Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

Somatic embryogenesis from zygotic embryos and shoot-tips of the Chilean tree <Emphasis Type="Italic">Gomortega keule</Emphasis>

The endangered Chilean tree Gomortega keule (Mol.) Baillon produces edible fruit, making it a potential crop. However, its cultivation from seed or cuttings is extremely difficult. This paper reports the induction of somatic embryogenesis and the initiation of liquid cultures in this species. Callus was induced from zygotic embryos and field-collected shoots. Somatic embryogenesis on zygotic embryos occurred at a low frequency. Induction of somatic embryogenesis was accomplished on micropropagated shoots after 6.5 months on semi-solid Murashige and Skoog (MS) medium with 30 g/l sucrose, 1.0 mg/l 2,4-dichlorophenoxyacetic acid and 1.0 mg/l 6-(γ,γ-dimethylallylamino) purine (2iP). Liquid cultures of compact callus and small aggregates were obtained and showed optimum proliferation in MS medium with 20 g/l sucrose, 0.01 mg/l α-naphthaleneacetic acid and 0.1 mg/l 2iP. The proliferation of friable embryogenic callus was observed in liquid medium and will allow the propagation of selected genotypes of this tree on a large scale. Genetic variation in two embryogenic genotypes cultured in vitro was not detected in an assessment using microsatellites; this approach is suitable for tracing genotypes.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures. Received: 14 July 1998 / Revision received: 2 November 1998 / Accepted: 6 November 1998     

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.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.     

Somatic embryogenesis from <Emphasis Type="Italic">Arabidopsis</Emphasis> shoot apical meristem mutants

Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.     

Induction,protein composition and DNA ploidy level of embryogenic calli of silver fir and its hybrids

Somatic embryogenesis was initiated from immature zygotic embryos of intraspecific cross ofAbies alba and interspecific combination ofAbies alba x Abies nordmanniana. The most responsive explants for induction of embryogenic calli were zygotic embryos in precotyledonary stage of development. Biochemically the compared embryogenic lines were uniform irrespective of their morphology or embryogenic potential. The flow cytometric analysis of single embryogenic lines suggests that no changes in ploidy levels occur during induction and culture of embryogenic callus inAbies alba what confirms the convenience of this system for propagation of valuable seed material.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

Somatic embryogenesis in elm

We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4-dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0.1 mg l(-1) BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely.     

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.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra- and inter-species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four-cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l^?1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.     

Histodifferentiation of somatic embryos in cotyledons of pineapple guava (Feijoa sellowiana Berg)

Summary Somatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than mature zygotic embryos. At the time of explantation, cotyledonary cells were rich in storage proteins and lipids but no starch was found. After the first 5 days of culture most of the reserves had been mobilized in cotyledons of germinating embryos, but were still present in large amounts in cotyledons undergoing embryogenie induction. In contrast to cotyledons following the normal pattern of development, cells of embryogenically-induced cotyledons accumulated starch, especially those cells not involved in the embryogenie process. Two patterns of somatic embryo differentiation were observed: (1) from single epidermal cells or (2) from groups of meristematic cells near the adaxial surface. Comparative observations on cotyledons from germinating embryos and those undergoing embryogenesis suggest that the meristematic layer arises as the result of successive divisions of cells that, under normal conditions, would form the palisade parenchyma. These were the only mesophyll cells that showed mitotic divisions during the normal development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FAA formalin/acetic acid/ethyl alcohol - PAS periodic acid-Schiff     

小麦组织培养中体细胞胚胎发生的细胞胚胎学及淀粉消长动态的研究

小麦幼胚在附加2,4-D 2 mg/L+KT0.5 mg/L+LH 300 mg/L+3%蔗糖的MS 培养基上诱导出愈伤组织,继代2—3次后,降低2,4-D 的浓度,该愈伤组织约以60%的频率转变为胚性愈伤组织,继而形成不同发育期的体细胞胚。这些体细胞胚是起源于愈伤组织表层或近表层的单个胚性细胞,体细胞胚经球形胚、梨形胚、盾片胚、成熟胚而成再生植株。在胚性细胞内淀粉粒大最积累,淀粉颗粒大而密集。随着胚性细胞的分裂,淀粉逐渐被消耗,到多细胞原胚期,胚体细胞内淀粉粒趋于消失。球形胚期淀粉粒又开始积累,但到成熟胚期,淀粉积累只限于生长点区域,尔后随着胚体萌发,淀粉含量又趋于减少。这一消长动态过程与小麦合子胚发生中淀粉代谢动态基本一致。