Increased isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa by elicitors

378-382Cell cultures of Pueraria tuberosa were established in modified Murashige and Skoog medium and challenged with yeast extract (YE), methyl jasmonate (MeJA) and salicylic acid (SA). Maximum isoflavonoids production was recorded at 48 h of YA incorporation at the stationary phase. 20 μM of MeJA and SA was most effective in isoflavonoids induction. Higher concentrations of these elicitors were negatively correlated with the isoflavonoids production. YE at 150 mg L⁻¹ was optimal for isoflavonoids production, yielding 10 mg L⁻¹ isoflavonoids, which was ~20% higher over the yields at optimal concentrations of MeJA and SA. YA incorporation can be used as a trigger to induce high yield of isoflavonoids

Studies on somatic cell variability in <i>Commiphora wightii</i> (Arnott.) Bhandari for guggulsterone production

532-536More than 125 clones were established in Commiphora wightii (Arnott.) Bhandari by aggregate cloning from stock cultures maintained on MS medium supplemented with 0.25 mg/l 2,4,5-trichlorophenoxyacetic acid and 0.1 mg/l kinetin. A few high productive clones were isolated while majority (>80%) were low productive. In the present paper, the somatic cell variability in callus cultures of C. wightii and their growth in shake flasks and bioreactor for high yield of guggulsterones has been reported. A high productive clone yielded 80μg/l guggulsterones when grown in cell suspension culture in shake flasks and 2 litre stirred tank bioreactor. The colour variations of the cultures (due to anthocyanin accumulation) were also explored to isolate high guggulsterone producing lines; however, no correlationship could be established between anthocyanin accumulation and guggulsterone contents of the clones

Effect of chemical factors on production of isoflavonoids in <i style="">Pueraria tuberosa</i> (Roxb.ex.Willd.) DC suspension culture

1063-1067Suspension cultures of Pueraria tuberosa, a woody legume, have been established and using different concentrations of growth regulators, sucrose, ammonium and nitrate nitrogen, attempts have been made to improve their isoflavonoid content. The cell cultures grew well on all the treatments. Up to ~8 folds increased isoflavonoids content was recorded in the cultures grown in MS medium modified with nitrogen and supplemented with 1mg l-1 of kinetin

Development of resin canals during somatic embryogenesis in callus cultures of <i style="">Commiphora wightii</i>

267-270Formation of resin canal in somatic embryos of Commiphora wightii (Arnott.) Bhandari has been reported. Resin canal formation is a prerequisite for obtaining resin canal based secondary metabolites and resin canal formation in callus cultures is not known. Resin canals were observed in torpedo shaped and cotyledonary stage embryos. Early stages were devoid of it. Resin canals formed in somatic embryos were comparable with those formed in the stem. This provides large quantities of aseptic resin canals in organized structures, which can be used for the production of guggulsterones through biotechnological methods relieving pressure on natural resources

Isoflavonoids production in callus culture of <i style="">Pueraria tuberosa,</i> the Indian kudzu

1012-1017Isoflavonoid contents of different plant parts and callus tissues of the Indian Kudzu, Pueraria tuberosa (Roxb.ex.Willd.) DC are presented. The initial cultures were slow growing, associated with browning of the tissues. The production of four isoflavonoids (puerarin, genistin, genistein and daidzein) in the callus cultures of P. tuberosa was studied by manipulating the plant growth regulators and sucrose concentration in the medium. Organogenesis was not recorded in callus on any of these treatments. Tuber and stem accumulated puerarin, a glycoside of daidzein, at high amounts, 0.65% and 0.054% respectively. However, the daidzein content of the callus tissues grown on Murashige and Skoog medium containing BA (20.9 µM) and sucrose (60 gl-1) was significantly higher (0.056%) than in vivo plant material (0.02%) and other comparable culture systems like Genista and Pueraria lobata

Assessment of variability in the regenerants from long-term cultures of ‘safed musli’ (<i style="">Chlorophytum borivilianum</i>)

527-534Somatic embryogenesis in long-term calluses of seedling and leaf explants, obtained from in vitro grown plants of Chlorophytum borivilianum—an endangered medicinal herb, has been achieved. Large number of plants were established in the field and evaluated for variability among the regenerants. The plantlets obtained through seedling-derived embryonic callus showed high level of morphological and cytological variations, which increased with the increase in age of cultures. Occasionally, variegated plants were also observed. Variation in leaf size, stomatal number, epidermal cell size and chromosomal number was observed in regenerants. Chromosomal variation was the least (3X-3 to 3X+3) in regenerants from 1 to 4-month-old cultures and increased (5X-1 to 7X) with the age in regenerants from cultures older than 6 months. Alternatively, regenerated plants from somatic embryos recurrently obtained from leaf explants showed very little variation (0.62%) in RAPD fingerprinting. However, further improvement in somatic embryogenesis is required for domestication of the plant using biotechnological method of propagation

Somatic embryo proliferation in <i style="">Commiphora wightii</i> and evidence for guggulsterone production in culture

217-222 A method for obtaining somatic embryos consistently in Commiphora wightii (Arnott.) Bhandari has been developed. Somatic embryos when sub-cultured on modified MS medium containing ABA (10 µg L-1) produced more embryos directly by meristematic activity of the epidermal and subepidermal cells at the hypocotyl region or also from the root tip region. Embryo proliferation was also observed on auxin-cytokinin free modified MS medium supplemented with different concentrations of activated charcoal, abscisic acid, mannitol and sucrose. Modified MS hormone-free medium containing activated charcoal 0.5 g L-1 and sucrose 10 g L-1 was most suitable for multiplication and maturation of embryos. Embryo formation was also recorded from callus produced by earlier formed somatic embryos (indirect). Embryos obtained from various maturation treatments when desiccated under laminar airflow bench for 5-9 d showed maximum germination percentage. Guggulsterone-E and -Z contents of in vitro embryonic cultures were ¼ - and ½ -fold of that found in zygotic embryos, but several fold higher than callus cultures, therefore, hold promise for the production of bioactive guggulsterones. </smarttagtype

Morphactin and cytokinin promote high frequency bulbil formation from leaf explants of Curculigo orchioides grown in shake flask cultures

520-525High frequency bulbil formation from leaf explants of an endangered monocot herb, Curculigo orchioides, grown in shake flask cultures has been achieved. The leaf explants were obtained from in vitro grown plantlets maintained on MS solid medium containing BA (0.1 mg/L) and IBA (0.1 mg/L) for the past 8 years. Inoculum density of one leaf explant (~1 cm²) per two mL liquid medium (50 explants 100 mL⁻¹) was recorded optimal for bulbil formation. Increase in bulbil number per explant was recorded with increase in IBA concentration (from 0.1 mg/L to 1 mg/L) in the medium; level of substituted urea in the medium did not alter this response. Maximal percent explant response, number per explant and yield of bulbils was recorded in the medium supplemented with 1.0 mg/L IBA with 0.1 mg/L substituted urea. The number of bulbils per explant doubled during 4 to 8 wks growth while total yield (fresh biomass) increased several-folds. In IBA+2iP combination, optimal number of bulbils with maximum yield per litre was recorded in the medium supplemented with 1.0 mg/L IBA and 0.1 mg/L 2iP, at 8 wks growth. However, maximum fresh weight of bulbils (1027 g) was recorded in the medium containing 0.1 mg/L IBA and 1.0 mg/L 2iP. In morphactin and BA interaction, optimal number (38/explant), yield (19165/L) and FB (1450 g/L) of bulbils was recorded in the medium containing 1.0 mg/L BA and 0.1 mg/L morphactin followed by 0.01 mg/L and 1 mg/L of morphactin with the same concentration of BA. The use of this synthetic growth regulator in bulbil formation was new and very significant over other treatments. Formation of plantlets from bulbils was 80% and their survival was ~80% in the field conditions. These results will be helpful in developing technology for micropropagation of the plant using bioreactor

Optimization of guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari

525-531Commiphora wightii (Arnott.) Bhandari is an endangered, slow growing medicinal tree. Its hypolipidemic and hypocholesterolemic activity is due to the presence of two closely related steroidal ketones guggulsterone-E and guggulsterone-Z in the resin. The callus cultures derived from zygotic embryos and leaf explants were exploited for the optimal production of these bioactive molecules. The production of guggulsterone in callus cultures was maximal during 35-d growth on modified MS medium. A correlation has been observed between guggulsterone yield in callus and in vivo in the plant. Guggulsterone content of callus was maximum during January to July, the period of gum exudation in nature. The cultures, which were grown on modified MS medium (950 mg/L KNO₃, 825 mg/L NH₄NO₃ and 220 mg/L CaCl₂.2H₂O) containing 2, 4, 5-T (0.25 mg/L) and kinetin (0.1 mg/L) (referred to as CM2 medium) accumulated ~15 µg/g guggulsterone. The optimal level of nitrogen [NH₄NO₃ (1650 mg/L), KNO₃ (475 mg/L)], CaCl₂ (110 mg/L) KH₂PO₄ (170 mg/L) in different media resulted in maximum accumulation of total guggulsterone up to 19 µg/g dry weight basis. The significant increase in total guggulsterone accumulation (59 µg/g) was recorded in the tissues grown on CM2 medium containing 40 g/L maltose. Significant increase in guggulsterone content was recorded in the tissues grown on the production medium with either sucrose or maltose alone (40 g/L) or glucose:sucrose ratio (20:20 g/L). Combination of all the optimal concentrations of salts and sugars did not further increase the guggulsterone accumulation. It appears that guggulsterone production reached to its maximum possible level (~3 fold increase) with medium salt manipulation containing 40 g/L maltose or glucose: sucrose (~60 μg/g dry weight) and further increase was not possible within these condition

A method for large-scale multiplication of <i>Curculigo orchioides </i>through bulbil formation from leaf explant in shake flask culture

145-148An efficient method has been developed for large-scale multiplication of Curculigo' orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7×10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 μM), adenine (0.11 mM), indole butyric acid (1.0 μM) and polyvinyl pyrrolidone (250 mgL-1). About 95% explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 μM) and gibberellic acid (3.5μM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour