Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

Efficient micropropagation protocol of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. possessing strong antimalarial activity

Micropropagation has been achieved in a promising larvicidal asteraceous taxon Spilanthes acmella L. using seedling leaf explants. The explants were reared on a variety of growth regulators, namely 2,4-dichlorophenoxyacetic acid, 1-naphthalene acetic acid, Indole-3-butyric acid, N⁶-benzyladenine, and kinetin either alone or in combination on Murashige and Skoog’s (MS) medium. The best green and compact callus was obtained on 1 μM NAA and 10 μM benzyladenine (BA) in 15 d. The callus on subculture to the same but fresh medium after every 30 d differentiated an average of 12.90 ± 0.32 shoot buds in 50% cultures. Elongation in shoot buds occurred only if they were transferred to NAA lacking MS+BA medium. An average number of 4.22 ± 0.83 shoots and 15 ± 0.84 shoot buds per explant were obtained in 70.3% cultures on MS + 10 μM BA in 30 d. One hundred percent excised shoots rooted in MS(1/2) + 0.1 μM IBA within 2 wk. The plants were gradually hardened and established in soil where they flowered and set viable seeds. The regenerated plants were morphologically similar to the field grown plants and showed 100% larvicidal activity against malaria and filarial vectors.     

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.     

In vitro propagation for the conservation of a rare medicinal plant <Emphasis Type="Italic">Justicia gendarussa</Emphasis> Burm. f. by nodal explants and shoot regeneration from callus

Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.     

Efficient protocol for in vitro production of androgenic haploids of <Emphasis Type="Italic">Phlox drummondii</Emphasis>

Production of haploid plants has been restricted to only a few ornamental species. In this paper an efficient anther culture protocol has been devised for production of haploid plants of Phlox drummondii, a garden ornamental. Anthers with microspores at early- to late-uninucleate stages were inoculated on MS (Murashige and Skoog, Physiol Plant 15:473–479, 1962) basal medium containing 9% sucrose, 10 μM 2,4-D + 5 μM BA in the dark for callus induction. The callus (~2 mm) was transferred to MS medium containing 3% sucrose + 10 μM BA + 5 μM NAA under a 16 h photoperiod for multiplication. Anther-derived callus showed the greatest shoot differentiation (60% with greater than 3 shoots per culture) at 13 weeks after culture initiation when maintained on MS medium supplemented with 3% sucrose and cytokinin (7.5 μM BA). At 68 weeks, only 4.6% of cultures differentiated with less than one shoot per callus. Anther-derived shoots rooted readily on MS medium containing 7.5 μM IAA. Of 60 plants that regenerated from anther callus, 50% were haploid, 30% diploid, and 20% aneuploid. Developed protocol could be useful for the haploid induction of outcrossing ornamental plants for production of their homozygous double haploids.     

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

Summary The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N⁶-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Production of triploid plants of papaya by endosperm culture

Triploid papaya (Carica papaya) plants were obtained by immature endosperm culture. Visible callusing of the endosperm occurred 21 days after initiation of cultures. A continuously growing callus was observed and a maximum of 68.7% of callus induction frequency was obtained when immature endosperm with embryo was cultured on Murashige and Skoog (MS) medium containing 6.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.5 μM α-naphthaleneacetic acid (NAA) and 4.0 μM 6-furfurylamino purine (Kn). Shoot buds were produced when the callus was subcultured on a medium supplemented with 6-benzyladenine (BA) or thidiazuron (TDZ) along with NAA. Shoots were detached from the callus and transferred to the elongation medium supplemented with indole-3-acetic acid (IAA) and BA. The combination of 3.0 μM IAA and 1.5 μM BA was the best in terms of the number of cultures (93.8%) showing axillary shoot proliferation. The addition of 2.0 μM indole-3-butyric acid (IBA) to the 1/2 MS medium was most effective at inducing root formation with 90% of the shoots developing four to five roots. Healthy rooted plantlets were transferred to pots containing sterilized bed soil and perlite (3:1) mixture in the greenhouse and 78% of the micropropagated plants survived transplantation. The leaves from endosperm-derived plants showed larger stomata and more chloroplasts in guard cells than that from the parent plants. Over 75% of the endosperm-derived plants were triploid with chromosome number 2n = 3x = 27.     

In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

Adventitious bud formation in Alhagi graecorum

Various parts of seedlings and in vitro propagated shoots of Alhagi graecorum Boiss were cultured on different media with different 6-benzyladenine (BA) and kinetin (KIN) concentrations to compare their potential to regenerate shoots. Murashige and Skoog (MS) medium with 2.5 μM BA and hypocotyl gave the best results. Callus was obtained from stem segments on MS medium supplemented with 2.5 μM BA, 5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Shoot formation from callus occurred upon its transfer to MS medium supplemented with 2.5 μM BA. Mature explants which showed a relatively low potential for adventitious buds or callus formation, regenerated shoots abundantly using the tiny-mature-explant method. The regenerated shoots were rooted on half strength MS medium supplemented with 5 μM 3-indolebutyric acid (IBA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Shoot multiplication and adventitious regeneration in Sorbus aucuparia

In vitro mother plants initiated from a mature tree of Sorbus aucuparia, produced numerous propagules on a medium containing 2 μM 6-benzylaminopurine (BAP) and 0.2 μM 1-naphthaleneacetic acid (NAA). These were rooted on a medium containing 0.25 μM NAA and 0.25 μM indole−3-butyric acid. Adventitious shoots were produced on excised leaves and internodes on media containing 10 μM thidiazuron and 0.3–1.0 μM NAA. They formed by direct regeneration in the axils of leaflets of intact leaves. They also developed indirectly, from callus that developed on the rachis of intact leaves, and the cut ends of petioles and internodes. Somatic embryos were produced on cotyledons of zygotic embryos on medium containing 1 μM BAP, 1 μM kinetin, 0.5 μM NAA, 500 mg l⁻¹ casein hydrolysate and 250 mg l⁻¹ glutamine. On basal medium, 69% developed cotyledons and 20% germinated after pre-treatment at 4 °C on medium containing 30 g l⁻¹ maltose.     

In vitro plant regeneration of Mammillaria elongata normal and cristate forms

In the normal form of Mammillaria elongata, shoots were regenerated in vitro, through callus, from tubercle explants excised from the upper part of the branch and cultured on Murashige and Skoog medium (MS) with 1.07 μM α-napthaleneacetic acid (NAA) and 22.20 μM 6-benzylaminopurine (BA). A high percentage of tubercles explants of the M. elongata cristate form, excised from the tip of the branch and cultured on MS with 0.54 μM NAA and 0.44 μM BA or 1.07 μM NAA, responded by initially forming an inflated cristate shoot, which gave cristate and normal shoots, without callus intervention, when transferred on basal MS. Callus formed on cristate tubercles explants gave both cristate and normal shoots when transferred onto basal MS. Normal and cristate shoots were rooted in vitro on MS with 9.84 μM or 0.98 μM indole-3-butyric acid, respectively, and established ex vitro. In both normal and cristate form, the differential response appeared to be associated with the site of the explant excision. The formation of shoots was influenced by the season of culture; i.e., explants excised in October had a higher shoot formation rate than those excised February. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde

Caulogenic responses of various explant types from 12-month-old plants of Hemidesmus indicus were tested. Second and third visible nodes (0.5 cm) from the apex and root segments (0.5 cm) were the most and least regenerative respectively, with the formation of 9.37 and 2.6 shoots in 4 weeks on half strength MS medium supplemented with 2.22 μM BA and 1.07 μM NAA and 4.44 μM BA and 2.69 μM NAA respectively. Caulogenic ability of the nodes decreased with increasing maturity. Shoot buds initiated upon the young nodes on day 10 developed into 7.2 cm long shoots within 4 weeks thereby making a shoot elongation phase unnecessary. Nodal explants of the in vitro raised shoots subcultured in the same medium produced 9.32 shoots of 7.1 cm length in 3–4 weeks, similar to those of the mature plant-derived nodes. Multiplication through subculture of the nodes up to 25 passages of 4 weeks each was achieved without decline. Shoot cultures were rooted in quarter salt strength MS medium containing 9.8 μM IBA and the rooted plants were hardened for establishment in pots at 96% rate. Four months after establishment, the micropropagated plants were stable and showed uniform morphological and growth characteristic. After 12 months of cultivation in the field, on an average micropropagated plant consisted of 4–5 shoots, 5–8 branches per shoot and increased root biomass (13.5 g) compared to the poor growth (single shoot and 2–3 branches) and root production (4.6 g) values obtained with plants raised from conventional rooted stem cuttings. The concentration of the root specific compound, 2-hydroxy 4-methoxy benzaldehyde per plant was 2–3 fold higher in micropropagated plants though on unit dry root biomass (0.12% per g dry wt) basis it remained the same between two sources of plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient plant regeneration from cell suspension-derived callus of East Indian rosewood (Dalbergia latifolia Roxb.)

A procedure is outlined for the establishment of a proliferating cell suspension culture of East Indian rosewood (Dalbergia latifolia Roxb.) and efficient plant regeneration from callus derived from such cultures. Callus was induced from hypocotyl segments derived from 1-week-old axenic seedlings on Murashige and Skoog (1962) medium (MS) containing 10.8 μM naphthaleneacetic acid (NAA) and 2.2 μM benzyladenine (BA). Calli were increased by subculturing on MS supplemented with same growth regulators and 10% coconut water (CW). Friable calli were used to initiate cell suspension cultures. Optimum cell proliferation occurred in MS containing 10.8 μM NAA, 2.2 μM BA and 10% CW, using an initial inoculum cell density of 2%. Cell clumps composed of 20–25 cells harvested from suspension cultures at the exponential growth phase readily formed callus within 3 weeks following plating on the semi-solid MS as above. High-frequency shoot-bud differentiation was induced in these calli on MS containing 2.7 μM NAA and 13.3 μM BA. The regeneration frequency declined at higher BA concentrations. The organogenic potential of the cell suspensions was influenced by the age of the culture. Regenerated shoots were rooted on half-strength MS containing 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. The plantlets were acclimatized and established in soil. Received: 22 August 1997 / Revision received: 21 May 1998 / Accepted: 1 June 1998     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

An efficient in vitro regeneration protocol for Tagetes minuta

Tagetes minuta is a source of secondary products which are used as pharmaceuticals, pesticides and as flavour components in the food industry. Cotyledons and hypocotyls of T. minuta were cultured on MS medium with combinations of IAA or NAA and BA. Hypocotyl-derived callus developed adventitious shoots which failed to develop further. Cotyledon-derived callus, cultured on medium with IAA, regenerated adventitious shoots which developed into plantlets on MS medium or half-strength MS with 2.85 μM IAA. Cotyledons cultured on medium with 5.71 μM IAA + 44.4 μM BA and transferred to MS medium for shoot growth yielded the highest number of shoots. Nodal segments from developing shoots were micropropagated on half-strength MS medium with 2.58 μM IAA and 95% of plantlets produced adapted successfully to greenhouse conditions. In vitro plants micropropagated from nodes had many shoots whereas plants regenerated from shoot tips had only a single main stem. This difference in morphology was retained after two months growth in a greenhouse. There were no significant differences in leaf and shoot fresh and dry weights among the regenerated plants after two months growth. After six subcultures of cotyledon-derived callus on medium with IAA and BA all explants lost their ability to regenerate except those cultured on medium with 17.23 μM IAA and 44.4 μM BA. The methods of regeneration developed will facilitate selection of T. minuta plants more tolerant of environmental stress, their micropropagation, and the in vitro production of secondary products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.