High Frequency Somatic Embryogenesis Research Articles

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60days after being transferred to auxins and cytokinin (1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg Kinetin L-1) along with 200mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Optimizing culture conditions for high frequency somatic embryogenesis and plantlet conversion in Daucus carota L

The present investigation was carried out to establish a simple and efficient in vitro method for rapid propagation of Daucus carota L for different applications. Root, stem, leaf and seed explants were tested to induce callus on Gamborg medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) for cell suspension culture establishment. The leaf, stem, seed explants induced calluses were more embryogenic and viable when compared to callus derived from tap root explants. Plant regeneration potential of embryogenic callus from tap root showed very slow response producing only fewer shoots, whereas the response of other explants derived calluses like stem, leaves and seeds exhibited rapid multiple shoot formation within three weeks’ time. Tap root derived suspension culture showed more clumps with less viable embryogenic cells, in contrast to the stem and seed derived suspension cultures, where cells were more embryogenic and proliferative. Explant, concentration of carbohydrates, 2,4-D and amino acids were tested for embryo induction response and it was found that 3% sucrose, 1.0 mg/L 2,4-D and amino acid alanine had great influence on embryo induction. The somatic embryos were subjected to desiccation prior to plating on the regeneration medium. It was found that 1 h desiccation increased the regeneration percentage of the embryos and the conversion frequency of embryos to plants decreased on increased desiccation time. These optimized conditions will be very useful for studies involving genetic transformation, cell cycle, cell proliferation, synthetic seed production and other physiological studies of D. carota species.

Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L.)

Plant somatic embryogenesis (SE) has been defined as the formation of embryos from a single or group of haploid or somatic cells [1, 2]. Low frequency (LFSE) and high frequency somatic embryogenesis (HFSE) have been described. In the first type, somatic embryos are induced directly from pro-embryogenic cells of explants, while in the second, they originate from embryogenic callus [1]. It has been suggested that in LFSE the origin of somatic embryos is unicellular, whereas in HFSE has been described as unicellular or multicellular [3]. SE is a powerful biotechnological tool used to propagate elite plants or to conserve important genotypes [4]. Moreover, SE offers an efficient in vitro regeneration approach as a fundamental step in plant genetic improvement for studying basic aspects of ontogenesis of somatic embryos [5]. In Coffea spp., the first studies of SE have been reported at the beginning of 1970 [6]. Since then, a large quantity of LFSE and HFSE protocols have been optimized demonstrating that coffee is not a recalcitrant species for SE [4]. In the LFSE the somatic embryos are obtained faster (approximately 70 days) using only one medium meanwhile in HFSE several media are used and somatic embryo formation takes 9-10 months [4]. Although, in LFSE small number of somatic embryos are obtained (around 10 per explant) compared to hundreds of somatic embryos obtained per gram of embryogenic calli [4], the unicellular origin of somatic embryos in LFSE represents an advantage for the chemical and physical mutagenesis, genetic transformation and genetic editing, since prevents or reduces the appearance of chimeras [7]. In C. arabica and C. canephora many factors (such as genotype, explant type, the physiological state, age and growth conditions of the donor plants, the season of collection, nutrient composition of the medium, the volume of dissolved CO2 or O2 in the culture flask, and plant growth regulators) that affect LFSE induction have been studied [3, 8, 9, 10, 11, 12, 13]. However, few studies reported the effect of silver nitrate on LFSE using leaf explants of C. arabica L. and to the best of our knowledge it has not been analyzed using Caturra and Catuai, which are two economic important producer cultivars in Costa Rica. Since SE is genotype dependent, the culture medium need to be modified for the different genotypes [7].Therefore, the objective of this study was to determine the influence of the benzyladenine (BAP), indole-3-acetic acid (IAA), and silver nitrate (AgNO3) on low frequency somatic embryogenesis using leaf explants of Coffea arabica L. cultivars Caturra and Catuai.

Indirect somatic embryogenesis of Theobroma cacao L. in liquid medium and improvement of embryo-to-plantlet conversion rate

The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression steps conducted in 250 mL Erlenmeyer flasks were described for three genotypes selected for their agronomical traits (EET95, EET96, and EET103). The glucose and dissolved oxygen concentrations and the absorption of Murashige and Skoog medium macronutrients (nitrate, ammonium, potassium, sulfate, calcium, phosphorus, and magnesium) were monitored. The multiplication of the embryogenic calluses in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 mg L−1, initiated with an inoculation density of 20 g L−1 of callus, was achieved. The growth rate was characterized by two phases, with the second being concomitant with a depletion of phosphorus and magnesium, and a decrease in the embryogenic potential of the callus. The expression of the callus embryogenic capacity was conducted in an auxin-free medium. The embryo production starting from 1 and 5 g L−1 inoculation densities was compared. When placed in the optimal expression conditions in flasks, 1 g of callus produced 1000 to 1500 embryos within 5 to 7 wk. Finally, two paths for improving the plantlet regenerative capacities of cocoa SE produced in liquid medium were identified. Supplementing the expression medium with myo-inositol used as an osmotic agent at a concentration of 50 g L−1 increased the embryo-to-plantlet conversion rate from 13–16% to 40–48%. A 6-wk culture of the embryos on a maturation medium in Petri dishes optimized their subsequent development into plantlets.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook.

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses.

High frequency somatic embryogenesis and plant regeneration of interspecific ginseng hybrid between Panax ginseng and Panax quinquefolius

BackgroundInterspecific ginseng hybrid, Panax ginseng × Panax quenquifolius (Pgq) has vigorous growth and produces larger roots than its parents. However, F1 progenies are complete male sterile. Plant tissue culture technology can circumvent the issue and propagate the hybrid. MethodsMurashige and Skoog (MS) medium with different concentrations (0, 2, 4, and 6 mg/L) of 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and somatic embryogenesis (SE). The embryos, after culturing on GA3 supplemented medium, were transferred to hormone free ½ Schenk and Hildebrandt (SH) medium. The developed taproots with dormant buds were treated with GA3 to break the bud dormancy, and transferred to soil. Hybrid Pgq plants were verified by random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses and by LC-IT-TOF-MS. ResultsWe conducted a comparative study of somatic embryogenesis (SE) in Pgq and its parents, and attempted to establish the soil transfer of in vitro propagated Pgq tap roots. The Pgq explants showed higher rate of embryogenesis (~56% at 2 mg/L 2,4-D concentration) as well as higher number of embryos per explants (~7 at the same 2,4-D concentration) compared to its either parents. The germinated embryos, after culturing on GA3 supplemented medium, were transferred to hormone free ½ SH medium to support the continued growth and kept until nutrient depletion induced senescence (NuDIS) of leaf defoliation occurred (4 months). By that time, thickened tap roots with well-developed lateral roots and dormant buds were obtained. All Pgq tap roots pretreated with 20 mg/L GA3 for at least a week produced new shoots after soil transfer. We selected the discriminatory RAPD and ISSR markers to find the interspecific ginseng hybrid among its parents. The F1 hybrid (Pgq) contained species specific 2 ginsenosides (ginsenoside Rf in P. ginseng and pseudoginsenosides F11 in P. quinquefolius), and higher amount of other ginsenosides than its parents. ConclusionMicropropagation of interspecific hybrid ginseng can give an opportunity for continuous production of plants.

High-frequency somatic embryogenesis and artificial seeds for mass production of true-to-type plants in Ledebouria revoluta: an important cardioprotective plant

Scaly bulb of Ledebouria revoluta is a natural source of several cardiac glycosides and is traditionally used in Indian and South African ethno-medicinal system from ancient age. High-frequency indirect somatic embryogenesis protocol via callus culture had been developed from bulb scale explant. Optimum embryogenic calli was induced on Murashige and Skoog (MS) medium supplemented with 3.0 mg l−1 2,4-dichlorophenoxyacetic acid and 0.75 mg l−1 β-naphthoxyacetic acid. A maximum of 93.3 % of the cultures responded after 6 weeks of culture on MS medium containing 3.0 mg l−1 of thidiazuron, 0.75 mg l−1 α-naphthalene acetic acid and 1.75 mM spermidine forming 46.7 ± 0.58 somatic embryos per 500 mg callus. Individual somatic embryo was encapsulated in calcium alginate beads to produce artificial seeds (ASs). The ASs were stored at 4, 15, and 24 °C temperatures up to 180 days. The ASs showed 57.8 % germinability even after 4 months of storage at 15 °C. Plantlets were acclimatized with a survival rate of 96.0 % and after 13–14 months, 92.4 % of these plants produced flowers. Chromosomal studies revealed cytological stability of the regenerants containing 2n = 30 chromosomes in root tips and n = 15 chromosomes in pollen grains, same as parental plants. The meiotic behaviour of the regenerants is also similar to that of parental plants. Randomly amplified polymorphic DNA analysis revealed that there is no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirm the very reliable method for short-term conservation and large-scale production of true-to-type plantlets of L. revoluta, which can be applied in pharmaceutical industries.

Efficient Somatic Embryogenesis and Plant Regeneration from Immature Embryos of Tapiscia sinensis Oliv., an Endemic and Endangered Species in China

High-frequency somatic embryogenesis and plant regeneration were achieved from immature cotyledonary-stage embryos in the endangered plant, Tapiscia sinensis Oliv. Plant growth regulators with different concentrations and combinations on embryogenesis capacity were studied. The optimal explants for in vitro somatic embryogenesis were immature embryos in T. sinensis. A high callus induction rate of 100% was achieved on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg·Ll−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5% (w/v) activated charcoal. Alternatively, a high induction rate (96.16%) of somatic embryogenesis was obtained on MS basal medium supplemented with the combination of 0.05 mg·L−1 α-naphthaleneacetic acid (NAA) and 0.2 mg·L−1 6-benzylaminopurine (6-BA), and somatic embryos proliferated fastest on the mentioned medium supplemented with 0.5% (w/v) activated charcoal and 3% (w/v) sucrose, inoculation of explants proliferating 21 times in the 23-day subculture. Of the 100 plantlets transferred to field after the acclimation, 95 (95%) survived. Based on the histocytological observations, the development of somatic embryos was similar to that of zygotic embryos. There were two accumulation peaks of starch grains in the embryogenic calli and in the globular-stage embryos, both closely related to the energy supply, and the embryoids were of multicelluar origin.

High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.)

Gherkin (Cucumis anguria L.) is an important commercial vegetable crop. An efficient protocol for plantlet regeneration from hypocotyl and leaf explants through direct somatic embryogenesis was developed. High frequency of somatic embryos (21.6 and 34.0) were obtained from hypocotyl and leaf explants when cultured on Murashige and Skoog (MS) salts plus B5 vitamins (MSB5) supplemented with 5% sucrose, 1.5mgL−1 2,4-D, 0.5mgL−1 BAP and 150μM l-glutamine. Initial culturing of embryos in the dark conditions for two weeks, followed by four weeks under light resulted in a higher frequency of embryo formation when compared to continuous light conditions. Histological observation showed that the somatic embryos originated from single cells of the epidermal layer. Histological evidence on formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. Maturation of somatic embryos occurred on plant growth regulator-free MSB5 semi-solid medium containing 3% sucrose and 0.5% (w/v) activated charcoal (AC). Conversion of embryos into plantlets was achieved on MSB5 medium supplemented with 3% sucrose, 0.3% AC and 0.5mgL−1 gibberellic acid (GA3). Sucrose was found to be the best carbon source for SE induction, maturation and germination. Ninety percent of embryos were converted into normal plantlets. The plantlets were successfully acclimatized in the greenhouse with 95% survival rate and transferred to ex vitro conditions which developed with normal phenotypes. This regeneration protocol assured successful embryo induction and plantlet conversion. This is the first report for the induction of direct somatic embryogenesis from hypocotyl and leaf explants of C. anguria. The result of this study is beneficial for genetic transformation and mass clonal propagation.

In vitro Plant Regeneration through Somatic Embryogenesis in Medicinally Important leaf explants of Coleus forskohlii Briq.

High-frequency somatic embryogenesis and complete plant regeneration from leaf explants of Coleus forskohlii Briq had been established. The influence of plant growth regulators (PGRs) on the frequency of somatic embryogenesis and plant regeneration were studied. Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or both with 6-benzylaminopurine (BAP), were superior for somatic embryogenesis. Primarily, embryogenic callus was initiated from leaf explants of young plant on MS medium supplemented with 2,4-D (1.0mgl -1 ), 3% sucrose and 0.9% agar in three weeks. Embryogenic callus transferred on 2,4-D and BAP-supplemented medium favored induction of embryos in advance as compared to that on 2,4-D alone supplemented medium. The highest frequency (80 %) of direct somatic embryogenesis was obtained in the combination of BAP (1.0 mg/l) and 2,4-D (0.5 mg/l). Eighty percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS medium having α-naphthaleneacetic acid (NAA), with BAP. Embryo-derived plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.

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

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

English

A protocol for high frequency somatic embryogenesis (SE) of pigeonpea, Cajanus cajan (L.) Millsp. was worked out. The age of the source seedlings for explants and type of explants were found to influence the callusing response. Concentration of phytohormones and growth factors were optimized for developing embryogenic callus consisting of globular cells with dense cytoplasm. Embryo axis or callus derived from such explants, when exposed to dehydration stress imposed by polyethylene glycol and osmotic stress created by D-mannitol were found to produce high frequency somatic embryos. Embryo axis from 3-day old sprouts or 28-day old callus derived from the embryo axis explants incubated for 4 h either in 4% polyethylene glycol (w/v) or 0.7 M mannitol were found optimal for SE. After exposure to stress incubation, explants or callus were cultured on semisolid Murashige and Skoog (MS) + 2,4-dichlorophenoxy acetic acid (2,4-D) (5 µM) + glutamine (0.03 mM) and the cultures formed only globular and heart shaped somatic embryos. The early stage somatic embryos subsequently developed into torpedo and cotyledonary somatic embryos in MS liquid medium supplemented with 2,4-D (2 µM), abscisic acid (3 µM) and glutamine (0.03 mM). The somatic embryos were converted into plantlets in hormone-free half-strength MS semisolid medium and subsequently established in garden soil.   Key words: Abscisic acid, glutamine, osmotic stress, polyamines, water stress.

Effect of polyethylene glycol and mannitol on somatic embryogenesis of pigeonpea, Cajanus cajan (L.) Millsp.

A protocol for high frequency somatic embryogenesis (SE) of pigeonpea, Cajanus cajan (L.) Millsp. was worked out. The age of the source seedlings for explants and type of explants were found to influence the callusing response. Concentration of phytohormones and growth factors were optimized for developing embryogenic callus consisting of globular cells with dense cytoplasm. Embryo axis or callus derived from such explants, when exposed to dehydration stress imposed by polyethylene glycol and osmotic stress created by D-mannitol were found to produce high frequency somatic embryos. Embryo axis from 3-day old sprouts or 28-day old callus derived from the embryo axis explants incubated for 4 h either in 4% polyethylene glycol (w/v) or 0.7 M mannitol were found optimal for SE. After exposure to stress incubation, explants or callus were cultured on semisolid Murashige and Skoog (MS) + 2,4-dichlorophenoxy acetic acid (2,4-D) (5 μM) + glutamine (0.03 mM) and the cultures formed only globular and heart shaped somatic embryos. The early stage somatic embryos subsequently developed into torpedo and cotyledonary somatic embryos in MS liquid medium supplemented with 2,4-D (2 μM), abscisic acid (3 μM) and glutamine (0.03 mM). The somatic embryos were converted into plantlets in hormone-free half-strength MS semisolid medium and subsequently established in garden soil. Key words: Abscisic acid, glutamine, osmotic stress, polyamines, water stress.

High frequency somatic embryogenesis, regeneration and correlation of alkaloid biosynthesis with gene expression in Papaver somniferum

Opium poppy (Papaver somniferum) remains the most important source for several pharmaceutical benzylisoquinoline alkaloids including the narcotic analgesic morphine, the anti-tussive drug codeine. Selection, breeding as well as other molecular practices have produced large number of germplasm with modulated biosynthesis and altered accumulation of specific alkaloids. However, there is need to modulate biosynthetic pathways of these alkaloids through transgenic approach, which requires development of an efficient regeneration system for genetic transformation. We studied response of different explants for somatic embryogenesis and developed an efficient protocol for P. somniferum somatic embryogenesis, regeneration as well as successful transplantation in fields. In our system, embryogenic callus from root explants was induced by 2,4-D and kinetin followed by maturation with gibberellin and abscisic acid and regeneration by hormone free half-strength MS media. Though root regeneration from somatic embryos has been shown as limiting factor in established protocols, we observed efficient root and shoot regeneration through somatic embryos in our study. We analyzed alkaloid content in in vitro callus and field grown plants and concluded developmental control of alkaloid biosynthesis. Absence of transcripts of some of the genes and specific alkaloids also suggests regulation at transcriptional level of alkaloid biosynthesis pathway.

High frequency somatic embryogenesis and synthetic seed production in Clitoria ternatea Linn

An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn., an important medicinal climber species. Optimum embryogenic callus (75 %) was induced on Murashige and Skoog (MS) medium supplemented with 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D). On subculturing the callus on MS medium supplemented with 2 mg/l 6-benzyladenine (BA) and 0.5 mg/l α-naphthalene acetic acid (NAA), 61 % of cultures responded with a mean number of 22 somatic embryos per gram callus at different stages of development after 45 days of culture. The addition of higher concentrations of sucrose and abscisic acid (ABA) significantly increased the embryogenic response. MS medium supplemented with 2 mg/l BA, 0.2 mg/l NAA and 4 % of sucrose resulted in 76 % of cultures responding with a mean number of 28 embryos per one gram callus. The highest embryogenic response, frequency of 83 % and mean number of 37 embryos per gram callus, was observed when the MS medium was supplemented with 2 mg/l BA, 0.2 mg/l NAA, and 3 mg/l ABA. Synthetic seeds were produced by encapsulating embryos in calcium alginate gel. The gel contained MS medium with 3 % of sucrose, 1.0 mg/l BA and 0.2 mg/l NAA. The synthetic seeds germinated on MS medium supplemented with BA (1.0-4.0 mg/l) alone or in combination with NAA (0.1–0.7 mg/l) or indole-3-butyric acid (IBA; 0.1–0.7 mg/l). The highest synthetic seed germination (92 %) was observed on MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA. The synthetic seeds were stored at 4 °C and lab conditions (25 ± 2 °C) up to 5 months. The synthetic seeds kept at 4 °C showed 86 % viability even after 5 months of storage. Both somatic embryos and synthetic seeds germinated and were transferred to soil successfully.

Calcium and calcium ionophore A23187 induce high-frequency somatic embryogenesis in cultured tissues of Coffea canephora P ex Fr

High-frequency somatic embryogenesis was achieved in Coffea canephora using calcium ionophore A23187, which influences the influx of calcium into a cell. With 100 μM calcium ionophore and 5 mM calcium, 85% and 70% of cultures produced embryogenic tissue, with 105 ± 7 and 95 ± 8 primary embryos from each callus mass respectively. Medium supplemented with 100 μM EGTA (calcium chelator) or 1 mM verapamil (calcium channel blocker) significantly reduced somatic embryogenesis. Calcium imaging studies were done to determine the relationship between morphogenetic response and the cellular calcium levels. The calcium ionophore/calcium treatment was very effective in driving cellular machinery toward embryogenesis. The embryos were regenerated into plantlets when cultured on MS medium supplemented with 5 mM calcium/100 μM calcium ionophore A23187. Somatic embryogenesis-derived plants were successfully transferred to soil and grown to maturity in the field.

Plant Regeneration Through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

A newly efficient protocol has been established for high frequency somatic embryogenesis through callus culture of Coelogyne cristata. The best frequency of callusing was obtained from leaf segments (3-5 mm) cultured on the MS medium supplemented with 2 mg?L–1 2,4-D and 2 mg?L–1 BA combination. A negative effect of coconut water was ob-served on the callus induction medium. When callus lines no. 4, 6 and 8 induced from leaf segments were sub-cultured separately on ½ MS and MS media containing AC (1-3 g?L–1), formation of somatic embryos was found. However, percentages of embryo formation and the number of embryos per explants were strongly affected by media and callus lines used. The effect of 1/2 MS media is definitely better than MS medium for somatic embryogenesis from the selected lines of leaf derived callus. Among the callus lines, line no. 4 is the best for somatic embryogenesis followed by line no. 6 and 8. The somatic embryos converted into healthy plants with well developed shoots on the same media. The plant-lets were transferred to 1/2 MS medium containing 1 g?L–1 AC for plant regeneration until 8 weeks of culture and successfully acclimatized in the greenhouse.

High-frequency somatic embryogenesis from germinated zygotic embryos of Schisandra chinensis and evaluation of the effects of medium strength, sucrose, GA3, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l−1 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA3), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA3 negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l−1 BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.

An efficient somatic embryogenesis system for velvet bean [Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug

The Mucuna pruriens Linn. is an important medicinal legume cover crop. Almost all the parts of the plant are reported to contain l-3,4-dihydroxy phenylalanine (l-DOPA), a non-protein amino acid that acts as precursor for the neurotransmitter dopamine. Here we report a rapid and reliable protocol for high fidelity regeneration of M. pruriens plants via somatic embryogenesis. Embryogenic callus was induced from cotyledon segments of in vitro grown seedlings on Murashige and Skoog medium supplemented with 6.7 μM 2,4-dichlorophenoxy-acetic acid (2,4-D). High-frequency somatic embryogenesis was achieved after transfer of embryogenic callus clumps to MS medium supplemented with 2.3 μM Kinetin (Kn) and 5.4 mM α-naphthaleneacetic acid (NAA) supplemented with 13.6 μM adenine sulphate. The maximum number of cotyledonary-stage embryos (60.5 ± 12.7) was obtained after 10 weeks. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. The protocol should provide an efficient means for large-scale cultivation and in vitro manipulation of M. pruriens, an important green manure cover-crop with medicinal properties.

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l−1 thidiazuron (TDZ) and 1 mg l−1 α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation.