Somatic embryogenesis from cell suspension cultures in Carica candamarcensis

Cell suspension cultures of Carica candamarcensis derived from hypocotyl calli were tested concerning their in vitro embryogenic capacity to improve asexual propagation rates in this species. Somatic embryos developed in culture from cells in suspension or from microcalli. Responses were affected by nutrient media and phytohormones used. Best results were obtained by growing the cells in suspension in Nitsch and Nitsch medium containing naphthaleneacetic acid and then plating them upon the same medium containing benzyladenine, or combinations of both hormones.     

Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures

A proliferating embryonal-suspensor mass (ESM) was initiated from immature embryos of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), 4–5 weeks after fertilization, on modified MS medium with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and N⁶-benzylaminopurine (BAP). ESMs were maintained for over 6 months as cell suspension cultures on modified DCR media with low 2,4-D and with kinetin and BAP. The development of individual somatic embryos was initiated in suspension culture by the gradual reduction of plant growth regulators and by addition of abscisic acid. The early stages of zygotic embryogenesis in Douglas-fir are unique among conifers and cleavage polyembroyogenesis is unknown. In somatic embryogenesis, characteristic stages of zygotic embryonic development were recapitulated and complete embryos were recovered by inhibiting cleavage polyembryony with abscisic acid and culturing individual embryos without growth regulators. Histological examination confirmed bipolar organization of somatic embryos. While conversion is low, plantlets with multiple cotyledons have been transferred to soil and continue to grow with production of a new shoot.     

Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum

Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space. Received: 26 July 1997 / Accepted: 9 September 1997     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L

Summary Embryogenic cell suspension cultures and somatic embryos of five genotypes of beech, were obtained from aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2, 4-dichlorophenoxyacetic acid and N6-benzyladenine. The origin of somatic embryos was traced from single cells. Embryos remained arrested at the globular stage on liquid media, further development was achieved after plating embryogenic aggregates on Murashige and Skoog's medium with half strength major salts supplemented with glutamine and low levels of growth regulators. Cultures of different genotypes showed significant differences in maturation frequency which was not affected by the hormone treatments assayed. The frequency of conversion of embryos into plantlets was low. This frequency increased after cold storage of embryos for up to 7 months.Abbreviations BA N6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOH ethanol - GA₃ giberrellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - WPM woody plant medium (Lloyd and McCown, 1980) - Z zeatin     

Somatic embryogenesis from cell suspension and protoplast cultures ofCapsella bursa-pastoris (L.) Medic

Summary Rapidly growing cell suspension cultures of shepherd’s purse (Capsella bursa-pastoris L. Medic.) were established from leaf-derived calli. These suspensions remained unorganized in the presence of 2,4-D, but underwent extensive root organogenesis in a growth regulator-free liquid medium. Attempts to induce direct embryogenesis in liquid cultures were unsuccessful, but numerous embryos were obtained from cells plated onto growth-regulator-free solid medium. These embryos were frequently abnormal, and secondary embryogenesis was problematic for plant recovery but fertile plants were recovered. Viable protoplasts could readily be isolated from these cell suspensions. After 1 wk of culture, protoplast viability was 62%, and 7% of the cells had divided. Embryogenesis was observed from protoplast-derived microcolonies, plated on growth-regulator-free medium. Although these somatic embryos were difficult to root, plants were recovered. New cell suspensions were more recently established, which were only 4 to 6 mo. old when plant regeneration was attempted. Numerous shoots were obtained when these cells were plated onto growth-regulator-free solid media. However, these shoots differed from the embryos previously obtained in that they readily rooted and rapidly developed into plantlets. This system may allow the use of shepherd’s purse as a gene source for introgression of agronomically interesting traits intoBrassica crop species through protoplast manipulation and somatic hybridization.     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

Somatic embryogenesis and regeneration from milkweed cell cultures

Whole plants of two milkweed species (Asclepias syriaca, A. tuberosa) were regenerated from cell cultures via in vitro embryogenesis. This process could provide a means for rapid selection of superior varieties of milkweed for hydrocarbon crop development.     

Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan)

Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos. The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham

Summary A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO₃ on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1⁻¹ (117 mM) sucrose, 0.05 mg 1⁻¹ (0.22 μM) 6-benzylaminopurine, and 10 mg l⁻¹ (58.8 μM) AgNO₃. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO₃ at concentrations below 10 mg l⁻¹ (58.8 μM). A semi-solid medium containing 1.5 g l⁻¹ Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.     

Somatic embryogenesis in suspension cultures of Gossypium klotzschianum anderss

Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Somatic embryogenesis of maize hybrids: histological analysis

The immature zygotic embryos of reciprocal maize hybrids (CHI-31 x GF1 and CHI-31 × GE2) were used as the initial material for induction of somatic embryogenesis in vitro. Histological analysis of somatic embryogenesis revealed high developmental variability. The arising formations were classified into 5 groups: A) somatic embryos phenotypically similar to zygotic embryos, B) polyembryos, C) formations with radicle but without meristematic plumule, D) formations with radicle without differentiated plumule, and E) formations with plumule without radicle. The formatioms A and B regenerated directly into plants. Plant regeneration from formations E required preculture on the rooting medium. Formations C and D failed to develope into plants possibly because of early loss of meristematic cell character during the embryo axis differentiation. The reverse sequence of radicle and plumule differentiation in somatic embryos in comparison with zygotic ones was noted. The epigenetic character of the scutellum, coleoptile, coleorhiza and leaves primordia development was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of palmarosa grass (Cymbopogon martinii)

A cell suspension culture was established from nodal callus ofCymbopogon martinii (Roxb.) Wats in a liquid medium containing Murashige and Skoog (1962) basal salts, vitamins, 100 mg 1^–1 myo-inositol and 20 g l^–1 of sucrose (MS) that was supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid and 1.15 M kinetin. An initial inoculum density of 2 x 10⁴ cells ml^–1exhibited optimum cell growth. Calli were obtained 12–15 days after the suspension was plated onto semisolid medium of a similar composition. When calli were transferred to semisolid regeneration medium containing MS + 6.7 M N ⁶-benzyl-adenine + 1.15 M kinetin, somatic embryogenesis and plantlet regeneration occurred after 10–25 days. There was no significant decrease in the regeneration potential of the calli even when the cultures were initiated from 47-week-old cell suspensions. Chromosome counts of cells in suspensions, calli and somatic embryos derived from cultures of different ages revealed the presence of diploids, tetraploids and octaploids. However, the 33 regenerated plants tested were all diploid, indicating that only diploid cells were capable of regeneration in vitro.Abbreviations MS Murashige and Skoog (1962) basal salts with vitamins (100 mg1^–1 myo-inositol, 20 g1^–1 sucrose) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N ⁶-benzyl-adenine - Kn kinetin - MSC MS + 13.6 M 2,4-D + 1.15 M Kn - MSR MS +6.7 M BA + 1.15 M Kn     

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.

Summary Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.     

Somatic embryogenesis in cell cultures of Thapsia garganica

Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l^–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EtOAc ethyl acetate - FDA fluorescein diacetate - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - 2-iP 2-isopentenyladenine - NAA 1-Napthaleneacetic acid     

Somatic embryogenesis in cell cultures of Glycine species

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Rajeli (AAB), an endangered banana cultivar

Rajeli (AAB), a commercially valuable Indian banana cultivar, is presently under the threat of extinction due to various diseases, warranting development of the strategies for its conservation and incorporation of disease resistance. This elite genotype was successfully regenerated in vitro via establishment of cell suspensions followed by somatic embryogenesis. The immature male flower buds were cultured on gelled Murashige and Skoog medium supplemented with 4 mg/l 2,4-D, 1 mg/l each of IAA and NAA, D-Biotin, 100 mg/l Malt Extract, 100 mg/l L-Glutamine and 30 g/l sucrose. Embryogenic calli with translucent somatic embryos were observed after 4 months of male flower bud culturing. Fine embryogenic cell suspensions were established in the liquid medium of same composition but with 45 g/l sucrose. Of the various sugars tested, maltose in the maintenance medium yielded better growth of plated cells as compared to sucrose, fructose and dextrose. Embryos differentiated at a high frequency and mature somatic embryos developed into plantlets on MS medium supplemented with 0.5 mg/l BAP. Approximately 40 % of the torpedo stage somatic embryos germinated and developed into complete plants. The regenerated plants exhibited normal phenotype during acclimatization in the greenhouse. The technique can be employed for conservation of this endangered cultivar and its improvement via mutagenesis and genetic transformation.     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium