Highest Number Of Somatic Embryos Research Articles

Somatic embryogenesis, encapsulation, and plant regeneration of Rotula aquatica lour., a rare rhoeophytic woody medicinal plant

Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.

Somatic embryogenesis, organogenesis, and regeneration from leaf callus culture of Decalepis hamiltonii Wight & Arn., an endangered shrub

A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N6-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA3) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.

In vitro propagation of the rare medicinal plant Ceropegia candelabrum L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52mM 2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52mM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45mM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien , a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l−1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l−1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l−1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.

Somatic embryogenesis in Citrus sinensis, C. reticulata AND C. nobilis x C. deliciosa

Most of the plant regeneration processes in citrus, through tissue culture, involve indirect somatic embryogenesis. The optimization of these processes is important for the development of in vitro plant improvement and micropropagation studies. Studies to evaluate the effect of different carbohydrates in somatic embryogenesis were conducted using calli from 'Ponkan' mandarin (Citrus reticulata, Blanco), 'Cravo' mandarin (C. reticulata), 'Itaboraí' sweet orange (C. sinensis L. Osbeck.), 'Valencia' sweet orange (C. sinensis) and 'Kinnow' mandarin (C. nobilis Loureiro x C. deliciosa Tenore). The culture medium used was MT supplemented with sucrose, galactose, glucose, maltose or lactose with the following concentrations of 18, 37, 75, 110, and 150 mM. The culture medium used for the maturation of somatic embryos had 0, 15, 29, 44, 58 and 73 mM of sucrose, in presence or absence of 0.5 g L<FONT FACE=Symbol>-</FONT>1 of activated charcoal. Seventy-three mM of sucrose with 0.1 mg L<FONT FACE=Symbol>-</FONT>1 of GA3 in the presence or absence 0.5 g L<FONT FACE=Symbol>-</FONT>1 of activated charcoal was also tested. Overall, the carbohydrates galactose or lactose induced a higher number of somatic embryos. Sucrose concentrations of 58 and 73 mM generated a higher number of plantlets from mature embryos of 'Ponkan' mandarin and 'Valencia' sweet orange.

Triazole treatment of explant source provides stress tolerance in progeny of Geranium (Pelargonium hortorum Bailey) plants regenerated by somatic embryogenesis

Thehypothesis that chemically induced stress tolerance in plants can betransferredto a larger clonal population regenerated by somatic embryogenesis wasevaluatedusing the triazole compound paclobutrazol as a chemical inducer of stresstolerance in Geranium (Pelargonium horturum Bailey). Seedswere imbibed in 3.4, 10.2 or 17.0 μM (1, 3, 5 mgL−1) paclobutrazol for 24 h and germinatedfor 7 days. Hypocotyl explants were cultured in vitro toinduce somatic embryogenesis. Plants regenerated from somatic embryos wereexposed to heat stress at 56 °C. Explants treated with3.4 μM paclobutrazol yielded a substantially higher number ofsomatic embryos compared with untreated explants. In contrast, 17.0μM paclobutrazol treatment inhibited embryogenesis producing asignificantly lower number of somatic embryos. There was no difference in theembryo number between control and 10.2 μM treatment. Somaticembryos derived from 3.4 and 10.2 μM paclobutrazol treatedexplants developed into plants at a faster rate than the control and 17.0μM treatments. Plants derived from paclobutrazol-treatedexplants displayed a greater tolerance to heat stress compared with thecontrols. Observations in this study provide a technique for regeneratingplantsin tissue/cell culture with additional desirable traits such as stresstolerancewith minimal chemical contamination of the environment.

Optimized somatic embryogenesis in Pinus strobus L.

Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH4 + and NO3 − ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 μM) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH4 + and NO3 − concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 μM) enhanced the conversion of somatic embryos to plantlets.

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and α-naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA3) and BA. Treatment with zeatin (22.8 μM) and BA (10.0 μM) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.

Induction of somatic embryogenesis in soybean: physicochemical factors influencing the development of somatic embryos

The embryogenic capability of five soybean cultivars (Renascença, IAS-5, IAC-17, BR-16 and FT-Cometa) was studied at different auxin concentrations (8, 10 and 12 mg/l naphthalene acetic acid, NAA), at different pHs (5.8 and 7.0) and at low (8-12 muEm-2 s-1) and high (27-33 mEm-2 s-1) light intensities. The experimental design was completely randomized with four replications. Immature cotyledons 4-6 mm in length were placed in the six induction mediums evaluated and submitted to two light intensities. Twenty immature cotyledons per cultivar were placed on each Petri dish, which was considered to be one replication. The number of somatic embryos per treatment per replication was counted. The results showed genotype influence on somatic embryogenic capability of each cultivar, with the most embryogenic cultivars being BR-16, FT-Cometa and IAS-5. Auxin concentration and pH value also influenced somatic embryo production, with 10 mg/l NAA being the best auxin concentration and 7.0 the best pH value. The interactions cultivar x auxin, auxin x pH and pH x light were significant, while other double interactions were not. All triple and quadruple interactions were significant, except cultivar x pH x light. No significant differences in somatic embryo production were observed in medium with different pHs or when the Petri dishes containing immature cotyledons were exposed to the two light intensities evaluated. However, a higher number of somatic embryos was produced when the medium pH was adjusted to 7.0.

Induction of somatic embryogenesis from leaves of Sadao Chang (Azadirachta excelsa (Jack) Jacobs)

The second pair of leaves from the tip of Sadao Chang seedlings were collected and cultured on Murashige and Skoog (MS) or Nitsch and Nitsch (NN) media containing various concentrations of auxins and cytokinins to induce embryogenic callus. Embryogenic suspension cultures were established in MS, medium for Hevea (MH), Schenk and Hildebrandt (SH) and woody plant medium (WPM) with various auxins and cytokinins. Somatic embryos or embryoids were fractionated and germinated onto the above mentioned media supplemented with various auxins and cytokinins or a hormone-free medium. The results showed that MS containing 1 μM α-naphthaleneacetic acid (NAA) and 10 μM kinetin (KN) gave the best result in induction of friable embryogenic callus (FEC). The highest fresh weight (453 g) and number of somatic embryos (81 per explant) were both obtained in MS medium supplemented with 10 μM NAA and 1 μM KN. The highest number of somatic embryos, 30 embryos per 1 ml packed cell volume (PCV), was obtained in SH medium containing 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 μM 6-benzyladenine (BA). Globular-staged embryoids was obtained on SH medium with 0.1 μM NAA and 2 μM BA. In some cases, somatic embryos developed only shoots. Hence, they were isolated and rooted at 100% in 1 2 MS supplemented with 15 μM NAA.

Somatic embryogenesis and plant regeneration in Acacia farnesiana and A. schaffneri

Efficient plant regeneration systems via somatic embryogenesis have been developed for Acacia farnesiana and Acacia schaffneri [Leguminosae (Mimosoideae)]. The protocol used in this study consisted of placing immature, zygotic embryos of these species in Murashige and Skoog semi-solid basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.65 μM kinetin to induce callus. Some parts of the callus were used for direct embryo differentiation and others for establishment of cell suspension cultures. In the first case, somatic embryos were produced on semi-solid differentiation media without growth regulators or with abscisic acid (ABA). The higher number of somatic embryos, 345 and 198 embryos per g callus in A. farnesiana and A. schaffneri, respectively was obtained in media without growth regulators, but adding ABA increased the percentage of embryos that reached more advanced differentiation stages. The production of somatic embryos was achieved starting from cell suspensions only when these suspensions were plated into the semi-solid differentiation medium. Somatic embryos germinated on medium containing 217 μM adenine sulfate with efficiencies of 69% in A. farnesiana and 47% in A. schaffneri. Some somatic embryos that developed into plantlets were acclimatized in the greenhouse, and they grew into normal plants.

A Basic Chitinase-like Protein Secreted by Embryogenic Tissues of Pinus caribaea acts on Arabinogalactan Proteins Extracted from the same Cell Lines

Embryogenic cell lines of Pinus caribaea consist of a high number of somatic embryos. We have previously characterized the embryogenic state by studying the proteins and glycoproteins ionically bound to the cell surfaces of preglobular somatic embryos. The embryogenic tissues and nonembryogenic calli produce proteins of 48 kDa or 56 and 25 kDa, respectively. All of these proteins cross-react with several classes of tobacco chitinases (CHs). These CH-like proteins express a potential chitinase activity on SDS-PAGE gels overlaid with glycol chitin as a synthetic substrate. When an arabinogalactan protein (AGP) fraction from embryogenic tissues substitutes for glycol chitin on gels, only the 48 kDa embryo-specific CH-like protein acts on this substrate, indicating that an interaction between this protein and a specific set of AGPs might exist within embryogenic tissues of Carribean pine.

Morphological and Physiological Changes during Thidiazuron-Induced Somatic Embryogenesis in Geranium (Pelargonium x hortorum Bailey) Hypocotyl Cultures

Somatic embryogenesis in geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl explants on media supplemented with either thidiazuron (TDZ) or indol-3-yl-acetic acid (IAA) and 6-benzylaminopurine (BAP). The physiological relationships between the morphological changes that lead to complete development of somatic embryos and the endogenous levels of plant growth regulators were studied. TDZ induced similar but more pronounced and earlier morphological changes in cultured tissues than did IAA and BAP, but after 21 d in culture, both treatments promoted the formation of somatic embryos of different developmental stages that were similar to zygotic embryos. TDZ induced a higher number of somatic embryos, with similar but greater increases in the endogenous levels of auxins and cytokinins, than did IAA and BAP. This may indicate that TDZ may be a more potent plant growth regulator in inducing physiological and morphological changes than combined auxin and cytokinin, during the process of somatic embryogenesis in geranium.

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tucker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 μM was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 μM) and of BA (5 μM) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 μM) as the sole exogenous growth factor. Some embryos isolated mechanically or ‘in situ’ on the callus developed as far as the later cotyledonary stage.

Role of high sugar concentrations in inducing somatic embryogenesis from cucumber cotyledons

We examined the effects of sucrose concentrations in initiation media with a constant carbohydrate molarity, and of osmotic potential provided by glucose or mannitol, on somatic embryogenesis from cotyledon explants of Cucumis sativus L. cv. ‘Shimoshirazu’. In constant carbohydrate molarity (0.25 M), the percentage embryo formation and the yield of embryos were enhanced by increasing sucrose concentrations. Sucrose (0.25 or 0.50 M) and glucose (0.50 M) both gave high frequencies of somatic embryogenesis; no embryos were formed when mannitol was the sole carbon source. When initiation media contained 0.25 M sucrose, raising the osmotic potential with either mannitol or glucose increased embryo yield and significantly promoted direct embryo formation. The percentage of bipolar embryos was also enhanced by increasing the molarity of carbohydrates in the initiation medium by the addition of glucose or mannitol. Glucose was a more effective sugar supplement than mannitol in inducing somatic embryos. Addition of 0.15 M glucose to the initiation medium containing 0.25 M sucrose resulted in the highest number of somatic embryos. Glucose had a specific carbon effect as well as an osmotic role. The specific carbon effect plays a major role and osmotic potential a minor and supplementary role in cucumber embryogenesis.

Spatial and Temporal Changes in Endogenous Polyamine Levels Associated with Somatic Embryogenesis from Different Hypocotyl Segments of Eggplant ( Solanum melongena L.)

Summary In Solanum Melongena L. Cv. Pusa Purple Long, Significant Differences Were Observed For Embryogenic Potential Within A Single Explant (Hypocotyl System). Terminal Hypocotyl Segments (Apical and Basal) Yielded A Higher Number Of Somatic Embryos Than The Medial (Sub-Apical and Sub-Basal) Segments. High Levels Of Conjugated Spermidine Along With High Levels Of Total Polyamines (Primarily Free Fraction) Could Be Correlated With The Formation Of Somatic Embryos In Terminal Segments. Temporal Changes In Endogenous Levels Of Free, Conjugated and Bound Putrescine, Spermidine and Spermine Were Analyzed At Critical Stages Of Somatic Embryogenesis From Four Different Hypocotyl Segments. Within 3 Days Of Culture, There Was A Sharp Decline In Free (and Total) Putrescine and An Increase In Conjugated Spermidine Levels. All Hypocotyl Segments Attained Similar Levels Of Free Spermidine, Irrespective Of Their Subsequent Embryogenic Response. As The Tissues Become Committed For Embryogenesis, Free, Conjugated and Total Putrescine and Spermidine Reach Uniformly Minimal Levels For All Segments. Just Before The Onset Of Embryogenesis, There Was A Dramatic Increase In All Fractions Of Putrescine, and In Free and Conjugated (Along With Total) Spermidine Levels. Intermediate Levels Of Conjugated and Total Putrescine and Spermidine Were Reached In Tissues With Good Embryogenic Potential: tissues with poor embryogenic potential attained lower or higher levels. After the embryos were formed, their levels fell sharply, and continued to decline. Free putrescine, however, reached another peak when embryos had fully matured. The changes in bound putrescine and spermidine, and in spermine (all forms) could not be correlated with somatic embryogenesis.

Plant regeneration through somatic embryogenesis in peanut (Arachis hypogaea L.)

Somatic embryos were induced from immature cotyledons and immature embryonal axis ofArachis hypogaea L. on L-6 basal medium supplemented with NAA, picloram or 2,4-D at 5–50 mg 1-1. Immature embryonal axis produced a higher number of somatic embryos in comparison with immature cotyledons. The highest number of responding cultures was produced on medium supplemented with NAA (50 mg 1-1), while the highest average number of somatic embryos per culture was produced on medium with 2,4-D (10 or 20 mg 1-1) and picloram (30 mg 1-1) from cotyledons. The somatic embryos developed into plants on basal medium supplemented with activated charcoal and about 100 plants were successfully transferred to the field.

High-frequency direct somatic embryogenesis in thin layer cultures of hybrid seed geranium (Pelargonium ×hortorum)

Thin layer explants from the hypocotyls of young seedlings of geranium (Pelargonium ×hortorum Bailey) produced somatic embryos in response to thidiazuron (TDZ) or a combination of indoleacetic acid and N6-benzylaminopurine, but the number of embryos was much less in the latter than with TDZ. The development of somatic embryos in cultures of thin layers was rapid and the number of embryos was about eightfold higher than in cultures of whole hypocotyl explants. Thin layer explants of 1-week-old seedlings produced a high number of somatic embryos than those obtained from older seedlings. The cultivar Scarlet Orbit Improved showed better regeneration potential than 'Sprinter Scarlet'. There were no visible differences in total number of somatic embryos produced in the medium containing sucrose or glucose. The addition of glutamine to the induction medium further improved the number of somatic embryos that developed. Regenerated somatic embryos developed into complete plantlets within 6 to 8 weeks of culture initiation. Key words: Pelargonium ×hortorum, geranium, somatic embryogenesis, thin layers, thidiazuron.