleneacetic acid (NAA) on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L⁻¹ 2,4-D (70.83%). Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L⁻¹ 2,4-D and 3% sucrose (9.47 ± 0.4667 mL). The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L⁻¹ yeast extract (9.275 ± 0.082 mg L⁻¹) that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 µM tryptophan and harvested at 6 days (13.226 ± 1.98 mg L⁻¹).