In vitro development of globular zygotic wheat embryos

Summary We have established in vitro culture conditions for globular zygotic wheat embryos (Triticum aestivum L.). Their nutritional requirements have been systematically investigated. The initial sucrose concentration, as well as the sucrose concentration during the culture, a 6-benzylaminopurine supplement, the use of nitrates and ammonium as nitrogen source have a major influence on the embryo development. Proline has an inhibitory effect on the germination. A double layer system with different media was used to give a continuous variation of the medium composition with time. These culture conditions allowed normal direct embryogenesis in up to 47% of the globular embryos.Abbreviations BAP 6-benzylaminopurine - MES 2-N-morpholinoethane-sulfonic acid - MS Murashige and Skoog (1962)     

In vitro culture of coffee zygotic embryos for germplasm preservation

Immature zygotic embryos of Coffea arabica L. Cv. Cauvery (Catimor) were cultured on Murashige and Skoog (1962) (MS) medium supplemented with abscisic acid (ABA) at 0, 0.4, 3.8, 18.9, 37.8 and 75.6 μM., L-cystein hydrochloride at 50 mg 1^-1 and sucrose at 3%. Cultures were preserved in parafilm sealed Petri dishes in dark at a temperature of 25 ± 1 °C for up to two years. The preserved embryos were taken out from the media at 6 month intervals in order to test their viability by germination on MS + NAA (0.5 μM) + BA (4.4 μM). On the preservation media devoid of ABA or with a low concentration (0.4 μM) of ABA, the embryos germinated and showed higher mortality with increasing duration of storage. In contrast, the embryos became increasingly dormant with increasing concentrations of ABA and a 74.2% survival was found even after 2 years on medium supplemented with 18.9 μM or 37.8 μM of ABA. The results suggest that embryos can be preserved with a little loss of viability in the presence of ABA even at the normal room temperature (25 + 1 °C) up to two years without any transfer. Application of this technique for germplasm preservation of coffee is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PK_c and PK_p, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PK_c and PK_p over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PK_c from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PK_p was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 12, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PK_c. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PK_p and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PK_c was not only far less abundant relative to PK_p, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PK_c and PK_p. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.Abbreviations IgG immunoglobulin G - IU international unit - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PK(s) pyruvate kinase(s) - PK_c cytosolic pyruvate kinase - PK_p plastidic pyruvate kinase - PYR pyruvate Plant Research Centre contribution No. 1374We wish to thank Ms. Kathryn Hovey and Ms. Suzanne Belliveau (Agriculture Canada) for their expert assistance in the culturing and harvesting of microspore-derived embryos of canola. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.     

Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Microspore-derived embryos from Quercus suber anthers mimic zygotic embryos and maintain haploidy in long-term anther culture

Microspore-derived embryos produced from cork oak anther cultures after long-term incubations (up to 10-12 months) were analysed in order to determine the genetic variability and ploidy level stability, as well as morphology, developmental pattern and cellular organisation. Most of the embryos from long-term anther cultures were haploid (90.7%), corresponding to their microspore origin. The presence of a low percentage of diploid embryos (7.4%) was observed. Microsatellite analysis of haploid embryos, indicated different microspores origins of the same anther. In the diploid embryos, homozygosity for different alleles was detected from anther wall tissues, excluding the possibility of clonal origin. The maintenance of a high proportion of haploid embryos, in long-term anther cultures, is similar in percentage to that reported in embryos originating after 20 days of plating (Bueno et al. 1997). This suggests that no significant alterations in the ploidy level occurred during long incubations (up to 12 months). These results suggest that ploidy changes are rare in this in vitro system, and do not significantly increase during long-term cultures. Microscopical studies of the microspore embryos in various stages revealed a healthy and well developed anatomy with no aberrant or chimeric structures. The general morphology of embryos appearing at different times after plating, looked similar to that of earlier embryos, as well as the zygotic embryos, indicating that they represent high quality material for cork oak breeding.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.     

Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised from them

The influence of sucrose or mannitol on in vitro zygotic embryo germination, seedling development and explant propagation of olive tree (Olea europaea L.) was compared. Embryos germinated without sucrose in the medium but for adequate development of the seedlings to yield viable plants, a carbohydrate supply was necessary; both sucrose and mannitol were equally suitable for this purpose. However, when explants obtained from in vitro germinated embryos were cultured with mannitol or sucrose, then the polyalcohol promoted significantly more growth than sucrose by increasing shoot length, pairs of leaves formed, and breaking apical dominance. This improved the in vitro culture of olive plant material, thus allowing new olive clonal lines to be obtained in shorter times. This will assist in future breeding experiments with the species.     

Purification of adenine phosphoribosyltransferase from Brassica juncea

Adenine phosphoribosyltransferase was purified from Brassica juncea leaves approximately 4000-fold, to homogeneity. The native enzyme is a homodimer, with a Mr of 54,000. The purification involved (NH4)2SO4 fractionation, differential ultracentrifugation, and anion-exchange, hydrophobic, dye-ligand, and affinity chromatography. The purified enzyme has a pH optimum of 9.15 and a temperature optimum of 60 degrees C. Activity of the enzyme is stimulated by Mg2+ and is inhibited by sulfhydryl reagents. At the optimum pH and 37 degrees C, the apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate were 3.8 and 15 microM, respectively. Analysis of the purified protein by isoelectric focusing revealed the presence of two isozymes with approximate isoelectric points of 5.3 and 5.4.     

Growth of zygotic flax embryos in vitro and influence of kinetin

Embryos of flax (Linum usitatissimum L. cv. Viera) may be cultured in vitro from the globular stage of their development to germination. Kinetin added to the culture medium at the concentration of 5×10-8 mol/l accelerates embryo development, including the transition from greater expansion in width to greater expansion in length during the heart stage.     

Somatic embryogenesis from peach palm zygotic embryos

The factors affecting the induction and development of somatic embryos and plantlet acclimatization of peach palm (Bactris gasipaes Kunth) were evaluated to establish an efficient regenerative protocol based on somatic embryogenesis. Mature zygotic embryos were cultured in Murashige and Skoog (MS) medium supplemented with 0–40 μM of picloram (4-amino-3,5,6-trichloropicolinic acid) and 0 or 5 μM of 2-isopentyladenine (6-dimethylaminopurine) (2-iP). After 5 mo. in culture embryogenic callus arose from primary calli. Picloram (10 μM) was effective in inducing embryogenic calli in 9.8% of the explants. The use of 1 μM of AgNO₃ enhanced embryogenic competence. Embryogenic calli showed an organized structure, a globular aspect, and were white to yellowish in color. Histological analyses showed that cell proliferation arose from subepidermal cells adjacent to vascular bundles, resulting in primary callus formed by a meristematic zone from which somatic embryos arose. Protein profile analyses revealed two high molecular mass bands in these embryogenic calli, but not in other tissues. Embryogenic calli were transferred to a culture medium containing 40 μM of 2,4-dichlorophenoxyacetic acid, 10 μM of 2-iP, plus 1 g l⁻¹ of glutamine, hydrolyzed 0.5 g l⁻¹ casein, and activated 1.5 g l⁻¹ of charcoal. Morphogenetic responses achieved in this medium were the development of somatic embryos, rooting, and loss of embryogenic capacity. Somatic embryos were converted to plantlets on MS medium plus 24.6 μM of 2-iP and 0.44 μM of naphthalene acetic acid. Plantlets were maintained in MS medium with activated charcoal (1.5 g l⁻¹) until they were 6 cm tall, and then acclimatized. After 16 wk, 84.2 ± 6.4% survival was observed. M. P. Guerra and C. R. Clement are Fellows of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, DF.     

Influence of in vitro culture conditions on glucosinolate composition of microspore-derived embryos of Brassica napus

Microspore-derived embryos (MDEs) of Brassica napus were used to study the influence of sucrose, jasmonic acid (JA), and abscisic acid (ABA) on dry weight and total glucosinolate (GSL) content as well as on specific GSLs. An improved procedure was developed to enable the detection of alkenyl and indole GSLs in single MDEs although they were cultured in medium containing 13% sucrose, where the accumulation of GSL is very low. A sucrose content of 2% and below in the culture medium of the embryos was necessary to significantly increase the total GSL content in embryos of three different rapeseed cultivars. The increase in total GSL content was caused predominantly by higher contents of the indole GSL glucobrassicin (GBC). Contents of 4-hydroxy-3-indolyl glucosinolate (4OH), neoglucobrassicin (NEO), and 4-methoxyglucobrassicin (4ME) were also increased. Alkenyl GSL content remained largely unaffected and increased significantly only in embryos cultured at the lowest tested sucrose concentration of 1%. Growing the embryos in the presence of JA did not change the alkenyl GSL content but led to a 7-fold increase in the indole GSL content. Significant increases were found for GBC, 4OH and NEO, whereas 4ME content was not affected. The JA treatment did not affect the morphology or dry weight of the MDEs. In contrast, a treatment with ABA significantly reduced the dry weight and the indole GSL content of the embryos. In the combined JA and ABA treatment, the stimulative effect of JA on indole GSL biosynthesis could not override the inhibitory effect of ABA on growth and indole GSL biosynthesis of the embryos.     

In vitro morphogenetic responses from cotyledonary explants of immature zygotic embryos of Pterocarpus santalinus

Plant Cell, Tissue and Organ Culture (PCTOC) - Pterocarpus santalinus is an endangered species with high therapeutic and commercial value that needs urgent protection. The study developed an easy...     

Recovery of cassava (Manihot esculenta Crantz) plants from culture of immature zygotic embryos

An embryo culture protocol using immature cassava seeds has been developed to enhance successful seed germination and reduce time for population establishment. Embryonic axes were excised from seeds 40 days after pollination and placed on 1/3 MS medium supplemented with growth factors. Fruits were either air-dried at 20 °C to aid dehiscence, or dissected immediately after harvest. Culture of embryonic axes from seeds obtained from mature fruits (90 days after pollination) served as control. Average percent germination and plantlet recovery rate were higher for embryos cultured from non air-dried immature seeds than from air-dried immature seeds. Immature seeds that were air-dried before germination had ≥50% reduction in germination rate and ≥75% reduction in plantlet recovery rate, indicating that cassava immature zygotic embryos are susceptible to osmotic pressure changes. Genotypic effects were observed in shoot elongation, formation of internodes, and vigor of cultures from both mature and immature seeds. The high percentage of plants recovered from immature seeds through embryo culture opens up opportunities for genetic stock development in cassava that has been previously unexplored. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos

Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ngxmL(-1) oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ngxmL(-1) oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine