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.     

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.     

Direct somatic embryogenesis and synthetic seed production from<Emphasis Type="Italic"> Paulownia elongata</Emphasis>

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, -naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l^-1 casein hydrolysate and 10 mg l^-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl₂. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.Abbreviations BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - TDZ ThidiazuronCommunicated by H. Lörz     

Propagation of three orchid genera using encapsulated protocorm-like bodies

Summary Protocorm-like bodies (plbs) of the orchid Dendrobium ‘Sonia’ were obtained from fractionated plb explants on Murashiga and Skoog (1962) medium (MS) supplemented with 4.44 μM 6-benzylaminopurine (BA). The developmental stage of plbs to be encapsulated, concentrations of sodium alginate (2–5%) and calcium chloride (25–100 mM), and concentration of MS salts in the matrix were assessed. Fractionated plbs 13–15 d after culture were at the leaf primordia stage and found suitable for encapsulation studies. The best encapsulation response was observed with 3% sodium alginate upon complexation with 75 mM CaCl₂.2H₂O. An encapsulation matrix prepared with MS medium (three-quarter strength) supplemented with 0.44 μM BA and 0.54 μM α-naphthaleneacetic acid gave 100% conversion of encapsulated plbs to plants after storage. A viability of >85% of encapsulated plbs of Dendrobium ‘Sonia’ was observed for 75 d at 4°C. Encapsulated plbs of orchids Dendrobium, Oncidium, and Cattleya were stored at 4°C for 75, 60 and 30 d, respectively, with >88% germination. All plants survived potting in media having either wood charcoal pieces alone or in combination with brick pieces.     

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.     

In vitro response of encapsulated somatic embryos of Lagerstroemia indica L

A method to produce encapsulatable units for synthetic seeds was developed in L. indica. Somatic embryos were harvested from leaf derived embryogenic callus on Murashige and Skoog's basal medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 0.5 mg/l), 6-benzyl amino purine (BAP, 1 mg/l) and ascorbic acid (AA, 50 mg/l). The embryos were encapsulated in alginate beads and dehydrated. Germination ability of the artificial seeds were investigated. The frequency of regeneration from the encapsulated embryos was significantly affected by (i) the concentration of alginate (ii) the duration of storage, and (iii) the effect of different types of media. A 2% sodium alginate concentration on MS salts resulted in significantly higher germination frequencies than at other concentrations. L. indica showed maximum germination on MS medium (93.84%) after 6 weeks of culture. The germinated synthetic seeds with well developed roots and shoots were transferred successfully to green house. This is the first report on artificial seeds in Lagerstroemnia indica.     

Role of polarity in de novo shoot bud initiation from stem disc explants of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn. and its encapsulation and storability

The occurrence of strong polarity towards shoot bud induction and the effect of cytokinin(s) on each segment of stem axis, encapsulation and storability of de novo Shoot buds of Curculigo orchioides Gaertn. (Hypoxidaceae) have been documented in the present communication. Maximum number of shoot buds arising de novo from the stem discs (cross section) explanted from proximal end on MS medium fortified with BAP and KIN 1 mg/L each. Stem discs from distal end were less efficient in shoot bud induction. A combination of two cytokinins (BAP and KIN) as a synergistic effect on shoot buds induction from each segment of stem axis. Stem discs in inverted position produced shoot buds from the lower surface, showing strong polarity within the explant. Further, storability and shoot development of sodium alginate encapsulated shoot buds of Curculigo orchioides were tested on half-strength Murashige and Skoog (MS) basal medium fortified with coconut water (10% v/v). The frequency of regeneration from encapsulated shoot buds was affected significantly by concentration of sodium alginate and the duration of exposure to calcium chloride. Shoot buds encapsulated with 2.5% sodium alginate dissolved in MS basal salts solution recorded significantly higher shoot development than other treatments. A relatively short (5 min) incubation with calcium chloride solution provided uniform encapsulation of shoot buds that gave the highest percentage (68%) of shoot development. Encapsulated shoot buds could be stored at 4°C for 50 days without reduction in viability as oppose to non-encapsulated shoot buds, which showed 9.5% viability after 20 days at 4°C. Encapsulated shoot bud developed into normal shoots. Based on the present observations an improved protocol may be developed for the rapid multiplication and conservation of the endangered species—C. orchioides.     

Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.)

Interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.) cotyledonary somatic embryos were encapsulated in sodium alginate. Somatic embryo viability was retained, but germination occurred at a reduced frequency compared with the equivalent zygotic embryos. The addition of 0.5% (w/v) activated charcoal to the alginate capsule significantly enhanced root development and germination for somatic embryos but not for zygotic embryos. The possibility of developing an artiflcal endosperm was also investigated, by addition of Litvay (Litvay et al. 1981) nutrients with or without 90 mM sucrose to the alginate-charcoal capsule. This treatment significantly enhanced root development for all embryo categories with the exception of black spruce somatic embryos. Encapsulated and non-encapsulated somatic embryos survived one month cold storage at 4 °C without reduction in germination frequency.NRCC No. 35895     

A novel encapsulation technique for the production of artificial seeds

A novel technique for the encapsulation of plant material in calcium alginate hollow beads was tested. The technique involves suspending plant material (i.e. plant cells, tissues, organs, shoot tips, somatic embryos) in a solution containing carboxymethylcellulose and calcium chloride and then dripping it into a stirred sodium alginate solution. In initial experiments with Daucus carota (carrot), it was found that after 14 days of cultivation, 100 % of seeds encapsulated in calcium alginate hollow beads would germinate in the liquid core and that 13% would burst the capsules. Embryogenic calli developed inside hollow beads and formed somatic embryos while calli in conventional calcium alginate beads became detached from the beads early in development, and no somatic embryogenesis occurred. With Solanum tuberosum (potato), development of calli was observed in 50% of hollow beads. Eighty-one percent of shoot tips encapsulated in hollow beads sprouted and grew out of the capsules. Received: 28 October 1999 / Revision received: 11 February 2000 / Accepted: 22 February 2000     

Encapsulation of <Emphasis Type="Italic">Pannonibacter phragmitetus</Emphasis> LSSE-09 in alginate–carboxymethyl cellulose capsules for reduction of hexavalent chromium under alkaline conditions

Cr(VI) was efficiently reduced to Cr(III) by Pannonibacter phragmitetus LSSE-09 encapsulated in liquid-core alginate–carboxymethyl cellulose capsules under alkaline conditions. Taking into account the physical properties of the capsules, the activity of encapsulated cells, and total Cr(III) concentration in the supernatant, optimal conditions (0.5% w/v sodium alginate; 2% w/v sodium carboxymethyl cellulose; 0.1 M CaCl₂; 30-min gelation time) for LSSE-09 encapsulation were determined. At optimal conditions, a relatively high reduction rate of 4.20 mg g_{(dry weight)}⁻¹  min⁻¹ was obtained. Total Cr(III) concentration in the supernatant was significantly decreased after reduction, because 63.7% of the formed soluble organo-Cr(III) compounds compared with those of free cells were captured by the relatively smaller porous structure of alginate capsules. The optimal pH value (9.0) for Cr(VI) reduction was not changed after encapsulation. In addition, encapsulated LSSE-09 showed no appreciable loss in activity after eight repeated cycles at 37°C, and 85.7% of its initial activity remained after 35-day storage at 4°C. The results suggest that encapsulated LSSE-09 in alginate–carboxymethyl cellulose capsules has potential biotechnological applications for the detoxification of Cr(VI)-contaminated wastewater.     

Encapsulation of Immature Somatic Embryos of <i>Coffea arabica</i> L. for <i>in Vitro</i> Preservation

The present study aimed to develop a protocol for somatic embryogenesis and encapsulation of coffee embryos (Coffea arabica L.), for the conservation of genotypes with characteristics of commercial interest. Somatic embryos were induced from leaf explants in Murashige and Skoog medium (MS) supplemented with 1 mg · L⁻¹ of 2,4-dichlorophenoxiacetic acid (2,4-D) combined with 2 mg · L⁻¹ of benzyladenine (BA). Somatic embryos (SE) at the globular stage were encapsulated in a sodium alginate matrix; two treatments were tested: MS + 5 mg · L⁻¹ BA + 1 mg · L⁻¹ NAA + 3% (w/v) alginate, and MS + 7 mg · L⁻¹ BA + 5.7 mg · L⁻¹ indoleacetic acid (IAA) + 3% (w/v) alginate. Alginate was complexed with 100 mM calcium chloride (CaCl₂). Viability of the encapsulated SE was determined by staining with 0.01% fluorescein diacetate (FDA) after 0, 15, 30, and 45 days of storage at 4°C. Embryo viability was 100% in both treatments.     

Phosphomannose-isomerase (<Emphasis Type="Italic">pmi</Emphasis>) gene as a selectable marker for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of rapeseed

Nodal segments (4 ± 1 mm long) of Hibiscus moscheutos (hardy hibiscus) were excised from in vitro proliferating microshoots and utilized to evaluate initial factors involved in bulk alginate encapsulation. The factors evaluated were; storage vessel type, volume and multiple rinse effects of CaCl₂ solutions, and sodium alginate concentrations (2.5, 2.75, 3.0 or 3.25%) for bulk alginate encapsulation. Results indicate that vessels utilized for bulk alginate encapsulation should have a lower base area (L × W) to height ratio to reduce the amount of alginate matrix shrinkage resulting in exposure of nodal segments. Increased volumes and multiple 50 mM CaCl₂ solution rinses did not have an effect on alginate solidification. Shoot length, root number, and root length significantly decreased in a linear manner from nodal explants stored for 4 weeks with increasing concentrations of sodium alginate. This research suggests an innovative technique for alginate encapsulation of H. moscheutos utilizing bulk methods as an alternative to single bead alginate encapsulation.     

Short-chain dehydrogenase/reductase,PsDeHase, from opium poppy: putative involvement in papaverine biosynthesis

Embryogenic synseeds were prepared in Albizia lebbeck by encapsulating cotyledon stage somatic embryos derived from in vitro maintained embryogenic cultures in different types of Ca-alginate beads. The germination rate of somatic embryos was affected significantly by the bead type, matrix composition and germination substrate. A matrix made of 3% Na₂-alginate complexed with 100 mM CaCl₂·2H₂O for a hardening period of 20 min provided uniform encapsulation of somatic embryo. Among different types of synseeds, type IIA, wherein somatic embryos encapsulated in a single layer of Ca-alginate matrix composed of MS medium supplemented with 2 g L^?1 activated charcoal and 1.0 µM gibberellic acid (GA₃) as reconstituted endosperm, was found to be the most efficient type having maximum germination rates (88.6?±?0.51%). Incorporation of GA₃ in the alginate beads stimulated greater germination of somatic embryos as against GA₃ supplementation in the germination substrate. Further, viability studies on short term cold (4 °C) storage of different types of embryogenic synseeds revealed that double layered synseeds (DLS) were found comparatively more robust to withstand longer storage durations than single layered synseeds as evident by greater germination rates of the former after 4–8 weeks of refrigerated storage. Also, the elevated levels of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and leaf proline content in the plantlets derived from DLS reveals the possible role of alginate coatings in conferring alleviation to low temperature stress generated during different storage durations. Similar Inter simple sequence repeat profiles of embryogenic synseeds derived plantlets and mother tree nullifies the possible occurrence of somaclones, thereby establishing the efficacy of synseed technology for clonal propagation of A. lebbeck germplasm.

     

Effects of encapsulation on Citrus reticulata Blanco somatic embryo conversion

Preliminary experiments on calcium-sodium alginate encapsulation of somatic embryos from anther culture were performed, in order to evaluate the effect of this technique on the recovery of plantlets, and the applicability of the synthetic seed technology to Citrus reticulata Blanco. Frequencies of conversion of somatic embryos on plant growth medium were 15.0%, 26.7% and 50.0%, respectively, when the somatic embryos were sown non-encapsulated, encapsulated with a hormone-less artificial endosperm or encapsulated with an artificial endosperm containing GA₃. Encapsulation with GA₃was also useful for storage of somatic embryos at 4 °C for one month; when sown on perlite medium, conversion frequencies were 25% with encapsulated somatic embryos against 5% with non-encapsulated embryos. Sowing on soil mix medium did not result in satisfactory conversion. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancing carrot somatic embryos survival during slow dehydration, by encapsulation and control of dehydration

In order to obtain dry artificial seeds, carrot somatic embryos were encapsulated and dehydrated. Encapsulation in some hydrogels delayed the dehydration and preserved the water content of carrot somatic embryos. In particular, a matrix made of alginate with gellan gum was found to be the most efficient in maintaining a high water activity (a_w) around somatic embryos. By delaying dehydration, and also rehydration, encapsulation seemed to protect somatic embryos against desiccation and imbibition damages, giving better germination and emergence of cotyledons. Matrices made of alginate mixed with kaolin or gellan gum were particularly adapted to protect the embryos during the dehydration. Apart from the matrix composition, the control of dehydration speed enhanced the survival and regeneration of encapsulated-dehydrated somatic embryos. Using a slow dehydration protocol (95-15% RH—relative humidity into the chamber—in 11.5 days), it was possible to exert different dehydration speeds. Slowing the dehydration between 70 and 45% RH stabilized the water activity (a_w) of the encapsulation matrix, and enhanced the survival and regeneration frequencies of encapsulated-dehydrated embryos. In the absence of any maturing pretreatment, alginate-gellan gum encapsulated carrot somatic embryos, dehydrated to 15% RH, and rehydrated in moistured air (90% RH), germinated up to 72.9%. Therefore, encapsulation in alginate-gellan gum, combined with a slow dehydration, leads to enhance the somatic embryos' desiccation tolerance.     

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.     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl. (Orchidaceae)

This article demonstrates the single bead alginate-encapsulation and conversion (complete plantlet regeneration) from protocorm-like bodies (PLBs) of Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. (AV) (a monopodial orchid hybrid) for the first time. PLBs, induced from leaf segments of AV were isolated from in vitro proliferating PLB clusters. Individual PLBs (4?±?1?mm diameter) were encapsulated in calcium alginate beads to manage mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3?% sodium alginate and 75?mM calcium chloride (CaCl₂·2H₂O). Highest percentage of germination (98.1?%) and conversion (96.2?%) of encapsulated PLBs (capsules) was obtained on plant growth regulator-free half-strength MS (Murashige and Skoog, Physiol Plant 15:473?C497, 1962) medium. Successful storage of capsules, until 180?days, was achieved at 25?°C under zero-irradiance with germination and conversion frequency of 76.9 and 70.2?%, respectively. Plantlets regenerated from capsules were acclimatized successfully with 92?% survival rate.     

Enhancement of synthetic seed conversion to seedlings in hybrid rice

Somatic embryos of hybrid rice (Oryza sativa L.) were encapsulated in sodium alginate matrix provided with MS nutrients, growth regulators and protectants as synthetic endosperm to enhance germination and conversion capacity. The synthetic seeds with synthetic endosperm constituents of MS nutrient; sucrose (3%), IAA (0.5 mg l^–1), NAA (0.5 mg l^–1), BA (0.5 mg l^–1) and charcoal (1.25%) gave a maximum germination (30%) and conversion (27%) of synthetic seeds. The inclusion of protectants, bavistin and streptomycin as constituents of synthetic endosperm had no effect on germination and conversion. The application of self-breaking gel beads technology increased the germination (52%) and conversion (47%) of synthetic seeds.