Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

在1/3海水培养基上筛选豆瓣菜耐盐变异体

The responses of stem segments of watercress ( Nasturtium offtcinale R. Br. ) to 6-BA, NAA and 2,4-D were studied. MS medium supplemented with 2.0 mg/L 6-BA, 0.2 mg/L 2,4-D was used for callus initiation and maintainance. MS medium supplemented with 4.0 mg/L 6-BA was suitable for plant regeneration and MS medium without plant hormone supplement was used for rooting and plant propagation. For screening of salt. tolerant calli, stem segments of watercress were plated onto callus initiation medium containing 1/3 natural seawater. Seventeen out of the 325 plated explants produced calli. The growth curves demonstrated that the growth rate of salt-tolerant calli on saline medium almost matched that of the control calli on normal medium. Some of the salt-tolerant calli were transferred to the normal regeneration medium or saline regeneration medium to induce plant regeneration. In the first case, buds and shoots were regenerated in the same way as those of control calli on normal regeneration medium. More than 1 000 regenerated shoots were obtained of which 83 regenerated shoots were cut and transferred to saline MS base medium. At first, all shoot growth was inhibited, but 40 days after the transfer, rapid-growing axillary shoots were observed on 16 of the original shoots but none on the control shoots on saline MS base medium. Moreover, green spots appeared on most calli 10 days after they were transferred to saline medium, however buds appeared only on 5 calli from the 30 transferred calli and at the end only 2 rapid-growing shoots were obtained from two calli. In total, 18 variant lines were obtained through propagation of the salt-tolerant shoots on saline MS base medium. RAPD analysis was performed in 10 of the 18 salt-tolerant variant lines and DNA variation was detected in all the tested variant lines.     

In vitro propagation of Cuphea procumbens Orteg. and Evaluation of genetic fidelity in plantlets using RAPD marker

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cuphea procumbens Orteg. using cotyledonary node explants excised from 15?days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at a concentration of 2.5???M was effective in inducing multiple shoots. The highest number of multiple shoots (9.33?±?0.60) and maximum average shoot length (4.16?±?0.44?cm) was standardized on MS medium supplemented with 2.5???M BA alongwith 0.5???M NAA. Addition of 200?mg/l Casein hydrolysate (CH) to the shoot induction medium enhanced the growth of regenerants. Rooting of in vitro regenerated shoots was best achieved on 1/2 strength MS medium. The in vitro raised plantlets with well developed shoots and roots were hardened, successfully established in earthen pots containing garden soil and maintained in greenhouse with 80% survival rate. Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate the genetic stability among in vitro regenerated progenies. All RAPD profiles from the micropropagated plants were monomorphic and similar to control plant. These results suggests that the culture conditions used for the axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with genetic integrity of in vitro regenerated plants. The described method can be successfully employed for large-scale multiplication and in vitro conservation of C. procumbens.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mg L⁻¹ of 6-benzyladenine (BA) and 2 mg L⁻¹ of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.     

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05–2.5 μM) was used as a supplement to the Murashige and Skoog’s (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.     

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg∙L ^-16-BA与0.5 mg∙L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

南瓜高效再生体系的建立

南瓜(Cucurbita moschata)再生率较低, 为建立高效的南瓜再生体系, 以南瓜子叶为外植体, 进行35组不同激素浓度的不定芽诱导研究。结果表明, 南瓜再生受培养基中激素浓度和配比的影响, 适宜浓度6-苄氨基腺嘌呤(6-BA)能有效促进不定芽形成; 单独使用脱落酸(ABA)诱导使南瓜子叶发黄, 但与6-BA组合使用可显著提高外植体的再生能力, 1.0 mg?L ^-16-BA与0.5 mg?L ^-1ABA组合南瓜芽再生率高达90.26%。将不定芽置于MS培养基中进行生根培养, 再生苗移栽易成活。从子叶接种到苗再生约需70天。     

A submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’)

Echinodorus ‘Indian Red’ is an underwater plant, used worldwide for aquarium ornamentation. An efficient method for in vitro propagation and plantlet acclimatization of this popular aquarium plant was standardized. Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations. The combination of 2.5 mg L⁻¹ 6-benzylaminopurine and 1.0 mg L⁻¹ α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L⁻¹ indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.

     

High Frequency Plant Regeneration from Nodal Explants of Paulownia elongata

Abstract: High frequency of plant regeneration from Paulownia elongata was obtained on Murashige and Skoog (MS) medium and Woody Plant Medium (WPM), with appropriate supplements of growth regulators. Leaves, leaves with petioles, internodes and nodes excised from 3-month-old non-aseptically grown P. elongata were used as explants. The highest shoot regeneration efficiency (93.7 %) was obtained from the nodes of P. elongata on MS medium supplemented with 0.1 mg/ml naphthaleneacetic acid (NAA) and 1 mg/ml 6-benzylaminopurine (BAP). The highest root formation efficiency (100 %) from the regenerated shoots was obtained on WPM supplemented with 1 mg/ml indolebutyric acid (IBA). Rooted plantlets were transplanted to soil with a survival efficiency of almost 100 %. The regeneration system reported here could be useful for rapid multiplication of elite genotypes of P. elongata in a short period of time.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin

A mass in vitro propagation system devoid of growth regulators for Bacopa monniera (L.) Wettst., a traditional Indian medicinal plant, has been developed. Direct shoot bud regeneration was induced by culturing internode and leaf explants on Murashige and Skoog's (MS) medium supplemented with an antibiotic (trimethoprim) or a fungicide (bavistin). Bavistin showed a marked cytokinin-like activity, as evident from high number of shoot buds induced in node, internode and leaf explants. Optimum adventitious shoot buds induction occurred at 300 mg/l bavistin from internode explants. In vitro regenerated shoots were elongated and rooted before transferred to field with 85% survival. The regeneration protocol developed in this study illustrates the usefulness of additives for mass propagation and germplasm conservation of B. monniera.Authors Vaibhav Tiwari and Kavindra Nath Tiwari contributed equally to this work     

Regeneration of plants from leaflet explants of tissue culture raised safed siris (Albizia procera)

Shoot bud regeneration was obtained from isolated leaflets of Albizia procera cultured on MS medium with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). The highest numbers of adventitious buds were obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. The replacement of 7 g l-1 Difco bacto agar with 2.6 g l-1 Phytagel in the medium enhanced adventitious bud regeneration. Further, addition of 15 μM silver nitrate promoted callus-free shoot regeneration from leaf explants. The regenerated shoot buds were elongated on MS medium containing 0.01 μM BA and 1 μM NAA. Rooting was obtained on modified MS medium supplemented with 2 μM IBA. To our knowledge this is the first report of direct regeneration of shoots from leaflet explants in A. procera, and should help facilitate genetic transformation in this species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

绿巨人白掌不同外植体组织培养研究

本文研究白鹤芋属观赏品种绿巨人白掌的茎顶、叶片、叶柄和幼花序的组织培养和快速繁殖技术。茎顶培养以0.2mg／LNAA 3．0mg／L6-BA培养效果较好;叶片诱导适宜的培养基为0．5mg／LNAA 5．0mg／L6-BA,分化培养基为0．2mg／LNAA 3．0mg／L6-BA;叶柄以0．5mg／L2,4-D 3．0mg／L6-BA诱导效果最好,分化适宜培养基为0．5mg／LNAA 3．0mg／L6-BA;幼花序胚状体的诱导则以0．5mg／L2,4-D 5．0mg／L6-BA效果最好,成苗培养基为0.5mg／LNAA 3．0mg／L6-BA;255mg／L的KH2P04比较／比较适合于绿巨人白掌丛芽的增殖;生根培养基以1／2MS 0.50mg／LNAA较适宜。     

In vitro propagation of four saponin producing <Emphasis Type="Italic">Maesa</Emphasis> species

A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.