Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Artemisia judaica L.: micropropagation and antioxidant activity

Artemisia judaica L., an Egyptian medicinal plant used in the treatment of gastrointestinal disorders, was mass-propagated and grown using solid, paper-bridge-support liquid, liquid-flask and bioreactor cultures. The liquid-flask culture using 50 ml MS liquid medium in 250 ml flask yielded significantly greater shoot proliferation than either solid cultures or paper-bridge-support liquid cultures. Increasing flask capacity from 100 to 500 ml improved shoot proliferation and growth. Mass-propagation efficiencies of various bioreactor systems, viz. temporary immersion reactors and bubble column reactors, were also compared. The temporary immersion bioreactor was found to have significant advantages for A. judaica shoot proliferation. The shoot cultures from the temporary immersion reactor formed complete plantlets when subcultured onto a medium containing 1 micromoll(-1) indole-3-butyric acid (IBA), and mature plants were established, acclimatized and thrived in standard greenhouse conditions. Assays of antioxidant activity and total flavonoid content of in vitro and in vivo grown tissues were evaluated as gross parameters of medicinal efficacy. Significantly higher antioxidant activity and flavonoid contents were observed in the tissues of mature greenhouse-grown plants. The efficient in vitro production systems developed in this study provided sterile, consistent tissues for investigation of bioactivity and germplasm conservation of A. judaica.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

Shoot culture of Bacopa monnieri: standardization of explant,vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5 mg l⁻¹) and IAA (0.01 mg l⁻¹) with 3 % sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5 cm explant size and 20 explant/40 ml (1 explant/2 ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100 ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250 ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l⁻¹ medium as compared to control (938 shoots l⁻¹). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.     

Studies on production of ajmalicine in shake flasks by multiple shoot cultures of Catharanthus roseus

The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low concentration of BA (2.22 microM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 microM) and a high concentration of BA (8.90 microM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 microM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 microM) supplemented medium compared with the high concentration of BA (8.90 microM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 microM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.     

Rapid clonal propagation of Vitex trifolia

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.     

白Pian体细胞胚悬浮培养的动力学研究

白(ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ.ｅｔＷｉｌｓ.)是我国特有的云杉属树种,在林业生产和环境绿化中均具有重要地位。其体细胞胚胎发生的研究,一方面可用于优良种质的大规模快速繁殖,为植树造林和园林绿化提供优质苗木;另一方面可作为遗传转化的再生系统,进行树种遗传...     

In vitro multiplication of Peganum harmala--an important medicinal plant

The frequency of shoot regeneration and multiplication of P. harmala was influenced by the type of explant and kind and concentration of hormones. Of the various seedling explants, cotyledonary node exhibited maximum shoot regeneration frequency from axillary region on MS medium supplemented with 5 microM BAP. Addition of 0.1 microM NAA enhanced the efficacy of BAP for multiple shoot regeneration as well as improved the growth of shoots. BAP (5 microM) in combination with NAA (0.1 microM) was found to be the optimal for inducing an average of 4-5 shoots per explant in 75% of the cultures within 5 weeks. Replacement of BAP with other cytokinins at equimolar concentration of BAP i.e. 5 microM was not effective in inducing multiple shoots. Regenerated shoots were rooted on MS medium containing IBA (8 microM) with 80% efficiency. The plantlets were successfully established in soil where 80% of them developed into morphological normal plants.     

Multiple shoots induction in wild cotton (<Emphasis Type="Italic">Gossypium bickii</Emphasis>) through organogenesis and the analysis of genetic homogeneity of the regenerated plants

Fertile plants were regenerated via organogenesis from cultures of Gossypium bickii Prokhanov cotyledonary nodes devoid of cotyledons. Cotyledonary nodes from 7-day-old seedlings yielded the maximum number of shoots (9.12 shoots per explant) using MSB medium [MS medium (Murashige & Skoog 1962) and B5 (Gamborg et al. 1968) vitamins] supplemented with the combination of 4 mg/L BAP (N6-benzyladinine) and 0.1 mg/L TDZ (thidiazouron), and the subculture was made on a medium containing 2 mg/L BAP. Elongation of multiple shoots was obtained on MSB medium containing 0.05 mg/L GA₃ (gibberellic acid). For rooting, a modified half-strength MS medium was utilized. Some regenerants were transferred to a greenhouse, and most of them were morphologically normal and fertile. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants. Flow cytometric analyses and chromosome counting suggested that the regenerants and their offsprings were diploid. DNA content of regenerants and their offsprings was homogeneous and similar to that of seed-derived plants with unchanged chromosome number (2n = 26). Random amplified polymorphic DNA (RAPD) analysis confirmed the genetic homogeneity of the regenerants and their offsprings with the diploid parent.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

In vitro propagation of Dendrobium macrostachyum Lindl.--a threatened orchid

In vitro propagation of Dendrobium macrostachyum, a threatened and endemic species was achieved through nodal explants. The nodal explants were cultured on Murashige and Skoog (MS) basal medium and MS medium supplemented with N6-benzyladenine (BA-2.22, 4.44 and 8.88 microM), Kinetin (KN-2.32, 4.65, and 9.29 microM) and Coconut water (CW, 5, 10 and 15%) individually or in combination with 2.69 microM alpha-naphthalene acetic acid (NAA). Axillary shoots were induced directly from nodal explants in medium containing BA, KN or CW. Optimal shoot induction (6 shoots/explant) was attained from nodal explants cultured on medium supplemented with 15% CW. Well developed shoots rooted at an average 5 roots per shoot in half strength MS medium devoid of any growth regulators.     

Micropropagation ofPenstemon serrulatus and iridoid formation in regenerated plants

A method for the micropropagation ofPenstemon serrulatus Menz. from shoot tips or nodal segments was developed. Multiple microshoot cultures (up to 20 shoots from a single explant) were obtained by maintenance of shoot tip explants on Schenk & Hildebrandt medium (SH) supplemented with 4.4 µM benzyladenine (BA) or 8.9 µM BA and 0.57 µM indole-3-acetic acid (IAA). Microshoots developed into numerous, normal shoots when explants were transferred to SH medium containing 2.9 µM IAA or 2.5 µM indole-3-butyric acid (IBA). Shoot cultures were also established from nodal segments (max. 6.8 shoots per segment) when they were placed on SH medium with 0.49 µM IBA and 2.2 µM BA. Rooting of shoots was better on SH medium containing auxin (IBA, NAA or IAA) than on SH medium without growth regulators. The plantlets were then transferred to pots and grown in the greenhouse. Four-month-old regenerated plants demonstrated similar iridoid content (leaves contained 3.83% dry wt. penstemide and 1.8% dry wt. serrulatoloside) as the original plants.     

Adventitious shoot regeneration from in vitro cultured leaves of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd<Emphasis Type="Italic">.</Emphasis>)

Adventitious shoots were successfully regenerated from leaf explants of in vitro cultures of Platanus acerifolia Willd. The leaves of three clones (genotypes), designated as PH1, PH2 and PC, respectively, were wounded by three to four transverse cuts through the midvein and cultured on 26 media based on Murashige and Skoog (MS) basal medium, containing different concentrations of 6-benzylaminopurine (BAP) in combination with different concentrations of indole-3-butyric acid (IBA). The highest regeneration rate (>90%) and the largest number of shoot clumps per regenerating leaf (>4 shoot clumps/explant) were obtained with leaves of genotype PH2 cultured on MS basal medium supplemented with 17.76 microM BAP and 4.92 microM IBA. The other two genotypes, PH1 and PC, showed very low capability of shoot regeneration (<10%) on all the media tested. Shoots on leaf explants originated mainly from callus that developed around the cut end of petioles and along the cuts across the midvein. The regenerated shoots were micropropagated, rooted and transplanted to the soil successfully.     

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.     

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.     

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill

Mature leaf explant derived callus of Tylophora indica (Burm. f.) Merrill yielded somatic embryos on MS medium supplied with BA(1-2 mg/L) or kinetin(1-5 mg/L) or kinetin/BA (1-2 mg/L) used along with IAA(0.1-1 mg/L). Maximum somatic embryos (30) could be recovered from 100 mg of embryogenic callus within 60 days at an optimum concentration of 2 mg/L of BA which was also best suited for providing the maximum conversion rate (90%) of embryoids to plantlets. Kinetin (1-5 mg/L), used as the sole growth hormone, induced the development of embryoids showing either shoot or root primordia in 30% of the cultures. However, embryoids with shoot primordia developed roots upon transfer to medium containing IAA(0.1 mg/L) and kinetin(2 mg/L). Embryoids from all cultures germinated in the initiation medium and were transplanted to sterile vermiculite for hardening. After two weeks of hardening, the plantlets were transferred to the green house where they grew and established well showing a high rate of survival (90%).     

Production of 2-hydroxy 4-methoxy benzaldehyde using root cultures of Hemidesmus indicus

Nodal explants of Hemidesmus indicus plants cultured in half strength Murashige and Skoog medium fortified with 2 mg indole-3-butyric acid l–1 in the dark produced 10–12 roots (1–2 cm) with minimal callusing in 10 days. Roots, 20 mg dry wt, cultured in Gamborg et al. medium supplemented with 2 mg IBA l–1, sucrose (4% w/v), pH (5.6) and agitation (70 rpm) in the dark yielded 550 mg root dry wt/flask and 0.18% 2-hydroxy 4-methoxy benzaldehyde in the roots after 30 days. The maintenance of normal morphology of the roots, consistent biomass and product yields over a 21-month period indicated the stability of the cultures. © Rapid Science Ltd. 1998