An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.     

Propagation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.     

Cryopreservation of somatic embryos from <Emphasis Type="Italic">Petiveria alliacea</Emphasis> L. by different techniques based on vitrification

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.     

Enhancement of somatic embryogenesis and plant regeneration in Japanese larch (<Emphasis Type="Italic">Larix leptolepis</Emphasis>)

Different nitrogen sources, abscisic acid (ABA), gellan gum at various concentrations, and osmotica were evaluated for their effects on maturation of somatic embryo (SE) in Japanese larch (Larix leptolepis). Different concentrations of l-glutamine or casein hydrolysate (CH) in the medium were also compared. The highest number of matured embryos was obtained with ½ Litvay (LM) medium supplemented with 1.71 mM l-glutamine and 250 mg l^?1 CH. In terms of osmoticum effect, the highest number of cotyledonary SEs was produced in medium containing 0.2 M maltose. As for the effects of ABA and gellan gum concentration, the highest number of cotyledonary SEs was achieved on a medium containing 60 μM ABA and 0.8% gellan gum. In addition, the best plantlet conversion frequency (35.5%) was obtained with SEs derived from the treatment with 60 μM ABA and 0.8% gellan gum.     

Isolation and functional characterization of two 5-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferases related to anthocyanin biosynthesis from <Emphasis Type="Italic">Freesia hybrida</Emphasis>

Kelussia odoratissima Mozaff. (or Kelus) is a medicinal plant native to the Zagros Mountains in Iran. This plant is widely used as a food flavoring and for its health-promoting properties. It has been considered an endangered species by the United Nations Development Programme. In this study, a somatic embryogenesis (SE) method was developed for mass propagation of Kelus. The green globular embryogenic callus was induced on cotyledonary leaves using the Murashige and Skoog (MS) medium supplemented with 1 mg/l 2,4-dichlorophenoxyaceticacid (2,4-D) and 0.25 mg/l Kinetin. Different treatments were assayed for proliferation of the embryogenic callus. The calli remained embryogenic in an MS medium containing 2,4-D (1 mg/l). The light treatments and carbon source showed significant effects (P?≤?0.05) on the proliferation and development of somatic embryos. These treatments improved the conversion rate of the cotyledonary-stage embryos by 100%. The average numbers of embryos in the globular, heart, torpedo, and cotyledonary stages decreased by the addition of 3 g/l case in hydrolisate. The genetic stability among tissue culture-derived plants and the mother plant were assessed using the amplification fragment length polymorphism. No polymorphic band was observed among all the plants, exhibiting the genetic stability during in vitro multiplication. This research provides a promising approach for true-to-type plant multiplication of K. odoratissima through SE.     

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.     

Optimization of culturing conditions for production of somatic embryos and lignins of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill

Schisandra chinensis (Turcz.) Baill. is a valuable medicinal plant species increasingly used in phytotherapy worldwide. This study systematically detected the lignin content and production during somatic embryogenesis of S. chinensis. The effect of various culture parameters on biomass accumulation and lignin production were also examined to optimize the accumulation of lignins in SEs in bioreactors, including the culture method, inoculum density, aeration volume and photoperiod. An inoculum density of 20 g L^??1 embryogenic calli enhanced production of lignin, while 30 g L^??1 embryogenic calli increased the biomass of somatic embryos. During somatic embryo induction, an aeration volume of 0.2 vvm and photoperiod of 16 h day^??1 were found to be optimal for biomass accumulation and lignin production. An approximately threefold increase in the biomass production rate and a fourfold increase in the total lignin production rate in SEs were achieved in bioreactors than on solid medium. The present study indicated, therefore, that the culturing of S. chinensis somatic embryos in bioreactors is an effective method for the industrialized production of lignin in vitro.     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

In vitro clonal propagation and stable cryopreservation system for <Emphasis Type="Italic">Platycladus orientalis</Emphasis> via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P?<?0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l^?1 abscisic acid (ABA), 50 g l^?1 maltose, and 100 g l^?1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P?=?0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.     

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.     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

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.     

A clonal propagation system for Atlantic white cedar (<Emphasis Type="Italic">Chamaecyparis thyoides</Emphasis>) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l^?1 producing more embryos than 0 g l^?1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l^?1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.     

Brassinosteroid increases the cytokinin efficiency to induce direct somatic embryogenesis in leaf explants of <Emphasis Type="Italic">Coffea arabica</Emphasis> L. (Rubiaceae)

The combination of different plant growth regulators can result in beneficial effects in the induction of in vitro morphogenetic pathways. The present study reports the effect of 24-epibrassinolid (24-epiBR; brassinosteroid) when added alone and in association with N⁶-(2-isopentnyl) adenine (2-iP; cytokinin) in the induction of direct somatic embryogenesis in Coffea arabica. Leaf explants were cultivated in a modified Murashige and Skoog (MS) medium with 0 or 10 µM 2-iP and different concentrations (0.01, 0.10 or 1.0 µM) of 24-epiBR. Explants cultured on MS medium supplemented with 1.0 µM 24-epiBR in association with 2-iP produced 6.8 times more somatic embryos than the explants cultured with only 2-iP. Histological analyses also provided evidence that the supplementation of brassinosteroids in the culture medium could have influenced somatic embryogenesis differentiation. Somatic embryos obtained in the presence of brassinosteroid and cytokinin were better structured morpho-histologically as compared to those obtained in the medium with just cytokinin. This study opens new perspectives for the use of brassinosteroids in the somatic embryogenesis of C. arabica, so as to optimize the in vitro regeneration systems used in genetic improvement programs in C. arabica productive systems.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Somatic embryogenesis from stem thin cell layers of <Emphasis Type="Italic">Dendrobium aqueum</Emphasis>

An efficient in vitro regeneration protocol through somatic embryogenesis was established from stem transverse thin cell layers (tTCLs) of Dendrobium aqueum Lindley, an imperiled orchid. This study outlines the induction and successive maturation stages of D. aqueum somatic embryos (SEs). The tTCLs (~ 0.5 mm thick) cultured on halfstrength Murashige and Skoog (MS) medium containing cytokinins and auxins, either individually or in combination, produced embryogenic callus (EC). Treatment with 0.5 mg dm^-3 zeatin induced EC in 41.42 % of tTCLs. As many as 42.66 globular SEs per tTCL were formed in the presence of 1.5 mg dm^-3N⁶-(2-isopentyl) adenine (2iP) but only on 10.33 % of explants. The combined treatment of 2iP (1.5 mg dm^-3) and 0.5 mg dm^-3 6-benzyladenine resulted in 34 globular SEs on 14.7 % of tTCLs whereas the combination of 2iP and 1.0 mg dm^-3 indole-3-butyric acid (IBA) induced 7.4 globular SEs on 52.33 % of tTCLs. Supplementation of activated charcoal, amino acids, and antioxidants alleviated browning at all the concentrations tested, but the EC response declined. The addition of 0.5 mg dm^-3 polyvinylpyrrolidone to 1.5 mg dm^-3 2iP and 1.0 mg dm^-3 IBA produced 24 SEs on 19.89 % of tTCLs suggesting that the EC and SEs can be effectively induced by individual cytokinins whereas the synergistic treatments with other compounds can only enhance the induction of EC. Histological observations of EC showed the formation of globular SEs from sub-epidermal regions. Successive developmental stages of globular SEs and the intermediate stage of protocorm like bodies until the formation of plantlets were observed. The plantlets obtained through SEs showed no morphological variations, and inter simple sequence repeat profiles also confirmed the genetic fidelity of in vitro-derived progeny with high monomorphism (97.78 %). In conclusion, the use of stem tTCLs is an effective method to produce SEs through indirect somatic embryogenesis in D. aqueum.     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower <Emphasis Type="Italic">Griffinia liboniana</Emphasis>

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0?±?1.9%) and numbers of differentiated buds (3.7?±?0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L^?1 2,4-D and 1.0 mg L^?1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9?±?2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L^?1 6-BA and 1.5 mg L^?1 2,4-D, with an average of 28.0?±?2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.     

Improvement of in vitro embryo maturation,plantlet regeneration and transformation efficiency from alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) somatic embryos using <Emphasis Type="Italic">Cuscuta campestris</Emphasis> extract

Developmental deficiency of somatic embryos and regeneration to plantlets, especially in the case of transformation, are major problems of somatic embryo regeneration in alfalfa. One of the ways to overcome these problems is the use of natural plant regulators and nutrients in the culture medium of somatic embryos. For investigating the influence of Cuscuta campestris extract on the efficiency of plant regeneration and transformation, chimeric tissue type plasminogen activator was transferred to explants using Agrobacterium tumefaciens, and transgenic plants were recovered using medium supplemented with different concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR. Somatic embryos of Medicago sativa L. developed into plantlets at high frequency level (52 %) in the maturation medium supplemented with 50 mg 1^?1 C. campestris extract as compared to the medium without extract (26 %). Transformation efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and without the extract, respectively. HPLC and GC/MS analysis of the extract indicated high level of ABA and some compounds such as Phytol, which can affect the somatic embryo maturation. The antibacterial assay showed that the extract was effective against some strains of A. tumefaciens. These results have provided a scientific basis for using of C. campestris extract as a good natural source of antimicrobial agents and plant growth regulator as well, that can be used in tissue culture of transgenic plants.     

Regeneration of soapnut tree through somatic embryogenesis and assessment of genetic fidelity through ISSR and RAPD markers

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants.