Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Embryogenic cultures of the leguminous tree Albizia julibrissin and recovery of plants

A somatic embryogenic system was developed and plants regenerated in mimosa (Albizia julibrissin Durazz). Development of somatic embryos in the species has not previously been reported. Immature seeds, embryo cotyledons and embryo axes (cotyledons removed) at defined developmental stages were placed on induction media with different concentrations of 2,4-D. Two distinct embryogenic responses occurred: either proembryo masses or cotyledonary-stage embryos. Twenty five percent of all embryo axes cultured on basal medium produced cotyledonary somatic embryos. Six percent of in ovulo immature seed explants generated proembryo masses. These masses proliferated in liquid culture in the dark. Proembryos developed further when transferred to a growth-regulator-free semisolid medium in the light. Somatic embryos derived from either proembryo suspensions or cotyledonary embryo cultures on semisolid medium germinated to form plants that continued to grow vigorously following transfer to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.     

Somatic embryogenesis and plant regeneration from leaflets of peanut,Arachis hypogaea

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) B5 medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6benzylaminopurine - NAA 1-naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis

Summary A plant regeneration methodvia somatic embryogenesis of severalHelianthus annuus L. genotypes was developed. Starting from cotyledonary explants high frequency embryo induction was obtained following several subcultures on defined media. An appropriate cotyledon developmental stage was identified. Etiolated explants and darkness treatment were necessary to obtain somatic embryos in all tested genotypes. After 20–25 days on somatic induction medium containing an auxin:cytokinin ratio of 1:1, the germination of embryos was induced by a reduction of the hormonal ratio (1:2). Shoots were excised from callus and transferred onto a medium containing various vitamins. The range of embryogenesis frequency was 33–72%, depending on the genotype. High frequency of rooting (49–82%) was obtained using a medium supplemented with 0.5 mg/L of ancymidol and by a reduction of photoperiod. A large percentage of somatic embryos developed into normal regenerated plants producing viable seeds.Abbreviations MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - BAP benzylaminopurine - GA₃ gibberellic acid - EIM embryo induction medium - GM germination medium - VM vitamins medium - RA2 ancymidol rooting medium - EtOH ethanol     

Rapid multiplication of adventitious somatic embryos in peach and nectarine by secondary embryogenesis

Somatic embryos were multiplied by secondary embryogenesis in cotyledonary cultures of peach and nectarine (Prunus persica L.) using a simplified culture medium for immature seeds. A three-stage process with an initial callus phase was established in darkness on a medium containing basal salts (modified MS) supplemented with 2,4-D (5 mg/l), Kn (2 mg/l) and BAP (2 mg/l) and casein hydrolysate (500 mg/l). This was followed by a growth regulator-free medium with activated charcoal for the adventitious and direct multiplication of somatic embryos under continuous light. Somatic embryos (10–15) originated from the epidermal layer of primary somatic embryos of 4–6 mm size. The incidence of morphologically abnormal embryos was reduced by subculturing every 20 days. Calli which were isolated and grown on a 2,4-D medium were more embryogenic than those on NAA. These embryos multiplied continuously for more than 10 months by a repetitive somatic embryogenic process. A third stage medium, supplemented with BAP (2 mg/l), was required for axis elongation, germination and transfer to soil.     

A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−¹ casein hydrolysate, 9 μ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 μ M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μ M 2,4-D and 4.7 μ M kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata -based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa , with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Advances in somatic embryogenesis and plant production of black locust (Robinia pseudoacacia L.)

Summary Black locust (Robinia pseudoacacia L.) immature seeds of different developmental stages were tested for the ability to initiate embryogenic cultures. Best results (average of 12% embryogenic cultures) were obtained when seeds collected 2–3 weeks post-anthesis were cultured for 3 weeks on modified Finer and Nagasawa medium containing 2,4-D (45–90 M) and BA (2.2 M) and then transferred to the same medium without growth regulators. Embryo conversion was obtained from naked or encapsulated somatic embryos derived from a long-term embryogenic line. Without cold treatment, 71% of naked embryos and 41% of the encapsulated embryos converted into plants. Fifteen days of cold treatment increased conversion rates up to 95% for naked embryos and 80% for encapsulated embryos. Recovered plantlets were acclimatized and grown in the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FM modified Finer & Nagasawa (1988) medium - MS modified Murashige & Skoog (1962) medium - PEM proembryogenic mass - SH modified Schenck & Hildebrandt (1972) medium     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Somatic embryogenesis in Encephalartos cycadifolius

Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg l^–1 2,4-D and 1 mg l^–1 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg l^–1 ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid