Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of <Emphasis Type="Italic">Melia azedarach</Emphasis> (Meliaceae)

Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor tree (Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation when the concentration of sucrose was higher than 50 g l⁻¹. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos.     

Growth regulators influence the fatty acid profiles of in vitro induced jojoba somatic embryos

Inducing somatic embryogensis from jojoba [Simmondsia chinensis (Link) Schneider] explants to produce artificial seeds in the laboratory (in vitro) may prove highly profitable, as the seeds contain a characteristic liquid wax of economic importance in industry, nutrition and medicine. Thus, there is a need to examine the effect of the factors involved in the in vitro process on the quality and quantity of the synthesized fatty acids in comparison with those naturally produced in vivo. Immature zygotic embryos and mature leaf explants were cultured on Murashige and Skoog basal medium (MS) supplemented with various levels of 2,4-D, BA and sucrose. Embryogenic calluses developed from the zygotic embryos and leaf explants over a period of 2–4 weeks with the highest response at 0.4 μM 2,4-D, 2.2/4.4 μM BA and 117 mM sucrose (4%). Following induction, the zygotic embryo derived somatic embryos developed to the globular, heart, torpedo, and cotyledon stages. Direct somatic embryogenesis was observed with some of the zygotic embryo explants. Leaf-derived embryogenic calluses did not mature on any of the maturation/germination media examined up to 4 weeks of culture. Analysis of fatty acids indicated that the mature seeds are characterized with long chain saturated fatty acids C22:0 behenic Acid. The zygotic embryo-derived somatic embryos (SE-Z) and leaf-derived somatic embryos (SE-L) are characterized with the induction of the essential polyunsaturated fatty acid C18:2 (omega-6) linoleic acid, (omega-3) alpha-linolenic acid (ALA), with higher values of long chain saturated fatty acids C16:0 palmitic acid and monounsaturated fatty acid C18:1 oleic acid. These results indicate that manipulating the growth regulators in the induction media influenced the fatty acids synthesis and hence the fatty acids profile in jojoba somatic embryos.     

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog’s (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 — 2.0 mg dm⁻³ N⁶-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm⁻³ BA + 0.5 mg dm⁻³ abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Changes in protein profiles associated with somatic embryogenesis in peanut

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

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

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

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.     

Effects of different amino acids on somatic embryogenesis of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Comparison between somatic and zygotic embryo development in Quercus robur L.

ABSTRACT

Somatic embryogenesis from juvenile explants as an efficient way for oak clonal propagation is drastically limited by the low rate of embryo germination. A comparison of the development of immature somatic and zygotic embryos, and a study of the changes in sugar content and lignin accumulation during somatic versus zygotic embryo development were conducted in view of understanding the effect of reserve substance deficiency upon somatic embryo maturation. A morphological comparison of somatic and zygotic embryos led to the identification of 4 to 7 similar developmental stages in both types of embryos, thus indicating that the accumulation phase in both zygotic and somatic embryos occurs at the same stage, when the cotyledons became thicker and opaque. Carbohydrate analysis showed the presence of glycerol, inositol, mannitol, galactose, trehalose, xylose, arabinose, glucose, fructose and sucrose in all stages of zygotic and somatic embryo development, but in different amounts. The amount of glycerol, inositol, glucose and sucrose during the early stages is larger in zygotic embryos than in somatic ones, but the time course of their accumulation is similar in both types of embryos. Lignin content, which increased continuously during development, showed a similar behaviour in zygotic and somatic embryos. In somatic embryos which were able to germinate, lignin content was higher than in nongerminating embryos at the same stage.     

Somatic embryogenesis from pea embryos and shoot apices

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

低温预处理对水曲柳体细胞胚胎发生的影响

用4℃低温预处理未成熟的水曲柳种子0-30d,取出种子内的合子胚为外植体诱导体胚发生,研究低温预处理影响体胚发生的结果表明：低温预处理过的外植体其体胚发生总数和子叶胚发生数均低于未作低温预处理的;随着预处理时间的延长,畸形胚发生数和发生比率与总的体胚发生数和发生率的变化趋势基本相同;处理20d的正常胚发生数和发生比率的绝对数虽然很低,但远高于不作低温预处理的。说明4℃低温预处理对水曲柳体胚发生没有促进作用,对畸形胚的发生也不能控制,总的来讲,适当的低温处理有一定的改善正常体胚发生的潜力。     

Control of in vitro somatic embryogenesis of the spindle tree (Euonymus europaeus L.) by the sugar type and the osmotic potential of the culture medium

 In vitro somatic embryogenesis was achieved from zygotic embryo explants of a woody angiosperm species, the spindle tree, cultivated on various culture media differing in their sugar type and concentration, or in the applied osmotic potential. The highest frequency of somatic embryogenesis was obtained with a 350 mM sucrose, or a 89 mM glucose concentration in the culture medium. Experiments with culture media differing only in osmotic potential indicated that a minimal threshold osmotic potential is required to stimulate the emergence of somatic embryos. Elevated concentrations of glucose have an inhibitory effect, independent of their osmotic effect, while elevated concentrations of sucrose mainly act osmotically, stimulating the emergence of numerous somatic embryos. Received: 15 July 1997 / Revision received: 27 October 1997 / Accepted: 24 March 1998     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Development of a cyclic somatic embryogenesis regeneration system for leek (Allium ampeloprasum L.) using zygotic embryos

Summary In leek (Allium ampeloprasum L.) a cyclic system of somatic embryogenesis was developed. Somatic embryos used for cyclic embryogenesis were able to develop the same type of embryogenic callus as zygotic embryos in the primary cycle. For the first time a comparison of the efficiencies of both expiants was made. Ten families were investigated for somatic embryogenesis. There was a genetic relationship with respect to somatic embryo production between the reciprocal crosses. From each family one genotype was selected for investigating cyclic somatic embryogenesis. Different levels of somatic embryo production were found between the expiants of zygotic and somatic embryos. The two best genotypes, 92.001-03 and 92.002-33 produced twice as many somatic embryos as the overall average. On average, 56% of the somatic embryos finally developed into greenhouse plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MS medium Murashige and skoog (1962) basal medium     

Somatic embryogenesis in wild cherry (Prunus avium)

Indirect somatic embryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic. Embryos at different stages of development were produced. Among cotyledonary-stage embryos, 50% had two cotyledons and a distinct hypocotyl, 43% had one or more than 2 cotyledons and 7% had fused cotyledons. Most of the embryos were translucent and conversion into plantlets was very rare. Secondary embryos could be observed to occur with low frequency from cultured somatic embryos and from embryos emerging from calluses. Indirect somatic embryogenesis was also induced from immature zygotic embryos. From one donor tree, 51% of the explants were embryogenic when cultured on a medium containing 0.9 μM kinetin, 0.9 μM BA and 0.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis in Araucaria angustifolia

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.