Enhancement of somatic embryogenesis in Norway spruce (Picea abies L.)

Summary Embryogenic callus developed in 55% of the mature embryo explants of Norway spruce (Picea abies L.) growing on a LP medium minus the amino acids and sugars (except sucrose). This is the highest reported yield of embryogenic callus from mature embryos of P. abies that has ever been reported. Callus induction from either the middle or the end of the hypocotyl of the embryos began after 2–3 weeks. Three types of calli were recovered: (a) globular, (b) light green-compact, (c) white mucilaginous. Only the white mucilaginous calli were embryogenic. The globular and light green-compact calli never become embryogenic, even after several subcultures. The development of somatic embryos was accomplished on half-strength macro-elements of NSIII medium containing 1 M -naphthaleneacetic acid, 1 M abscisic acid, and 3% sucrose. The addition of 10^–7 M buthionine sulfoximine to the medium increased the development of somatic embryos by three fold. These results suggest that there is a great potential for increasing the frequency and development of somatic embryos in P. abies. Careful selection of the genotype and modification of the culture medium is required.     

Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.)

Interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.) cotyledonary somatic embryos were encapsulated in sodium alginate. Somatic embryo viability was retained, but germination occurred at a reduced frequency compared with the equivalent zygotic embryos. The addition of 0.5% (w/v) activated charcoal to the alginate capsule significantly enhanced root development and germination for somatic embryos but not for zygotic embryos. The possibility of developing an artiflcal endosperm was also investigated, by addition of Litvay (Litvay et al. 1981) nutrients with or without 90 mM sucrose to the alginate-charcoal capsule. This treatment significantly enhanced root development for all embryo categories with the exception of black spruce somatic embryos. Encapsulated and non-encapsulated somatic embryos survived one month cold storage at 4 °C without reduction in germination frequency.NRCC No. 35895     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

火炬松胚性细胞悬浮培养物的生长参数变化

以火炬松（PinustaedaL.）成熟合子胚来源的胚性愈伤组织为材料建立了胚性细胞悬浮系,测定了其培养物的鲜重、干重、细胞体积和胚数及培养液中的pH值、电导率和蔗糖浓度等生长参数在培养过程中的变化动态。结果表明,在培养周期内,培养液中的pH值、电导率和蔗糖浓度的逐步降低与培养物的鲜重、干重、细胞体积和胚胎数的逐步增加保持一致性。在培养至18—21d,pH值、电导率和蔗糖浓度均接近或降到最低点,而胚数及细胞体积的增长都达到最高点。     

Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr.

The effects of sucrose on maturation and of plant growth regulators on germination of soybean somatic embryos were investigated for the purpose of developing an efficient culture method for plant recovery. Somatic embryos produced on medium with a low sucrose concentration (5 gl^-1), less than 1 mm in length, 0.6 mg in fresh weight, and green in color, were grown for 2 weeks on MS medium containing 5 gl^-1 or 30 gl^-1 sucrose and then for another 5 weeks on MS medium containing 5–90 gl^-1 sucrose. The highest increase in fresh weight of somatic embryos was obtained in the treatment of transferring from 30 gl^-1 sucrose (2 weeks) to 60 gl^-1 (5 weeks). With the increase in fresh weight, the somatic embryos gradually changed color from green to yellow, and finally to white, when they stopped growth. Soybean seed storage proteins (-conglycinin and glycinin) were accumulated in somatic embryos under tissue specific and stage specific control analogous to that in zygotic embryos. Exogenous gibberellic acid was effective in promoting precocious germination of premature soybean somatic embryos, but was not necessary for the germination of mature somatic embryos. The efficiency of somatic embryo germination was as high as 77% from semi-wild soybean and 60–64% from cultivated soybeans, showing that the plant regeneration system developed in this study was efficient and practical.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BR brassinolide - GA₃ gibberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS Sodium Lauryl Sulfate     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^&#x2212;1 sucrose. A sucrose concentration lower or higher than 30 gl^&#x2212;1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^&#x2212;1 tyrosine in the medium. Seedlings germinated in the presence of 0.2&#x2013;0.4 mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and 0.3&#x2013;0.5 mgl^&#x2212;1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60&#x2013;70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Toxicity of antibiotics on zygotic embryos of white spruce (Picea glauca) cultured in vitro

The toxicity of kanamycin, hygromycin B, geneticin, methotrexate and cefotaxime on zygotic embryos ofPicea glauca was studied. Embryos placed on bud induction medium produced approximately 20 adventitious buds per embryo under control conditions. Addition of antibiotics reduced the number of bud-forming embryos. Using the percentage of embryos with buds as an indication of antibiotic toxicity, two-day-old explants were found to be more sensitive than nine-day-old. Kanamycin toxicity was enhanced by cefotaxime and this effect increased with increases in concentration of either antibiotic. Although no morphological difference was observed after 21 days, embryos growing on medium containing 20 g ml^–1 kanamycin showed a decrease of 73% in dry weight and 23% in protein content per embryo when compared to control embryos. Similarly, a decrease of 38% in dry weight and 40% in protein content per embryo was found in embryos on medium containing 300 g ml^–1 cefotaxime.Abbreviations BA 6 benzyl-aminopurine - EDTA ethylenediamine-tetraacetic acid - PVP 10 polyvinylpyrrolidone (MW 10,000) - Tris-HCl Tris [hydroxymethyl] amino hydrochloride NRCC No. 30262     

Maturation of black spruce somatic embryos. Part I. Effect of l-glutamine on the number and germinability of somatic embryos

Different concentrations of l-glutamine and different nitrogen sources in the medium were compared during maturation of black spruce (Picea mariana (Mill.) B.S.P.) somatic embryos. l-glutamine can be used as the sole nitrogen source for the maturation of Picea mariana somatic embryos at 2 to 3 gl^-1. A significantly lower number of somatic embryos was obtained on a medium prepared with only inorganic nitrogen. Compared with a medium supplement to inorganic nitrogen resulted in a twofold increase in the number of embryos for six genotypes. The nitrogen source and concentration in the maturation medium significantly affected the germination sensus stricto of somatic embryos (radicle appearance), but not their development into plantlets; at the time of epicotyl appearance, an effect of the nitrogen source was no longer found. A comparison of the development of somatic embryos into plantlets from seven genotypes showed that the genotype had more effect in terms of epicotyl appearance and in conversion rate than the nitrogen source present in the maturation medium.Abbreviations HLM-1 half-Litvays's medium with 10 M 2,4-D and 5 M BA - i only inorganic nitrogen in the medium - i+1 gG inorganic nitrogen plus 1 g l^-1 glutamine in the medium - SMM standard maturation medium - 2.5gG only 2.5 g l^-1 glutamine in the medium     

Influence of osmotica and abscisic acid on triglyceride accumulation in peanut somatic embryos

Attempts were made to determine the influence of sucrose, mannitol, sorbitol and abscisic acid on accumulation of triglycerides in peanut (Arachis hypogaea L.) somatic embryos. The results revealed that 0.584 m sucrose in the medium produced increased triglycerides in the embryos compared to the control. At 0.730 m sucrose, embryos were necrotic although the triglyceride content was high. Sorbitol at 0.6 m or abscisic acid at 20 μm were effective in increasing triglycerides in the embryos. The increase in triglycerides on a fresh-weight basis observed with increasing concentration of osmoticium was not apparent when determined in terms of dry weight. However, an increase in triglyceride as percent fresh weight observed in the presence of 20 μm abscisic acid remained unaltered when determined in terms of percent dry weight. An increase in storage lipid did not improve conversion of peanut somatic embryos. Received: 4 April 1997 / Revision received: 15 February 1998 / Accepted: 2 March 1998     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Production of Gammarus pulex L. (Amphipoda) in a small Danish stream

Quantitative samples of Gammarus pulex L. taken from a small Danish stream during 1975 showed mean annual population densities varying from 500 m^–2 in early May to 5 500 m^–2 in late September. The mean annual biomass was 1.5 g dry weight m^–2. No discrete cohorts could be distinguished from the size frequency distributions. Annual production, estimated by the size-frequency method, was 3.9 g dry weight M^-2 and P/B ratio was 2.6. The contribution to trout energetics may have been as much as 17%.     

Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze)

Addition of betaine to the inductionmedium significantly enhanced the rapid formation ofsomatic embryos directly without callusing from maturefresh seeds of tea within two weeks of cultureinitiation. The induction response was furtherenhanced when ABA (7.5 mgl^–1) was co-supplementedwith betaine in the induction medium. The rate ofinduction of somatic embryogenesis increased linearlywith external betaine concentration. Globular somaticembryo-like structures (embryoids) were observed in 4-week old cultures when inoculated on the inductionmedium without ABA and betaine. The positive effectof ABA on the induction process was found to bedependent on the presence of betaine in the medium. ABA alone in the medium could not bring the inductionstimulus in the explants; on the contrary, it provedinhibitory. The optimum response of ABA was observedwhen the medium was supplemented with 500 to1000 mgl^–1 of betaine. Primary somatic embryosobtained in the presence of ABA and betaine were ableto produce secondary embryos. A conversion rate of15–20% was achieved upon transfer of somatic embryosof size 3–5 mm in diameter to the basal medium consistof half strength of macro nutrients, full strength ofmicro nutrients and vitamins of MS. Medium wasfurther supplemented with 100 mg l^–1 each ofadenine hemisulfate sulphate and L-glutamine, 30 gl^–1 sucrose, gelled with 7 gl^–1 bitek agar. The plantlets regenerated by this procedure did notshow any visible abnormalities. This report for thefirst time details the potential use of betaine inplant tissue culture.     

Chemotaxis towards sugars inChlamydomonas reinhardtii

Chemotaxis ofChlamydomonas reinhardtii 137c cells towards maltose and sucrose was observed by capillary assay. The threshold concentration was approximately 10^–5 m for maltose and 10^–3 m for sucrose. The peak accumulation of cells occurred at 10^–3 m for maltose and 10^–2 m for sucrose. A selection procedure for chemotaxis mutants was developed. Mutant strain CHE-1 was not attracted by maltose; mutant strain CHE-2 was not attracted by sucrose. Addition of attractant fully inhibited photoaccumulation of cells. After a period of time the photoresponse of cells recovered. Under the conditions of our experiments the period of adaptation lasted 15–20 min in wild-type cells and 4–5 min in mutant cells on the given sugar. Glucose and acetate did not attract cells ofChlamydomonas. Added to the medium, these compounds had no effect on the photoaccumulation of cells.     

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Summary Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl ^–1 of fructose and 2.5gl ^–1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h^–1 and yielded 0.31gl ^–1h^–1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h^–1.     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Growth and accumulation of storage reserves by somatic embryos of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was the assessment of in vitro growth of embryogenic cultures of Ocotea catharinensis Mez. (Lauraceae) on Woody Plant Medium (WPM) supplemented with 22.7 g l^–1 sorbitol, 2 g l^–1 Phytagel, 20 g l^–1 sucrose and 400 mg l^–1 glutamine and the biochemical analysis of somatic embryos at different developmental stages (globular, early cotyledonary, cotyledonary and mature). The embryoids underwent repetitive embryo-genesis and developed non-synchronously, throughout the culture period. Dry weight increased 12.6- and 26.8-fold after 3 and 5 weeks, respectively. During the 5 week culture period a reduction in the frequency of embryoids at the globular stage and increasing frequencies of embryoids at early cotyledonary, cotyledonary and mature stages were observed. Embryoids at the mature stages presented a small but significantly higher water content than those at the globular stage. Therefore, embryoid expansion at the latter stages of development was mainly due to water uptake. Embryoids at the globular stages accumulated higher levels of total proteins while those at the cotyledonary and mature stage showed higher levels of soluble sugars and starch (physiological markers of embryo maturation). Thus, significant differences in the profiles of accumulated storage reserves were detected in the embryoids at different developmental stages in the culture conditions tested.     

In vitro culture of embryos of Cucumis spp.: heart-stage embryos have a higher ability of direct plant formation than advanced-stage embryos

Summary The ability of embryos at different developmental stages to form plants in vitro has been studied in cultivated Cucumis sativus L. and in the wild species C. zeyheri 2 x Sond. and C. metuliferus Naud. On MS medium containing 3.5% sucrose, 0.1 mg 1^–1 kinetin (Kn) and 0.01 mg 1^–1 indoleacetic acid (IAA), proembryos (0.03–0.05 mm) and early globular embryos (0.05–0.08 mm) from the wild species developed into plants in low frequencies of 8% and 21%, respectively. These embryos should be surrounded by the embryo sac tissue. On the same medium late globular (0.08–0.1 mm) and early heart-stage embryos (0.1–0.3 mm) developed into plants in moderately high and high frequencies of 48% and 83%, respectively. The presence of the embryo sac at these stages was still beneficial, but no longer a prerequisite. Late heart-stage embryos (0.3–0.8 mm) also showed high frequencies of plant formation, 63%, if Kn was applied at a concentration of 1 mg 1^–1. From the early cotyledon stage onwards, the frequency of plant formation gradually decreased, reaching a minimum at the late cotyledon stage. Subsequently it began to increase again up to the late maturation stage. The poor plant formation shown by the intermediate-aged embryos could be improved slightly by lowering the sucrose concentration to 0.5% and by increasing the Kn concentration to 10 mg 1^–1. Relative to the wild species, embryos of C. sativus showed lower percentages of plant formation. The optimum sucrose concentration was 2% for the heart-stage C. sativus embryos. In all three species the ability to form plants strongly decreased with increasing embryo age, from early to late cotyledon. This is thought to be caused by the increasing tendency of the embryos at these stages to continue in vitro the normal embryo development.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Summary Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150g ml^–1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm^–1 (960 F). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.NRCC No. 30491