Compact Callus Research Articles

Indirect Organogenesis through Seedling-Derived Leaf Segments of Ficus Religiosa - a Multipurpose Woody Medicinal Plant

The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.

MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS

Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture.Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4-D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC).Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%±1.9, 80.60%±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealed its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C. albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis.Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens.

Growth response of leucaena embryogenic callus on embryo age differences and Auxin 2,4-Dichlorophenoxyacetic acid

<p>Leucaena (<em>Leucaena leucocephala</em> cv. Tarramba) is a source of protein from the legume family. Tarramba varieties able to adapt well in tropical area such as West Nusa Tenggara and East Nusa Tenggara. This study aimed to identify the growth response and embryogenic callus morphology of lamtoro (<em>L. leucaena</em> cv. Tarramba) in embryos different ages and auxin 2,4-D levels. This research was used explants derived from lamtoro (<em>L. leucocephala</em> cv. Tarramba) as much 400 explants. this study conducted of 16 treatments with two factors, the first factor is the provision of PGR 2,4-D concentration of 0.5 mg L<sup>-1</sup>, 1 mg L<sup>-1</sup>, 1.5 mg L<sup>-1</sup> and 2 mg L<sup>-1</sup>. The second factor was the type of embryo such as mature embryo, cotyledon, heart, and pre-globular. Each treatment was repeated 25 replications. This study uses a randomized complete design (CRD) with two factor. Data were analyzed using analysis of variance and if there was significant difference, data were further analyzed using Duncan’s multiple range test. Analysis of variance showed that PGR 2,4-D at a concentration of 1.5 mg L<sup>-1</sup> and the type of optimum embryo was mature embryo callus on parameters as height, diameter increment callus, and callus color scores were significantly different (P <0.05). callus texture parameter indicates results that are uniform throughout the treatment that was compact callus. Lamtoro plant embryogenic callus (<em>L. leucocephala </em>cv. Tarramba) indicate an optimal response at the concentration of PGR 2,4-D 1.5 mg L<sup>-1</sup> and the type of embryo was mature embryo. </p>

Production and characterization of lectin from Bauhinia holophylla (Fabaceae:Cercideae) calli

Bauhinia holophylla is a woody plant found in the Brazilian Cerrado used in folk medicine to treat diabetes. Lectins are glycoproteins with several biotechnological applications and play important roles in plant defenses and in medicine. Lectins have been produced in vitro using plant tissue culture techniques. This study shows the production and characterization of lectin from B. holophylla by callus culture. Calli were obtained from leaf segments placed on woody plant and Murashige and Skoog media supplemented with 2,4-dichlorophenoxyacetic acid (0, 4.52, 9.05, 18.10 µM) or 6-benzylaminopurine (0, 4.44, 8.88, 17.75 µM), in the presence or absence of light. The highest concentrations of lectin expressed by hemagglutination activity were observed in green and compact callus induced in woody plant medium supplemented with 6-benzylaminopurine in the presence of light, which showed specificity by all types of erythrocytes tested. The highest concentrations of lectin (128 HU mg−1) and fresh weight of callus were observed in the stationary phase (41st–60th day). Lectin was not detected in subcultured calli. d-Galactose promoted the highest inhibitory effect on the lectin activity in human erythrocytes ABO system, especially B-type. Lectin has been shown to be a stable protein, maintaining its hemagglutination activity after incubation at a wide range of temperatures, pH values and ethylenediaminetetraacetic acid concentrations.

Anti-metastatic Potentiality of Purified Anthocyanin from Osbeckia aspera (L.) Blume. and O. reticulata Bedd. Against Selected Human Cancer Cell Lines

To evaluate the anti-metastatic potentialities of purified anthocyanin from Osbeckia aspera (L.) Blume. and O. reticulata Bedd. against selected human cancer cell lines such as HT29 colon, MG63 bone and HeLa cervical by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and DNA fragmentation test. Anthocyanin was extracted from the in vitro callus culture of the Osbeckia species, purified using amberlite column chromatography and fractionated by LC-MS/MS. Anthocyanin producing callus cultures were trialed on MS medium fortified with various combinations of phytohormones and sucrose. Significant callus formation in O. aspera was initiated in cultures containing 0.5 mg/L of 2, 4-D and 0.5 mg/L BA, while that in O. reticulata was initiated with 1.2 mg/L BA and 1.4 mg/L NAA. The same hormonal combinations on sub-culturing turned white friable callus into red compact callus. Purified anthocyanins obtained from O. aspera and O. reticulata contained Malvidin-3 -diglucoside, delphinidin, cyanindin aglycone and Peonidin. Osbeckia species displayed differential responses against the HT29 colon, MG63 bone and HeLa cervical cancer cell lines in terms of IC50 values of toxicity. O. aspera was more effective against HeLa cervical cell lines (23.7μg/ml) followed by HT29 colon (64.7μg/ml) as compared to O. reticulata. Poor selectivity index was noticed with bone cancer cell lines. The results were substantiated by apoptotic analysis and DNA fragmentation results. The overall results suggest that the purified anthocyanin of O. aspera and O. reticulata was excellent as antimetastatic and warrant further studies to isolate novel compounds for chemotherapeutic use.

Establishment of a Protocol for in vitro Seed Germination and Callus Formation of Ficus religiosa L., an Important Medicinal Plant

Background: Ficus religiosa is known as a pure source in traditional medicine for the treatment of diabetes, asthma, diarrhea, gastric problems, epilepsy, sexual, infectious, and inflammatory disorders. Despite the fact that many studies have authorized its traditional medicinal uses, yet these utilized raw extracts have not been yet characterized. Therefore, there is a necessity for standardizing its phytochemical features and recognizing bioactivity, guided by bioactive metabolites. Objectives: In this study, the effect of light and dark condition, different strengths of Murashige and Skoog (MS) medium, and their interactions on seed germination and the effect of plant growth regulators on callogenesis of F. religiosa via leaf, petiole, root, and internode explants were investigated. Results: The results demonstrated the highest seed germination percentage was achieved at one-tenth strength of MS medium under the light condition. Also, the highest callus fresh weight was obtained from media supplemented with 0.5 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.05 mg/L 6- benzyl amino purine (BAP) in all explants. The MS medium containing indole-3-butyric acid (IBA) produced greenish and compact calli while yellowish and compact calli was achieved in α-naphthalene acetic acid (NAA) and finally 2,4-D produced yellow-brownish calli. Conclusions: To the best of the author’s knowledge, this study was the first report of seed germination and callus induction through immature explants of F. religiosa. The seed germination and callogenesis system established in this study could be applied in the future for the enrichment of certain secondary metabolites as well as the production of new secondary metabolites, with the purpose of understanding and use of medicinal properties for this valuable germplasm.

In vitro shoot regeneration and enhanced synthesis of plumbagin in root callus of Plumbago zeylanica L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47 ± 0.63 mg g−1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS + 5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08 ± 1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3 ± 1.36 roots with an average length of 15.31 ± 2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L−1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.

In Vitro Adventitious Shoot Regeneration through Direct and Indirect Organogenesis from Seedling-derived Hypocotyl Segments of Ficus religiosa L.: An Important Medicinal Plant

Ficus religiosa is an important industrial, medicinal, and ornamental plant, so in vitro regeneration is of high paramount in this valuable germplasm. Two efficient protocols were developed for indirect and direct shoot organogenesis through hypocotyl explants. In the first experiment, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and indole butyric acid (IBA) (0.5, 1.0, and 1.5 mg·L−1) in combination with 6-benzyl amino purine (BAP) (ratio 10:1, respectively) were used for callus formation. Two types of callus were obtained from different concentrations of plant growth regulators (PGRs). Also, 2,4-D produced yellow-brownish and friable callus (Type I), whereas the green and compact callus (Type II) was achieved in IBA. The highest callus fresh weight (2.43 g) was observed in Murashige and Skoog (MS) medium containing 0.5 mg·L−1 2,4-D plus 0.05 mg·L−1 BAP. In the later experiments, various concentrations of thidiazuron (TDZ), 6-furfuryl amino purine (KN), and BAP (0.25, 0.5, 1.0, and 1.5 mg·L−1) in combination with IBA (ratio 10:1, respectively) were applied for shoot regeneration (direct and indirect organogenesis). In shoot regeneration from callus, the highest regeneration frequency (86.66%) and shoot number per callus (4.13) were achieved in MS medium supplemented with 1.5 mg·L−1 BAP plus 0.15 mg·L−1 IBA from type I calli. However, no regeneration was observed in type II calli. In direct shoot regeneration, the highest regeneration frequency (96.66%) and shoot number (6.26) were obtained in the medium mentioned previously. In root induction experiment, different concentrations of naphthalene acetic acid (NAA) and IBA alone or in combination were applied, and MS medium containing 2.0 mg·L−1 IBA along with 0.1 mg·L−1 NAA was the best hormonal balance for root induction. The rooted plantlets’ survival rate was more than 95% in the acclimatization stage. These results demonstrated that the direct regeneration method provides more shoot regeneration frequency and take a less time for shoot organogenesis than the indirect regeneration method. Based on our knowledge, this study is the first report of direct and indirect shoot organogenesis of F. religiosa via hypocotyl from in vitro–grown seedling.

VALIDATION OF HEPATOPROTECTIVE AND ANTIOXIDANT ACTIVITY OF FOLK LORE KNOWLEDGE OF PURIFIED ANTHOCYANIN FROM CELL SUSPENSION CULTURE OF CLERODENDRON INFORTUNATUM L.: A SEARCH

ABSTRACT Objective: To validate the hepatoprotective and antioxidant activity of purified anthocyanin extracted from the cell suspension culture of Clerodendron infortunatum Linn. Methods: A protocol has been developed for the induction of callus proliferation from leaf and nodal explants of C. infortunatum. The explants were inoculated on MS medium supplemented with diverse combinations of 2,4-D and BAP for triggering callus formation. Subsequently, the green compact callus has been sub-cultured in the medium fortified with 2,4-D and Kinetin for anthocyanin synthesis. Cell suspension culture was also established and the elicitor, salicylic acid was used for triggering anthocyanin synthesis. Three different chromatographic columns (solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7 + Sephadex LH 120 sorbents) were employed to purify the in vitro synthesized anthocyanin from cell suspension cultures. For purity evaluation HPLC and molar absorptivity assay were used. Further, hepatoprotective and antioxidant activity was evaluated comparing with silymarine, as standard in rats. In vitro antioxidant scavenging activity was analysed by DPPH, FRAP and ORAC assay.Results: After 1 month, the leaf explants yielded remarkable green compact callus on MS medium containing 2.0 mg/l BAP and 0.5 mg/l 2,4-D. Salicylic acid enhanced anthocyanin synthesis. The mean purity values obtained by HPLC were 90.9% ± 1.9 and 80.60% ±2.3 for Oasis MCX, Amberlite XAD-7 + Sephadex LH-20 column respectively. However, the purity calculated by molar absorptivity was found to be less. The highest purity achieved using molar absorptivity analysis was with MCX cartidges i.e., 85.9 ± 3.8%. HPLC yielded 12 anthocyanin fractions. Remarkable antioxidant scavenging activity was noticed as revealed by DPPH, FRAP and ORAC assay. The hepatoprotection activity (25, 50, 100 mg/100g b.w) was compared with silymarine (25 mg/kg b.w) against CCl4 induced toxicity. Anthocyanin extract improved the AST, ALT and recovered the activity of kidney function by decreasing the urea and creatinine content. In addition, the administration of anthocyanin significantly inhibited the oxidative stress via its scavenging of the reactive oxygen species formed by CCl4 stress. Further, a decrease in the MDA, H2O2, NO accumulation and increase of GSH content were noticed. Similarly, improved lipid profiles, LDL and HDL levels were also observed suggesting that anthocyanin significantly suppress the toxicity via its activation of antioxidant enzymes (GST, CAT and SOD).Conclusion: The overall results showed that the purified anthocyanin of C. infortunatum function as an antioxidant and there by hepatoprotective protection against CCl4 inducedtoxicity in animal models.

Analysis of genetic variations in Musa paradisiaca cv Karibale-Monthan using AFLP markers

In the present study, the genetic variations of leaf calli regenerants of Musa paradisiaca cv Karibale-Monthan has been analyzed using AFLP markers. The highest (86.66%) frequency of compact callus formation was obtained when leaf explants were cultured on MS media, containing 3mg/L 2,4-D and 0.5 mg/L BAP. The optimum shoot formation and multiplication (5.70±1.49) was achieved from the leaf derived callus on MS medium fortified with 3mg/L BAP+0.5mg/L TDZ. The 3-4 cm small shoots produced distinct roots on MS media containing 0.5mg/l NAA with 0.2% activated charcoal. The AFLP analyses produced 1012 monomorphic bands from total 1094 bands with 7.5% polymorphism between mother plant and callus derived regenerants. The percentage of monomorphism for individual primer combinations was very high and varied from 90.3% (EcoRI-AG×MseI-CCG) to 97.6% (EcoRI-AT×MseI-CGA). The result showed lack of polymorphism is due to genetic integrity of the plants that was not altered indifferent to the hormonal treatment.

In vitro propagation of Xao tam phan (Paramignya trimera (Oliv.) Guill.)

Paramignya trimera (Oliv.) Guill., a woody climber commonly known as "Xao tam phan", has been used in Vietnamese folk for the treatment of numerous cancers. Due to word of mouth about the anticancer properties of this plant, its stems and roots have been overexploited leading to the serious decline of this species in Phu Yen, Khanh Hoa and Ninh Thuan provinces. The aim of the study was to establish an in vitro propagation protocol for the conservation of P. trimera. In this research shoot clusters (5–8 shoots/cluster) were regenerated from axillary bud explants of 1–3 year-old trees after 3 months of cultures on the WPM (woody plant medium) supplemented with STS 3 and BA 5–7 mg/L. STS (silver thiosulfate) was used to prevent the leaf abscission. These shoot clusters grew slowly and reached 1–3 cm in heights after 4 months of the cultures. These shoot clusters did not form any roots after 2 months of culture on the rooting media with IBA and/or NAA 1–5 mg/L. However, there was 51 % of the treated shoot clusters acclimatized and produced new stem and leaves after 2 months growing in greenhouse. WPM supplemented with STS 3, BA 5 and IBA 5 mg/L showed the best response for callus induction in leaf explants after 3 months of cultures. Among the callus types, the milky white compact calli were induced at the cut surface of leaf explants after 3 months of the cultures and became the compact and nodulated calli within 4 weeks later.

Morphogenetic Responses in Anthurium Andreanum (Hort) Cultivars

In this study, in vitro morphogenetic responses have been studied in popular Anthurium andreanum cultivars Tropical Red, Acropolis, Sun Glow, Esmeralda, Chaco, Pistachio, Neveda and Safari employing explants of various types and growth stages, through indirect organogenesis. Creamy and compact callus initiated in cut ends along the veins of Leaf lamina with mid rib (LLMR) and petiole with leaf (PL) explants of various cultivars in 30-120 days of inoculation, it was sub cultured and incubated in dark for proliferation. Leaf tip, candle and petioles did not show any kind of response, turned brown and died later. Explants from young pale green leaves with smooth structure showed early response in all cultivars. Callus proliferation rate was 3.0-5.0 times 30-35 days of incubation in various cultivars. Shoot bud initiation and elongation observed after 45-60 days incubation in dark. Number of shoot buds per explants ranged from 30-60 per explants and 8-10 buds attained 1.5-2.5 cm in height. Then, shoots were separated, inoculated in shoot elongation medium and incubated in light. Remaining callus was sub cultured in callus multiplication medium and incubated in dark. Callus and shoot multiplication ratio was 1-2 and 2.5-4.2 times respectively. Shoots attained 4-5.3 cm in height in 45-55 days, rooted and ready for hardening in 65-80 days. Rooted plantlets were hardened in 1:1 ratio soil rite and coco-peat mix in portrays, transferred to beds prepared with coconut coir-pith, 95% plants survived, appeared normal and flowered. In this paper, materials, methodology, results will be discussed.

Use of compact and friable callus cultures to study adaptive morphological and biochemical responses of potato (Solanum tuberosum) to iron supply

The efficient utilization, storage and buffering of iron (Fe) is necessary to prevent the development of iron deficiency or toxicity related stresses in plants. It is of practical interest to develop effective in vitro plant tissue culture schemes for generating chlorosis-tolerant and/or Fe-biofortified plants. The objectives of this study were to induce the formation of compact and friable calli from leaf explants of potato and to investigate adaptive responses to Fe nutrition using these in vitro cell culture-based systems. The severity of chlorosis of the callus cultures increased with the withdrawal of Fe supplementation from the growth medium and the culture duration. Ferric chelate reductase (FCR) activity was found to be higher in the compact compared to the friable potato callus cultures. Chlorophyll and carotenoids content in compact calli increased with an increase in Fe supply but in friable calli carotenoids content declined and the chlorophyll level was unaffected. Phenolic content in Fe-deficient compact calli was significantly higher than in friable ones but a reverse trend was observed as the culture period was extended. Peroxidase activity was reduced in both compact and friable calli cultured under Fe-deprived condition compared to those on Fe-sufficient medium. Friable and compact calli displayed differential morphological and biochemical responses to Fe deficiency. These calli also appeared to vary in their sensitivity to Fe-deficiency and the response to Fe nutrition seemed to be related to the culture duration.

Role of Explants and NAA on Callus Induction of Potato (<i>Solanum tuberosum</i>)

An experiment was conducted to study the role of NAA and different explants on callus induction of potato varieties. There were three factors such as variety (Diamant, Heera and Cardinal), explants (leaves and internodes) and NAA levels (0, 2, 3, 4, 5 mg L^-1). The experiment was laid out in complete randomized design with three replications. Minimum 6-8 days was required for callus initiation of internode of Cardinal in 3 mg L^-1 NAA while the maximum 22-25 days was required for internode of Cardinal in 5 mg L^-1 NAA. Both the leaf and internode of Heera produced 100% callus while Cardinal produced minimum callus (37.7 and 20.8% for leaf and internode, respectively) in 5 mg L^-1 NAA. Neither leaf nor internode produced callus without NAA. Internode of Diamant, Heera and Cardinal produced 100% compact calli in 4, 3 and 5 mg L^-1 NAA respectively. Leaf of Diamant without NAA produced the highest weight (0.667 g) of callus after one month. After two months, internode of Cardinal showed the highest weight (0.205 g) of calli in 3 mg L^-1 NAA. Therefore, the present protocol has the potential for the rapid multiplication of true-to-type clones without changing the genetic fidelity.

Improvement of shoot proliferation and comparison of secondary metabolites in shoot and callus cultures of Phlomis armeniaca by LC-ESI-MS/MS analysis

Phlomis armeniaca Willd. is a medicinal plant in the Lamiaceae family endemic to Turkey. The present study describes efficient plant regeneration and callus induction protocols for P. armeniaca and compares phenolic profiles, total phenol and flavonoid contents, and free radical scavenging activity of in vitro-derived tissues. Stem node explants from germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with 75 plant growth regulator (PGR) combinations. The highest shoot number per explant, frequency of shoot proliferation, and frequency of highly proliferated, green, compact callus were obtained on MS medium containing 0.25 mg L−1 thidiazuron (TDZ) and 0.25 mg L−1 indole-3-acetic acid (IAA). The best root formation was on MS basal medium (control). Methanol extract of leaves obtained from regenerants contained higher total phenol and flavonoid contents than the callus extract. The callus extract showed stronger free radical scavenging activity than leaves with IC50 [concentration inhibiting 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical] values of 4.30 ± 0.08 and 2.21 ± 0.04 mg g−1 dry weight in leaves and callus, respectively. Apigenin, caffeic acid, p-coumaric acid, luteolin, rutin hydrate, vanillic acid, ferulic acid, salicylic acid, sinapic acid, and chlorogenic acid were detected by liquid chromatography–electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS/MS) analysis in in vitro-grown leaves and callus tissue. Rutin hydrate, p-coumaric acid, and vanillic acid were found at approximately tenfold higher levels in callus than in leaves. This new micropropagation protocol, the first for P. armeniaca, could be used in industrial production for new herbal tea and germplasm conservation.

Callus culture and in vitro production of anthraquinone in Gynochthodes umbellata (L.) Razafim. & B. Bremer (Rubiaceae)

Gynochthodes umbellata is a rare medicinal plant used in traditional systems of medicine for curing various ailments. It is a woody climber belonging to the family Rubiaceae. Its over collection from the wild population resulted the rapid depletion of this species. There is an urgent need to conserve this species and also establish an alternative system for the production of bioactive compound in vitro without harming the wild population. The present investigation was carried out to analyse secondary metabolite (anthraquinone) production in callus cultures of G. umbellata. In vitro production of anthraquinone through callus cultures of G. umbellata was standardized using in vitro leaves, derived from the nodal explant cultures maintained in Murashige and Skoog solid medium containing 2mg/l 6-benzylaminopurine and 3% sucrose. Leaves were harvested from the healthy shoots of axenic cultures and culture conditions were optimised for the establishment of callus. In vitro derived leaves (1–2cm2) were inoculated onto Murashige and Skoog medium containing different individual concentrations of 2, 4-Dichlorophenoxyacetic acid (0.5–5mg/l), indole-3-butyric acid (0.5–5mg/l), indole-3-acetic acid (0.5–5mg/l) and 1-naphthaleneacetic acid (0.5–5mg/l). Of the different auxins tried for callus induction, 2, 4-Dichlorophenoxyacetic acid was found to be the most suitable auxin with different concentrations for the induction of friable yellow callus. 1-Naphthaleneacetic acid doesn’t show any callus formation while indole-3-butyric acid and indole-3-acetic acid produced greenish white compact callus. A maximum fresh weight (1.7953±0.61g) and dry weight (0.1149±0.04g) of the friable yellow callus and highest amount of anthraquinone content (18.1875±0.58mg/g dw) were recorded in MS medium supplemented with 1mg/l 2, 4-Dichlorophenoxyacetic acid. From the present study it revealed that the callus culture is an effective method for the production of anthraquinone in vitro in G. umbellata. Anthraquinone production through in vitro leaf segment derived callus culture is a viable system for the production of this pharmacologically and industrially important natural plant pigment.

Callus induction and plant regeneration from lateral shoots of herbaceous bamboo Mniochloa abersend

ABSTRACTCallus induction and plant regeneration of Mniochloa abersend via lateral shoots were conducted in this study. Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L naphthaleneacetic acid (NAA) was effective for compact callus induction. Remarkably, calli on the MS medium with 0.1 mg/L 2,4-D yielded the highest folds of proliferation (8.01), and showed a high potential capacity to differentiate 1 year after subculture. In addition, the compact calli possessed 100% differentiation rate and generated more shoots that were green and strong in 1.0 mg/L kinetin and 1.0 mg/L NAA. Vigorous roots were generated in the 1/2MS supplemented with 0.5 mg/L indole-3-butyric acid, and the resultant plantlets exhibited 90% survival rate after they were hardened and transplanted. The established regeneration system of M. abersend provides a promising platform for bamboo gene function study.

UVC-priming mediated modulation of forskolin biosynthesis key genes against Macrophomina root rot of Coleus forskohlii⿿A tissue culture based sustainable approach

Coleus forskohlii is susceptible to root rot disease that reduces yield of root specific metabolite forskolin. In this communication, we first time reported sustainable management of Macrophomina phaseolina root rot in C. forskohlii via Ultraviolet-C (UVC)-hormesis using tissue culture. Compact calli were developed using 2,4-dichlorophenoxy acetic acid (2.0mgl⿿1) and tolerance level of UVC exposure was optimized. Calli exhibited 88% viability, 8.4% relative growth in biomass and higher absorbance in 2,3,5-triphenyl tetrazolium chloride analysis subsequent to 10min of UVC exposure. Pathogenicity of M. phaseolina was reduced in UVC-primed calli-regenerated plants compared with un-primed ones under controlled condition. Plant height, leaf number, shoot and root biomass significantly improved in UVC-primed calli-regenerated plants over un-primed ones when challenged by M. phaseolina in glasshouse. Significant elevation of economically important forskolin via up-regulation of key forskolin biosynthesis genes GGPP synthase, CfTPS2 and CfTPS3 highlighted the potentiality of UVC-priming to boost plant⿿s defence mechanism and protect from drastic economic loss.

Callus induction and bioactive phenolic compounds production fromByrsonima verbascifolia(L.) DC. (Malpighiaceae)

This study developed a methodology for callus induction in leaf segments of B. verbascifolia and evaluated the bioactive phenolic compounds production. Leaf explants were cultured in MS medium with 30 g L-1 sucrose, solidified with 7 g L- 1 agar supplemented with 2,4-D (0; 4.52; 9.05; 18.10 µM) and BAP (0; 4.44; 8.88; 17.75 µM ) in the presence and absence of light. Forty-five days after inoculation we assessed the percentage of callus induction, color, consistency, fresh and dry matter, total phenols, flavonoids, tannins contents, and chromatographic profile by HPLC-DAD method. Callus induction occurred only in medium with growth regulators. Maximal induction (100%) was found in medium containing 2,4-D combined with BAP in the presence and absence of light. We obtained friable and compact callus in yellow, green, and red. Culture media containing 4.52 µM 2,4-D + 4.44 µM BAP induced 100% of friable callus with higher fresh and dry weight in the absence of light. The callus produced higher amounts of total phenols and flavonoids than the initial explant. Total tannins were detected only in callus induced in media containing 17.75 µM BAP and 4.52 µM 2,4-D + 17.75 µM BAP, and were not found in the initial explant. The bioactive phenolic compounds detected are derived from benzoic, p-coumaric, cinnamic, gallic acids, and catechins.

Establishment of in vitro regeneration from petiole explants and assessment of clonal fidelity by ISSR markers in Luffa acutangula L.Roxb

An improved in vitro organogenesis was achieved from petiole explants of Luffa acutangula L.Roxb, a medicinally and economically important cucurbitaceous member. Morphogenetic response of petiole explants excised from in vitro grown plants has been studied with different concentrations of auxins and cytokinins. Higher percent of callus induction was occurred on Murashige and Skoog (MS) medium supplemented with 2mg/L BAP and 0.2mg/L NAA from the cut ends of petiole explants after 4 weeks of culture. Multiple shoots (8.28±0.85 shoots per explant) were induced after 2-3 weeks from the green compact callus upon on to MS medium with 30g/L sucrose, 2.0mg/L BAP along with 0.2mg/L IAA. The elongation of proliferated shoots was achieved on same medium supplemented additionally with 0.3 mg/L GA3. The elongated shoots (1cm) were rooted on MS medium supplemented with 0.5 mg/L IBA. Rooted plants were acclimatized in the greenhouse and subsequently established in soil with 85% survival rate. Clonal fidelity of in vitro raised plantlets was determined using ISSR marker technique. The results showed the similar banding pattern with different ISSR primers ranging from 250bp to 1000bp and indicated the absence of polymorphism in donar mother plant and as well in in vitro regenerated plants.