Auxin-stimulated somatic embryogenesis from immature cotyledons of white clover

Cotyledons from immature embryos of white clover (Trifolium repens L.) cv. Osceola were exposed to 2,4-D or NAA to induce somatic embryogenesis. NAA at 10 or 20 mg 1^–1 was very inefficient at stimulating embryogenesis, while concentrations of 30 or 40 mg 1^–1 resulted in death of the explant tissue. Continuous exposure of cotyledons to 40 mg 1^–1 2,4-D resulted in somatic embryos which were arrested at the globular stage, or which underwent cycles of secondary embryogenesis, never proceeding beyond the globular stage. A 10 day exposure time to 2,4-D at the same concentration led to formation of somatic embryos, most of which had poorly developed cotyledons. Almost 10% of the somatic embryos converted into plants following transfer to medium devoid of growth regulators. Attempts to improve morphology of somatic embryos by using shorter exposure times to 2,4-D at 40 mg 1^–1, or by maintaining the 10 day exposure time while varying the concentration of 2,4-D, were not successful. Plants were obtained from all parents evaluated, although at different frequencies.     

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 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     

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

Summary A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l^–1 kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l^–1 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine     

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.     

Direct somatic embryogenesis and plant regeneration from ray florets of chrysanthemum

Direct somatic embryogenesis from ray floret explants of five chrysanthemum cultivars has been obtained within 12 – 15 d on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Scanning electron microscopic observation also confirmed the direct origin of somatic embryos from explants. Somatic embryos developed asynchronously on the adaxial surface of explants. Among the five cultivars tested, Birbal Sahani was best responding (40 % explants responded on 4 mg dm^–3 2,4-D and 2 mg dm^–3 BA supplemented medium). Precocious germination of somatic embryos was noticed on the same medium. The best sucrose concentration in the medium was found to be 60 g dm^–3 where 70 % explants responded while 55 % embryogenic response was obtained on medium supplemented with 400 mg dm^–3 inositol. Plants developed from somatic embryos were transferred to soil and produced true-to-type flowers.     

Somatic embryogenesis in mature zygotic embryos of <Emphasis Type="Italic">Picea likiangensis</Emphasis>

Somatic embryogenesis (SE) was successfully induced from mature zygotic embryos of seven families of Picea likiangensis (Franch.) Pritz after 20 weeks culture on initiation medium. Three basal media (one-half strength LM medium, one-half strength LP medium and improved LP medium) with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (6-BA) were tested but only one-half strength LM medium supplemented with 2,4-D and 6-BA was successful for the embryogenic cultures (EC) initiation. The initiation frequencies of EC varied greatly from different families when culturing on the same initiation medium. The highest frequency (41.3%) was induced from one of the families on one-half strength LM medium supplemented with 3 mg L⁻¹ 2,4-D and 1.5 mg L⁻¹ 6-BA and 16.83% on average for seven families. EC were subcultured and proliferated on the same medium as the initiation one every 10 days. 3 lines of EC induced from the same family were applied in maturation experiment. Cotyledonary somatic embryos were observed after EC were transferred to maturation media of one-half strength LM medium containing 20-80 mg L⁻¹ abscisic acid and 7.5% polyethylene glycol (PEG-4000). However, one-half strength LM medium supplemented with 40 mg L⁻¹ or 60 mg L⁻¹ ABA and 7.5% PEG gave the best maturation and the 3 lines showed different ability in maturation. Over 80% cotyledonary somatic embryos germinated normally on DCR medium containing 0.2% activated carbon. The success on SE induction of the species has provided an effective clonal propagation method for this important tree’s genetic improvement.     

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.     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

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     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

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     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

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 from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.