Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system

Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro culture of cells and tissues from the mosquito,Anopheles quadrimaculatus say

Summary Tissues and cells from various life stages and ages ofAnopheles quadrimaculatus were cultured in several media. Pupal tissue maintained in Schneider's (1) medium was the most successful system. These cells remained viable for 210 days. Xix morphologically distinguishable cell types were observed: spherical cells, two type of neuronlike cells, musclelike cells, epithelial-like cells, and polymorphic cells.     

Individual human tumors in short-term micro-organ cultures: Chemosensitivity testing by fluorescent cytoprinting

Summary Using innovative approaches, we addressed several problems often associated within vitro chemosensitivity testing of individual human tumors: 1) obtaining a high rate of evaluability; 2) excluding participation of nonmalignant stromal and vascular components usually present in tumor specimens; 3) preserving cell-to-cell interactions present in the original tumor; 4) assessing drug-induced cytotoxicity without sacrificing the tumor culture. To circumvent these problems, tumor specimens were processed as follows: i) tissue (fresh or cryopreserved) was mechanically or enzymatically dissociated under mild conditions into cellular clusters (termed micro-organs); ii) large micro-organs were separated by a brief decantation, resuspended, and then exposed to fluorescein acetate to visualize (under naked eye) viable micro-organs; iii) fluorescent (i.e., viable) micro-organs were collected using a Pasteur pipette, and then planted on a solid support made of cellulose fibers impregnated with collagen. Since tumor micro-organs have been previously shown to consist solely of malignant cells, the procedure described here not only preserves a critical portion of the tumor architecture but eliminates at the onset necrotic tissue and nonmalignant cellular components that could interfere with the chemosensitivity testing. Drug-induced cytotoxicity was measured by “fluorescent cytoprinting”, a novel, nondestructive procedure for assessing micro-organ viabilityin situ. The key feature of fluorescent cytoprinting is that cytotoxic effects arenot measured against control cultures but against a baseline provided by a cytoprint of the same culture before drug addition. Using three experimental designs, we tested the potential of the method for clinical applications. The results using 469 human malignant tumors showed that the micro-organ culture assay can distinguish individual tumor chemosensitivity profiles with an overall success rate of 96%. For three commonly used chemotherapeutic drugs, the observed frequency of responding tumors was found to be comparable to previously reported clinical results using single agents. This work was partially supported by Brown University Research Foundation.     

Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut

Localized etiolation of branches in the crown of a 30-year-old chestnut tree produced plant material that responded much better to establishment and multiplication in vitro than unetiolated material, whose cultures were very difficult to maintain (response being measured in terms of the percentage of cultures established, the mean number of shoots formed per explant, the number of 8-mm segments per new shoot, the length of longest shoot in each culture, and the coefficient of multiplication). Only 22% of the initial explants from unetiolated material were successfully established, as against 79% for etiolated material, with similar differences between the coefficients of multiplication of the two lines in successive cultures. Accordingly, partial etiolation of branches is proposed as a suitable pretreatment for in vitro propagation of selected mature trees, when physiologically juvenile materials such as stump sprouts, epicormic shoots or root suckers are not available.     

Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

香椿离体快繁技术研究

６～７月取香棒当年生半木质化、具腋芽的茎段为外植体,诱导腋芽萌发,进行繁殖培养。通过大量试验,筛选出各阶段适宜的培养基组成。萌芽诱导阶段培养在ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ中,腋芽萌发早,生长快;在继代和增殖培养中以ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ效果较好,ＭＳ＋ＺＴ０．２ｍｇ／Ｌ＋ＧＡ３２．０ｍｇ／Ｌ次之。培养３０天繁殖倍数３．１～４．０,在仅含ＭＳ有机元素。铁盐减半、蔗糖１５ｇ／Ｌ的固体培养基中,附     

Epidermis generated in vitro: practical considerations and applications

The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.     

The rat-tail artery maintained in culture: An experimental model

Summary The rat-tail artery was maintained in vitro for 2 weeks to investigate its suitability as an experimental model. The criteria were that (a) it should retain the overall histological organization with normal ultrastructural appearance of the smooth-muscle cells; (b) stored neurotransmitter which could be activated by experimental treatment should be absent; and (c) smooth-muscle ion transport mechanisms should fall within normal range. Vessels were maintained in Falcon tissue-culture dishes in Dulbecco's modified Eagle's medium. Either 2% or no serum supplement was found to be more suitable than 10% serum due to the high rate of cell proliferation induced by the latter. Light and electron microscopy of cross sections of the vessels indicated that the overall normal vessel architecture was retained, and the ultrastructural features predicted normal function. There were no discernible differences dependent on the length (up to 8- to 10-cm lengths) of the cultured vessel. Preliminary experiments with fluorescent microscopy showed that stored neurotransmitter in the nerves of the vessel wall was no longer present after 48 hr. Ultrastructural examination revealed that storage vesicles in vitro lost their dense cores, representing noradrenalin, between 41 and 48 hr in culture. Normal ion transport mechanisms were retained in the smooth-muscle cells of the arteries in vitro for up to 2 weeks when tested with ion-specific electrodes. Morphological and physiological evidence support the suitability of the rat-tail artery as a model for experimental testing of vascular tissues. This work was carried out with the aid of grants from the British Columbia Heart Foundation.     

光照和温度对红花槭限制生长保存的影响

该文主要探讨了光照时间、光照强度、温度及昼夜温差等保存条件对卓越红花槭(Acer rubrum cv. ‘Somerset’)限制生长保存的影响。结果表明, 在为期182天的保存过程中, 离体材料前期以分化生长为主, 后期以营养生长为主, 并呈现一定的低温适应性。温度对离体材料生长的影响达极显著水平(P<0.01); 光照时间和光照强度影响持久, 二者交互作用达显著水平(P<0.05); 昼夜温差对平均出芽数和生根率都有显著影响。研究表明, 保存效果最好的条件是T3 (25°C, 12小时光照, 62.50 μmol·m ^-2·s ^-1)和T7 (25°C, 12小时光照, 31.25 μmol·m ^-2·s ^-1)处理组, 但最佳保存条件的选择标准并不唯一, 其核心是保证种质材料的分化能力。     

大叶蚁塔组织培养技术

以大叶蚁塔茎尖作为外植体诱导出无菌苗;再以无菌苗的嫩叶诱导不定芽发生;研究了其离体培养和不定芽再生的过程;成功建立了低成本的快速繁殖技术体系。结果表明：(1)无菌苗增殖培养基为MS+BA 1.0 mg·L^-1+NAA 0.1 mg·L^-1;培养30 d;增殖率稳定为3.80;(2)叶片可直接诱导再生出不定芽;其最佳培养基为MS+ZT 1.0 mg·L^-1+NAA 0.1 mg·L^-1;诱导率达95%;分化幼苗众多;(3)生根培养基为1/2MS+NAA 1 mg·L^-1+蔗糖20 g·L^-1时;生根率达95%。     

锡金海棠子叶再生体系的建立

锡金海[Malussikkimensis（Wenz．）Koehne]是我国稀有植物和优良苹果砧木种质资源。本研究探讨了影响锡金海棠离体再生的条件,建立了锡金海棠子叶高效离体再生体系。结果表明,子叶再生体系的最佳诱导培养基SC＋0．1mg·L-1 NAA＋2．0mg·L-1 TDZ,最佳分化培养基SC＋0．1mg·L-1 NAA＋1．5mg·L-1 TDZ,最佳继代增殖培养基SC＋2．0mg·L-1 6-BA＋0．2mg·L-1 NAA,最佳生根培养基为1／2MS＋I．0mg·L-1 IBA。炼苗后,移入珍珠岩、花生壳与草炭（1：1：1）的基质中,20d移栽成活率高达90％。     

‘香槟’月季的组织培养和试管开花诱导

本文对‘香槟’月季（80sachinensis‘Xiangbin’）的组织培养技术和诱导试管开花进行了研究。结果表明：以茎段为外植体能诱导获得无菌苗,适宜的启动培养基为MS＋6-BA1．0mg-L-1＋IBA0．1mg·L-1,幼芽继代增殖的最佳培养基是MS＋6．BA1．0mg·L-1。＋IBA0．1～0．2mg·L-1,诱导生根的适宜培养基为1／2MS＋NAA0．3mg·L-1,生根率达80．0％。诱导试管开花的适宜培养基为MS＋6．BA0．5mg·L-1＋NAA0．1mg·L-1最适宜的诱导试管开花的蔗糖含量是30g·L-1;在三角瓶中培养,试管花可以正常开放,在培养瓶中培养花芽不能正常开放;MS培养基中增加2倍磷的含量,可以提高花芽诱导率,为25．O％;诱导试管开花的最适培养条件为温度21℃,光照强度80～100μmol·m-2．s-1,光照时间16h—d-1。     

枸杞髓组织离体培养及高频率植株再生的研究

枸杞髓组织在４种ＭＳ培养基上都能诱导出愈伤组织,诱导率５３．７％～１００％。在培养基ＭＳ＋６－ＢＡ０．１ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ获得的愈伤组织,呈颗粒状,分散性能好,胚性细胞多．将其转移到ＭＳ＋６－ＢＡ０．５ｍｍ／Ｌ＋ＮＡＡ０．０１ｍｇ／Ｌ的分化培养基上获得大量绿色小芽,小芽在ＭＳ＋６－ＢＡ０．２ｍｇ／Ｌ的培养基上得到快速繁殖,繁殖系数５０～１５０株／芽·月。丛生芽在ＭＳ＋ＮＡＡ０．２ｔｍｇ／Ｌ的培养基上形成完整植株