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.     

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.     

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.     

Effects of capsule hardness on germination frequency of encapsulated somatic embryos of carrot

Summary Somatic embryos of carrot were encapsulated in calcium alginate beads to provide artificial carrot seeds. Alginate capsules with a hardness of 0.2 to 0.5 kg/cm² were found to be suitable for germination of encapsulated somatic embryos. The germination frequencies were more than 95%, when grown aseptically on polyester fiber supports loaded with hormone-free Murashige-Skoog medium.     

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.     

Storability and germination of sodium alginate encapsulated somatic embryos derived from the vegetative shoot apices of mature <Emphasis Type="Italic">Pinus patula</Emphasis> trees

Storability and germination of sodium alginate encapsulated somatic embryos derived from vegetative shoot apices of mature Pinus patula trees were tested on half strength DCR basal medium without growth regulators. The germination percentage of encapsulated somatic embryos was affected significantly by the concentration of sodium alginate and the duration of exposure to calcium chloride. Somatic embryos encapsulated with 2.5 sodium alginate dissolved in DCR basal salts gave significantly higher germination (89) than other treatments. Short (5 min) incubation of the alginate encapsulated embryos in calcium chloride solution proved to be the best encapsulation procedure and the embryos subsequently gave the highest germination (89). Synthetic seeds could be stored at 2 °C for 120 days without a reduction in germination as opposed to non-encapsulated somatic embryos which showed only 9 germination after 20 days at 2 °C. Germinated synthetic seeds produced normal plantlets. This study reports for the first time the storability of encapsulated somatic embryos generated from vegetative shoot apices of mature Pinus patula trees. This has potential for application in forestry.     

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.     

Production,storability, and regeneration of shoot tips of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) encapsulated in calcium alginate hollow beads

Summary Shoot tips, of four potato cultivars (Désirée, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Désirée), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Désirée), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.     

Plant regeneration from encapsulated embryoids and an embryogenic mass of pistachio, Pistacia vera L.

Pieces of an embryogenic mass (EMS) induced in culture from immature fruits of pistachio, Pistacia vera L., were encapsulated into calcium alginate beads. Somatic embryos were also encapsulated individually into calcium alginate beads to produce synthetic seeds. The viability of the encapsulated EMS and somatic embryos was investigated immediately following encapsulation, and after storage for 60 days at 4°C. The encapsulated-stored EMS fragments recovered their original proliferative capacity after two months storage following two sub-cultures, but non-encapsulated-stored EMS failed to recover. The conversion frequency of synthetic seeds to seedling plants was 14% after storage for 60 days at 4°C, from which it may be concluded that encapsulation is a practical procedure for short-term storage of embryogenic pistachio tissue, and may be applicable to the preservation of desirable elite genotypes.Abbreviations BAP Benzylaminopurine - EMS(es) Embryogenic mass(es) - MS Murashige and Skoog medium (Sigma M-0404) - PGR(s) Plant growth regulator(s)     

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.     

High frequency plant regeneration of garlic (Allium sativum L.) calli immobilized in calcium alginate gel

Calli obtained from a shoot-tip of garlic,Allium sativum L., were encapsulated using a calcium alginate gel. Some of the encapsulated calli were cultured on a 1/2 MS medium supplemented with 3% sucrose, 10⁻⁵ M kinetin, and 5×10⁻⁶ M NAA, whereas the remainder was stored for 40 days at 4°C. All the naked calli regenerated on the solid medium, while 95% of the encapsulated calli regenerated, and 88% of the encapsulated calli regenerated after 40 days of storage at 4°C. The capsule matrix delayed the germination time of the encapsulated calli, yet activated the shoot formation of the artificial garlic seeds. The shoot length of the encapsulated garlic calli was much longer than that of the naked garlic calli. The encapsulated garlic calli were dried in a laminar airflow cabinet and the conversion frequency of the dried artificial garlic seeds on a 1/2 MS medium remained at 93% with a water loss of less than 50%.     

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.     

In vitro Storage of Strawberry and Raspberry in Calcium-Alginate Beads at 4°C

Three-millimeter-long shoot tips of strawberry 'Senga Sengana' and raspberry 'Norna' encapsulated in calcium alginate were stored in vitro at 4 °C in the dark. The cultures which were donors for the shoot tips were grown before encapsulation on shoot multiplication media (Boxus medium with 2.2 µM BAP and 2.46 µM IBA for strawberry, and MS medium with NH₄NO₃ and KNO₃ reduced by 50%, and with 3.55 µM BAP and 0.49 µM IBA for raspberry) as well as on these media supplemented with 10 g l^–1 mannitol or paclobutrazol (1.7 µM for strawberry and 3.4 µM for raspberry). Sodium alginate was dissolved in water, water with sugar or in a culture medium without growth regulators. Regrowth ability of the stored explants and in vitro multiplication in three successive subcultures were evaluated. The encapsulated shoot tips could be stored for 9 months in beads containing sugar or a culture medium. The pre-conditioning of the donor cultures on a mannitol containing medium was beneficial for regrowth ability. The multiplication rate of strawberry and raspberry shoots in the first subculture after storage was lower than that of non-stored cultures. Particularly low multiplication was obtained for strawberry which had been stored for 9 months and for raspberry stored for 3 and 6 months, in combinations where the beads were prepared by dissolving sodium alginate in water. Multiplication of strawberry in the second subculture was generally higher than in non-stored cultures, but multiplication of raspberry was lower also in the second subculture, with the exception of the combination stored for 9 months and pre-cultured on mannitol. In the third subculture, shoot multiplication in both species was similar to that in non-stored cultures.     

Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Plant regeneration from encapsulated somatic embryos of Carica papaya L.

Carica papaya L. (papaya) single somatic embryos (2.0 mm diameter) produced in a high-frequency liquid production system were encapsulated in two different synthetic encapsulation compounds. The frequency of regeneration from encapsulated embryos was significantly affected by (1) the concentration of sodium alginate, (2) the presence or absence of nutrient salts in the capsule, and (3) the duration of exposure to calcium chloride. A 2.5% sodium alginate concentration in a half-strength MS salts base resulted in significantly higher germination frequencies than other treatments. A relatively short (10 min) exposure to CaCl₂ provided uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Germinated artificial seeds produced normal plantlets. Received: 12 March 1997 / Revision recieved: 24 June 1997 / Accepted: 18 July 1997     

Physico-chemical properties of the encapsulation matrix and germination of carrot somatic embryos

Carrot somatic embryos were encapsulated in alginate gel beads. To improve the quality of a "synthetic seed" coating, the rheology and dehydration properties of different matrices were tested. By increasing alginate and CaCl(2) concentrations, additional mineral elements were shown to increase resistance to rupture, and to depress the germination of somatic embryos. A polysaccharide addition was found to slow the alginate matrix dehydration; alginate-gellan gum and alginate-kaolin matrices could preserve the viability of somatic embryos at low relative humidities (30% to 35% germinations at 50% relative humidity) to a greater extent than other matrices. (c) 1995 John Wiley & Sons, Inc.     

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.     

Large Scale Production and Storability of Encapsulated Somatic Embryos of Mothbean (Vigna aconitifolia Jacq)

Storability and germination of sodium alginate encapsulated somatic embryos of Vigna aconitifolia (Jacq.) cv. BMB-43 were tested on half strength Murashige and Skoog (MS) basal medium fortified with coconut water (10% v/v). The frequency of regeneration from encapsulatd embryos was affected significantly by concentration of sodium alginate and the duration of exposure to calcium chloride. Embryos encapsulated with 2.5 % sodium alginate dissolved in MS basal salts solution recorded significantly higher germination than other treatments. A relatively short (5 min) incubation with calcium chloride solution provided uniform encapsulation of embryos that gave the highest percentage (65%) of germination. Synthetic seeds could be stored at 4üC for 50 days without reduction in viability as opposed to non - encapsulated somatic embryos which showed 6% viability after 20 days at 4°C. Germinated synthetic seeds produced normal plantlets.     

Entrapment of biological control agents applied to entomopathogenic nematodes

Summary Hydrogels of alginate, phospho guar gum, carboxymethyl guar gum, k-carrageenan and cellulose sulphate, respectively were tested to find easily redissolvable gels. The entomopathogenic nematode, Heterorhabditis sp., was entrapped in calcium alginate beads, calcium alginate hollow spheres and foils made from different hydrogels. Emigration from calcium alginate beads after 7 days of storage was 100 % at room temperature and was lowered to 6 % at 6 °C, whereas no emigration from calcium alginate hollow spheres was found at either temperature. Highly concentrated polymer foils produced on gauze showed reduced emigration with a survival of 80 % after 24 h compared to foils produced on glass slides. Calcium alginate beads can be used for a controlled release of the nematode into the environment, while hollow spheres and foils are suitable for storage.Dedicated to Prof. Dr. F. Wagner on the occasion of his 65th birthday     

Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Artificial seeds were obtained through encapsulation of protocorm-like bodies (PLBs) of Dendrobium densiflorum in calcium alginate beads. This paper demonstrates the alginate-encapsulation and conversion (complete plantlet regeneration) from PLBs, the effect of storage conditions (at different temperature; 4, 8, 16 °C, RT and duration; 15, 30, 45, 60, 75, 90 days) on viability of encapsulated plant materials as well as the assessment of genetic fidelity of the regenerants. Individual PLBs were encapsulated in calcium alginate beads for mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3 % sodium alginate and 100 mM calcium chloride (CaCl₂·2H₂O). The highest percentage of conversion (100 %) of encapsulated PLBs (capsules) was obtained on MS2 medium (MS medium + 2 mg/l BAP). Capsules were successfully stored till 60 days at 8 °C with conversion frequency of 95.5 %. Plantlets regenerated from encapsulated beads were acclimatized successfully with 95 % survival rate. A total of 40 primers were screened, out of which 10 primers successfully generated 39 scorable bands, ranging from 0.2 to 1.3 kb amplicons. The uniform RAPD banding profile among the plantlets derived from encapsulated PLBs following 60 days of storage confirmed genetic fidelity.