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.

     

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.     

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.     

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.     

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 via organogenesis and embryogenesis of <Emphasis Type="Italic">Cyrtanthus mackenii</Emphasis>: a valuable threatened medicinal plant

Efficient and simple, organogenesis (direct and indirect) and somatic embryogenesis (cell suspension) systems were developed for in vitro propagation of Cyrtanthus mackenii, a valuable economic plant from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of sucrose, plant growth regulators (PGRs), glutamine, phloroglucinol (PG) and 6-(2-hydroxy-3-methylbenzylamino) purine (PI55). MS medium solidified with 8 g L^?1 agar (MS_S) containing 40 g L^?1 sucrose, 10 µM picloram, 2.5 µM benzyladenine (BA) and 20 µM glutamine produced a higher number of shoots from white nodular callus. This was however, not significantly different to direct shoot regeneration on media containing 10 µM picloram, 2.5 µM BA and a reduced concentration of sucrose and glutamine. The regenerated shoots were rooted best with MS_S medium incorporating 10 µM PG. The number of somatic embryos (SEs) were significantly higher using liquid MS medium containing 30 g L^?1 sucrose, 0.5 µM picloram, 1 µM thidiazuron or BA and 3 µM glutamine or gibberellic acid. The embryos were germinated in PGR-free MS_S medium. All plantlets were successfully acclimatized in the greenhouse. Histological studies confirmed the different developmental stages and bipolar structure of SE. The organogenesis and somatic embryogenesis protocols provides a system for large scale propagation and germplasm conservation. Developed protocols can be used for clonal production and pharmacological and genetic transformation studies.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

In vitro propagation and the acclimatization effect on the synthesis of 2-hydroxy-4-methoxy benzaldehyde in Decalepis hamiltonii Wight and Arn.

The present study reports a high frequency in vitro propagation protocol through apical bud sprouting and basal organogenic nodule formation in shoot tip explants of Decalepis hamiltonii, an endemic and endangered medicinal liana. Among different combinations of plant growth regulators (PGRs) and growth additives, maximum of 8.20 shoots per explant with mean shoot length of 6.54 cm were induced on Murashige and Skoog’s medium (MS) supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-acetic acid (IAA) + 30.0 µM adenine sulphate (ADS) through apical bud sprouting. On single cytokinin treatment explants did not exhibit good multiplication but showed nodulation (N₁) from the basal cut end similar to cytokinin–auxin combination (N₂). Between two types of nodular tissues, N₂ was proved to be better for maximum shoot regeneration (15.40 shoots per explant) and shoot length (4.56 cm) when cultured on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 30.0 µM ADS and 1.0 µM gibberellic acid (GA₃). Microshoots were efficiently rooted on half-strength MS medium supplemented with 2.5 μM α-naphthalene acetic acid (NAA). After successful acclimatization in Soilrite, 95.10 % plantlets were survived in field conditions. Histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Acclimatized plantlets were studied for the estimation of chlorophyll and carotenoid content as well as the net photosynthetic rate (PN) during subsequent days of transfer to ex vitro condition. Moreover, acclimatization had a significant effect on biomass production and the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2HMB). Maximum fresh weight (3.78 gm/plant), dry weight (0.39 gm/plant) of roots and 2HMB content (15.94 µg/ml of extract) were noticed after 8 weeks of acclimatization.     

Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique

Limonium ‘Misty Blue’ is an interspecific hybrid of Limonium latifolium and L. bellidifolium and has a huge demand in floriculture business as both fresh and dry flowers with stunning purple-blue blooms. The propagation only through vegetative means restrict the popularization of this plant to the flower growers. We therefore optimized an efficient micropropagation protocol for direct organogenesis from root explants, as leaf is not conducible to respond in culture. 61.43% of root explants directly formed shoot buds on their surface after 4-weeks of culture in media containing ½ MS, 43.82 mM sucrose 2.22 µM BA and 1.07 µM NAA. The shoot buds failed to differentiate into healthy shoots unless the previous medium was replaced by full strength MS, and 87.64 mM sucrose along with 0.44 µM BA and 1.07 µM NAA. Encapsulations of juvenile shoots were carried out by 3% sodium alginate and 100 mM CaCl₂ which were again successfully stored at 4?°C for 30 days along with 56.79% of plant recovery in MS?+?0.44 µM BA?+?4.5 µM IBA?+?87.64 mM sucrose containing medium. 150 synthetic seed derived full grown plants were successfully acclimatized in green house, where a total of 101 plants survived after secondary hardening. The ISSR analysis revealed genetic homogeneity of synthetic seed derived hardened plants.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

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.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

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.     

Somatic embryogenesis of <Emphasis Type="Italic">Byrsonima intermedia</Emphasis> A. Juss.: induction and maturation via indirect approach

Byrsonima, especially the species Byrsonima intermedia, is an endangered Brazilian plant that has been widely used as food and for its therapeutic characteristics. However, this species faces challenges with sexual propagation, and somatic embryogenesis has emerged as a viable alternative option for propagation. Therefore, this study aimed to establish a protocol for inducing somatic embryogenesis in B. intermedia. For the induction of callus from in vitro seedling leaves, different subcultures (three subcultures, 60 days each) and concentrations of different cytokinins (BAP, TDZ, Kin and ZEA) combined with varying NAA solutions were tested. Different concentrations of NAA were also analyzed in the induction of pro-embryogenic calli. For the induction of embryogenic calli and somatic embryos, the pro-embryogenic calli were subcultured on MS medium without adding growth regulators. The somatic embryos that originated were inoculated on a maturation medium containing different concentrations of gibberellic acid (GA₃). The formation of secondary embryos was also analyzed using different concentrations (0, 2.88, and 8.66 µM) of GA₃ and different types of lids (Conventional lid, Biossama® commercial lid and conventional lid with membranes). The results show that for the induction of somatic embryos, the use of kinetin with NAA presented the formation of somatic embryos in the second (4.76 µM CIN?+?0.54 µM NAA) and third (5.17 µM CIN?+?10.54 µM NAA) subcultures. The use of 28.87 µM GA₃ favored the formation of seedlings. The Biossama lid and 2.88 µM GA₃ showed higher formation of secondary embryos.     

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of <Emphasis Type="Italic">Allamanda cathartica</Emphasis> L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00?±?1.30) and shoot length (7.50?±?0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50?±?0.16 mean roots/shoot with 4.05?±?0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.     

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

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

Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 µM GA₃ resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 µM BAP?+?2.7 µM NAA), indirect organogenesis of shoot buds (0.54 µM NAA?+?9.08 µM TDZ), somatic embryogenesis (2.2 µM BAP?+?5.4 µM NAA) and somatic embryo germinated plantlets (H-MS?+?1.4 µM GA₃?+?2.2 µM BAP?+?5.4 µM NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS?+?8 µM NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 µM BAP?+?0.54 µM NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65?±?0.04 pg, 2.62?±?0.06 pg and 2.68?±?0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.