In vitro culture of Ginkgo

Summary Ginkgo biloba L. is an important landscape tree, is resistant to insect, fungi and other pests, and produces a number of chemicals that have pharmaceutical properties (termed ginkgolides). Studies were initiated to establish an in vitro culture protocol for Ginkgo. Explants (intact embryos, embryos with cotyledons removed, and cotyledon tissue) were removed from disinfested seeds and cultured on Murashige and Skoog minimal organics medium with various combinations of either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and either kinetin or benzyladenine (BA). Cultures were incubated in the light and morphological development was recorded. Both embryo and cotyledon explants produced callus (cotyledon tissue produced the most callus). Ginkgolides A and B were detected in callus tissue extracts. Intact embryo cultures initiated on media with 2,4-D plus NAA for 5 wk produced shoots and roots when transferred to media with 4.5 μM 2,4-D alone for an additional 5 wk. Plants were transferred from the 2,4-D media to pots and maintained in the greenhouse.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

In vitro culture of Trichobilharzia ocellata

Cercariae and schistosomula of Trichobilharzia ocellata were cultured to the organogeny stage in vitro in a medium based on Earle's saline (gassed with 5% CO₂ in air) and containing lactalbumen hydrolysate and duck or chicken serum together with homologous red cells. Similar results were obtained irrespective of whether cercariae, schistosomula recovered from the skin of ducklings or chickens, or schistosomula recovered after cercariae had penetrated gelatine membranes were used to initiate cultures. A lipid coat developed around the body of each worm during the first 2–3 days in vitro, but subsequently it became dislodged. The worms ingested red cells, and the caeca became dark with haematin after a few days but by day 10 they were generally translucent. Maximum development was achieved at day 12; by this time males had attained a length of 2·1 mm and females 1·4 mm. Worms cultured for 7 and 9 days were injected into the leg veins of ducks and patent infections were established.     

In vitro culture of bovine preantral follicles

Bovine preantral follicles (40-100 microm diameter at collection) were collected from ovaries of slaughtered cows and cultured in vitro with one of the four treatments: follicle stimulating hormone (FSH; 100 ng/ml) alone; FSH plus epidermal growth factor (EGF; 100 ng/ml); FSH plus insulin-transferrin-selenium (ITS; +1%) or FSH plus hypoxanthine (4 mM) in tissue culture medium (TCM 199) supplemented with 10% fetal calf serum (FCS), 0.1 mg/ml sodium pyruvate, 100 IU/ml of penicillin and 100 microg/ml streptomycin. The control culture medium was TCM 199 with supplements without any treatments. Follicles of each size were cultured separately in groups of one to three in 24-well multidishes each containing 500 microl of the appropriate culture medium. Culture commenced at follicle recovery (day 1) and continued for 10 days (harvested on day 11). In each case, half the medium was removed and replaced by fresh medium every third day. Follicle diameters were recorded on days 1, 5 and 11 of the experiment. At the end of the 10-day culture period, half of the follicles were stained with trypan blue to assess their potential viability and half were stained with bisbenzimide plus propidium iodine to estimate various morphological features of the follicles. Follicles of all initial sizes, on all culture treatments, increased in diameter during in vitro cultures with the greatest increases, both in absolute and proportional size, occurring between days 1 and 5 of culture. All of the culture medium supplements caused greater increases in follicle diameters than control medium at both days 5 and 11 of culture for all initial sizes of follicles (p<0.01). The most effective culture supplements for follicles of 40-, 60- and 80-microm initial diameter were FSH alone and FSH+EGF. The size of these follicles at both days 5 and 11 of culture on both the treatments was significantly larger (p<0.01) than follicles cultured in the presence of the other two supplementary treatments. The growth of follicles of 100-microm initial diameter did not differ between culture medium supplements. None of the culture media caused follicle size to increase to the initial diameters of the next larger size category during the 10 days of culture although follicles of 100-microm diameter achieved a diameter of 120 microm, after 4 days of culture.The overall follicular viability and morphology were better with treatments than the controls in all cases; however, there was no significant difference (p>0.05) among them.From this experiment, FSH and FSH plus EGF may be recommended for in vitro culture of smaller (40, 60 and 80 microm) follicles.     

In vitro culture of growing mouse oocytes

A method is described for in vitro culture of naked growing mouse oocytes for at least four days. Tests for metabolic function indicate that the oocytes increase in volume, accumulate glucose-6-phosphate dehydrogenase, and maintain a steady level of 3H-uridine and 3H-leucine incorporation.     

In vitro culture of silver fox embryos

This experiment was designed to establish in vitro culture methods for silver fox embryos in order to develop the methods for evaluation of the post-thaw viability of frozen embryos in future studies. Artificially inseminated silver fox females were killed humanely on predetermined days after insemination and oviducts and uteri were flushed for embryos. The embryos were cultured in modified TCM 199 or in the same medium supplemented with silver fox oviductal tissue suspension for varying periods, from 6 days to 3 weeks. A total of 60 embryos was recovered. Only embryos beyond the 8-cell stage up to expanded blastocysts developed in vitro (28 % of all embryos). Early stage blastocysts developed most reliably and were of the best quality.     

In vitro culture of lemon juice vesicles

This study explores the possibility of culturing Citrus limon (L.) Burm. f. cv. Eureka (lemon) juice vesicles as isolated intact tissues capable of retaining their unique growth structure in vitro. Isolated juice vesicles either attached to or detached from the endocarp/mesocarp (albedo) tissue of origin were grown on various nutrient media using several physical environments. Various growth responses achieved in vitro from cultured vesicles are described. Intact vesicles attached to endocarp/mesocarp tissue were found to grow up to 6 months in culture as a distinct tissue with a minimum of adverse callus proliferation. Callus formation from some cultured explants occurred on all media and physical environments tested. Callus production eventually obliterated and irreversibly altered the normal development of juice vesicles. Inherent vesicle physiology rather than the tissue culture environment was the major determining factor affecting culture growth. Reducing the concentration of carbohydrates (fructose, glucose or sucrose) added to media from 3 to 0.01% or 0.0% reduced, but still did not totally prevent, callus production. Treatment of vesicles with 1000 mg/l ascorbic acid or citric acid, or 0.1 mg/l indole-3-acetic acid or abscisic acid enhanced the occurrence of normal appearing vesicles.Mention of a trade name or proprietary product or vendor does not constitute approval or guarantee of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

In vitro culture of chicken bursal epithelium

An in vitro organ culture method was used to produce alymphoid bursal epithelium. When fragments of 2-week-old chicken bursas were cultured, the following changes were observed in the follicular structures. The cortical lymphoid cells disappeared early in the culture, while medullary cells seemed to remain intact. The specific follicular epithelium disappeared and the medullas were opened to the plical surface. The openings became gradually wider, and the medullary cells were emptied through them to the plical surface. The basement membrane-associated epithelium at the corticomedullary border proliferated markedly, and when the medullary openings became wider, this layer became gradually a part of the epithelium covering the fragment. After 14 days of culture, when the fragments were alymphoid, they were transplanted onto the CAM of 10-day-incubated, histocompatible chick embryos. Eight days after transplantation the epithelium of the grafts was richly infiltrated by lymphoid cells. These findings show that alymphoid bursal epithelium can be produced by in vitro culture of bursal fragments. The exact characterization of the lymphoid infiltration in the transplanted fragments is yet to be determined.     

Ecological and morphological characteristics of the endoparasitoids of larval Acronicta rumicis (Lepidoptera: Noctuidae)

The Hymenopterans Glyptapanteles liparidis, Microplitis sp. and Diadegma sp. were found to be larval parasitoids and koinobionts of Acronicta rumicis (Lepidoptera: Noctuidae). Mesochorus semirufus is believed to be a new unreported hyperparasitoid of G. liparidis, which, along with M. semirufus, is a gregarious parasitoid. In contrast, the parasitoids Microplitis sp. and Diadegma sp. are solitary. All of the hymenopteran parasitoids are multivoltine insects that emerge from A. rumicis more than once. Compcilura concinnata, Euexorista sp. and Exorista sp. of the Diptera were found to be larval–pupal parasitoids, solitary parasitoids and koinobionts. These three species are univoltine, and emerge only once from A. rumicis. Morphological and life cycle data were collected for G. liparidis, and for the parasitoids of that species found in this study. The major and minor axes of an egg of G. liparidis were 0.10 and 0.02 mm, respectively, while the mean clutch size of G. liparidis was 67.71 ± 39.36 individuals. The body length of female and male G. liparidis were 2.25 ± 0.06 and 2.21 ± 0.12 mm, respectively, and the longevity of an adult was 2.93 ± 0.96 days. Among the parasitoids, the mean body length of an adult Microplitis sp. was 3.5 mm and adults lived for an average of 8.13 ± 3.54 days. The adult Diadegma sp. was larger (mean body length 6.5 mm) but lived for a shorter interval (3.33 ± 1.32 days). The body lengths of female and male M. semirufus were 3.16 ± 0.11 and 3.10 ± 0.23 mm, respectively, greater than the body lengths of female and male G. liparidis. The body lengths of adult C. concinnata, Euexorista sp. and Exorista sp. were 9.5, 9.53 and 8.68 mm, respectively. All of their pupae were dark brown.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

In vitro culture of Plasmodium yoelii blood stages

Lewis-Hughes P. H. and Howell M. J. 1984. In vitro culture of Plasmodium yoelii blood stages. International Journal for Parasitology14: 447–451. Plasmodium yoelii infected reticulocytes were cultured for 72 h at either 37 or 20°C in MEM (Eagle's modification) medium containing, in addition, glucose, para-aminobenzoic acid and 5% foetal calf serum, buffered at pH 7.3 with sodium bicarbonate/ HEPES and maintained under 10% CO₂ in air. Red blood cell numbers were more stable at 20°C than at 37°C. Culture at both temperatures resulted in an increase in parasitaemia of the reticulocyte population over the initial 36 h at 37°C and for at least 72 h at 20°C. The effects of different temperatures appeared to be related to the continued presence of target cells. Parasites were not detected after 72 h culture at 37°C, but persisted for up to 120 h at 20°C. Increasing parasitaemia at both temperatures was associated with changes in the numbers of some parasite development types. Early falls in schizont numbers were associated with an increase in the numbers of ring forms. Trophozoite numbers tended to remain constant throughout the culture period. Viability of parasites cultured for 36 h was confirmed by their infectivity to CBA mice. In addition, parasites progressively incorporated H³-leucine into TCA-precipitable material over the initial 36 h of culture.     

In vitro culture of Geigeria aspera Harv.

Callus-, root-, shoot-, and rooted shoot cultures of Geigeria aspera were established on Murashige and Skoog's nutrient medium. Organogenesis of roots and shoots was induced with treatments containing BA or KN in combination with NAA, IAA, IBA or 2, 4-D. Rooted plantlets were successfully transplanted into soil.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.