Efficient organogenesis and plantlet regeneration in the timber species <Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis> (Lamb.) Hook

Two efficient regeneration systems were developed in Cunninghamia lanceolata, the most important conifer for industrial wood production in China. Cotyledons and hypocotyls derived from greenhouse-grown seedlings were used as initial explants in our research. A high frequency (95.1?±?1.84%) of adventitious buds were initiated directly from cotyledons cultured on Douglas-fir cotyledon revised (DCR) medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.1 mg l^?1 α-naphthaleneacetic acid (NAA), and 0.004 mg l^?1 thidiazuron (TDZ) with a maximum mean number of adventitious buds per cotyledon explant of 3.76?±?0.08. In contrast, a high percentage (93.73?±?0.55%) of adventitious buds regenerated via callus produced from hypocotyls cultured on DCR medium supplemented with plant growth regulators with a maximum number of adventitious buds per explant (16.71?±?0.34). Adventitious buds elongated on DCR medium supplemented with 0.2 mg l^?1 BA and 0.02 mg l^?1 NAA. After liquid pretreatment with 50 mg l^?1 indole-3-butyric acid (IBA), over 95% of the shoots successfully rooted on ½ DCR medium supplemented with 0.3 mg l^?1 IBA. The innovated systems reported in this study will be useful tools for future genetic manipulation of C. lanceolata and may be adapted for large-scale propagation in other conifers.     

In vitro plant regeneration from leaf callus of Grindelia robusta Nutt

Abstract

A regeneration protocol from leaf explants of Grindelia robusta Nutt. was developed. The combination of 0.5 mg l^?1 IBA plus 0.5 mg l^?1 or 1 mg l^?1 BA added to Murashige-Skoog (MS) medium resulted in the best callus induction frequency; the combination of 0.4 or 0.9 mg l^?1 BA plus 1.2 mg l^?1 GA₃ resulted in the best shoot regeneration. Rooting was successful on MS medium supplemented with 0.5 mg l^?1 IBA. Hardening of G. robusta plants was accomplished in 30 days with 85% survival rate.     

Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense

A protocol for regenerating and subsequent in vitro flowering of an economical important and endangered medicinal orchid, Dendrobium huoshanense, was established mainly via indirect protocorm-like body (PLB) formation. A four-step method was developed to induce successful plant regeneration on 1/2 MS medium supplemented with suitable plant growth regulators (PGRs). Step 1 (callus induction): the root tip explants (1 cm long) were cultured at 1 mg l^?1 2,4-D + 1 mg l^?1 TDZ for 3 months. Step 2 (callus proliferation): the calli were subcultured with a 1-month interval at 1 mg l^?1 2,4-D + 1 mg l^?1 TDZ. Step 3 (PLB induction): the calli were cultured at 2 mg l^?1 NAA + 1 mg l^?1 BA for 2 months. Step 4 (plantlet conversion): the 2-month-old PLBs were cultured at 0.1 mg l^?1 IBA for 4 months. It took at least 6 months to produce well-rooted regenerated plantlets with an average of 3.2 roots and 3.6 leaves from the initial callus. The 6-month-old rooted plantlets were transferred onto PGR-free 1/2 MS medium for 6 months, and then potted with Sphagnum moss for acclimatization. After 2 month of culture, the survival rate was 100 %. The in vitro flowers were obtained on the 8-month-old plantlets at 1 mg l^?1 IBA, 5 mg l^?1 IBA and 0.1 mg l^?1 NAA, but the flowers showed a lack of the gynandrium. The abnormity was overcome by the aid of 5 mg l^?1 TDZ, and subsequently, the capsules formed without artificial pollination. This protocol provides the basis for further investigation on cell suspension, micropropagation, in vitro flowering and breeding programs in Dendrobium huoshanense.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

In Vitro Propagation of Endangered Temperate Himalayan Medicinal Herb Picrorhiza kurroa Royle ex Benth using Leaf Explants and Nodal Segments

The present study describes the micropropagation of Picrorhiza kurroa, (commonly known as kutki) an endangered medicinal herb of the temperate Himalayas and a source of hepatoprotective picrosides. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segments and leaf tissue. For shoot regeneration, the hormone combinations kinetin (2.0 mg l^?1) and Kinetin + Indole-3-butyric acid (IBA) (2.0 mg l^?1 + 0.50 mg l^?1) with leaf explant was found superior. Interestingly, the basal MS medium gave 99.94 % response (direct proliferation) with nodal explant. The medium supplemented with IBA (1.0 mg ^?1) was found best for rooting of regenerated shoots. Nodal segments plated on the medium supplemented with TDZ + IBA (0.11 mg ^?1 + 0.50 mg ^?1) formed somatic embryos, however further regeneration could not be achieved. The in vitro raised plantlets were hardened and successfully established in the glass house conditions.     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers

Two efficient morphogenetic pathways for micropropagation of Bletilla striata (Thunb.) Reichb. f. have been established through the callus-mediated and direct formation of protocorm-like bodies (PLBs) from protocorms and shoot tips. Green calli were induced from the basal surface of protocorms and the cut-end of shoot tips on Vacin and Went (VW) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene acetic acid (NAA) after 3–5 weeks, with the highest frequency of explants forming callus (48.0 %) from protocorms at 1.0 mg l^?1 2,4-D. The calli obtained from all plant growth regulator (PGR) treatments could proliferate and differentiate PLBs on the PGR-free medium. NAA and 2,4-D significantly enhanced the growth of callus. The fastest growth rate of callus was achieved at the combination of 1.0 mg l^?1 2,4-D and 1.0 mg l^?1 TDZ with 46.2-fold within 3 months. The regeneration of PLBs from callus was significantly improved by 6-benzyladenine (BA), and a mean number of 48.4 PLBs was produced from 100 mg calli at 1.0 mg l^?1 BA within 3 months. BA and thidiazuron (TDZ) promoted the direct formation of PLBs from explants. The highest frequency of direct PLBs formation (76.0 %) and the highest mean number of PLBs per explant (30.2) were observed in protocorms cultured with 0.5 mg l^?1 BA. Assessment of clonal fidelity by inter-simple sequence repeat (ISSR) markers revealed similarity ranges of 99.8–100.0 % between the regenerants and their mother plants and 99.5–100.0 % among the regenerants, which suggested the micropropagation protocols were genetically stable.     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> 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.     

Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N⁶-benzyladenine, 6-furfurylaminopurine, N-phenyl-N ^??-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5?mg?l^?1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8?%) was observed as early as 8?days of culture and an average of 25 PLBs was obtained from 1?cm² leaf segment after 40?days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96?% conversion (with well-developed shoots and roots) was achieved within the next 30?days of culture, when well developed PLBs were transferred in MS medium supplemented with 1?mg?l^?1 BA, 0.5?mg?l^?1 IBA plus 60?mg?l^?1 adenine sulphate. After 60?days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90?% well-acclimatized plantlets were recovered.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

Abstract

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36?h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0?μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0?μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5?μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15?μM TDZ for 24?h) explants was achieved on WPM augmented with 6.0?μM TDZ and 0.5?μM NAA after 8?weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (～0.5?g) and were transferred onto WPM fortified with different concentrations (2.0–9.0?μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0?μM) and gibberellic acid (GA_3, 0.5–3.0?μM). Maximum shoot number (10.4?±?0.74) and average shoot length (4.75?±?0.71?cm) were observed in WPM enriched with 2.0?μM BAP, 1.0?μM IAA and 1.5?μM GA₃ after 8?weeks of incubation. The developed shoots (4?cm) were excised, pulse-treated for 24?h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0?μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60?days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.