Nutrient alginate encapsulation of nodal segments of Althaea officinalis L., for short-term conservation and germplasm exchange

Abstract

In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl₂.2H₂O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved Soilrite^TM (Keltech Energies, Bangalore, India).     

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.     

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Direct plant regeneration from encapsulated nodal segments of Vitex negundo

An efficient protocol for encapsulation of nodal segments of Vitex negundo L. has been developed for the production of non-embryogenic synthetic seeds. The encapsulations of nodal segments were significantly affected by the concentrations of sodium alginate and calcium chloride. A 3 % Na₂-alginate with 100 mM CaCl₂ has been found to be optimum concentration for the production of uniform synthetic seed. For germination, the synseeds were cultured on Murashige and Skoog (MS) basal medium supplemented with kinetin (KIN) and α-naphthalene acetic acid (NAA) either singly or in various combinations. MS medium containing 2.5 μM KIN in combination with 1.0 μM NAA was found to be the optimum for maximum (92.6 ± 3.71 %) plantlet conversion frequency. Well developed regenerated plantlets were hardened, acclimatized and established in field, where they grew well without any detectable variation.     

Optimization of culture medium components and culture period for production of adventitious roots of <Emphasis Type="Italic">Echinacea pallida</Emphasis> (Nutt.) Nutt

An efficient short term storage protocol was developed for Ansellia africana, a vulnerable medicinal orchid of Africa using encapsulated protocorm-like bodies (PLBs) induced from nodal segments of seedlings with highest response recorded on MS medium supplemented with 10 µM TDZ and 5 µM NAA. The gel matrix containing 3% sodium alginate and 100 mM calcium chloride was the best for the production of viable synthetic seeds. In the present study, the effects of meta-topolin (mT) and its derivatives i.e. meta-Topolin riboside (mTR) and meta-methoxy topolin 9-tetrahydropyran-2-yl (memTTHP) were studied on the viability of synthetic seeds, maintained at different temperatures (4, 8 and 25 °C) for varying duration (15, 30, 45, 60, 75 and 90 days). The highest response percentage (88.21%) of encapsulated PLBs was recorded in those cultivated on medium supplemented with 7.5 µM memTTHP. The alginate beads were successfully stored for 75 days at 8 °C with a recorded conversion frequency of 86.21%. Synergistic effect of auxin (IBA or IAA) and the phenolic elicitor phloroglucinol (PG) were tested on root induction and proliferation. The highest rooting frequency was achieved using 15 µM IBA and 30 µM phloroglucinol resulting in successful acclimatization of the plantlets. The clonal fidelity of the regenerated plantlets was also ascertained using inter-retrotransposon amplified polymorphism and start codon targeted markers which revealed a high degree of genetic homogenity amongst the in vitro raised plants. The study also documents the role of mT, mTR and memTTHP on the regeneration of artificial seed-derived plantlets in orchids. The regeneration protocol, would be helpful in reducing stress on fragmented natural habitats of A. africana and can also be extended to conserve other orchids which are encountering threats of extinction.     

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.     

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.     

High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in <Emphasis Type="Italic">Gymnema sylvestre</Emphasis>

The present study describes the first attempt of exploiting encapsulation technology for high plantlet recovery, short-term storage and conservation of Gymnema sylvestre—an antidiabetic liana. Indole-3-butyric acid (IBA) pretreated nodal segments (NS) were encapsulated in sodium alginate (Na₂-alginate) matrix and the optimal culture conditions were evaluated in terms of maximum conversion capacity of synseeds into complete plantlets. Highest conversion frequency of synseeds was obtained on Murashige and Skoog’s (MS) medium supplemented with 5.0 µM 6-benzyladenine (BA). Augmentation of Na₂-alginate matrix with plant growth regulators (PGRs) and additive further improved in vitro conversion rates and the synthetic endosperm composed of 3% Na₂-alginate in MS?+?2.5 µM BA?+?2.5 µM gibberellic acid (GA₃)?+?50 µM adenine sulphate (AdS) stimulated maximum recovery (88.2?±?0.48%) of complete plantlets from synseeds. Studies on short term cold storage of synseeds showed that nutrient encapsulation maintains the viability of NS for a storage period of 8 weeks. Ex-vitro conversion of synseeds was also carried out on soilrite and vermicompost (3:1) mixture under culture room conditions. Monomorphic DNA profiles produced through Inter-Simple Sequence Repeat (ISSR) markers confirmed the genetic uniformity between synseed derived and mother plantlets.     

Conservation,ex vitro direct regeneration,and genetic uniformity assessment of alginate-encapsulated nodal cuttings of <Emphasis Type="Italic">Sphagneticola calendulacea</Emphasis> (L.) Pruski

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L^?1 N₆-benzyladenine plus 0.25 mg L^?1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To by-pass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L^?1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.     

Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn.

An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl₂·2H₂O). A gelling matrix of 4?% Na-alginate and 100?mM CaCl₂·2H₂O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00?±?2.09?%) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0???M 6-benzyladenine (BA), 0.5???M indole-3-acetic acid (IAA) and 30.0???M adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5???M ??-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

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.     

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 internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Effect of activated charcoal on multiplication of African yam (Dioscorea cayenensis-rotundata) nodal segments using a temporary immersion bioreactor (RITA®)

In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA^®) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA^® bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4–6.6 cm long compared to 3.2–3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5–20 nodal segments per RITA^® bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0–17.6 μM), 0.44 μM induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA^® bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

In vitro conservation of Mandevilla moricandiana (Apocynaceae): short-term storage and encapsulation–dehydration of nodal segments

In vitro conservation of Mandevilla moricandiana was performed by slow-growth storage and encapsulation–dehydration. For slow-growth storage, half- and full-strength Murashige and Skoog (MS) medium and Woody Plant Medium, with or without sorbitol, mannitol, or glucose, were used to test the development of nodal segments and maintenance of plant viability after 6 mo. Recovery was performed using MS medium. The basal medium and carbon source did not interact, and only the carbon source had a significant effect on slow-growth storage and recovery. Sorbitol and glucose, individually or in combination, promoted development of plants with a low multiplication rate during the slow-growth period and a high multiplication rate during the recovery period. For encapsulation–dehydration, nonencapsulated and sodium alginate-encapsulated nodal segments were evaluated to determine their viability after storage at different temperatures. Nonencapsulated nodal segments gave 16.6% recovery after 60 d at 25°C. The effects of preculturing encapsulated nodal segments in MS medium with 0.4 or 0.75 M sucrose followed by dehydration were also tested. Capsules precultured for 48 h in the presence of 0.40 M sucrose and dehydrated to 40% moisture content showed 93.3% recovery. These conditions were then used to store capsules under different temperatures and for different lengths of time. The precultured capsules showed ca. 30% recovery after storage for 30 d at 4°C. Well-developed plantlets regenerated from encapsulated, stored nodal segments were rooted and acclimatized successfully, with 100% survival.     

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.