Genetic fidelity of in vitro regenerants,encapsulation of shoot tips and high diosgenin content in <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L., a potential alternative source of diosgenin

Dioscorea bulbifera L. containing the pharmaceutically important compound, diosgenin, was regenerated in vitro through nodal segments on supplemented Murashige and Skoog medium (MS). Diosgenin was at 12 mg g⁻¹dry wt in 12-week-old plantlets raised on MS with various growth hormones. Random amplified polymorphic DNA (RAPD) analysis showed genetic fidelity of regenerants. Encapsulation of shoot tips in 3% (w/v) calcium alginate for storage and germplasm exchange was achieved.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

Micropropagation of an elite Darjeeling tea clone

Shoot cultures of Camellia sinensis (L.) O. Kuntz var. T-78, an elite Darjeeling tea clone, were established from cotyledonary nodes and shoot tips of germinated seedlings as well as from nodal explants of field grown plants. Shoot multiplication rate ranged from 4x in nodal explants to 35x in cotyledonary nodes after 18 weeks of culture. Rooting was achieved in 80–90% micro-shoots by either placing them on an inductive medium for 10 d and then transferring shoots to hormone-free medium, or by treating micro-shoots with a chronic dose of IBA (500 mg/l) for 30–40 min. Rooted plants were established in soil under glasshouse condition at 60% frequency after hardening phase of 4–6 weeks. The regenerated plants show a constant chromosome number of 2n=30 and are morphologically true to type. This procedure can be applied for conservation and utilisation of an elite clone of Darjeeling tea.     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in <Emphasis Type="Italic">Cedrela fissilis</Emphasis> Vellozo (Meliaceae)

Improvements to in vitro organogenesis are essential for optimizing shoot development and understanding basic physiological processes. The addition of polyamines (PAs) to the culture medium has been used to modulate organogenesis in plants, and this work evaluated the effects of exogenous PAs on direct organogenesis from apical and cotyledonary nodal Cedrela fissilis explants as well as the effects of putrescine (Put) on endogenous PA levels and variations in protein abundance. The effects of exogenous Put, spermidine, and spermine at 0, 0.5, 1, 2.5, or 5 mM on shoot development were tested. The comparison of the tested PAs to the control treatment revealed that 2.5 mM Put significantly increased the length of shoots from cotyledonary nodal explants, which are more sensitive than apical nodal explants, and treatment with 2.5 mM Put significantly increased the endogenous total free-PA and free-Put levels in shoots compared with the control (no Put). A comparative proteomic analysis of shoots indicated that 2.5 mM Put significantly changed the abundance of proteins, primarily metabolic and cellular proteins associated with stress and energy processes such as cell division. These results show that Put functions in endogenous PA metabolism and alters protein abundance, thereby contributing to shoot development in C. fissilis.     

Micropropagation of camphor tree (Cinnamomum camphora)

Multiple shoots were induced from shoot tips and nodal segments of a 12-year-old tree of Cinnamomum camphora on Woody Plant Medium (WPM) supplemented with BA and kinetin. The nodal segments from the in vitro developed plantlets could be induced again to produce a large number of harvestable shoots. Harvested shoots were rooted in vitro in WPM supplemented with activated charcoal (AC) and IBA. The plantlets were transferred to thermocol cups after which they were replanted into polybags and then to field. These plants survived with over 90% success under field conditions and exhibited vigorous growth. This system could be utilized for large-scale multiplication of C. camphora by tissue culture.     

Storage and conversion of Eclipta alba synseeds and RAPD analysis of the converted plantlets

The encapsulated shoot tips and nodal segments of Eclipta alba were stored at 4, 12 and 20 °C under irradiance of 1.5 gmmol m⁻² s⁻¹ and high conversion was observed in synseeds stored at 4 °C for 8 weeks. Duration of storage was extended up to 12 weeks by decreasing sucrose concentration in the alginate matrix from 3 to 1 or 2 % and conversion frequency was 71.2–76.1 %. Synseed-derived plantlets survived by 100 % in ex vitro conditions. RAPD analysis revealed uniform amplification profile in donor and synseed derived plantlets.     

In vitro germplasm conservation of high Δ<Superscript>9</Superscript>-tetrahydrocannabinol yielding elite clones of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. under slow growth conditions

Germplasm conservation of a high Δ⁹-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl₂. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ⁹-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pluronic F‐68 Enhanced Shoot Regeneration in Micropropagated Citrus Rootstock and Passiflora Species

The promotory effects have been studied of the non‐ionic surfactant, Pluronic F‐68, on bud induction/shoot regeneration in epicotyl and cotyledon explants of Citrus depressa and on shoot regeneration from leaf segments of 4–6 week‐old axenic nodal segment‐derived in vitro plants of Passiflora mollissima, P. giberti and P. edulis var. flavicarpa. For epicotyls of C. depressa, supplementation of agar‐solidified MS‐based bud induction/shoot regeneration medium with 0.5% [w/v] Pluronic F‐68 significantly (P < 0.05) increased mean fresh weight gain of cultures, percentage of explants giving shoots and number of shoots per explant. The same Pluronic concentration also enhanced the mean percentage of cotyledons exhibiting bud induction and the number of buds regenerated per cotyledon explant. Fresh weight gain was unaffected across the range of concentrations (0.001–0.5% w/v) of Pluronic F‐68 evaluated for this latter explant source. For leaf explants from axenic shoot cultures of P. mollissima, supplementation of NN‐based medium, containing 3 mg/l 6‐benzyladenine and 2.0 mg/l kinetin with 0.001–0.5% [w/v] Pluronic F‐68, significantly (P < 0.05) increased mean (± s.e.m.) biomass gain by a maximum of 2.7 ± 0.1 g fresh weight (g f.wt.) over the control. Similarly, for leaf explants of P. giberti, 0.001–0.5% [w/v] Pluronic F‐68 in MS‐based medium, containing 1.0 mg/l 6‐BAP and 0.5 mg/l kinetin significantly (P < 0.05) increased mean percentage of explants undergoing shoot regeneration. For P. edulis leaf explants, mean f.wt. gain was also significantly (P < 0.05) higher with Pluronic F‐68 at 0.001–0.5% [w/v].     

A micropropagation protocol forCinnamomum camphora

Summary A micropropagation protocol was developed forCinnamomum camphora (L.) Sieb., using as initial explants 3–5-mm shoot tips from newly emerged laterals of 2-yr-old trees. Performance of small shoot tips was compared with that of 2.0-cm nodal segments during subculture. Murashige and Skoog medium (MS) supplemented with different concentrations of N₆-benzyladenine (BA) or thidiazuron (TDZ) was used to examine shoot proliferation. In separate experiments, MS was supplemented with 1-naphthaleneacetic acid (NAA) for rooting of shoots, and the commercial preparation EM₂ for prevention of hyperhydricity. BA stimulated shoot formation and callus development, whereas TDZ promoted only callus development. Both cytokinins induced hyperhydricity when small shoot tips were used, with severity being directly related to concentrations. Hyperhydricity was avoided in subcultures by using larger nodal segments. EM₂ did not alter degree of hyperhydricity but suppressed callus development and strongly promoted shoot multiplication. The number of new shoots after a 6-wk subculture was 9 per nodal segment when supplemented solely with 4.4 μM BA and 18 per segment when further supplemented with 1000 mg EM₂ per I. Rooting of shoots occurred best when supplemented solely with 0.54 μM NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12–25°C

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.Abbreviations BAP 6-Benzylaminopurine - KIN 6-Furfurylaminopurine     

Micropropagation of Lippia junelliana (Mold.) Tronc.

A method for the micropropagation of Lippia junelliana (Mold.) Tronc. from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on full strength Murashige and Skoog medium (MS) supplemented with 4.4 μM benzyladenine (BA) or 0.04 μM indolebutyric acid- (IBA) plus 4.4 μM BA. The rooting of shoots was better on full-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. The shoot cultures showed a lower essential oil accumulation in comparison with parent plants. Essential oil accumulation is closely related with growth and shows a negative correlation with shoot proliferation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation of soybeans using an embryonic tip regeneration system

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l^–1 N⁶-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l^–1 BAP and 0.2 mg l^–1 indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.Abbreviations GUS -Glucuronidase - T-DNA Transferred DNA - BAP N⁶-Benzylaminopurine - IBA Indolebutyric acid     

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoog's medium containing either N ⁶-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil. Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998