In vitro development of encapsulated shoot tips of cardamom

Summary Shoot tips of cardamom var. Malabar isolated from multiple shoot cultures were encapsulated in 3 % (w/v) sodium alginate with different gel matrices. Maximum conversion of the encapsulated shoot tips into plantlets was on White's medium and the plantlets were successfully grown in soil.     

Propagation of Morus indica L. (Mulberry) by encapsulated shoot buds

Axillary buds of mulberry (Morus indica L) were encapsulated in alginate and agar to produce individual beads. The beads could be stored at 4°C for 45 days without loss of viability. Amongst the encapsulating agents tested, sodium alginate was found to be a better matrix. Encapsulated buds regenerated complete plantlets on an appropriate medium. This technique would provide an easy and novel propagation system for the elite as well as difficult-to-root species of mulberry.Abbreviations BAP benzylaminopurine - MS Murashige and Skoog (1962).     

Micropropagation of kiwifruit using non-axenic shoot tips

Kiwifruit (Actinidia chinensis Planch.) shoot tips were subjected to a standard surface sterilization procedure and cultured on a Murashige and Skoog basal medium in the presence of two surviving bacterial contaminants. The fresh weight increase of the cultures and the number of shoots produced were greater in liquid medium than in medium solidified with 0.4 or 0.8% agar. A greater number of shoots was obtained with 125 ml than with 50, 250, or 500 ml Erlenmeyer flasks. A concentration of 2 mgl^-1 N⁶-benzylaminopurine (BAP) gave a greater increase in fresh weight than either 0 or 4 mgl^-1.Shoots cut from proliferating cultures were dipped in 0.05% indolebutyric acid (IBA) and rooted directly in a peat: vermiculite: perlite mix. Over 93 % of 907 plantlets produced were successfully acclimatized. The productivity of the method was comparable to that reported for the axenic culture of meristems. The contaminants which survived the initial surface sterilization procedure thus presented no major obstacle to the in vitro propagation of kiwifruit.     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

Adventitious shoot production from calloid cultures of banana

Isolated tips (approx. 2 mm long) from aseptic, multiplying shoot cultures of the triploid dessert banana clone Highgate were tested for their morphogenetic responsiveness to hormone treatments on semisolid media. Medium containing Murashige and Skoog (1962) salts, p-chlorophenoxyacetic acid, and kinetin produced a compact calloid mass. Protuberances disclosed by SEM as rounded, button-shaped, and pointed outgrowths resembling fasciated shoots were formed in profusion. Sections showed many meristematic regions, some associated with distinct leaf primordia. Formation and growth of successive leaves yielded small, elongated, adventitious shoots with constricted bases. Transferral to a basal MS medium with 1 mg/l 1-naphthaleneacetic acid (NAA) led to the formation of rooted plantlets.     

Cryopreservation of isolated mint shoot tips by vitrification

Shoot tips isolated from a mint clone, Mentha aquatica x M. spicata, were gradually exposed to a mixture containing 35% ethylene glycol, 1 M dimethylsulfoxide and 10% polyethylene glycol-8000 and then immersed into liquid nitrogen. Cooling and warming rates were approximately 4800°C/min and 9000°C/min respectively. Survival after liquid nitrogen treatment ranged from 31% to 75% among experiments. There was no obvious reason for this variation. In many cases the treated shoot tip directly developed into a shoot without any or with only slight callus formation.Abbreviations DSC differential scanning calorimetry - DMSO dimethylsulfoxide - EG ethylene glycol - PEG-8000 polyethylene glycol - MW avg. 8000 - LN liquid nitrogen - IBA indolebutyric acid - BA benzyladenine     

Plant regeneration from shoot tips and callus of papaya

Summary Two methods of in vitro culture were employed to regenerate papaya plants. One involved regeneration of plants from callus and the other, production of multiple plants from single shoot-tip explants. Callus was induced from stem sections of papaya seedlings in a medium containing 1 mg per 1 NAA and 0.1 mg per 1 kinetin. The callus regenerated shoots and/or embryoids when transferred to a medium of lower auxin, 0 to 0.05 mg per 1 IAA, and higher cytokinin, 1 to 2 mg per 1 kinetin Multiple shoots were produced when the excised shoot-tip explants were cultured in a medium supplemented with 0.05 mg per 1 IAA and either 5 mg per 1 kinetin or 0.5 to 1.0 mg per 1 benzyladenine. Root formation of the shoots or embryoids that derived from callus or shoot tips occurred in a medium containing 5 mg per 1 IAA and in a light intensity of 3000 to 4000 Ix. The rooted plants could be established in soil and under standard greenhouse conditions after they had been acclimated by initially growing them in moist vermiculite contained in polyethylene-covered pots. This research was supported by the National Science Council, Republic of China.     

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     

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Effect of jasmonic acid on cold-storage of <Emphasis Type="Italic">Taraxacum pieninicum</Emphasis> encapsulated shoot tips

Jasmonic acid is involved in plants response to various abiotic and biotic stresses. The aim of this study was to examine physiological response of Taraxacum pieninicum to JA-treatments during shoots micropropagation and during cold storage at 4 °C under reduced light or in the darkness. Obtained results indicated that JA during preculture reduced significantly growth of shoots without impact on proliferation rate. During cold storage JA (24–72 µM) limited effect of cold stress what was manifested by reduced accumulation of proline and TBARS. Growth inhibition of the stored tissue was more effective when JA was inside synthetic seeds structure than after preculture on medium supplemented with JA. Shoot proliferation and rooting were effective during regrowth after cold storage combined with JA exposure. Only elongation of roots was inhibited after storage in this conditions. However, length of roots did not affect acclimatization of plantlets to ex vitro conditions.     

Micropropagation of Withania somnifera from germinating seeds and shoot tips

Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. using low concentrations of 6-benzyladenine (BA), viz. 2.2, 4.4 and 8.9 M. Maximum number of shoots were obtained when 2.3 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.5 M indolebutyric acid (IBA) was added to medium containing 4.4 M BA during initiation of shoot multiplication, but not when added later. Direct multiple shoot initiation was also obtained from germinating seeds in the presence of BA alone. Rooting was successful in excised shoots grown on growth regulator-free MS medium. Rooted shoots were successfully established in soil in a greenhouse.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

人参果茎尖脱毒培养与快速繁殖

对人参果(Solanum muricatum Ait)茎尖进行离体培养及快速繁殖.结果表明:适于外植体生长分化的培养基是不加任何激素或只加0.2 mg*L-1 NAA的MS培养基;用MS 0.5 mg*L-1 NAA培养基能促进组培苗茎尖快速伸长,有利于茎尖的剥离和脱毒培养;适于茎尖愈伤组织形成和分化的培养基为MS 0.2 mg*L-1 NAA 2.0 mg*L-1 6-BA;适于壮苗快繁的培养基为MS 0.5 mg*L-1 NAA 0.1 mg*L-1 PP333.生物学和电镜检测结果表明,利用0.2～0.3 mm茎尖培养的人参果试管苗已脱除病毒.     

Biosynthesis of ent-kaurene in cell-free extracts of Pisum sativum shoot tips

Soluble enzyme preparations from pea shoot tips incorporated mevalonic acid-2-¹⁴C into ent-kaurene-¹⁴C, squalene-¹⁴C and other products. The assay for either ent-kaurene or squalene is quite direct; both products can be obtained apparently free of radioactive contaminants by TLC on silica gel G in hexane. The enzyme system is dependent upon added ATP and Mn²⁺ or Mg²⁺, with Mn²⁺ being a more effective activator than Mg²⁺ under the experimental conditions. Reduced pyridine nucleotide had no effect on ent-kaurene production but stimulated squalene synthesis. The accumulation of both ent-kaurene and squalene was stimulated by dithiothreitol and carbon monoxide and was reduced by the addition of particulate cell components. AMO-1618 inhibited ent-kaurene production and had no effect on the synthesis of squalene. Enzyme extracts from shoot tips are much less active in ent-kaurene synthesis than extracts from the cotyledons of immature seeds on either a fresh weight or protein basis.     

Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Cryopreservation of shoot tips from the endangered endemic species Tuberaria major

Tuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed.