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)     

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 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     

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.     

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.     

Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi baker

Somatic embryos of Asparagus cooperi were encapsulated as single embryos approximately 4–6 mm in diameter to produce individual synthetic seeds. The frequency of conversion of artificial seeds to plants was 34%. This frequency was affected by the concentration of calcium chloride, the commercial sources of sodium alginate, and the nutrient medium. The conversion frequency of artificial seeds to seedling plants was 8.3% after storage for 90 days at 2°C.Abbreviations NAA -Naphthaleneacetic acid - ABA Abscisic acid - GA₃ Gibberellic acid - Kn 6-Furfurylaminopurine - MS Murashige and Skoog (1962) medium - SH Schenk and Hildebrandt (1972) medium - WM White (1963) medium     

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.     

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     

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.     

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.     

Field planting of alfalfa artificial seeds

Summary Encapsulated somatic embryos (artificial seeds) and naked (uncoated) somatic embryos of alfalfa (Medicago sativa L.) were planted directly into the field to demonstrate the feasibility of using artificial seeds for direct sowing. Various row coverings that provided protection for the somatic embryos during conversion (plant formation) in the field and encapsulation methods were investigated. The highest conversion obtained in the field was 25% when naked somatic embryos were planted under the protective covering of inverted styrofoam cups. In comparison, 60% conversion was obtained when embryos were planted in potting mix in a growth chamber. Somatic embryos encapsulated by the thin-coat method converted at 23% under cups in the field and 40% in potting mix in the growth chamber. Naked somatic embryos had an average of 13 and 9% conversion in the field under plastic and cloth coverings, respectively, whereas encapsulated embryos converted at 5 and 14%, respectively. Direct-planted embryos (no row covering) converted at 1% in the field. Successful conversion of coated and naked somatic embryos planted in the field supports the concept of artificial seeds serving as a substitute for natural seeds.     

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.     

Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species

One of the alternative methods adopted in recent years is to use biotechnological approaches for improving the tree species. The synthetic seeds offer several advantages, e.g., easy handling, storability, reduced size of propagules, and transportability. Germplasm can be effectively stored in the form of synthetic seeds. A protocol has been developed for plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb. Nodal segments collected from basal sprouts of mature trees were encapsulated in calcium alginate beads. Inability of nodal segments entrapped in calcium alginate beads to form root was a major problem. To avoid this problem, an appropriate root induction treatment was given to nodal segments for 10 days, prior to encapsulation to allow formation of root primordia. For synthetic seeds production and subsequent conversion into plantlet, nodal segments with root primordia were encapsulated using sodium alginate and calcium chloride as gelling matrix. The best gel complexation was achieved using 3% sodium alginate and 75 mmol/L CaCl2 2H2O. Maximum percentage response (85%) for conversion of encapsulated nodal segments into plantlets was achieved on 1/2-MS medium without plant growth regulators, after 25 days of culture. The frequency of conversion of encapsulated nodal segments into plantlets affected by the concentration of sodium alginate, and the presence or absence of 1/2-MS nutrients in calcium alginate beads. Plantlets with well developed roots and shoots were transferred to pots containing autoclaved mixture of peat moss and soil (1:1). Plants were also established in pots. The conversion of encapsulated nodal segments into plantlets also occurred when calcium alginate beads having entrapped nodal segments were directly sown in autoclaved peat moss moistened with 1/2-MS0 medium. Out of 60 encapsulated nodal segments, in each experiments, stored at 4 degrees C for 30 days, 44 plants developed under in vitro conditions, and 27 on peat moss moistened with 1/2-MS0.     

Encapsulation of microcuttings for propagation and short-term preservation in Ruta graveolens L.: a plant with high medicinal value

Synthetic seeds technology is a potential tool for an efficient and cost-effective clonal propagation system. In the present study, synthetic seeds were produced by encapsulating nodal segments (synthetic or synseeds) of Ruta graveolens in calcium alginate gel. The best gel complex was achieved using 3 % sodium alginate and 100 mM CaCl₂.2H₂O. Maximum conversion response of synthetic seeds into plantlets was obtained on MS medium supplemented with 10 μM 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA). Encapsulated nodal segments could be stored at low temperature (4 °C) up to 4 weeks with a survival frequency of 86.7 %. The regenerated shoots rooted on MS medium containing 0.5 μM indole-3-butyric acid (IBA). Well-developed plantlets with proper root and shoot system from encapsulated nodal segments were hardened off successfully with 90 % survival rate. The high frequency of plant re-growth (conversion) from alginate-coated nodal segments coupled with high viability percentage after 4 weeks of storage is highly encouraging for the exchange of R. graveolens genetic resources.     

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.     

High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita

An efficient protocol for plant regeneration through somatic embryogenesis was established from in vivo leaf explants of Swertia chirayita, a critically endangered medicinal herb. The highest frequency (76%) of embryogenic callus was induced on Murashige & Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (Kn) from in vivo leaf explants. Globular somatic embryos were induced and further matured from such embryogenic calli by subsequent culture on the same medium. The highest number of somatic embryos (48.83 ± 4.6) was recovered from embryogenic calli derived from leaf explants after 6 weeks of culture. Synthetic seeds were produced by encapsulating of torpedo stage embryos in sodium alginate (4% W/V) gel, dropped into 100 mM calcium chloride (CaCl₂ · 2H₂O) solution. The synthetic seeds were germinated on MS medium. The highest frequency of synthetic seed germination (84%) was observed on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. Regenerants were successfully acclimatized under ex vitro condition. This is the first report on synthetic seed production of S. chirayita. Application of these protocols would be helpful in reducing stress in natural habitat, and in long-term storage of elite genotypes through synthetic seed production.     

Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring

Cork oak (Quercus suber) somatic embryos were coated with alginate for the production of synthetic seeds and their storability for commercialization was investigated. Also, the automatic monitoring of somatic embryo growth with a digital system of image capture was tested. A power regression model was fitted between size and fresh weight (Adjusted R-squared = 0.96). This method permitted growth assessment without contamination risk and opens the possibility of an automated control of culture growth for the future up scaling of plant production. Conversion rate of synthetic seeds was higher on medium supplemented with mineral nutrients than on medium without nutrients. Also, when the somatic embryos were coated without mineral nutrients added to the capsule, conversion rate was significantly lower. The addition of sucrose to the capsule had no significant effect on the conversion rate. No differences were recorded between 50 and 100 mM CaCl₂ for capsule complexation. Synthetic seeds were cold stored at 4°C for two months without significant loss of conversion capacity. The present study reports the first attempts to determine optimal storage time and conditions for conversion of encapsulated somatic embryos of cork oak.     

Advances in somatic embryogenesis and plant production of black locust (Robinia pseudoacacia L.)

Summary Black locust (Robinia pseudoacacia L.) immature seeds of different developmental stages were tested for the ability to initiate embryogenic cultures. Best results (average of 12% embryogenic cultures) were obtained when seeds collected 2–3 weeks post-anthesis were cultured for 3 weeks on modified Finer and Nagasawa medium containing 2,4-D (45–90 M) and BA (2.2 M) and then transferred to the same medium without growth regulators. Embryo conversion was obtained from naked or encapsulated somatic embryos derived from a long-term embryogenic line. Without cold treatment, 71% of naked embryos and 41% of the encapsulated embryos converted into plants. Fifteen days of cold treatment increased conversion rates up to 95% for naked embryos and 80% for encapsulated embryos. Recovered plantlets were acclimatized and grown in the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FM modified Finer & Nagasawa (1988) medium - MS modified Murashige & Skoog (1962) medium - PEM proembryogenic mass - SH modified Schenck & Hildebrandt (1972) medium     

In vitro propagation and conservation of Indian sarsaparilla, Hemidesmus indicus L. R. Br. through somatic embryogenesis and synthetic seed production

A protocol has been developed for achieving somatic embryogenesis from callus derived from nodal cuttings and production of synthetic seeds in Hemidesmus indicus L. R. Br. a highly traded ethnomedicinal plant. Proembryogenic, friable, light yellowish callus was induced from the basal cut end of the nodal cuttings on Murashige and Skoog (MS) medium supplemented with 3 μM indole-3-butyric acid (IBA). The highest rate of somatic embryogenesis (92 %) was observed when the callus was subcultured on half strength MS medium supplemented with 2 μM IBA. On induction medium somatic embryos were developed up to the torpedo stage. Further elongation and germination of somatic embryos were obtained in MS medium supplemented with 4 μM 6-benzylaminopurine (BA) in combination with 1.5 μM gibberellic acid (GA₃). Somatic embryos were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V) dropped into 75 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds and later transferred to MS medium for germination. The synthetic seeds were successfully germinated on medium even after 120 days of storage at 4 °C. The plantlets were eventually transferred to soil with 92 % success.