Growth Regulator-free Medium Research Articles

Somatic embryogenesis and shoot organogenesis in peanut cv. 'Georgia-12Y' and successful transfer to the soil.

An efficient regeneration system was established through somatic embryogenesis and shoot organogenesis using mature embryos explants of peanut cultivar 'Georgia-12Y'. The role of plant growth regulator combinations was investigated for embryogenic callus and somatic embryo induction. Results showed that Murashige and Skoog (MS) medium supplemented with 20 μM picloram (4-amino 3, 5, 6-trichloropicolinic acid), casein hydrolysate (0.2 g/L), sucrose (30 g/L) and sorbitol (10 g/L) supported callus induction in dark and higher number of somatic embryos in light. No somatic embryos were induced at 0.1 μM to 10.0 μM of 2,4-Dichloro phenoxy acetic acid (2,4-D) and picloram individually. The highest regeneration frequency of 90% was recorded on 40 μM 2,4-D + casein hydrolysate (0.2 g/L), sucrose (30 g/L) and sorbitol (10 g/L). The plantlets regenerated via somatic embryogenesis did not exhibit any morphological abnormalities. Double staining with acetocarmine and Evans blue distinguished between embryogenic and non-embryogenic callus. Histological observations confirmed distinct developmental stages of somatic embryos. On the other hand, highest number of shoots were induced in response to MS + 15 μM thidiazuron (TDZ) among various treatments tested. Incubation of shoots on plant growth regulator free MS medium induced in-vitro flowering after 12 weeks under light conditions. The induction of embryogenic and morphogenic callus and production of fertile peanut plants using manipulations of various plant growth regulators is reported on peanut cultivar 'Georgia- 12Y'.

Multiplication of lavender (L. angustifolia) and lavandin (Lavandula x intermedia) in explant culture

Abstract Lavender plants are preferably propagated vegetatively. The aim of the work was to propose the composition of nutrient media for in vitro multiplication of plants of Lavandula angustifolia (var. Krajová and Beta) and Lavandula x intermedia (var. Budrovka and Grosso). Explant cultures were initiated from apical or nodal segments. Apical segments were more suitable and regenerated the highest number of shoots in MS medium supplemented with 1 mg/L BAP with or without 0.1 mg/L IAA in all used varieties. Nodal segments are suitable for var. Budrovka, where the number of shoots was not statistically different between the apical or nodal segments and among the media tested. Shoot multiplication of L. angustifolia varieties was the most effective in the MS media with 0.5 or 1 mg/L BAP. For variety Grosso, the optimal medium was MS with 1 mg/L BAP, while var. Budrovka regenerated a similar number of shoots in all media supplemented with 0.5 or 1 mg/L BAP with or without 0.1 mg/L IAA. All genotypes rooted with the highest frequency on growth regulator-free medium with a half dose of MS salts. After transplanting into the soil and ex vitro acclimatization, plantlets survivability after 10 weeks was 78.9 – 87.7%, except for var. Beta with a lower survivability of 50%. The proposed procedures enable a rapid plant multiplication of varieties Budrovka, Grosso and Krajová. With the Beta variety, it is advisable to continue working on the optimization of nutrient media to verify whether the procedures can be further optimized.

In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant

Aims: C. spinosa (Family Rubiaceae) is a valuable medicinal since years ago. A protocol was developed including best sterilization and best medium for callus induction of C. spinosa using leaf discs as explants.
 Methodology: Sterilization protocol optimized using different concentrations of Carbendazim® (0.2,
 0.3%) and Clorox (10, 15%) exposing to different time intervals (10, 15 min). Percentage survival and contaminations were calculated. Best medium was optimized using different concentrations of 6-Benzyl Amino Purine (BAP) and Naphthalene Acetic Acid (NAA) (1.0-6.0 mg L-1). Growth regulators free Murashige and Skoog (MS) medium was used as control. Completely randomized design was followed with ten replicates in each concentration. Days taken to initiate calli, morphological characteristics and mean dry weights of calli were evaluated after 3 months of incubation.
 Results: Leaf discs sterilization with 0.3% Carbendazim for 10 min, 10 % Clorox for 10 min and 70% ethanol for 30 sec followed by two washings in sterile distilled water was found to be best sterilization protocol. It recorded lowest percentage contamination (13.34%) and highest percentage survival (86.66%) after 8 weeks. No observable changes were found in calli grown in growth regulators free MS medium. Calli growth and morphologies were significantly affected by the type of growth regulator and their concentrations in MS medium. Color of the calli varied from white opaque to yellow brown to green and the texture from foamy, loose, and friable to compact. Best medium with 1.0 mg L-1 BAP and 3.0 mg L-1 NAA produced green friable calli with 0.0969±0.01 g mean dry weight after 3 months. Some traits were found to be depended on synergetic effect of growth regulators and genotypic characteristics of explant.
 Conclusion: The study provides a better sterilization protocol and medium for in vitro calli induction and growth of C. spinosa.

Somatic Embryogenesis and Plant Regeneration from Sugi (Japanese Cedar, Cryptomeria japonica D. Don, Cupressaceae) Seed Families by Marker Assisted Selection for the Male Sterility Allele ms1.

One of the possible countermeasures for pollinosis caused by sugi (Cryptomeria japonica), a serious public health problem in Japan, is the use of male sterile plants (MSPs; pollen-free plants). However, the production efficiencies of MSPs raised by conventional methods are extremely poor, time consuming, and resulting in a high seedling cost. Here, we report the development of a novel technique for efficient production of MSPs, which combines marker-assisted selection (MAS) and somatic embryogenesis (SE). SE from four full sib seed families of sugi, carrying the male sterility gene MS1, was initiated using megagametophyte explants that originated from four seed collections taken at one-week intervals during the month of July 2017. Embryogenic cell lines (ECLs) were achieved in all families, with initiation rates varying from 0.6% to 59%. Somatic embryos were produced from genetic marker-selected male sterile ECLs on medium containing maltose, abscisic acid (ABA), polyethylene glycol (PEG), and activated charcoal (AC). Subsequently, high frequencies of germination and plant conversion (≥76%) were obtained on plant growth regulator-free medium. Regenerated plantlets were acclimatized successfully, and the initial growth of male sterile somatic plants was monitored in the field.

Micropropagation of Cleome dendroides (Cleomaceae), an endemic Brazilian species, as a source of glucosinolates

This is the first study related to micropropagation and phytochemical analyses of Cleome dendroides, an endemic species of the Brazilian Atlantic Forest, a biome affected by human activity. Among the classes of substances produced by the genus Cleome, the glucosinolates are used as chemotaxonomic markers. They have a wide structural diversity, which affords, for example, antineoplastic bioactivity. The micropropagation was established using explants from two sources: seedlings and in vitro plants. The morphogenic response was evaluated on Murashige-Skoog (MS) medium with different concentrations of benzyladenine (BA). Hypocotyl explants from seedlings and stem explants from in vitro plants showed higher capacity of shoot regeneration (56.3-100%). Shoot proliferation (75-100%) was maintained when hypocotyl explant-derived shoots and stem explant-derived shoots were subcultured three times a month. Hypocotyl explant-derived shoots maintained on MS + 2.0 mgL−1 BA induced the highest shoot production along the subcultures. Shoots were elongated and rooted on growth regulator-free MS medium. Plantlet establishment was successful in the hardening medium composed of Plantmax® substrate, reaching 85%survival. The phytochemical studies indicated two glucosinolate derivatives, highlighting the presence of 5,6-dimethyltetrahydro-1,3-oxazine-2-thione, identified for the first time in plants. This work demonstrates an efficient method for in-vitro production of C. dendroides plants, which can be used as a source of glucosinolates.

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures.

Elicitation is a possible aid to overcome various difficulties associated with the large-scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator-free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor-specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.

Development of an efficient protocol for ‘Brazos’ blackberry in vitro multiplication

In vitro culture is a very important technique in plant mass propagation, as well as an auxiliary tool to germplasm conservation and gene transfer. The present study developed an efficient multiplication protocol for 'Brazos' blackberry. Here, its multiplication ability was evaluated using in vitro shoots grown on medium supplemented with different growth regulators: thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin (KIN), zeatin (ZEA) and isopentyladenine (2iP) in two concentrations (5 and 10 μM) plus growth regulator free medium and five different basal media: Murashige and Skoog (MS), Murashige and Skoog with half-strengthened salts (MS/2), Anderson (AND), Quoirin and Lepoivre (QL) and woody plant medium (WPM). Number of shoots formed, shoot length and number of leaves per shoot were evaluated. Shoots were evaluated and subcultured three times, at every four weeks into a fresh medium. Among the different cytokinins, BAP in both concentrations was the best for shoot multiplication with 4.4, 6.1 and 7.9 shoots per explant with 5 μM and 4.3, 4.7 and 4.8 shoots per explant with 10 μM, in the three subcultures. Thidiazuron was not viable because of shoot malformation and great callus formation. Among the medium salts tested, MS medium was slightly better, reaching 3.9, 4.3 and 2.5 shoots per explant in the first, second and third subculture. The results of this investigation indicate that blackberry multiplication is viable and represents a potential technique for rapid and efficient multiple shoot induction and plantlet recovery. In vitro multiplication and a culture medium MS supplemented with BAP 5 µM can be recommended for 'Brazos' blackberry.

Structural and ultrastructural variations in in vitro and ex vitro rooting of microcuttings from two micropropagated Leucospermum (Proteaceae)

Abstract In vitro rooting of two Leucospermum cultivars (L. cordifolium ‘Flame Spike’ and L. ‘Tango’) microcuttings was studied with different indole-3-butyric acid (IBA) treatments: A) including in a solid medium (0, 0.2, 1 and 2 mg L−1), B) by dipping the base of the shoots in 500–2500 mg L−1 for 5 s and C) including in a liquid medium (12.5 and 25 mg L−1) for 24–96 h (induction step). After the treatments B and C, shoots were transferred to an auxin-free root elongation medium containing 20 g L−1 sucrose and solidified with 7 g L−1 agar. The long-term presence of IBA in the culture-medium inhibited root development. The culture of shoots in a liquid medium with higher IBA concentrations and the application of IBA pulses at the base of the microcuttings proved to be suitable treatments to induce rhizogenesis. Culturing microcuttings in a growth-regulator free solid medium after IBA treatments (B and C) triggered root initiation and differentiation. The results show that the microcuttings cultured in a liquid medium with IBA had the highest percentages of rooting. The sequence of anatomical changes during rooting was similar in vitro and ex vitro, the origin of the adventitious root in the vascular cambium. In vitro roots with an organized tissue system emerged from the microcutting stems 6 days after the root induction treatments. The acclimatized plantlets showed a suitable root system. Roots were formed ex vitro from the stem vascular cambium and connections were established between the stem vascular bundles and the roots, allowing root elongation. Some modifications were also observed in the basal rooting zone of stems in L. ‘Tango’.

Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability.

Plant growth regulator interactions in physiological processes for controlling plant regeneration and in vitro development of Tulbaghia simmleri

The endogenous auxin and cytokinin contents of in vitro regenerated Tulbaghia simmleri maintained on applied plant growth regulators in Murashige and Skoog (MS) medium were investigated using UHPLC–MS analysis. The highest number of shoots (27.6 per leaf) were produced in MS medium supplemented with 2.5 μM thidiazuron. A higher number of these shoots were rooted with 10 μM 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55, cytokinin antagonist). Production of somatic embryos (SEs: 16.4–4.6, globular to cotyledonary stages) improved significantly with liquid MS medium containing 2.5 μM picloram, 2.5 μM phloroglucinol (PG) and 1.5 μM gibberellic acid or 1.5 μM PI-55 and 1.0 μM trans-zeatin. SEs (torpedo and cotyledonary stages) germinated (100%) in plant growth regulator free MS medium. The plantlets were acclimatized and all survived in the greenhouse. Higher levels of endogenous auxin, 2-oxindole-3-acetic acid (oxIAA, 371.52 pmol/g DW) and indole-3-acetylaspartate (IAAsp, 141.56 pmol/g DW) were detected in shoots from PG treatments. The roots of garden-grown mother plants possessed the highest level of indole-3-acetic acid (IAA, 630.54 pmol/g DW) and oxIAA (515.26 pmol/g DW). Cytokinins [CKs: trans-zeatin-O-glucoside (tZOG), cis-zeatin (cZ) and N6-isopentenyladenosine-5′-monophosphate (iPRMP)] levels were relatively high in shoots and roots of plantlets in vitro. However, PI-55 treatments influenced the development of plantlets promoting a higher biosynthesis level of iPRMP (418.06 pmol/g DW in root) and cZRMP (904.61 pmol/g DW in roots and 1427.83 pmol/g DW in shoots). The presented protocols offer organogenesis and somatic organogenesis systems for rapid plant regeneration of T. simmleri. In addition, the importance of exogenous and endogenous hormonal effects on in vitro plant growth and development as well as endogenous hormone metabolism signalling and transport related to the physiological processes of CK metabolism and transport are illustrated for in vitro development of T. simmleri.

Propagation of Drosera rotundifolia and Drosera capensis in an in vitro Culture System

Drosera rotundifolia and Drosera capensis (Droseraceae) are carnivorous plants grown as ornamentals and sources for homeopathic medicine. The aim of this study was to optimize nutrient and growth regulator concentrations for the in vitro propagation of these species. Half strength MS medium (1/2MS) was supplemented with kinetin (0.5, 2, 5 mg/l) or 6-benzyladenine (3, 5 mg/l) and plantlets were transferred to 1/2MS with or without cytokinins. After 8 weeks rosette diameter, plant height, number of roots, root length were recorded and plants were cultured in full strength MS, 1/2MS or 1/2MS with 0.5 mg/l α-naphthaleneacetic acid for the same period of time. Afterwards, plant characteristics (number of roots, root length, number of shoots, number of flower stalks) were assessed. For D. rotundifolia, shoot development and rosette diameter increased significantly in the medium with 0.5 mg/l kinetin and 3 mg/l 6-benzyladenine, while root development decreased. Plant growth regulator free medium was more suitable for root development than medium with α-naphthaleneacetic acid and thus supported the formation of significantly more flower stalks. For D. capensis, kinetin was detrimental for shoot development, the optimum medium for both shoot and root formation being MS without plant growth regulators.

Detection of viruses of Bangladeshi and Japanese garlic and their elimination through root meristem culture

A number of viruses cause considerable yield loss and quality deterioration in garlic. Root meristems of virus infected plants are known to be free from detectable viruses. This potentiality could be exploited to obtain virus free clones at a high frequency by culturing excised root meristems in vitro. We have developed efficient methods of direct and somatic embryo derived shoot regeneration from root meristems of garlic. The objectives of this work were to detect viruses infecting Bangladeshi and Japanese garlic clones and find an easy and efficient method of eliminating the viruses for the improvement of both yield and quality of garlic. At first, we confirmed the presence of detectable viruses in three Bangladeshi and one Japanese clones. The clones were infected with four different types of viruses: Garlic viruses (GarVs), Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), and Garlic common latent virus (GCLV). To eliminate those viruses, as per our previous method, root meristems were cultured on MS medium supplemented with 1.0 µM NAA and 10.0 µM BA. Shoot primordia developed from the cultured explants within 1 month. The regenerated individual shoot buds (2-5 mm) were separated from the mother explants and transferred to growth regulators free medium. RT-PCR confirmed that the viruses present in the mother garlic plants were absent in the shoots found after two-step culture. The regenerated shoots were rooted on growth regulator free medium and transferred to pots. Results indicated that the plants remained free from LYSV. Virus elimination through root meristem culture emerged as an efficient novel technique for the eradication of multiple viruses as confirmed by RT-PCR in this study. This technique has the potential for the production and supply of virus free propagules (plants/bulblets) for the yield and quality improvement of garlic.Progressive Agriculture 28 (2): 55-63, 2017

Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L.

Semecarpus anacardium L. is a tree species which produces secondary metabolites of medicinal importance. Roots of the plant have been traditionally used in folk medicines. Different strains of Agrobacterium rhizogenes (A4, ATCC15834 and LBA 9402) were used for induction of hairy roots in in vitro grown tissues of the plant. Hairy root initiation was observed after 25–30 days of infection. Optimum transformation frequency of 61% was achieved on leaf explants with ATCC15834 strain. Infection time of 30 min resulted in greater transformation frequency compared to 10 and 20 min, respectively. The hairy roots cultured in growth regulator-free semi-solid woody plant medium differentiated into callus. Whole shoots infected with ATCC 15834 were found to produce more transformants upon co-cultivation for 4 (65%) and 5 (67%) days. Induction of hairy roots in stem explants infected with ATCC 15834 was lower (52%) compared to leaves (62%) after 4 days of co-cultivation. In A4 and LBA9402 strains transformation efficiency was 49 ± 2.8% and 36 ± 5.7% in shoots after 4 days of co-cultivation. Transformation frequency was higher in ATCC15834 strain, irrespective of explants. The hairy roots of S. anacardium elongated slowly upon transfer to half-strength liquid medium. After 3–4 passages in liquid medium slender hairy roots started differentiating which were separated from the original explants. Visible growth of the roots was observed in hormone-free liquid medium after 2–3 months of culturing. Polymerase chain reaction with gene-specific primers from rol A, B and C genes confirms the positive transformation events.

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.

In vitro morphogenic responses of African yam bean (Sphenostylis stenocarpa (Hochst ex. A. Rich.) Harms) accessions to plant growth regulators

Morphogenic responses of two accessions of African yam bean to different concentrations of plant growth regulator supplements to Murashige and Skoog basal medium was investigated to develop a more efficient regeneration system. Mature embryo explants were cultured on growth regulator-free and BAP + NAA supplemented media. Nodal cuttings excised from 4-week old shoots of the regenerated embryos were cultured on media containing varying concentrations and combinations of 6-benzyl aminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Growth regulator-free medium favored embryo regeneration and growth over supplemented media and both enhanced shoot regeneration and rooting, but could not induce multiple shoot formation on embryo explants. Multiple shoots were produced by nodal explants and the highest average number of shoots (5.3 ± 2.3), leaves (7.7 ± 3.6), roots (3.7 ± 2.9) and root length (3.1 ± 0.0 cm) were obtained on a medium with 0.6 mg l−1 BAP + 0.03 mg l−1 NAA for accession TSs154, while in TSs5, highest number of shoots (3.2 ± 2.5) and leaves (5.9 ± 1.5) were induced by 2.0 mg l−1 Kinetin + 0.05 mg l−1 NAA. Such differential morphogenic responses to culture media underline the genotypic control of in vitro propagation of this crop. Embryo and nodal explants rooted directly on shoot regeneration media, and regenerated plantlets were successfully acclimatized. The efficient regeneration system obtained will enhance genetic improvement of African yam bean by facilitating molecular genetic transformation for advanced breeding.

Assessment of Genetic Fidelity Using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats Markers of Lawsonia inermis L. Plants Regenerated by Axillary Shoot Proliferation

An efficient, reliable and reproducible plant regeneration protocol was developed for Lawsonia inermis L. using mature nodal explants. Shoot proliferation (81.6 %) with 7.8 shoots/explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 1.0 mg l−1 6-benzyladenine. Shoot numbers were up-scaled by inducing multiple shoots from axenic nodal segments derived from the primary shoots on the shoot regeneration medium. Thus the authors could achieve ca. 129–134 shoots from single nodal explant. Ninety-three percent rooting of in vitro regenerated shoots was achieved on growth regulator free half-strength MS medium. Regenerated plantlets were successfully transferred to soil with 85 % survival rate. Genetic stability analyses of the in vitro regenerated plants using random amplified polymorphic DNA and inter simple sequence repeat markers revealed a homogeneous amplification profile for all micropropagated plants. This is the first report that evaluates the use of molecular markers to establish genetic fidelity of micropropagated L. inermis for the rapid clonal multiplication and true-to-type production of plant for attaining the ever increasing demand in pharmaceutical industries.

Glandular trichomes and essential oil characteristics of in vitro propagated Micromeria pulegium (Rochel) Benth. (Lamiaceae).

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 N(6)-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 10μM 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.

Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

AbstractStudy describes protocols for

Effect of growth regulators on rapid micropropagation and antioxidant activity of Canscora decussata (Roxb.) Roem. & Schult. – A threatened medicinal plant

Abstract Objective An efficient in vitro plant regeneration protocol for Canscora decussata Schult. (Gentianaceae) (C. decussate), a threatened medicinal herb used in Ayurvedic system of medicine was developed. Seed germination was achieved on MS growth regulator free medium. Methods The nodal explants were excised from the in vitro raised seedlings and inoculated on MS medium supplemented with various plant growth regulators such as BAP, KIN, TDZ and Zeatin individually and in combinations with or without GA3. BAP (2 mg/L) was proved to be effective for multiple shoot induction (30.20 ± 6.53) among the cytokinin tested individually. Addition of NAA (1 mg/L) to cytokinin containing medium resulted in callus, KIN (3 mg/L) with NAA (1 mg/L) produced highest percentage of callus (82%) per explant. Results Among the various combination of cytokinin tested, BAP (0.5 mg/L) in combination with KIN (2 mg/L) induced highest number of multiple shoots (72.10 ± 1.05 shoot per explant). Addition of 1 mg/L of GA3 to the above medium induced highest number of shoots (100.80 ± 3.20) with an average shoot length of 6.98 ± 0.66 cm. Rooting was optimized in half-strength MS medium supplemented with IBA at 1.0 mg/L. The plantlets were successfully transferred to hardening medium containing vermiculite with 83% survival rate. Among the antioxidant activity of methanol extract of wild-grown plants and in vitro regenerants tested, half-MS medium supplemented with NAA (0.5 mg/L) derived callus has promising activity for total phenolics, DPPH, ABTS, FRAP and phosphomolymbdenum assays. Total flavonoid content was found to be high in callus derived from MS medium supplemented with KIN (2 mg/L) in combination with NAA (1 mg/L). Conclusion Our present study suggest that in vitro derived callus of C. decussata represent a promising alternative source to meet the pharmaceutical demands for commercial formulations and the protocol could effectively be applied for the conservation of C. decussata Schult.

Micropropagation of Salvia wagneriana Polak and hairy root cultures with rosmarinic acid production

Salvia wagneriana Polak is a tropical species native to Central America, well adapted to grow in the Mediterranean basin for garden decoration. Micropropagation has been assessed from axillary shoots of adult plants using a Murashige and Skoog basal medium, with the addition of 1.33-μM 6-benzylaminopurine for shoot proliferation; the subsequent rooting phase occurred in plant growth regulator-free medium. The plants were successfully acclimatised with high survival frequency. Hairy roots were induced after co-cultivation of leaf lamina and petiole fragments with Agrobacterium rhizogenes and confirmed by PCR. The establishment and proliferation of the selected HRD3 line were obtained in hormone-free liquid medium and the production of rosmarinic acid (RA) was evaluated after elicitation. The analysis of RA was performed by LC-ESI-DAD-MS in the hydroalcoholic extracts. The addition of casein hydrolysate increased the RA production, whereas no enrichment was observed after the elicitation with jasmonic acid.