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     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana

Somatic embryogenesis is an obvious experimental evidence of totipotency, and is used as a model system for studying the mechanisms of de-differentiation and re-differentiation of plant cells. Although Arabidopsis is widely used as a model plant for genetic and molecular biological studies, there is no available tissue culture system for inducing somatic embryogenesis from somatic cells in this plant. We established a new tissue culture system using stress treatment to induce somatic embryogenesis in Arabidopsis. In this system, stress treatment induced formation of somatic embryos from shoot-apical-tip and floral-bud explants. The somatic embryos grew into young plantlets with normal morphology, including cotyledons, hypocotyls, and roots, and some embryo-specific genes (ABI3 and FUS3) were expressed in these embryos. Several stresses (osmotic, heavy metal ion, and dehydration stress) induced somatic embryogenesis, but the optimum stress treatment differed between different stressors. When we used mannitol to cause osmotic stress, the optimal conditions for somatic embryogenesis were 6-9 h of culture on solid B5 medium containing 0.7 m mannitol, after which the explants were transferred to B5 medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 microm), but no mannitol. Using this tissue culture system, we induced somatic embryogenesis in three major ecotypes of Arabidopsis thaliana-Ws, Col, and Ler.     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

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。     

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.     

Plant regeneration through direct somatic embryogenesis from leaf explants of <Emphasis Type="Italic">Dendrobium</Emphasis>

A protocol for induction of direct somatic embryogenesis, secondary embryogenesis and plant regeneration of Dendrobium cv. Chiengmai Pink was developed. Thidiazuron (TDZ) at 0.3, 1 and 3 mg dm⁻³ induced 5–25 % of leaf tip segments of in vitro grown plants to directly form embryos after 60 d of culture, and 1 mg dm⁻³ TDZ was the best treatment. Somatic embryos mostly formed from leaf surfaces near cut ends, and occasionally found on leaf tips. Higher frequency of embryogenesis was obtained in light than in darkness. During subculture, secondary embryos developed from outer cell layers of primary embryos. All combinations of NAA (0, 0.1, 1 mg dm⁻³) and TDZ (0, 0.3, 1, 3 mg dm⁻³) increased the multiplication rate of embryos. It takes about 8 months from embryo induction, plantlet formation to eventually acclimatization in greenhouse.     

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l⁻¹) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l⁻¹) + thidiazuron (1 mg l⁻¹). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons

An in vitro protocol has been developed for direct somatic embryogenesis of zygotic cotyledons from mature sugarbeet (Beta vulgaris L.) embryos. Explants were sequentially cultured on modified Murashige and Skoog (MS) medium supplemented with different combinations of 2,4-D, NAA, BAP and TIBA. Somatic embryogenesis was induced within 4 weeks of culture on embryogenesis induction medium which contained MS medium supplemented with BAP and TIBA. Proliferation of somatic embryos was observed on embryo proliferation medium, which contained MS medium supplemented with BAP and NAA within 4 weeks of culture. Plants were regenerated on hormone free half; strength MS medium containing a low sucrose concentration. With some sugarbeet lines, high frequencies of plant regeneration in excess of 90percnt; were observed. The incorporation of TIBA in the media was essential for successful regeneration.     

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.     

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。     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH₄ ⁺ and NO₃ ^– ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 M) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH₄ ⁺ and NO₃ ^– concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 M) enhanced the conversion of somatic embryos to plantlets.     

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     

马尾松幼胚体细胞胚胎发生研究

本论文首次报道了马尾松（Pinus massoniana Lamb.）幼胚体细胞胚胎发生的完整发育过程,并对影响马尾松胚性愈伤组织诱导的因素如球果采种期、球果冷藏处理时间、外植体处理方式等进行了探讨,统计胚性愈伤组织诱导率,进行增殖评价,探讨ABA浓度梯度对马尾松体细胞胚分化成熟的影响,试验数据用SPSS16统计分析软件进行方差分析、差异显著性检验。结果表明：1）2008~2009连续2年内15个采种期得到的幼胚,胚性愈伤组织诱导和增殖有显著性差异,最适宜的马尾松球果采种期是6月下旬至7月下旬,诱导率在9.66%~22.59%之间;2）球果冷藏处理时间,对胚性愈伤组织诱导有显著性差异,其中4℃冷藏球果15d有利于幼胚胚性愈伤组织诱导;3）雌配子体包含幼胚的接种处理方式是可取的;4）胚性愈伤组织经稳定增殖培养后,转入分化成熟培养基,得到体细胞胚状体＂爆发式＂分化成熟,数量多,质量好。适宜体胚成熟转化的培养基为：成熟LP培养基添加ABA5.0mg·L-1＋60.0g·L-1蔗糖,并附加L-谷氨酰胺和水解酪蛋白;5）成熟体细胞胚在无激素萌发型LP培养基上正常萌发,并转化为结构完整的小植株。本研究首次建立了马尾松幼胚体细胞胚胎发生技术平台,为马尾松遗传改良种质创新、缩短育种周期奠定了研究基础。     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf.): histology and improvement by AgNO3

Immature zygotic embryos of durum wheat cv Ardente were cultured vitro on 2,4-D to induce somatic embryogenesis. Five days after culture initiation, somatic proembryos were directly initiated from the scutellum of immature embryos. After 28 days, somatic embryos were fully developed with a scutellum-like structure. Histological observations between the first and the eighty day in culture showed a clear unicelllar origin for a few of these somatic embryos, whilst most of them originated from a meristematic multilayer. Furthermore, estimation of the mitotic index of outer epithelium, subepithelium and inner epithelium of the scutellum during the first week of culture, showed a strict epidermal origin of these early developed structures. The addition of 1 mg·L^–1 of AgNO₃ enhanced the induction of direct somatic embryogenesis (a more than 22 fold increase), affecting both the percentage of embryogenic explants and the number of somatic embryos per explant, suggesting the possible involvement of ethylene.     

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.     

Plant regeneration through somatic embryogenesis from zygotic embryo-derived callus of <Emphasis Type="Italic">Areca catechu</Emphasis> L. (Arecaceae)

Summary An in vitro culture procedure was established for somatic embryogenesis and plant regeneration from callus cultures of the important palm ‘betel nut’ (Areca catechu L.). Segments of zygotic embryos of Areca catechu L. were cultured on Murashige and Skoog basal medium supplemented with dicamba (9.05, 18.1, 27.15, and 36.2 μM). After 7–8 wk in darkness, wounded regions of explants formed callus with yellow, soft, glutinous structures. Proliferation and maintenance of callus was on the same dicamba-containing medium. With regular subculture every 8 wk, the callus showed pale yellow, compact and nodular structures. During subculture, somatic embryos were formed spontaneously from nodular callus tissues within 2–4 mo. The embryos developed into plantlets after 10 wk of culture on basal medium free of plant growth regulators. After subculturing every month for 3 mo., the plantlets were transferred to containers for acclimatization in the greenhouse. The survival rate was 24%.