In Vitro Divisions of Unfertilized Central Cells and Other Embryo Sac Cells in Nicotiana tabacum var macrophylla

The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var. macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10% mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with 1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water, and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to develop into small cell clusters.     

Transfer of defined numbers of chloroplasts into albino protoplasts by subprotoplast/protoplast microfusion: chloroplasts can be “cloned”, by using suitable plastome combinations or selective pressure

Summary Defined numbers (1–5) of (donor) chloroplasts were transferred into (acceptor) protoplasts of plastid albino mutants by subprotoplast/protoplast microfusion. Single transferred plastids gave rise to new organelle populations in the progeny of the fusion products when suitable combinations of plastomes were used or when selective pressure for the plastome transferred was applied. This process is termed chloroplast cloning and is the first reported case of cloning a cell organelle. The plastome combination and the presence or absence of selective pressure were found to influence the frequencies with which cell lines, containing both plastomes or acceptor or donor only, were obtained, and the number of cell generations needed for complete segregation — as measured by the duration of culture before the green donor plastome could be detected. The high frequency of cell lines and regenerated shoots recovered with donor plastome only, even when only a single chloroplast was transferred, leads to the conclusion that all organelles present in the fusion product contribute to the organelle population of the progeny, i.e. organelle death or loss are not regularly occurring events during plant regeneration from protoplasts in Nicotiana tabacum.Some of the results reported here were presented at the 8th International Protoplast Symposium, Uppsala 1991     

A microculture chamber and improved method for combined light and electron microscopy of filamentous fungi

A microculture chamber has been developed which enables specific fungal hyphae to be followed in their living state before being processed and longitudinally sectioned for electron microscopy. Photomicrographs and their corresponding electron micrographs are presented which illustrate the effectiveness of this approach. Examples of the application of the method are presented.     

Individual selection,culture and manipulation of higher plant cells

Summary Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts — mainly performed with cells of the crop plant Brassica napus L. — are presented.This article is dedicated to the memory of H.-G. Schweiger, initiator and mentor of the experimental single cell approach reviewed herein     

烟草受精后胚囊和合子的分离及合子的离体分裂

以酶解－振荡、酶解－解剖及酶解－研磨３ 种方法分离出烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）受精后生活胚囊。其中以第三种方法效果最好。将分离的胚囊经再次酶解并结合显微解剖，进一步分离出合子、胚乳细胞及其原生质体。以微室饲养法培养离体合子，启动了第一次分裂。     

Isolation of Fertilized Embryo Sacs and Zygotes and Triggering of Zygote Division in Vitro in Nicotiana tabacum

Three methods were established to isolate fertilized embryo sacs in Nicotiana tabacum, i. e. enzymatic maceration combined either with shaking, microdissection or grinding respectively. Living fertilized embryo sacs of various developmental stages after fertilization could be isolated successfully by these methods. Each method had its own adoptation to the materials of different developmental stages. Among them the method of enzymatic maceration combined with grinding was the best:Ovules were first treated in enzymatic mixture (1% cellulase R-10, 0.5% macerozyme R-10, 12% mannitol, pH 5.7) for about 30 min. Then droplets of the ovule suspension were gentlely grinded by a flat-headed glass rod. After grinding several droplets of mannitol solution (8%～ 10%) were added for releasing and washing embryo sacs. Compared with the other two methods this method was more convenent and had higher isolation efficiency. Isolation of fertilized embryo sacs offered a good means for microscopic observation on the postfertilization development including synergid degeneration, endosperm formation and zygotic changes without interference by the surrounding sporophytic tissue. Living zygotes and endosperm cells could be further isolated by a second enzymatic maceration procedure followed by brief micromanipulation. Several characters had been found to distinguish the protoplas'ts of free zygotes from those of other cell sources. Isolated zygotes were cultured in microchambers (Millicell-CM) feeded with macrocultured mesophyll protoplasts. The first division of zygotes was induced, resulting in proembryos consisting of two cells.     

Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture and their potential for transformation by microinjection

Summary In order to identify microspores, suitable for transformation via microinjection of DNA, single microspores of barley (Hordeum vulgare L.) were selected after initial preculture of anthers floating on liquid media and analysed for their development in individual culture in microdroplets of culture medium. Conditions for microculture and plant regeneration from single selected embryogenie microspores were established. The technical feasability of intranuclear microinjection was demonstrated by injecting the fluorescent dye Lucifer Yellow. All essential procedures for a transformation system of barley based on microinjection into microspores have thus been performed successfully. Further efforts to increase efficiencies of culture and microinjection procedures are necessary, however, in order to improve the suitability of this approach towards transformation of barley.Abbreviations MES 2 (N-morpholino) ethanesulfonic acid - PEG polyethylene glycol     

Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

Summary Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.     

Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological,genetic, and molecular characterization

Summary Somatic hybrid/cybrid plants were obtained by microfusion of defined protoplast pairs from malefertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile (cms), streptomycin-sensitive N. tabacum cms (N. bigelovii) after microculture of recovered fusants. Genetic and molecular characterization of the organelle composition of 30 somatic hybrid/cybrid plants was performed. The fate of chloroplasts was assessed by an in vivo assay for streptomycin resistance/ sensitivity using leaf explants (R₀ generation and R₁ seedlings). For the analysis of the mitochondrial (mt) DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA, with three DNA probes of N. sylvestris mitochondrial origin. In addition, detailed histological and scanning electron microscopy studies on flower ontogeny were performed for representative somatic hybrids/cybrids showing interesting flower morphology. The present study demonstrates that electrofusion of individually selected pairs of protoplasts (microfusion) can be used for the controlled somatic hybridization of higher plants.Abbreviations ac alternate current - BAP benzyl aminopurine - cms cytoplasmic male sterile - dc direct current - NAA naphthalenacetic acid - SEM scanning electron microscopy     

The pharmaceutical exploration of cold water ascidians from the Netherlands: a possible source of new cytotoxic natural products

Six ascidian species from the Dutch North Sea coast were screened for cytotoxic activity. Freeze-dried biological material was extracted with solvents of different polarity followed by determination of the cytotoxicity. The most active extracts were further separated using different chromatographic techniques. The microculture tetrazolium (MTT) assay was used to determine the cytotoxicity against two human tumor cell lines: COLO₃₂₀ (a colon adenocarcinoma) and GLC₄ (a small cell lung carcinoma). The two cell lines were selected for their different response towards known cytostatics. GLC₄ is sensitive and COLO₃₂₀ is resistant to most of the known cytostatics. Three of the species tested yielded interesting fractions. From the in colonial Didemnum lahillei we have isolated a compound which is more active against the COLO₃₂₀ cell line (IC₅₀:33 μg ml⁻¹) than the GLC₄ (IC₅₀:49 μg ml⁻¹). The structure is currently elucidated. Another colonial species, Aplidium glabrum, yielded a very cytotoxic fraction, with an IC₅₀ of 5 μg ml⁻¹ against the COLO₃₂₀ cell line. From our results it can be concluded that the North Sea could be another interesting source of new compounds with pharmaceutical potential.