Direct Shoot Organogenesis Research Articles

Direct shoot organogenesis from rhizomes of medicinal zingiber Alpinia calcarata Rosc. and evaluation of genetic stability by RAPD and ISSR markers

A simple and efficient protocol for direct in vitro shoot multiplication and plant regeneration was established for an important aromatic medicinal plant, Alpinia calcarata. Preinduction of rhizome segments in medium containing 8.8 μM 6-benzylamino purine (BAP) rescued the buds from dormancy in 60% of the cultures. An average of 6.2 shoots were produced from rhizomatous bud explants on Murashige and Skoog (MS) medium supplemented with 5 μM BAP, 10 μM kinetin, and 2.5 μM α-Naphthalene acetic acid (NAA). The mother cultures retained their morphogenetic potential upto four subcultures and a maximum of 1.77-fold increase in shoot multiplication was recorded after the 3rd subculture. Rooting was simultaneously induced during subculture on shoot multiplication medium eliminating an additional step for rooting induction. Rooted plantlets were successfully acclimatized in pots in the greenhouse and subsequently established in the experimental garden without any visible symptoms of wilting and necrosis. The genetic fidelity of regenerated plants was evaluated by adapting to two PCR-based DNA marker techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) which detected no variability in the in vitro multiplied plantlets of A. calcarata. This efficient method of clonal multiplication may be useful for commercial scale multiplication, and in situ and ex situ conservation of elite germplasm of A. calcarata.

In vitro plant regeneration in rough lemon (Citrus jambhiri Lush.) through epicotyl segments by direct shoot organogenesis

The effect of Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of growth hormones on direct regeneration from one month old epicotyl segments of in vitro grown rough lemon (Citrus jambhiri Lush.) seedlings was studied. The earliest bud induction in 7.5 days, highest bud induction frequency (98.50%), percent regeneration(90.53) were obtained on MS medium supplemented with 6-Benzylaminopurine (BAP) (1mglit-1) with an average number of 12.50 buds per explants. The epicotyls segments with proliferated buds were transferred to elongation media in order to improve the recovery of normal shoots. Maximum number of elongated shoots (8.50) was obtained on MS medium having BAP (0.5mglit-1) + Gibberellic Acid (GA3)(1.0 mglit-1).These elongated shoots were then rooted on MS medium containing Indole-3-butyric acid (IBA) (0.1mglit-1) + Indole-3-aceticacid(IAA)(0.5mglit-1) with highest rooting percentage(96%) and root number(5.0). Early (10.10 days) rooting was observed in MS medium supplemented with NAA1.0 mglit-1 + IBA0.5 mglit-1.The plantlet survival was 98.52%, when plantlets were transferred to plastic pots containing a mixture of garden soil and vermiculite (1:1). The hardened plants were successfully established in the soil. The present study developed protocol which can be reliably used for in vitro regeneration of rough lemon and for gene transfer studies in rough lemon, especially to induce salinity and Phytophthora tolerance.

Screening of Five and Six-Membered Nitrogen-Containing Heterocyclic Compounds as New Effective Stimulants of Linum Usitatissimum L. Organogenesis in Vitro

Linum usitatissimum L. (flax or linseed) is one of the oldest economically important crops used in biotechnology as a source for bioactive compounds. Flax fiber and seed have industrial significance as a source of cellulosic fiber for textile and paper industry and seed oil for pharmaceutical, cosmetic and food industry. The elaboration of new effective techniques for in vitro regeneration of Linum usitatissimum L. is very actual problem today. The influence of low molecular weight five and sixmembered nitrogen-containing heterocyclic compounds on organogenesis of Linum usitatissimum L. cultivar heavenly in vitro has been studied. It was found that heterocyclic compounds derivatives pyridine, pyrimidine, pyrazole and isoflavones revealed high stimulating effect on direct shoot organogenesis of Linum usitatissimum L. in vitro. The maximal shoot regeneration frequency was obtained on the MS media containing heterocyclic compounds at the concentration 10 -8 M/l as compared to lower regeneration frequency obtained on the control MS medium containing 1 mg/l BAP and 0.05 mg/l NAA. The maximal index of explants with regeneration (in %) that exceeded almost twice the same index in control MS medium was obtained on the MS medium containing heterocyclic compound derivative of isoflavones. At the same time, the index of explants with regeneration (in %) that exceeded one and a half times the same index in control MS medium and the maximal index of shoots elongated more than 1 cm (in %) that exceeded almost twice the same index in control MS medium were obtained on the MS media containing heterocyclic compounds derivatives of pyridine, pyrimidine and pyrazole. The current study confirms perspective of using of low molecular weight five and six-membered nitrogen-containing heterocyclic compounds at the concentration 10 -8 М/l of the MS medium as new effective substitutes of traditional growth regulators auxin NAA and cytokinin BAP for regeneration of Linum usitatissimum L. cultivar heavenly in vitro.

Efficient in vitro direct shoot organogenesis from seedling derived split node explants of maize (Zea mays L.).

Maize is one of the important cereal crops around the world. An efficient and reproducible regeneration protocol via direct organogenesis has been established using split nodes as ex-plants derived from 7 to 10 day old in vitro grown seedlings. Surface sterilized maize seeds were germinated on MS medium lacking plant growth regulators. Nodal sections of 7–10 day old seedlings were isolated, split longitudinally into two halves and cultured on regeneration medium containing different concentrations of 6-benzyladenine (2.20, 4.40, 6.60, 8.80, 11.0 and 13.2 μM) or kinetin (2.32, 4.65, 6.97, 9.29, 11.6 and 13.9 μM). Inclusion of 8.80 μM BA into MS supplemented medium triggered a high frequency of regeneration response from split node explants with a maximum number of shoots (12.0 ± 1.15) and the highest shoot length (3.0 ± 0.73) was obtained directly (without an intervening callus phase) within 4 weeks of culture. Further shoot elongation was achieved on medium containing 4.40 μM BA. The elongated micro shoots were rooted on MS medium fortified with 1.97 μM indole-3-butyric acid. The regenerated plantlets with roots were successfully hardened on earthen pots after proper acclimatization under greenhouse conditions. This new efficient regeneration method provides a solid foundation for genetic manipulation of maize for biotic and abiotic stresses and to enhance the nutritional values.

Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia W. T. Wang.

An efficient protocol providing a dual regeneration pathway via direct shoot organogenesis and somatic embryogenesis for an endangered species, Metabriggsia ovalifolia W. T. Wang, was established from leaf explants. When applied at 2.5 μM, the cytokinins 6-benzyladenine (BA) or thidiazuron (TDZ) and the auxins indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce shoots when on basal Murashige and Skoog (MS) medium. BA and TDZ could induce more adventitious shoots (19.1 and 31.2/explant, respectively) than NAA (4.6/explant), IBA (5.7/explant) or IAA (6.4/explant). BA and TDZ at 5–10 μM could induce both shoots and somatic embryos. A higher concentration of TDZ (25 μM) induced only somatic embryos (39.8/explant). The same concentration of BA induced both adventitious shoots (23.6/explant) and somatic embryos (9.7/explant). Thus, somatic embryogenesis in this plant needs a high cytokinin concentration (BA; TDZ), as evidenced by histology. Somatic embryos germinated easily when left on the same media, but formed adventitious roots in two weeks on MS supplemented with 0.5 μM NAA, 0.5 μM IBA and 0.1% activated charcoal. Over 93% of plantlets survived following acclimatization and transfer to a mixture of sand and vermiculite (1:1, v/v) in trays.

Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

Solanum trilobatum L. Is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. Trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 μM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.

Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas

Nardostachys jatamansi (D. Don) DC., a small, perennial, rhizomatous herb of immense medicinal importance since ancient times, is restricted to specialized habitats of alpine Himalayas ranging from 3000 to 5200 m asl. The species has been recently listed as critically endangered under IUCN Red list of threatened species due to over exploitation of its rhizomes for medicinal uses, habitat degradation, trade and other biotic and anthropogenic interferences. An efficient protocol using both indirect and direct shoot organogenesis has been optimized for N. jatamansi. Best callusing was achieved from the cut ends of leaf and petiole explants within 15 days of culture in MS medium supplemented with 1.5 mg/l α-naphthalene acetic acid and 1.0 mg/l meta-Topolin. Culturing the explants at low temperature (13 ± 1 °C) resulted in better callus growth, shoot regeneration, hyperhydricity control and improvement in photosynthetic pigment content in regenerated shoots. Also, direct organogenesis from shoot tip and petiole explants was achieved in MS medium containing 1.0 mg/l meta-Topolin. Optimum rooting was achieved in the same medium supplemented with 1.0 mg/l indole acetic acid wherein averages of 4.52 roots/shoot were induced. Genetic stability of in vitro-derived plantlets was assessed and compared to mother plant using molecular markers and flow cytometry. Intron Splice Junction (ISJ) and Start Codon Targeted polymorphism (SCoT) marker based profiling revealed uniform banding profile in case of direct shoot organogenesis (DSO)-derived plants while callus mediated organogenesis (CMO)-derived plants showed slight variations as compared to mother plant. The genome size of N. jatamansi was found to be 2C = 1.40 ± 0.01 pg and therefore 684.6 Mbp (1C). Although organogenic calli showed mixoploidy but no major phenotypic and genetic rearrangements were detected by flow cytometry in callus-derived plants. Significantly higher antioxidant activity was observed in callus-derived plants as compared to mother and DSO-derived plants. Plant parts, regeneration pathways and various solvent systems greatly affected the yields of total phenolics, flavonoids, alkaloids, tannins contents present in the in vitro raised plantlets.

Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos of Sapium sebiferum Roxb.

Sapium sebiferium Roxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) of S. sebiferium through direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence on in vitro morphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl−1 indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl−1 6-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement of S. sebiferium.

Direct-shoot organogenesis as an alternative protocol for in vitro regeneration of oil palm (Elaeis guineensis Jacq.)

In the present study, in vitro culture via direct-shoot organogenesis was firstly established as an optional route for clonal propagation in oil palm (Elaeis guineensis Jacq.). The shoot explant excised from zygotic embryo-derived seedling was cultured on half-strength MS (½MS) medium supplemented with two cytokinins, N6-benzyladenine (BA; 8.9, 17.8, 35.5 and 71.0μM) or 6-(γ,γ-dimethylallylamino) purine (2-iP; 9.8, 19.7, 39.4 and 78.7μM). The highest percentage of shoot induction (54.2%) with 5 shoots per explant was obtained when shoot explants were cultured on ½MS medium supplemented with 9.8μM 2-iP. The frequency of direct-shoot organogenesis expressed in the term of percentage of shoot induction was significantly influenced by the duration of incubation period on shoot induction medium. The optimal incubation period of shoot explants on the induction medium (½MS+9.8μM 2-iP) was found to be 4 weeks, which improved the frequency of shoot induction up to 2.6-fold, comparing to those obtained from incubation period of 8 weeks. Histological examination of induced shoots confirmed that newly developed shoot was directly regenerated via direct-shoot organogenesis. Oil palm plants derived from a defined regeneration regime in this study were successfully established in greenhouse. The study established the incubation of 4th week-developmental stage of seedlings as initial explant on shoot induction medium (½MS medium containing 9.8μM 2-iP) with incubation period of 4 weeks as the successful protocol for the direct-shoot organogenesis of oil palm.

Total phenol content and total antioxidant activity drop during Tacitus bellus direct shoot organogenesis

Changes in total phenolics content (TPC) and total antioxidant activity (TAA) in different morphogenic stages of Tacitus bellus (L.) Moran and J. Meyran (Crassulaceae) direct shoot organogenesis in vitro were examined. A HPLC method was used for the assessment of changes in phenolic profile. Significant decrease in TPC and TAA, as well as the decline of the amount of specific phenolic compounds coincided with the initiation of shoot organogenesis. Additional decrease in TPC and TAA, and the disappearance of procyanidin glycoside, occurred associated with shoot development. Performed correlation analysis suggests phenolics as the main component of nonenzymatic antioxidant system involved in T. bellus direct shoot organogenesis, it argues in favor of involvement of specific phenolics in the regulation of early stages of T. bellus direct shoot organogenesis and indicates different fine regulatory mechanisms of early and late stages of shoot organogenesis.

Erratum to: Phyto-molecular profiling and assessment of antioxidant activity within micropropagated plants of Dendrobium thyrsiflorum: a threatened, medicinal orchid

The escalating loss of biological diversity throughout the world has become a major concern for the conservation biologists. Like other threatened plant species, the natural populations of the orchids are also severely threatened. Dendrobium thyrsiflorum is one such representative of the family Orchidaceae whose natural populations are getting destroyed at an alarming rate and deserves special conservation attention. Both direct shoot organogenesis (DSO) and indirect shoot organogensesis (ISO) pathways were experimented and the highest regeneration frequency for DSO and ISO pathways were found to be 86.2 and 96 % respectively. The regenerated shoots were best rooted in half-strength MS medium supplemented with 1 mg/l indole butyric acid (IBA) and 0.5 mg/l phloroglucinol. The genetic stability of the acclimatized plants derived from ISO and DSO pathways was assessed using Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT) molecular markers. SCoT proved to be a superior marker over ISSR in detecting clonal variability. The phytochemical analysis of the micropropagated plants also revealed a comprehensively higher yield of various secondary metabolites with significantly higher antioxidant potentials in both ISO- and DSO-derived plants over the mother plant. However, the ISO-derived plants were more phytochemically enriched compared to the DSO-plants. The rapid multiplication rate, higher genetic stability and secondary metabolite production ensures the utility of this micropropagation method for D. thyrsiflorum in the ex situ conservation and commercial exploitation of other important orchid species.

On 2D generalization of Higuchi’s fractal dimension

We propose a new numerical method for calculating 2D fractal dimension (DF) of a surface. This method represents a generalization of Higuchi’s method for calculating fractal dimension of a planar curve. Using a family of Weierstrass–Mandelbrot functions, we construct Weierstrass–Mandelbrot surfaces in order to test exactness of our new numerical method. The 2D fractal analysis method was applied to the set of histological images collected during direct shoot organogenesis from leaf explants. The efficiency of the proposed method in differentiating phases of organogenesis is proved.

English

A reproducible protocol for direct shoot organogenesis of Cleome viscosa, an important medicinal weed herb was developed. The seed explants were collected primarily from field grown mature plants for in vitro germination on different strength of MS basal medium with or without selection of GA3 at various concentration (0.1 to 1.0 mg/L). The highest rate of seed germination (55.3%) was noticed on full strength MS basal medium fortified with 0.5 mg/L GA3 after 30 days of culture. The excised 7 to 10 days old cotyledonary leaf, cotyledonary node and hypocotyls explants cultured on MS medium fortified with different concentration of individual cytokinin (BA/KIN/TDZ) alone or BA+KIN or TDZ+KIN or TDZ in combinations with different auxins (IBA/NAA/IAA) influenced the frequency of adventitious microshoot formation. The rate of shoot multiplication was greatest (100%) in cotyledonary leaf explants cultured on 3.0 mg/L TDZ and 0.3 mg/L IBA tested medium after 45 days of culture. The individual microshoots were elongated well in 0.3 mg/L TDZ and 0.1 mg/L GA3 treated medium, but more number of adventitious micro roots were developed on half strength MS medium fortified with 0.1 mg/L NAA. The regenerated healthy plants were hardened in pots containing soil mix and well established into complete state similar to that of field grown plants under greenhouse condition.   Key words: Cleome viscosa L, medicinal weed plant, shoot multiplication, direct organogenesis.

Direct adventitious shoot organogenesis and plant regeneration from cotyledon explants in Neolamarckia cadamba

Neolamarckia cadamba is a fast-growing and deciduous tropical hardwood with anatomical, morphological, and chemical characteristics that make it suitable for building materials, pulp production, and m

High efficiency direct shoot organogenesis from leaf segments of Aerva lanata (L.) Juss. ex Schult by using thidiazuron.

An efficient protocol for direct shoot organogenesis has been developed for the medicinal plant Aerva lanata (L.) Juss. ex Schult. Regeneration was achieved from leaf segments of 20 days old in vitro plantlets raised on Murashige and Skoog (MS) medium containing 0.25–2.0 mg L−1 thiadiazuron (TDZ), 3% sucrose, and 0.8% agar. After 21 days of culture incubation, maximum number of shoot organogenesis (23.6 ± 0.16) was obtained on medium containing 1.0 mg L−1 TDZ. The shoots were able to produce in vitro flowers on medium containing 1.0 mg L−1 TDZ in combination with 0.25–0.5 mg L−1 α-naphthaleneacetic acid (NAA). Histological observation showed that the epidermal cells of the leaf explants exhibited continuous cell division led to formation of numerous dome shaped meristematic protrusions and subsequently developed into adventitious shoots. Upon transfer of shootlets to half strength MS medium containing 1.0 mg L−1 indole-3-butyric acid (IBA), around 86% of the regenerated shoots formed roots and plantlets. Rooted plants were hardened and successfully established in the soil at the survival rate of 92%. The regeneration protocol developed in this study provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large scale production of its medicinally active compounds and genetic transformations for further improvement.

Induction of direct shoot organogenesis and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L. cv. ‘Green long’)

In vitro flowering is an alternative breeding tool for generating hybrid Cucumis spp. as it is able to overcome limitations caused by interspecific incompatibility. The present study describes an efficient method for induction of multiple shoots and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L.). Shoot tip explants were excised from 7-day-old seedlings and cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 6-benzylaminopurine (BAP; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L kinetin (KIN). The highest frequency (93.1%) of multiple shoot formation with maximum number of shoots (15.2 shoots/explant) was achieved on MS medium supplemented with 1.0 mg/L BAP. For in vitro flowering, shoots were cultured on MS medium supplemented with 0.5 mg/L BAP and different concentrations of sucrose. Flowering occurred on about 95% of in vitro shoots cultured on MS medium fortified with 6% (w/v) sucrose and 0.5 mg/L BAP after 15 d. For rooting, shoots (>2 cm) were cultured on MS medium augmented with various concentrations of indole-3-butyric acid (IBA; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L KIN. Among the combinations tested, supplementation with IBA (1.5 mg/L) and KIN (0.5 mg/L) induced maximum rooting rates (95.4%) with 7.8 roots/shoot. Rooted plantlets were successfully transferred into plastic cups containing a mixture of soil and sand (1:1), established in the greenhouse, and subsequently acclimatized in the field. The in vitro flowering reported in this study may facilitate rapid hybridization in Cucumis species and offers a model system for studying the physiological mechanisms involved in flowering.

Field Evaluation and Genetic Stability Assessment of Regenerated Plants Produced Via Direct Shoot Organogenesis from Leaf Explant of an Endangered ‘Asthma Plant’ (Tylophora indica) Along with Their In Vitro Conservation

Adventitious shoot buds were induced directly without intervening callus phase from the cut edge of the ‘aged leaves’ explants as well as via nodular meristemoids from the cut edge of the ‘young leaves’ of Tylophora indica an endangered medicinal climber of the family Asclepiadaceae. Shoot primordia developed directly from the cut edge of the ‘aged leaf’ explant without any callus phase within 26–30 days and maximum 18.6 ± 0.36 shoot per explant were induced on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 6-benzyl-aminopurine (BAP). Whereas maximum 25.2 ± 0.57 shoots per explant were produced from ‘young leaf’ explant via nodular meristemoids on 2.0 mg/l BAP and 0.2 mg/l indole-3-acetic acid fortified medium within 40–45 days. Multiplied shoots were successfully rooted with maximum 8.3 ± 0.36 root per shoot with 15.5 ± 0.44 cm length on half strength MS medium supplemented with 0.2 mg/l indole-3-butyric acid. Finally, the rooted plants were hardened and transferred to soil with a 93.3 % success rate. After 18 months of field transfer, a 69.0 % of regenerated plants produce flowers and 28.5 % produce fruits. Seeds of these fruits show 78.3 % viability more or less similar with in vivo grown plants with 71.7 % viability on in vitro germination, which ensures the fertility of the regenerants. These regenerated plants were conserved in vitro for up to 21 months without sub-culturing in presence of osmoticums (Mannitol and Sorbitol) and best results found on MS medium supplemented with 3 % sucrose and 2 % mannitol, where 53.3 ± 3.33 % plants survive up to 21 months and 86.7 ± 3.33 % of these survived plants could turn to normal plants after a re-growth for 2 month which showed no obvious difference in morphology. Further, clonal fidelity assessment of the micropropagated plants was carried out using cytological studies as well as using randomly amplified polymorphic DNA (RAPD) markers analysis. All the micropropagated plants have shown normal diploid 2n = 22 chromosomes, same as that of the mother plant. A result of RAPD markers analysis indicates all the regenerants are true-to-type and there were no somaclonal variations among these regenerants.

Direct Shoot Organogenesis in Murraya paniculata (L.) Jack: A Prerequisite for Genetic Transformation

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L−1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L−1 BAP and 0.4 mg L−1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L−1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.

Shoot Organogenesis and Plant Regeneration from Leaf Explants of Lysionotus serratus D. Don

The gesneriaceous perennial plant, Lysionotus serratus, has been used in traditional Chinese medicine. It also has a great development potential as an ornamental plant with its attractive foliage and beautiful flowers. An efficient propagation and regeneration system via direct shoot organogenesis from leaf explant was established in this study. High active cytokinin (6-benzyladenine (BA) or thidiazuron (TDZ)) was effective for direct organogenesis of initial induction. Murashige and Skoog (MS) growth media containing 0.5 mg L−1 BA alone or with combination of 0.1 mg L−1 α-Naphthaleneacetic acid (NAA) were the most effective for shoot proliferation. High BA concentration (1.0 mg L−1) in the media caused high percentage of vitrified shoots though they introduced high shoot proliferation rate. Histological observation indicated that adventitious shoot regeneration on the medium containing 0.5 mg L−1 BA alone occurred directly from leaf epidermal cells without callus formation. Regenerated shoots rooted well on medium containing half-strength MS medium with 0.5 mg L−1 indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), and the plantlets successfully acclimatized and grew vigorously in the greenhouse with a 94.2% and 92.1% survival rate.