Propagation of banana through encapsulated shoot tips

Plants were regenerated from encapsulated shoot tips of banana. Shoot tips (ca 4 mm) isolated from multiple shoot cultures of banana cv. Basrai were encapsulated in 3% sodium alginate containing different gel matrices. The encapsulated shoot tips regenerated in vitro on different substrates. Use of White's medium resulted in 100% conversion of encapsulated shoot tips into plantlets. The plantlets were successfully established in soil.Abbreviations BA Benzylaminopurine - NAA Naphthalene acetic acid - DMSO Dimethyl sulphoxide     

Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species

A protocol was developed for plant regeneration from encapsulated shoot tips collected from in vitro proliferated shoots of Withania somnifera. The best gel composition was achieved using 3% sodium alginate and 75 mM CaCl2.2H2O. The maximum percentage response (87%) for conversion of encapsulated shoot tips into plantlets was achieved on MS medium supplemented with 0.5 mg/l IBA after 5 weeks of culture. The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads having entrapped propagules were directly sown in autoclaved soilrite moistened with 14-MS salts.     

Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species

Summary A method was developed for plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation was achieved using 3% sodium alginate and 75 mM CaCl₂·2H₂O. Maximum percentage response for conversion of encapsulated shoot tips into plantlets was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant growth regulator. The regrowth ability of encapsulated shoot tips was affected by the concentration of sodium alginate, storage duration, and the presence or absence of MS nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were transferred to pots containing an autoclaved mixture of soilrite and peat moss (1∶1). The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts. Encapsulation of vegetative propagules in calcium alginate beads can be used as an alternative to synthetic seeds derived from somatic embryos.     

Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella. S. K. Singh and M. K. Rai contributed equally to this work.     

Alginate-encapsulation of shoot tips of jojoba [Simmondsia chinensis (Link) Schneider] for germplasm exchange and distribution

Shoot tips excised from in vitro proliferated shoots derived from nodal explants of jojoba [Simmondsia chinensis (Link) Schneider] were encapsulated in calcium alginate beads for germplasm exchange and distribution. A gelling matrix of 3 % sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Best response for shoot sprouting from encapsulated shoot tips was recorded on 0.8 % agar-solidified full-strength MS medium. Rooting was induced upon transfer of sprouted shoots to 0.8 % agar-solidified MS medium containing 1 mg l⁻¹ IBA. About 70 % of encapsulated shoot tips were rooted and converted into plantlets. Plants regenerated from encapsulated shoot tips were acclimatized successfully. The present encapsulation approach could also be applied as an alternative method of propagation of desirable elite genotype of jojoba.     

葡萄试管苗热处理结合茎尖培养脱病毒效果的研究

对７个生食葡萄品种的试管苗热处理结合茎尖剥离培养脱除病毒的方法做了研究,结果表明,从热处理试管苗剥离的茎尖培养成活率为６１．２％,其中有１８．１％的成苗。各品种热处理试管苗剥离的茎尖分化再生能力不同：先形成愈伤组织的成苗率较低,而先分化芽的茎尖成苗率较高。这一方法对扇叶病毒和卷叶病毒的脱除率达９２％以上。     

Encapsulation-Dehydration and Encapsulation-Vitrification Based Cryopreservation of in vitro-Grown Shoot-Tips of Medicinal Plant Astragalus membranaceus

Shoot tips of Amembranaceus excised from in vitro grown axillary bud were encapsulated in calcium alginate beads. Subsequently, shoot tips were precultured in liquid MS medium enriched with 075mol·L-1 sucrose for 5d at 25℃ and then desiccated aseptically on dried silica gel for 5h to a water content of 231% (fresh weight basis) prior to immersion in liquid nitrogen (LN) for 1d. After rewarming at a 40℃ water bath for 2-3min and transferred to solid culture medium for shoot tip recovery. About 50% of cryopreserved shoot tips grew into shoots within 2 weeks after plating. Cryopreservation of Astragalus membranaceus (Fisch.) Bge. shoot tips by encapsulation vitrification has also been developed. Excised shoot tips were firstly encapsulated into alginate gel beads and then precultured in liquid MS medium containing 1mg·L-1 6 BA, 005mg·L-1 NAA and 075mol·L-1 sucrose at 25℃ for 3d. After loading for 90min with a mixture of 2mol·L-1 glycerol and 04mol·L-1 sucrose at 25℃, shoot tips were dehydrated with PVS2 for 120min at 0℃ prior to direct immersion in liquid nitrogen for 1d. After rapidly thawing at a 37℃ water bath for 2-3min, shoot tips were washed for 10min with liquid MS medium supplemented with 1mg·L-1 6 BA, 005mg·L-1 NAA and 12mol·L-1 sucrose at 25℃ and then post cultured on solid MS medium supplemented with 2mg·L-1 6 BA, 005mg·L-1 NAA. The regeneration rate of shoot tips amounted to nearly 80%. Both of plantlets regenerated from cryopreserved shoot tips were morphologically uniform, which both showed as that of control plants. Thus, this encapsulation dehydration and encapsulation vitrification technique appears promising as a routine method for the cryopreservation of shoot tips of Amembranaceus.     

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (<Emphasis Type="Italic">Ceratopetalum gummiferum</Emphasis>) shoot tips

Summary Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation-dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.     

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     

Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana?×?C. citriodora

A protocol was developed for short-term preservation and distribution of the plantation eucalypt, Corymbia torelliana × C. citriodora, using alginate-encapsulated shoot tips and nodes as synthetic seeds. Effects of sowing medium, auxin concentration, storage temperature and planting substrate on shoot regrowth or conversion into plantlets were assessed for four different clones. High frequencies of shoot regrowth (76–100%) from encapsulated explants were consistently obtained in hormone-free half- and full-strength Murashige and Skoog (MS) sowing media. Conversion into plantlets from synthetic seeds was achieved on half-strength MS medium by treating shoot tips or nodes with 4.9–78.4 μM IBA prior to encapsulation. Pre-treatment with 19.6 μM IBA provided 62–100% conversion, and 95–100% of plantlets survived after acclimatisation under nursery conditions. Synthetic seeds containing explants pre-treated with IBA were stored for 8 weeks much more effectively at 25°C than at 4°C, with regrowth frequencies of 50–84% at 25°C compared with 0–4% at 4°C. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile planting substrates. Highest frequencies (46–90%) of plantlet formation from pre-converted synthetic seeds were obtained by transferring shoot tip-derived synthetic seeds onto an organic compost substrate. These plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion of non-embryonic synthetic seeds is a novel technique that provides a convenient alternative to somatic embryo-derived artificial seeds.     

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.     

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.     

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     

Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.

An efficient short-term storage system of synthetic seeds, produced using in vitro shoot tips of the monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. (AV), was developed. In vitro shoot tips (3–4 mm) were successfully encapsulated, resulting in uniform spherical beads (capsules), using 3 % sodium alginate with 75 mM CaCl₂·2H₂O. Maximum (~100 %) conversion (into plantlets with shoot and root) of capsules (or synthetic seeds) was achieved on quarter-strength Murashige and Skoog regrowth medium, while full-strength MS medium was required for effective conversion of non-encapsulated shoot tips. The capsules showed distinct difference in their response to temperature during storage. The conversion efficiency declined upon storage duration at both 4 and 25 °C, with those stored at 25 °C being more tolerant to storage. Capsules stored at 4 °C had rapid deterioration and faced complete death within 160 days while those stored for 200 days at 25 °C showed relatively high conversion (71.6 %). An inter-simple sequence repeats fingerprinting approach, employed on indiscriminately chosen plantlets from converted capsules (following 4 and 25 °C of storage), ensured the post-storage genetic stability.     

Improved survival after cryogenic exposure of shoot tips derived from in vitro plantlet cultures of potato

In vitro plantlets were used for axillary shoot tip isolation to avoid microbial contamination which often occurred from use of greenhouse-grown plants. Periodic subculturing gave a supply of uniform plantlets necessary for obtaining shoot tips for cryogenic experiments. Previous results have shown that all cells within a shoot tip did not survive cryogenic exposure and the regrowth percentage depended upon the composition of the culture medium. A medium containing 0.5 mg/liter zeatin, 0.2 mg/liter gibberellic acid, and 0.5 mg/liter indoleacetic acid gave regeneration of a multiple-shoot mass from control and some frozen shoot tips. The two-step cooling procedure (0.2-03 degrees C/min to -35 degrees C followed by immersion in liquid nitrogen) gave high percentages of regrowth in the cultivars Norland and Red Pontiac. Shoots were obtained from treated materials in both cultivars.     

Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm

We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and alpha-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.     

Cryopreservation of wild Shih (<Emphasis Type="Italic">Artemisia herba</Emphasis>-<Emphasis Type="Italic">alba</Emphasis> Asso.) shoot-tips by encapsulation-dehydration and encapsulation-vitrification

Artemisia herba-alba, called Shih is a medicinal herbal plant found in the wilds. The biodiversity of this plant is heavily subjected to loss because of heavy grazing, land cultivation and collection by people to be used in folk medicine. In the current study, two cryopreservation dependent techniques to conserve the shoot-tips of in vitro grown Shih were evaluated: encapsulation- dehydration and encapsulation- vitrification. Shoot-tips of Shih were encapsulated into sodium-alginate beads. In encapsulation- dehydration, the effect of sucrose concentration (0.5, 0.75 or 1.0 M) and dehydration period (0, 2, 4 or 6 h) under sterile air-flow on survival and regrowth of encapsulated shoot tips were studied. Maximum survival (100%) and regrowth (27%) rates were obtained when encapsulated unfrozen Artemisia herba-alba shoot tips were pretreated with 0.5 M sucrose for 3 days without further air dehydration. After cryopreservation the highest survival (40%) and regrowth (6%) rates were achieved when Artemisia herba-alba shoot tips were pretreated with 1.0 M sucrose for 3 days without further air dehydration. Viability of Artemisia herba-alba shoot tips decreased with increased dehydration period. In encapsulation-vitrification, the effect of dehydration of encapsulated Artemisia herba-alba shoot tips with 100% PVS2 for various dehydration durations (10, 20, 30, 60 or 90 min) prior to freezing was studied. After cryopreservation the dehydration of encapsulated and vitrified shoot tips with 100% PVS2 for 30 min resulted in 68% survival and 12% regrowth rates. Further conservation techniques must be evaluated to increase both survival and regrowth percentages.     

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.     

Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)

Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube.     

Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for short-term preservation and distribution of the medicinal and timber plantation tree, Khaya senegalensis, using alginate-encapsulated shoot tips. The study assessed the effects of culture medium, storage temperature, auxin concentration and planting substrate on shoot regrowth or conversion into plantlets of four different clones. Optimal shoot growth was obtained, with high frequencies (92–100%) of shoot emergence, on Murashige and Skoog (MS) culture media containing 4.4 μM benzyladenine (BA). Encapsulated shoot tips survived longer at 25°C than at 4°C, with viability of 73–88% after 8 weeks. Conversion into plantlets was achieved on half-strength MS medium by pre-culture treatment of shoot tips with 49–490 μM indole-3-butyric acid (IBA) before encapsulation. Treatment with 245 μM IBA provided 52–98% conversion, and 90–95% of plantlets survived acclimatisation under nursery conditions. To eliminate the in vitro culture step after encapsulation, synthetic seeds were allowed to pre-convert before sowing directly onto a range of ex vitro non-sterile substrates. Highest frequencies of plantlet formation from pre-converted synthetic seeds (42–86%) were obtained by transferring synthetic seeds to organic compost, and these plantlets exhibited almost 100% survival in the nursery without mist irrigation. Pre-conversion is a novel and convenient method for producing synthetic seeds that are suitable for distribution to commercial nurseries.