Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in plant tissue culture media in the presence of activated charcoal

Somatic embryos and embryogenic callus were initiated from immature zygotic embryos of ginseng (Panax ginseng C.A. Meyer). These somatic embryos were multiplied by adventitious (secondary and tertiary) embryogenesis and their growth and development were dependent on growth hormones in the medium. Auxins, 2,4-d, NAA, and IAA at 1.0 mg l^-1 were effective in inducing secondary and tertiary somatic embryos, which proliferated directly from the apical or cotyledonary portions of the primary somatic embryos. Single somatic embryos or clusters or embryos developed from the explanted primary embryos. Cytokinin (Kn, BA) inhibited adventitious embryogenesis. Secondary somatic embryos developed to maturation and later regenerated into plantlets in two stage process; firstly elongation of the shoot axes on MS +1.0 mg l^-1 Kn, secondly formation of root on 1.0 mg l^-1 Kn+1.0 mg^-1 GA₃ medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA in-doleacetic acid - Kn kinetin - BA benzylaminopurine - PSE primary somatic embryo - SSE secondary somatic embryo - TSE tertiary somatic embryo     

Maturation of avocado somatic embryos and plant recovery

Avocado proembryonic masses from suspension cultures were used to develop a protocol for somatic embryo development and maturation. Avocado somatic embryos could develop from proembryonic masses both in liquid and on semisolid medium but only the latter could develop to maturity. Size and number of opaque somatic embryos were affected by gellan gum concentration, with the optimum response obtained on medium supplemented with 6–7 g l⁻¹ gellan gum. The optimum sucrose concentration for recovery of opaque somatic embryos was 90 g l⁻¹; however, the development of embryos was suppressed at this concentration. Consequently, recovery of cotyledonary, opaque somatic embryos was achieved on medium with 30 g l⁻¹ sucrose. Somatic embryo development from dedifferentiating proembryonic masses required media with a high ratio of NO ₃ ⁻ :NH ₄ ⁺ (1:0 and 3:1) as opposed to the standard ratio (2:1) of MS medium. Germination of somatic embryos was sporadic. In order to increase the frequency of plant recovery, shoots that developed from somatic embryos were micropropagated using standard protocols. This revised version was published online in June 2006 with corrections to the Cover Date.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

Comparative study of somatic embryogenesis from immature and mature embryos and organogenesis from leaf-base of Triticale

Immature and mature zygotic embryos of hexaploid, Triticale var. DT-46 formed an embryogenic callus, with subsequent somatic embryo formation upon subculture to MS (Murashige and Skoog, 1962) or N₆ (Chu et al., 1975) nutrient medium supplemented with various concentrations (9.0–22.5 M) of 2,4-dichlorophenoxyacetic acid (2,4-D). Of the two types of explants, embryogenic tissue from immature embryos responded at a higher frequency, to form somatic embryos over the callus surface. Leaf-base segment cultured on to 2,4-D-containing medium formed a tissue which did not form somatic embryos and instead differentiated into shoot-buds. N₆ medium proved to be more effective than MS in support of somatic embryogenesis or shoot-bud formation. Regeneration of plantlets occurred on 2,4-D-free basal medium. These in vitro-formed plantlets were successfully transferred to soil and set seed.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader)

 Indirect somatic embryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somatic embryogenesis. Another cycle of somatic embryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were cultured on media containing different auxin/cytokinin ratios. The auxin/cytokinin ratio had an influence on the intensity of embryo formation, germination and the capability to regenerate plants. Somatic embryogenesis was generally more intensive on the medium with lower concentrations of 6-benzylamino-purine. Further, the percentage of regenerated plants was higher when embryos were induced on high-cytokinin, low-auxin medium. Secondary somatic embryogenesis was induced on embryos by culture in liquid hormone-free medium. Similar to direct embryogenesis the efficiency of secondary embryogenesis depended on the medium used for the induction of the primary embryos. In contrast to the mostly low frequencies of conversion of secondary embryos into plants that has been observed in other species, the percentage of regenerated plants from secondary embryos of H. maximiliani was quite high, although slightly lower than that obtained in primary embryos. Received: 28 March 2000 / Revision received: 1 September 2000 / Accepted: 2 October 2000     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA₃, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.     

Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS_3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MS_WH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.     

Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber

Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA indolebutyric acid - MS Murashige & Skoog (1962) medium - SH Schenk & Hildebrandt (1972) medium - G Gamborg (1966, PRL-4-C) medium (macronutrients in mg l^–1: NaH₂PO₄·H₂O, 90; Na₂HPO₄, 30; KCl, 300; (NH₄)₂SO₄, 200; MgSO₄·7H₂O, 250; KNO₃, 1000, CaCl₂·2H₂O, 150) - PGR plant growth regulator     

Peroxidase activity of desiccation-tolerant loblolly pine somatic embryos

Summary Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 μM abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG₆₀₀₀). Scanning electron microscopy of desiccated somatic embryos showed that the size and external morphology of the desiccation-tolerant somatic embryos recovered to the pre-desiccation state within 24–36 h, whereas the non-desiccation-tolerant somatic embryos did not recover and remained shriveled, after rehydration. Peroxidase activity of desiccated somatic embryos increased sharply after 1 d of desiccation treatment at 87% relative humidity (RH), and desiccation-tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation-tolerant somatic embryos may have allowed them to catalyze the reduction of H₂O₂ produced by drought stress, and protected them from oxidative damage.     

Somatic embryogenic tissue establishment from mature Pinus nigra Arn. ssp. salzmannii embryos

Summary Somatic embryogenesis from mature zygotic embryos of salgare?o pine (Pinus nigra Arn. ssp. salzmannii) was induced after 2 wk of culture in L₁ medium [Murashige and Skoog mineral solution with 10.7 μM naphthaleneacetic acid (NAA) and 8.8 μM 6-benzyladenine (BA)] or L₂ medium [Gupta and Durzan mineral solution (DCR)] supplemented with the phytohormone combination as above. Four different combinations of growth regulators, NAA (2.6–10.7 μM) and BA (6.6–8.8 μM), were tried for subsequent passage. The best percentage of embryo induction and manifestation was obtained on DCR medium with 2.6 μM NAA and 6.6–8.8 μM BA. Transfer of isolated somatic embryos from the basal media to L₃ medium (Quoirin and Le Poivre modified mineral solution without hormones and supplemented with 0.3% activated charcoal), facilitated random embryo maturation and some development into plantlets.     

Induction,germination and shoot development of somatic embryos in cassava

Four Indonesian and two Latin-American cassava genotypes (Manihot esculenta Crantz), were evaluated for their ability to develop somatic embryos from young leaf lobes. All genotypes formed somatic embryos but they differed in the frequency of embryos induced. The best genotypes, M. Col 22 and Tjurug, produced germinating embryos (GE) on 81% (22.1 GE/initial leaf lobe) and 46% (4.3 GE/initial leaf lobe) of the cultured leaf lobes, respectively. Up to 57% of the germinating embryos of M. Col 22 and 12% of Tjurug produced either normal or malformed shoots. Most malformed shoots developed into shoots with normal morphology after prolonged culture. All shoots formed roots after transfer to medium without BAP. Roots of all normal and most malformed regenerants had the original ploidy level (2n=36). Regardless of whether the plants were multipliedin vitro (150 plants) or in the greenhouse (30 plants) there were no morphological differences compared to parent plants.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

The ability of Prunus avium× P. pseudocerasus `Colt' to form somatic embryos in vitro contrasts with the recalcitrance of P. avium

Somatic embryos were induced on roots excised from in vitro plants of Prunus avium× pseudocerasus `Colt'. On medium containing 6-benzylamino purine (BAP, 1.5 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D, 15 μM), a mean of 25 (s.e. ± 2.0) somatic embryos were produced on intact root systems and 15 (s.e. ± 1.7) on roots systems cut into 10 mm pieces. Most somatic embryos were formed directly on intact roots and indirectly (from callus) on sectioned roots. A mean of 2.5 (s.e. ± 0.25) secondary embryos per primary embryo were formed directly on primary embryos after they were transferred to medium containing BAP (1.5 μM), indole-3-butyric acid (10 μM) and 2,4-D (5 μM). After transfer to a medium containing BAP (2 μM) and gibberellic acid (GA<sub>3</sub>, 3 μM), shoots developed in 75% (s.e. ± 7.3) of the embryos. Somatic embryos were not induced on explants of in vitro roots or shoots of P. avium, and were induced infrequently on zygotic embryos, although a wide range of media were tested. Possible reasons for the contrasting embryogenic ability of `Colt' and P. avium are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.