Slow growth in vitro conservation of coffee (Coffea spp.)

The effects of reduced sucrose concentrations and low temperature on a collection of coffee microcuttings have been examined. Sucrose concentrations of 0.5 g l^-1 and 20 g l^-1 and temperatures of 20°C and 27°C were compared in three accessions: the Arabusta (interspecific hybrid) and Coffea arabica L. cv. Caturra amarillo and cv. Mokka de Tahiti. After six months, low sucrose concentrations reduced microcutting growth, rooting and survival rate. At 20°C, microcutting growth was also reduced, but leaf loss and survival rate were promoted. The genotypic differences at six months were minor. After one year without subculture, survival rate was influenced by sucrose concentration and by genotype. These two species can be cold-stored six months at 20°C on a medium containing at least 20 g l^-1 sucrose.Abbreviations BA 6-benzylaminopurine - MS Murashige & Skoog     

Strain Differences in Superovulatory Response, Embryo Development and Efficiency of Transgenic Rat Production

The differences between rat strains in superovulation response, in vitro and in vivo development of preimplantation embryos and overall transgenic efficiency was studied. The protocols for induction of superovulation using single injections of pregnant mare’s serum gonadotropin (PMSG) or minipumps with follicle stimulating hormone (FSH) were compared in Lewis (LEW), Wistar-Kyoto (WKY), and stroke-prone spontaneously hypertensive rats (SHRSP) or Sprague–Dawley (SD) and Wistar rats as representative inbred or outbred strains, respectively. The percentage of mated animals with positive superovulatory response was similar in all strains (60.0–100%). The mean number of ova per donor was not dependent on the kind of hormonal treatment used within each rat strain. In general, females from outbred SD and Wistar rats were more responsive to hormonal treatments than animals from inbred rat strains. In addition, SD female rats produced a significantly higher number of embryos per female in response to PMSG-treatment compared to all other strains. Between the inbred strains, SHRSP was the most effective for superovulation. In vitro development of intact zygotes to the blastocyst stage was not different between SD, Wistar and SHRSP rats. In contrast, in vitro development of WKY zygotes was significantly less efficient than in other strains. However, 2-cell stage embryos in vivo produced from SD, SD × Wistar and WKY animals showed no difference in competence to develop to blastocyst stage in vitro. The proportion of offspring developing after oviduct transfer of intact zygotes was similar in all strains (44.0–56.4%) with the exception of WKY rats (35.9%). We also compared the survival rate after injection, ability of manipulated zygotes to develop to term and overall transgenic efficiency in various rat strains. SD and SHRSP zygotes survived after microinjection better than the WKY and Lewis zygotes. No differences were found in the efficiency of transgene integration per newborn in different strains ranging from 5.7 to 16.7%. The results of this study demonstrate that different rat strains have varying responses to superovulation, sensitivity to microinjection, capability to develop in vitro until blastocyst stage or in vivo to term after transfer to foster mothers. Despite these differences all studied strains can be used for efficient transgenic rat production.     

Differential in vitro development of inflorescences in long and short day Lemna spp.: involvement of ethylene and polyamines

In vitro-development of Lemna inflorescences on minimal medium is known to differ in long day (LDP) and short day (SDP) plants (Z. Pfl, physiol. 77, 395). In LDP pistil growth predominates, while in SDP stamen growth predominates. This indicates that LDP and SDP inflorescences differ in endogenous hormones and depend for a balanced male-female development on different plant-supplied factors (Z. Pfl. physiol. 80, 283 and 298). Here inflorescences of the LDP L. gibba and the SDP L. aequinoctialis were tested for differences in ethylene-polyamine (PA) relations, as ethylene and PAs are inversely related (shared precursor, mutual inhibition of synthesis), and exogenous ethylene has been shown previously to restore male-female balance in SDP inflorescences (Z. Pfl. physiol. 80, 283). Promotion of pistil or stamen growth indicates a predominance of ethylene and PAs in LDP and SDP, respectively. Hence, in LDP, exogenous PAs and inhibitors of ethylene synthesis, and in SDP, an inhibitor of PA-synthesis, were applied to restore the male-female balance in vitro. In L. aequinoctialis (SDP), application of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of spermidine (SD) synthesis, resulted in near normal development via stamen inhibition and/or pistil promotion. In L. gibba (LDP), ethylene inhibition was effective, especially by aminoethoxyvinylglycine (AVG), which reduced pistil growth. Effects of alpha-aminooxyacetic acid (AOA) were less clear. Putrescine (PUT) promoted stamen growth under certain circumstances, perhaps acting as a precursor for the more active SD. SD effects were concentration-dependent for pistil and stamen. Most importantly, increases in SD turned pistil promotion into inhibition and almost normalised floral development. Spermine (SM) enhanced stamen growth. Results are conclusive that PA-ethylene relationships are involved in inflorescence development in a contrasting manner in LDP and SDP. It is apparent that in whole plants the LDP supplies the inflorescences with factors inhibiting ethylene and/or stimulating PA-synthesis. In SDP the converse is true.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

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.     

In vitro pollination of angiosperm ovules with gymnosperm pollen grains

Summary Pollen grains of the gymnosperm species Ephedra distachya and Pinus wallichiana germinated abundantly on the in vitro cultured placentae of the angiosperm species Nicotiana tabacum, Melandrium album and Allium moly. Some P. wallichiana pollen tubes entered the micropyles of M. album. Embryological observations of cross-pollinated M. album ovules 2 or 3 d after cross-pollination revealed the presence of pollen tube remnants within the embryo sacs. Karyological disturbances in the two- and three-celled proembryos and endosperm nuclei indicated their probable hybrid origin. In some crosses, gynogenetic haploid proembryos were also noticed.     

Changes in the distribution of mitochondria in mouse embryos blocked at the two-cell stage

Changes in the distribution of mitochondria in the two-cell mouse embryos preceding the developmental arrest in vitro, caused by a genetically determined two-cell block in vitro or genisteine treatment, were examined vitally using the mitochondrial-specific probe rhodamine 123 and conventional fluorescence microscopy. In the former case, serious disturbances in the localization of mitochondria appeared already from the middle of two-cell stage, long before the time corresponding to the 2nd cleavage division. Comparison of the behavior of mitochondria in the embryos successfully developing between the one- and two-cell stages and that in the embryos that ceased to cleave suggests that the developmental arrest was accompanied by aggregation of the mitochondria into clusters. There are many such clusters unlike in the cytoplasm of normally developing embryos. Intracellular localization of clusters observed in the genisteine-treated embryos differed radically from that observed in the embryos blocked in vitro at the two-cell stage.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 51–60.Original Russian Text Copyright © 2005 by Bogolyubova.     

Callose pattern and polarization phenomena in the ovules in the F2-hybrids betweenOenothera hookeri andOe. suaveolens

The pattern of callose formation in meiotic cell walls and the order of megaspore degeneration and polarity during embryo sac development are investigated in F₂-plants ofOe. hookeri ×suaveolens and the reciprocal cross. All investigated characters are variable between the ovules in the same ovary. Plants differ in the frequency of the types of callose pattern and polarity of the embryo sacs. In segregating progenies different combinations of both characters are found. The genetic basis of the polarity phenomena during the embryo sac development is discussed. In our material no correlation can be seen between the callose pattern in the surrounding wall of the meiotic cell and the development of polarity in the later stages.     

A study on floral biology of seedlings in vitro in Orychophragmus violaceus: Induction of flowers in seedlings of O. violaceus cultured in vitro

Seedlings of O. violaceus were cultured on MS media and treated at low temperature. Cold treatment at 5–7 °C for more than 7 days was needed for flower induction of seedlings in vitro originated from germinated seeds. When cultured on MS media supplemented with 2.5 mg l⁻¹ zeatin and 2 mg l⁻¹ gibberellin (GA₃), seedlings in vitro did initiate flowers without cold treatment. When MS media was used with a reduced amount of NH₄NO₃, flower induction of seedlings in vitro could be accelerated. This revised version was published online in June 2006 with corrections to the Cover Date.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

猪源乳酸杆菌与益生元组合筛选及其体外发酵特性

[目的]对3株乳酸杆菌和4种寡糖类益生元进行组合筛选,并探究其对猪结肠微生物体外发酵特性的影响.[方法]将3株乳酸杆菌(罗伊氏乳杆菌L45、植物乳杆菌L47和罗伊氏乳杆菌L63)分别添加至以菊粉(inulin)、低聚果糖(FOS)、低聚半乳糖(GOS)或乳果糖(lactulose)为唯一碳源的培养基中,结合菌株24 h...     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

Content of phenolics in mycorrhizal roots of Pinus sylvestris seedlings grown in vitro

The effect of Paxillus involutus, Laccaria laccata, Suillus luteus, S. bovinus, Hebeloma crustuliniforme and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) on the content of phenolic compounds: ortho-diphenols and total soluble phenols in roots of Pinus sylvestris seedlings grown in vitro was investigated. Mycorrhizal fungi varied significantly in the ability to influence the concentration of phenolics in Scots pine roots. Plants inoculated with S. luteus accumulated the lowest amount of the phenolics, whereas the amount of phenolics in pine roots inoculated with L. laccata was the highest.     

Micropropagation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees

In vitro propagation protocols were established for two leguminous trees, Bauhinia variegata and Parkinsonia aculeata. In each case axillary shoot proliferation was achieved from nodal explants from mature (6-2-8 years) trees using Murashige & Skoog's medium supplemented with 2.22–31.1 M of 6-benzyladenine. Subsequent rooting of the regenerated shoots was achieved on medium containing 2.46–14.8 M of indole-3-butyric acid. Successful transfer of the regenerants to soil has been accomplished.     

Differentiation of cotton fibers from single cells in suspension culture

Summary A cotton cell suspension culture has been developed that provides unique opportunities for plant biologists to investigate early developmental events regulating cotton fiber properties, plant cell elongation, and cell wall biogenesis. The suspension culture was derived from cells of cotton (Gossypium hirsutum L.) ovule callus. These cells undergo the stages of fiber development previously described for in vivo fiber development. Fibers range in length up to 11 mm and have secondary walls. Supported by the U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Laboratory, New Orleans, Louisiana, and Cotton Incorporated, Raleigh, North Carolina.     

Effect of abscisic acid and stratification on somatic embryo maturation and germination of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.)

Summary The effect of abscisic acid (ABA) was evaluated during the maturation and germination of holm oak (Quercus ilex L.) somatic embryos. The addition of ABA to the culture medium significantly reduced unwanted recurrent embryogenesis in mature somatic embryos without affecting the germination of embryos subjected to stratification at 4°C. Stratification at 4°C for 2 mo. was the most efficient for stimulating somatic embryo germination of holm oak. The addition of 90 and 450 mM sucrose also improved germination, while higher sucrose concentrations were inhibitory.     

Influence of Liquid Pulse Treatment with Growth Regulators on in vitro Propagation of Banana (Musa spp. AAA)

The effect of liquid pulse treatment of growth regulators on in vitro propagation of banana (Musa spp. AAA) was studied. Optimal shoot proliferation rates were achieved due to the pulse treatment of 6-benzylaminopurine (BA) and kinetin combination (1:1) at the concentration of 50 mg l^–1 for 60 min. Similarly high frequency of root induction was obtained due to pulse treatment with a NAA and IBA combination (1:1) at the concentration of 100 mg l^–1 for 60 min.     

Induction of embryogenic callus and cell suspension culture from shoot tips excised from flower stalk buds of Phalaenopsis (Orchidaceae)

Summary Embryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 μM α-naphthaleneacetic acid (NAA), 4.4 μM 6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented with 5.4μM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.     

Regeneration of fertile wheat (Triticum aestivum) plants from isolated zygotes using wheat microspore culture as nurse cells

A new method for growing isolated wheat zygotes by co-cultivation with microspores isolated from the same species has been developed. Although the mortality of isolated zygotes within 6 h after the transfer into the nurse culture was relatively high (70%) in this method, the majority of the structures which survived developed into fertile plants (61%). Zygotic structures grown in these cultures were morphologically not as regular as embryos grown in planta, but they resembled them closely in cytological properties and developmental pattern.     

Production of adventitious shoots and plantlets from the hypocotyl explants of Sesbania rostrata (Bremek & obrem)

Summary Adventitious shoots were induced from the hypocotyl explants derived from 12–15-d-old seedlings of Sesbania rostrata on Nitsch's medium (Nitsch, 1969) supplemented with 1 mgl⁻¹ (4.4 μM), of N⁶-benzylademine (BA). A maximum of 5.9±3.4 shoots per explant in 100% of cultures were obtained. The BA treatment for different time durations (1, 3, 5, 7, 10, 17, 21, or 30 d) exhibited significant variation in the caulogenic potential of the explants. BA treatment for 10–17 d proved optimum for the response. Although at all concentrations of kinetin the explants developed multiple shoots, they were malformed. Sucrose at 3% exhibited the development of the maximum of 3.5±0.9 shoots per explant with an average shoot length of 4.7±3.9 cm. Among the different carbon sources, i.e., fructose, galactose, maltose, mannose, and sucrose at 3% (w/v), sucrose supported the best caulogenic response. The role of various other factors (viz. size, orientation of explant, and seedling age) on the caulogenic response of the hypocotyl explants of S. rostrata were also studied. The shoot development in all cases was accompanied by the development of moderate to profuse callus at the basal cut end of the explant. The in vitro-regenerated shoots produced roots when transferred to half-strength MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and 1 mgl⁻¹ (4.9 μM) indole-3-butyric acid (IBA). The developed plantlets were transferred to the field after an initial acclimatization period of 3–4 mo. Such transferred plants produced flowers and fruits in the field and exhibited the development of prominent and organized stem nodules.