Green Compact Callus Research Articles

Scanning electron microscopic and morphological studies on in-vitro derived callus of low-chill peach (Prunus persica L. Batsch)

In this investigation the effect of collection seasons of explants (winter, spring and summer), type of explants (leaf disc and intermodal segments) and length of explants (0.5, 1.0 and 1.5 cm) for callusing in low-chill peach were standardized. The maximum callus induction (97.78%) in the low-chill peach was obtained from the intermodal segments of 0.5 cm in length used as an explant collected during spring season. The structural changes on the surface of the callus (5–7 weeks old yellowish green compact callus) during the progress of somatic embryogenesis of low-chill peach from the both intermodal segment as well as leaf disc derived callus were also examined with the use of scanning electron microscope (SEM). The SEM studies indicated that callus derived from internodal segment explant had the highest frequency of somatic embryos than callus from leaf discs. The SEM investigation, also demonstrated the sequential events/steps leading to low-chill peach somatic embryogenesis which was originating from somatic embryo mother cells through one unicellular pathway. Two types of calli were morphologically distinguished in both leaf disc and intermodal segment generated callus and these were the compact, well organized yellowish green embryogenic callus, containing large number of small, rich cytoplasmic, starch containing meristematic cells and soft and unorganized non-embryogenic callus containing sparsely cytoplasmic, vacuolated, and large cells devoid of metabolic reserves. The present SEM studies clearly demonstrated that somatic cells from peach explants generated callus could develop into fully differentiated somatic embryos through the characteristic embryological patterns of differentiation.

In vitro Propagation of Endemic Species Mahonia Jaunsarensis Ahrendt Through Callus Culture.

Mahonia jaunsarensis Ahrendt (Family Berberidaceae) an endemic species was successfully propagated in vitro. An efficient propagation protocol has been developed first time. The callus cultures were established from leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D; 1µM) and resulted 70% callus induction with green compact callus. When callus was transferred to MS medium containing Thidiazuron (TDZ; 0.75µM), maximum average number of shoot (3.06) produced but shoot length (3.37cm) and average leaf number (2.87) was increased upon transfer to MS medium containing N6-benzylaminopurine (BA; 6.0µM) plus α-naphthalene acetic acid (NAA; 0.5µM). In MS medium containing indole-3-butyric acid (IBA; 0.01µM), the maximum rooting percentage (56%) and average root number (2.56) per shoot and root length (3.33cm) were recorded. The rooted plantlets transferred in vermiculite + garden soil + farmyard manure (1:1:1) with maximum (55%) survival percentage under greenhouse condition. The phytochemical analysis of leaves obtained from tissue culture-raised plants revealed significantly higher levels of alkaloids (berberine and palmatine) than those obtained from wild plants. Similar trends were observed for antioxidant and antimutagenic activities. Results of this study offer a baseline for the conservation and sustainable utilization strategies forM. jaunsarensis.

Study on Induction and Culture of Callus of Citrus grandis cv. ‘Hongmianmiyou’

Citrus grandis cv. ‘Hongmianmiyou’ is mutated from ‘Guanximiyou’, which has been widely cultivated in Sichuan Province because of the attractive appearance, rich nutritional ingredients and trace elements. To effectively cultivate more Hongmianmiyou seedlingsfor production, we established the callus tissue induction system. The basic nutrient medium consisted of MS medium with 3% of sucrose and 0.8% of agar, adjusted to pH 5.8.Various concentrations of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic, as well as polyvinyl pyrrolidone and active carbon, and culture condition were carried out to developprotocols for the in vitro propagation of pummelo. The appropriate formula was MS mediumsupplemented with 1 mg•L−1of 2,4-D,0.5 mg•L−1of 6-BAand 0.05% of active carbon. Explants were cultured maintained under a 16 h photoperiod provided by cool white fluorescent light 1200 Lux after cultured at dark for 7 days. Light green compact callus induced from semi-lignified stems explant of spring shoots in ‘Hongmianmiyou’, reaching 97% induction rate.

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.

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.

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.

Hormone-Induced Indirect Regeneration Protocol for Scutellaria baicalensis Georgi (Huang-qin)

There is no report available to date on somatic embryogenesis or even callus formation and plant regeneration in Scutellaria baiclensis (Huang qin). Ours is the first report on somatic embryo development in Scutellaria baicalensis. Plant growth regulators Thidiazuron [TDZ: N-phenyl N'- (1,2,3-thidiazol-5-ylurea)], 6-benzyladenine (BA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf, internode, nodal portion, and root explants of Scutellaria baicalensis. The TDZ (4 mg/l) showed the highest percentage of calli-induction frequency in various explants, viz., leaf (42.2%), nodal stem portion (62.9%), and root (63.4%). The TDZ-derived calli were incubated on MS salt + B5 vitamin medium containing glutamine (1.5mM) (MSE) and fortified with various combinations of TDZ, BA, and 2,4-D for induction of somatic embryos. No somatic embryogenesis was recorded in any culture at this stage. On transferring the above friable calli onto hormone-free MSB medium and after 12 days of culture, several globular and heart-shaped embryos appeared over the surface of green compact callus. The mature somatic embryos were dissected out and cultured on hormone-free MS medium for germination. Plantlets were hardened in a growth chamber and, after 30 days, transferred to greenhouse and subsequently to pots and field. There was no morphological difference in plantlets regenerated by this procedure and normal seed-grown plant.

In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate β-cells regeneration and anti-diabetic activity in Wistar rats

A methanol extract of Gymnema sylvestre leaf and callus showed anti-diabetic activities through regenerating β-cells. Optimum callus was developed under stress conditions of blue light with 2,4-D (1.5 mg/l) and KN (0.5 mg/l), which induced maximum biomass of green compact callus at 45 days, as determined by growth curve analysis. Leaf and optimum callus extracts contains gymnemic acid, which was analyzed using TLC, HPTLC and HPLC methods. The research reported here deals with leaf and callus extracts of G. sylvestre, which significantly increase the weight of the whole body, liver, pancreas and liver glycogen content in alloxan-induced diabetic rats (Wistar rats). The gymnemic acid of leaf and callus extracts significantly increases the regeneration of β-cells in treated rats, when compared with the standard diabetic rats. It could have potential as a pharmaceutical drug for insulin-dependent diabetes mellitus (IDDM).

Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l−1 benzyladenine (BA) and 0.1 mg l−1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l−1 BA along with 1.0 mg l−1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l−1 thidiazurone (TDZ) and 0.1 mg l−1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l−1 BA and 0.1 mg l−1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.

Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium Sweet Sugar

Segments taken from flower-stalk internodes of Oncidium Sweet Sugar formed somatic embryos and shoot buds directly from wound surfaces or via nodular masses proliferation within 1.5 months, when cultured on a Gelrite-gelled 1/2-MS basal medium supplemented with thidiazuron (0.1–3 mg l−1) in darkness. In light, when subcultured, these nodular masses proliferated into green compact callus, and produced somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1–1 mg l−1 NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures. Somatic embryos that directly formed from wound surfaces of flower stalk explants usually developed into abnormal structures, but the callus-derived embryos could germinate into PLBs and eventually developed to normal plantlets on a hormone-free basal medium for 3–4 weeks. Both the embryo-and shoot bud-derived regenerants developed into healthly plantlets when potted in sphagnum moss and acclimatized in the greenhouse.

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N6-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.

A PROTOCOL FOR HORSEGRAM [MACROTYLOMA UNIFLORUM (LAM.) VERDC.] CALLUS INDUCTION

Calli of Macrotyloma uniflorum (Lam.)Verdc.) Co.1 and Bgm.1 were established from expiants of epicotyl, hypocotyl, cotyledon, and young leaf on MS medium supplemented with auxins and cytokinins (2, 4-D, IAA, NAA, BAP, KN, and CM). When they were cultured subsequently on various combinations and different concentrations, BAP and 2,4-D were found to yield green compact callus in all expiants. Yellow friable calli were obtained on MS medium supplemented with auxins (2,4-D or NAA or IAA). Browning of callus was observed at a higher concentration of (2.5 mg1−1) auxins after 25 days.

In VitroShoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgarisL.)

Procedures are described for producing axillary shoots from seedling apices and adventitious shoots from petioles and leaf derived callus of sugar beet cultivars. The rate of adventitious shoot regeneration from petioles was influenced by temperature, BAP concentration of the medium, and the time in culture of the seedling apices from which the petioles were excised. Petiole sectioning confirmed that adventitious shoots originated in the sub-epidermal parenchyma. Two distinct types of callus were produced from leaf explants, but only white friable callus was capable of shoot development. This callus developed from brown tissue and was composed of thin-walled cells with dense cytoplasm and prominent nuclei. Green compact callus with thick-walled lignified cells developed from green tissue, but did not produce shoots. Successful seed sterilization and shoot regeneration from petiole expiants and callus was cultivar-dependent. Adventitious shoots were rooted and successfully transplanted to potting compost under glasshouse conditions.

Optimized conditions for callus induction, plant regeneration and alkaloids accumulation in stem and shoot tip explants of Phyla nodiflora

The present study describes callus induction and the subsequent plant regeneration with alkaloids accumulation instem and shoot tip explants of Phyla nodiflora. Both explants were cultured on different media (MS, B5, SH and WPM) for callus induction. Stem explants showed better callus biomass (dry weight) than shoot tip explants with green compact callus when cultured on MS medium containing 1.5 mg L–1 α-naphthalene acetic acid. Shoots were regenerated from the callus on MS medium with 1.5 mg L–1 α-naphthalene acetic acid and 1.0 mg L–1 benzyl adenine. The rooting of all regenerated shoots was successfully performed on half-strength MS medium with 1.0 mg L–1 indole-3-butyric acid. The plantlets were acclimatized and established in soil (90%) and exhibited morphological characteristics similar to those of the mother plant. In addition, the alkaloids content was higher in regenerated callus than intact stem and shoot tip explants, which were analyzed by a gravimetric method, TLC (thin layer chromatography) and HPTLC (high performance thin layer chromatography). The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species

In vitro Regeneration and Flower Induction on Solanum nigrum L. from Pachamalai hills of Eastern Ghats

Explants of Solanum nigrum L., collected from Pachamalai hills callused successfully on MS basal medium supplemented with IAA and BAP. The highest frequency of green compact callus and multiple shoots were obtained on MS containing 2.0 mg/l IAA and 0.5 mg/l BAP. The callus when cultured on MS basal medium fortified with different concentrations of BAP (3.0 - 8.0 mg/l) and IAA (0.5 mg/l) showed multiple shoot formation. The highest frequency of multiple shoots was obtained on MS containing 6.0 mg/l BAP and 0.5 mg/l IAA. For in vitro flowering, the node explants were cultured on MS fortified with different concentrations of BAP (2.0 - 7.0 mg/l) and NAA (0.5 mg/l). The highest number of multiple shoots were obtained in MS supplemented with 6.0 mg/l BAP and 0.5 mg/l NAA. The in vitro flowering was observed on MS containing 2,4-D and BAP 1.5 mg/l, respectively. The best rooting was obtained on MS containing 0.5 mg/l IBA. The well-rooted plants were hardened and finally planted in the garden. Key words: In vitro studies, Medicinal plant, Solanum nigrum, Node, Callus D.O.I. 10.3329/ptcb.v18i1.3264 Plant Tissue Cult. & Biotech. 18(1): 43-48, 2008 (June)