Secondary somatic embryogenesis and plant regeneration in cassava

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

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 g/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 g morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 g morphine equivalents/g fr. wt.) by ELISA.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - HF phytohormone-free - NAA 1-naphthaleneacetic acid - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography     

Production of Fumonisins by Fusarium Verticillioides Strains on Solid and in a Defined Liquid Medium — Effects of L-Methionine and Inoculum

In order to evaluate the toxicological and carcinogenic effects of fumonisins, large amounts of fumonisins need to be purified, which requires optimal conditions for production in culture. Five strains of F. verticillioides were compared for their ability to produce fumonisins in solid and liquid media with and without the addition of methionine, a fumonisin precursor. Inoculations were made either with lyophilized cultures or a concentrated inoculum. Growth in liquid medium, measured by biomass, was directly correlated to total fumonisin production when a lyophilized inoculum was used. Fumonisin production was stimulated by the addition of 0.2% L-methionine to solid media for most strains. Levels ranged from 1500-3900 mg/kg in rice, and 2900-12500 mg/kg in maize cultures inoculated with lyophilized cultures; 200-4800 mg/kg in rice, and 1500-4200 mg/kg in maize cultures inoculated with concentrated inoculum. Strains that produced relatively high levels of fumonisins in solid media did not necessarily do so in liquid medium and vice versa. Total fumonisin levels in liquid medium ranged from 40-590 mg/l inoculated with lyophilized cultures and < 1-110 mg/l inoculated with concentrated inoculum, without adding the precursor. F. verticillioides strains therefore varied in their ability to produce fumonisins, and conditions for production need to be optimized individually for each strain.     

Development of an embryogenic suspension culture of soybean (Glycine max Merrill.)

A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1^–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration.     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Mass production of Siberian ginseng plantlets through large-scale tank culture of somatic embryos

Siberian ginseng (Eleutherococcus senticosus) is an endangered medicinal woody plant that is distributed throughout the cold regions of northeast Asia. Its propagation is very difficult. In the present article we describe the mass production of Siberian ginseng plantlets by large-scale tank cultures. High-frequency somatic embryogenesis was achieved through suspension culture of embryogenic cells in Murashige and Skoog (MS) liquid medium lacking 2,4-D. About 12,000 embryos were produced per 500-ml flask after 4 weeks of culture. Cotyledonary somatic embryos were germinated, and these converted into plantlets following a pretreatment of the embryos with gibberellic acid (20 µM) and subsequent transfer to a 10-l plastic tank. Low-strength MS medium (1/5-, 1/3-strength) was more effective than its full-strength counterpart for the production of plantlets. After 40 days, an initial 35-g inoculum of embryos was converted to 567 g fresh mass of plantlets in 1/3-strength MS medium. This result indicates that an efficient protocol for the mass production of Siberian ginseng plantlet can be achieved by tank culture of somatic embryos. The Siberian ginseng plantlets produced can be used as a source of medicinal raw materials.     

The influence of osmotic pretreatment and inoculum age on the initiation and regenerability of switchgrass suspension cultures

Summary Experiments were conducted to study the influence of osmotic pretreatment and inoculum (callus) age on the initiation and induction of somatic embryogenesis from suspension cultures of ‘Alamo’ switchgrass (Panicum virgatum L.). Embryogenic 10-, 20-, or 30-d-old calluses (hereafter referred to as inocula), produced from in vitro-cultured inflorescences, were used as explants to initiate the suspensions. Inocula were cultured for 30 h on MS solid medium containing 0.1, 0.2 or 0.3 M each of sorbitol and mannitol as an osmotic pretreatment. They were then transferred to liquid MS medium with 5 μM 6-benzylaminopurine and 9 μM 2,4-dichlorophenoxyacetic acid and cultured for 28 d in 2-ml Multiwell™ plantes. Individual multiwell contents were transferred to 125-ml Erlenmeyer flasks containing 20 ml of the above liquid medium and cultured for an additional 2 wk. Cultures initiated from the 10-d-old inoculum produced a higher embryogenic response than those initiated from 20- or 30-d-old inocula. Cultures initiated with 30-d-old inocula produced nonembryogenic clusters and the number of embryogenic cells was low. More embryos and a higher regeneration frequency were produced by 0.3 M than by 0.1 or 0.2 M of each of the osmostica.     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 μM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H₂O g⁻¹ dry weight.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

漏斗多孔菌液体菌种培养基及其栽培条件

为实现漏斗多孔菌资源化利用,设计单因素试验,以菌丝生物量、菌球密度和菌球直径为指标,获得漏斗多孔菌Polyporus arcularius液体菌种培养基配方为马铃薯（去皮）200g、玉米粉20.0g、蛋白胨5.0g、KH₂PO₄ 3.0g、K₂HPO₄ 1.0g,MgSO₄·7H₂O 1.5g,初始pH 5.0,并优化培养条件为装液量250mL/500mL,接种量2%,培养温度32℃,转速170r/min,种龄6d;依上述条件制备液体菌种,以固体菌种为对照,进行出菇试验,调查农艺性状,试验结果表明液体菌种接种后,菌袋满袋时间提前7d,子实体原基产生时间提前8d,头潮菇鲜重提高22.5%;配方试验结果表明,以细木屑为主料的配方产量最高,头潮菇鲜重为270.6g/袋。     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

Somatic embryos cultures of <Emphasis Type="Italic">Vitis amurensis</Emphasis> Rupr. in air-lift bioreactors for the production of biomass and resveratrol

Resveratrol are the most important bioactive compounds found in Vitis amurensis. In this study, a somatic embryo induction system for V. amurensis was established in air-lift bioreactors for the production of biomass and resveratrol. The somatic embryos biomass growth was low on solid medium (69.60 g L^?1) compared to in liquid medium in bioreactor (329.45 g L^?1). Bioreactor cultures were found to be superior compared with solid medium culture not only in terms of biomass but also resveratrol productivity. Various culture parameters, including culture method, inoculum density, carbon source, and organic compounds were optimized. An inoculum density of 20 g L^?1 embryogenic calli was found suitable for the accumulation of biomass and resveratrol production, whereas 10 g L^?1 embryogenic calli increased the amount of resveratrol per fresh weight in somatic embryos. For bioreactor culturing, sucrose was an optimum carbon source and 500 mg L^–1 casein hydrolysate acid was conducive to the biomass and resveratrol production. This result indicates that an efficient protocol for the large-scale production of resveratrol can be achieved by bioreactor culturing of V. amurensis somatic embryos and can be used as a source of medicinal raw materials.     

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.     

Somatic embryogenesis in the opium poppy, Papaver somniferum

A procedure is described for the induction of somatic embryos in the opium poppy. Papaver somniferum L. Callus was obtained from seedling hypocotyls on an agar solidified medium [Murashige and Skoog (1962) Physiol. Plant. 15: 473–497] containing 0.25 mg/l (1.2 μ M ) kinetin and 2.0 mg/l (10.7 μ M ) naphtalene acetic acid (NAA). Suspension cultures were initiated from callus using a liquid medium in which 2.0 mg/l (9.0 μ M ) 2,4-dichlorophenoxyacetic acid was substituted for NAA. Meristemoids, spheres of closely packed cells, developed in suspensions and on the surface of a few callus cultures. Differentiation of meristemoids into somatic embryos was accomplished by removing growth regulators from the liquid medium. Embryoids appeared morphologically normal and similar to torpedo stage embryos, however, they possessed mature tracheary elements and laticifers in areas that should have contained only procambium. Whole plants have been obtained by placing embryos in the light on solid medium that also lacked growth regulators.     

Effect of oleic acid on vegetative growth of the aphid-pathogenic fungus Erynia neoaphidis

Abstract The aphid-pathogenic fungus Erynia neophidis grew on a semi-defined medium containing 16 g·1⁻¹ glucose, 3 g·1⁻¹ yeast extract and 5 g·1⁻ mycological peptone only when the medium was supplemented with low concentrations of certain fatty acids. Of these, oleic acid fulfilled the growth requirement at a concentration of 0.02% (v/v), but higher concentrations were toxic, causing complete loss of viability of cultures at a concentration of 0.2% (v/v) in liquid medium and at 4% (v/v) on solid medium. The reduced viability of the fungus in liquid culture compared to that on equivalent solid medium, and at low inoculum density compared to high inoculum density in liquid medium, is explicable in terms of this toxicity.     

High frequency production of embryos inDatura innoxia from isolated pollen grains by combined cold treatment and serial culture of anthers in liquid medium

Summary This study concerns the development of pollen embryos as affected by various physical conditions of culture in media devoid of hormones. Freshly isolated pollen, from anthers ofDatura, failed to form embryos regardless of whether they were cultured on liquid or solid medium. In contrast, pollen isolated from anthers precultured on solid medium did form embryos and the response could be increased by prior cold treatment of anthers at 4 °C for 4 days. However, the best results were obtained when anthers were cultured from the very beginning in liquid medium and transferred serially to fresh medium. Under such conditions, the anthers dehisced, allowing spontaneous shedding of pollen grains. It was thus possible to have several fractions of shed pollen continuing their development into embryos. When serial culture was started with anthers from cold-treated buds not only were embryos formed in all the fractions of shed pollen but the frequency was also considerably higher than in any mode of culturing.     

Plant regeneration from somatic embryos in black pepper

Embryogenic callus derived from zygotic embryos of black pepper (Piper nigrum Linn.) were induced to form somatic embryos on solid and liquid Schenk and Hildebrandt basal medium. Callus proliferation, somatic embryo-genesis and germination of embryos were achieved in about 8 months in static cultures while it took only 8 weeks in liquid suspension cultures. The highest number of embryos and plantlets was produced from cells grown as suspension cultures raised in half-strength medium without growth regulators and sucrose level reduced from 3% to 1.5%. Regenerated plants were established in soil.