Cytological features of developing embryogenic callus and somatic embryos of Iris pumila L. and Iris setosa Pall

Abstract

Cytological examination during somatic embryogenesis in Iris pumila L. and Iris setosa Pall, were performed using light and electron microscopy. The first sign of the cellular differentiation in the initial embryogenic callus (EC; stage 1) of both Iris species was the formation of short and elongated cell types. After the onset of embryogenesis, short cells divided producing a mass of densely packed meristematic cells, closely connected with numerous plasmodesmata. Further differentiation into globular embryos (GE) led to a loss of plasmodesmata and cell separation. In vacuolated elongated cells, cytoplasm was located near the wall and around the nucleus. In both cell types amyloplasts and small mitochondria with poorly developed crystae were abundant.

Cell of GE (stage 2) contained an increased number of mitochondria and plastids comparing to those from stage 1, indicating further differentiation. Thylakoids and starch grains were observed within the plastids, while the number of cristae within the mitochondria was increasing.

In cells of embryos with coleoptile (ECl) (stage 3), plastids differentiated into chloroplasts with thylakoids. In all stages of cell differentiation, short and long cisternae of endoplasmic reticulum with ribosomes were seen. Activity of dictyosomes was increased in stages 1 and 2, then reduced in stage 3.

Ultrastructure of EC cells was identical to that of proembryogenic cells, i.e. of early GE. Ultrastructural appearance of GE cells was identical in both Iris species, but evident, and increasing, differences in mitochondria and plastids were observed between GE and ECl embryos.The presence of bi-, three- and eight- cell proembryos demonstrates that they originate from a single cell in both Iris species.     

Plant regeneration of Iris pallida Lam. and Iris germanica L. via somatic embryogenesis from leaves,apices and young flowers

Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somatic embryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants.Abbreviations AIB indolyl butyric acid - BAP 6-benzylaminopurine - Kin 6-furfurylaminopurine - KN Knudson's orchid Agar - MS Murashige & Skoog medium - NAA naphthaleneacetic acid - N6 Chu et al. medium - Pro proline - 2,4-D dichlorophenoxyacetic acid     

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl^-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.     

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Summary Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.     

Plant regeneration from highly embryogenic callus,cell suspension and protoplast cultures of Trifolium fragiferum

Using various media, tissue and protoplast cultures plant regeneration systems were developed for Trifolium fragiferum (2n=16). (L.). The best media for induction of embryogenic cultures were based on Kao (1977) or Kao and Michayluk (1975). Somatic embryogenesis was observed in cultures derived from green leaf mesophyll protoplasts of branching plants, somatic embryo protoplasts and cell suspension protoplasts, leaflets and various explants of immature zygotic embryos. The process of somatic embryogenesis was maintained for over two years on Murashige and Skoog's (1962) medium supplemented with 0.5 mg l^-1 benzyladenine and 0.05 mg l^-1 naphthaleneacetic acid. These long term cultures were capable of regenerating plants that were fertile and produced seeds. These results were compared with those from protoplast, tissue and organ culture of other species of the Trifolium genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

百合体细胞胚胎发生和植株再生

以切花百合(Lilium)品种‘黄天霸’(‘Manissa’)花器官为外植体诱导体细胞胚胎发生与植株再生。结果表明,不同花器官、不同激素配比对愈伤组织形成均具有显著影响。花丝为最佳外植体,激素对愈伤组织诱导的影响效应为NAA>6-BA>2,4-D,最适培养基为MS+1.0 mg.L-1NAA+0.2 mg.L-16-BA;激素诱导体细胞胚胎发生的影响效应为2,4-D>KT>6-BA,最佳培养基配方为MS+1.0 mg.L-12,4-D+0.2 mg.L-1KT+1.0 mg.L-16-BA;MS培养基添加IBA可促进体细胞胚萌发成苗,体细胞胚芽成苗的最佳培养基为MS+0.2 mg.L-16-BA+1.0 mg.L-1IBA。     

长白落叶松体胚发生再生体系优化

以长白落叶松(Larix olgensis)未成熟合子胚为外植体诱导胚性愈伤组织, 通过调节影响体胚发生的营养物质和植物生长调节剂配比, 进行愈伤组织的胚性恢复与保持以及体胚发生再生体系的优化。结果表明: 不同无性系之间胚性愈伤组织诱导率差异显著, 胚性愈伤组织在S+0.2 mg·L ^-1NAA+0.5 mg·L ^-1BA+0.5 mg·L ^-1KT+0.5 g·L ^-1谷氨酰胺+0.5 g·L ^-1水解酪蛋白+30 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶培养条件下, 可以恢复胚性并长久保持。在S+20 mg·L ^-1ABA+60 g·L ^-1PEG₄₀₀₀+60 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下分化培养6周, 体胚发生率可达100%。将正常发育的体胚先在WPM+ 6 mg·L ^-1间苯三酚+1.0 g·L ^-1活性炭+3.0 mg·L ^-1VB₁+20 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下培养2周, 再转接至B₅+ 0.4 mg·L ^-1NAA+1.0 mg·L ^-1IBA+0.5 mg·L ^-1GA₃+2.0 mg·L ^-1VB₁+1.0 g·L ^-1活性炭+20 g·L ^-1蔗糖及3.0 g·L ^-1植物凝胶条件下培养2周, 可见子叶舒展、下胚轴伸长且根系正常的体胚苗。该研究建立了长白落叶松胚性愈伤组织胚性恢复与保持方法, 并进一步优化了体胚发生的植株再生体系, 为林木资源快速繁育和遗传改良奠定了基础。     

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×10⁶ and 1×10⁷ protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×10⁵ / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.Abbreviations CMS Murashige & Skoog (1962) medium for cucumber - GLC gel-like callus - ESC established embryogenic suspension culture - 2,4-d 2,4-dichlorophenoxyacetic acid     

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。     

胡桃楸胚性愈伤组织诱导与体细胞胚胎发生

胡桃楸是东北东部山地阔叶红松林的重要组成树种。因其被大量采伐,资源日趋枯竭。体细胞胚胎发生是快速繁殖和人工种子研制的基础,对遗传改良有重要意义。为探讨不同外植体、植物生长调节物质种类及配比对胡桃楸培养物的影响,建立了胡桃楸体胚发生及再生植株体系。结果表明:合子胚为外植体时最易形成胚性愈伤组织,外植体最佳取材时期为5～6月。胡桃楸胚性愈伤组织最适诱导为MS+1.0mg·mL-12,4-D+0.5mg·mL-16-BA;体细胞胚的诱导、发育和分化的适宜的培养基为附加蔗糖60g.L-1、水解酪蛋白700mg·mL-1时不添加任何生长调节物质的MS培养基。     

Plant regeneration from protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Summary Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 μM 2,4-dichlorophenoxyacetic acid, 17.8 μM 6-benzyladenine, 18.6 μM kinetin, 500 mg l⁻¹ casein hydrolysate, and 500 mg l⁻¹ l-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radieles of mature zygotic embryos. Callus was subcultured on the callus proliferation medium, the same as the induction medium but with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO₃, Ca(NO₃)₂·4H₂O, NH₄NO₃, KCl, ZnSO₄·7H₂O, and MnSO₄·H₂O, 720, 1900, 400, 250, 25.8, and 25.35 mg l⁻¹, respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating the production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4–12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l⁻¹). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1∶1∶1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.     

Evaluation of a plant regeneration test of embryogenic cell suspension cultures of rice (Oryza sativa L. cv Taipei 309)

Summary In this paper we discuss the changes in the number of plants developed per embryogenic mass, the percentage of embryogenic masses that developed plants, the percentage of plant regeneration, and the index of vigor of regenerated plants for embryogenic masses that were induced from daily samples of a cell suspension culture of rice (Oryza sativa L. cv. Taipei 309). The results indicate that embryogenic masses that matured first and were induced from freshly subecultured cell suspension culture had the highest values for the four parameters. An index of vigor of the regenerated plants was defined.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

Plant regeneration from callus culture of Cymbidium ensifolium var. misericors

Totipotent calli of Cymbidium ensifolium var. misericors, a locally grown orchid of high commercial value, were induced from sections of pseudobulbs, rhizomes and roots of seed-derived plantlets on 1/2-strength Murashige and Skoog medium plus 10 mg/l 2,4-dichlorophenoxyacetic acid and 0.1 mg/l thiadiazuron. The calli could be maintained by subculturing in the same medium. The calli could be induced to develop further along one of three distinct morphogeneic routes: (1) production of rhizomes, (2) production of shoot buds, or (3) development of granular embryoids. Efficient mass propagation was possible via rhizome proliferation and embryoid formation. Received: 28 May 1997 / Revision received: 19 August 1997 / Acceptance: 2 September 1997     

Somatic embryogenesis in sugarcane—An addendum to the invited review ‘sugarcane biotechnology: The challenges and opportunities,’ in vitro cell. Dev. Biol. Plant 41(4):345–363; 2005

Summary Since the 1960s, numerous studies on sugarcane plant regeneration have been reported. Essentially, successful culture and regeneration of plants from protoplasts, cells, callus, and various tissue and organs, have been achieved in this crop. Although plant regeneration from callus cultures had been reported since the 1960s, definitive proof of somatic embryo development was not available until 1983. Since then, considerable progress has been made in understanding and refining somatic embryogenesis and plant regeneration in sugarcane, for which development of an efficient embryogenic system was critical for the application of transgenic technology. Recent research in Australia and South Africa has led to the development of direct somatic embryogenic systems, which may improve transgenesis in sugarcane.     

大蕉(Musa paradisiaca ABB Linn.)花序愈伤组织的诱导及其体细胞胚发生

大蕉未成熟雄花接种到胚性愈伤组织诱导培养基中,4～5个月后可诱导出胚性愈伤组织,并可在继代培养基上增殖.胚性愈伤组织转移到体细胞胚诱导培养基中可诱导出体细胞胚.体细胞胚在成熟培养基上培养2个月后转移到含有0.2mg·L-1 6-BA的分化培养基上可以萌发,进而形成再生植株.组织学切片证明所诱导的愈伤组织是胚性组织,其所产生的体胚具有典型的单子叶植物体细胞胚的组织结构.     

Plant regeneration in weeping lovegrass, (Eragrostis curvula) through inflorescence culture

Plant regeneration from four genotypes of weeping lovegrass (Eragrostis curvula (Schrad.) Nees), is reported via three developmental pathways: embryogenesis, organogenesis and direct regeneration. Organogenic and embryogenic callus cultures were initiated from young inflorescence segments on Murashige and Skoog's medium supplemented with 2,4-d and BA at different concentrations. The most suitable concentrations of 2,4-d for callus growth and development were 9 and 18 M combined with a BA concentration of 0.044 M. Genotypical differences were observed in the morphogenetic capacity. Direct regeneration was observed under similar culture conditions (culture medium, temperature and photoperiod) but with high light intensity (66 mol m^-2 s^-1). Young plants were successfully transplanted to pots and grown to maturity in the greenhouse.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid