J. Reinert

Isolation, culture, and induction of embryogenesis in protoplasts from cell-suspensions ofAtropa belladonna

SummaryProtoplasts isolated from actively growing cell-suspensions ofAtropa belladonna have been induced to divide repeatedly, and to undergo embryogenesis. An optimal protoplast yield of up to 80% was obtained in 4–5 hours by treating cell-suspensions with an enzyme mixture of cellulase R 10 (1%) and macerozyme R 10 (0.5%) in 0.6 M sorbitol at 30 °C. The protoplasts cultured at a density of 6 · 104/ml in a modifiedMurashige andSkoogs (1962) liquid medium supplemented with NAA (2 mg/l), kinetin (0.1 mg/l) and 0.5 M sorbitol, and incubated in the dark at 28 °C regenerated cell walls within 48 hours. They underwent first division in 3–4 days and formed cell clumps and colonies in 10 days, which when plated on an agar-solidified medium developed into masses of calli. After transfer to an auxin-free liquid medium these calli underwent embryogenesis within the next two weeks and eventually developed into plantlets.

Induction of haploid tobacco plants from isolated pollen

SummaryThe isolated pollen ofNicotiana tabacum cv. “Badischer Burley” obtained from coldtreated anthers (4 °C for 72 hours) cultured for 4 days, have been induced to undergo androgenesis on a synthetic medium enriched with amino acids. The advantages and potential of the culture of isolated pollen for the induction of haploids and mutation work are discussed.

Environmental control and evidence for predetermination of pollen embryogenesis inNicotiana tabacum pollen

SummaryThe effect of daylenght and temperature for the donor plants (Nicotiana tabacum var. Badischer Burley) on the formation of pollen competent for embryogenesis (P-pollen) by the three possible routes (during normal flower developmentin situ (pollen dimorphism), during cold-treatment of excised flower buds, in cultured anthers) was studied. In all three routes, P-pollen frequency (premitotic pollen, before 1. sporophytic division, PPF) was affected in essentially the same way. At 24 °C and long days, PPF was low and short days had only a slightly increasing effect. At 18 °C and long days, PPF was higher and short days further increased it. Correlated with PPF under the different growth regimes was the percentage of units with more than one vegetative-type nucleus (normal embryos + abortive embryos + multinucleate pollen) in 3 weeks old anther cultures. Under greenhouse conditions, PPF was generally higher than at 24° in growth rooms and showed a maximum in the winter months. Plant age did not affect PPF. These results give further evidence that pollen embryogenesis is predetermined before excision and culture of the pollen or anthers.

Selection of embryogenic pollen from cold-treated buds ofNicotiana tabacum var. badischer burley and their development into embryos in cultures

SummaryBy using density gradient centrifugation, employing 55% percoll and 4% sucrose as suspension medium, it is possible to select embryogenic pollen from buds after cold treatment at 10 °C for 8 or more days. These buds at the uninucleate stage of pollen were collected from plants grown in 8 hours photocycles at 18 °C and supplied with mineral salts. The embryogenic pollen are small, starch-free with a clear cytoplasm whereas large starch-filled ones are nonembryogenic. The embryogenic pollen regularly form embryos at a frequency of 2% on a mineral medium supplemented with glutamine, asparagine and sucrose at pH 6.5.These results demonstrate, for the first time, that it is possible to have embryos in appreciable frequencies in “ab initio” pollen cultures raised from cold treated anthers.

Subcellular aspects of origin and structure of pollen embryos ofNicotiana

SummaryEmbryogenic pollen ofNicotiana tabacum cv. Badischer Burley is smaller than gamete-forming pollen, and is characterised by an attenuated cytoplasm that is poor in ribosomes and with condensed mitochondria. In these respects, an embryogenic pollen grain resembles the angiosperm egg before fertilization. On culture, such pollen readily form embryos. During embryogenesis the first noticeable feature is the formation of a fibrillar wall around the pollen cytoplasm and within the intine. The significance of this wall is at present uncertain. Following wall formation, there is an increase in density of cytoplasm associated with an increase in the ribosome population. Also there is decrease in opacity of the matrix of the mitochondria and a change in the dilation of cristae. These changes are specific features of pollen embryogenesis, and reflect the transformation of pollen from a dormant to an active state. The other non-specific features of embryogenesis, which are similar to changes occurring in other cells undergoing differentiation, are the appearance of starch in amyloplasts and of lipid droplets.

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

SummaryPollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.

Differentiation of embryogenic pollen in cold-treated buds ofNicotiana tabacum var. Badischer burley and nutritional requirements of the isolated pollen to form embryos

SummaryEmbryogenic pollen were selectively isolated from buds after cold treatment at 10 °C for 10 days; it was immaterial whether the buds were taken from short day and low temperature (SD and LT; 8 hours light, 18 °C) or long day and high temperature (LD and HT; 16 hours light, 24 °C) plants. However, in buds from SD and LT plants the differentiation of embryogenic pollen could be detected as early as 7 days after the cold treatment, and pollen from these plants formed embryos at higher frequency (up to 4% of cultured pollen) than those from LD and HT plants (up to 1% only).The embryogenic pollen, in isolated buds, differentiated by way of pollen dimorphism. During cold treatment a fraction of pollen remained small, retained clear cytoplasm and was capable of embryogenesis in comparison to gametophytic pollen which enlarged and acquired granular cytoplasm. In our experiments cold treatment was a key factor in the induction of pollen dimorphism. This aspect of cold treatment in pollen embryogenesis is reported for the first time and was possible on the basis of selection of embryogenic pollen by density gradient centrifugation. The ratio of embryogenic pollen was about one fifth of the total population.The nutritional requirements of isolated pollen for embryogenesis were rather simple. These pollen formed embryos which readily developed into plantlets on a mineral medium supplemented with sucrose provided the pH was 6.8.

Cytological identification of colony formation of intergeneric somatic hybrid cells

SummaryColony formation of intergeneric somatic hybrid cells was obtained by culturing highly viable heterokaryocytes. The high viability of theAtropa belladonna × Petunia hybrida,A. belladonna × Nicotiana tabacum, andDaucus carota × hybrida, D. carota × N. tabacum heterokaryocytes was due to the use of a refined polyethylene glycol (PEG) treatment. The heterokaryocytes underwent continuous division and formed colonies when placed in contact with a nutrient medium which stimulated growth of the parental cells. To confirm the original observations which were made using vacuolar anthocyanin pigments and the presence of chloroplasts as phenotypic markers, the formation of colonies was investigated by using cytological procedures. Nuclear fusion was frequently observed during synchronous mitosis. Completion of the mitotic cycle led to the formation of real hybrid daughter cells, which in the course of further division formed hybrid colonies. The high mitotic activity of the cells during the first 10–14 days of culture enabled the identification of hybrid colonies up to the 20–30 cell stage. Random tests with macroscopically visible calli indicated that up to this stage the hybrid character is retained. All calli obtained from PEG treatedA. belladonna andP. hybrida protoplasts were exposed to environmental conditions which induced differentiation. 240 plants, which subsequently flowered have been regenerated, 235 of these so far were identified asP. hybrida plants by analysis of the karyogram of the root tip cells. In 53 instances theP. hybrida plants were tetraploid. Five tetraploidA. belladonna plants were also obtained. In addition, 33 shoots were regenerated which grow rather slowly and do not show phenotypic markers characteristic of eitherA. belladonna orP. hybrida plants.

Isolation, Culture, and Fusion Studies on Protoplasts from Different Species

SummaryProtoplasts obtained from cell suspensions of an anthocyanin synthesizing strain ofDaucus carota cv. “Rote Riesen”, cultured on a modified Murashige and Skoog medium (MS) have been induced to regenerate cell walls, and divide repeatedly to form masses of callus. Techniques have been substantially refined, and optimal conditions for the isolation of protoplasts have been established. An optimal protoplast yield of 80–90% was obtained by treating the cells with 1.5% cellulase (pH 5.0) for 4.5 hours in a gently shaking water-bath maintained at 33 °C.The protoplasts when plated in the agar-solidified medium regenerated cell walls within 2 days, and first division was observed within 3 days. In addition, they showed irregular elongation, budding and the formation of sub-protoplasts; another phenomenon was the formation of a chain of buds, which either separated from each other or formed a “coenocytic”, tube-like structure. On the agar medium which contained 0.425 M sorbitol, growth slowed down or stopped after 3–4 weeks. In order to obtain further growth, it was necessary to transfer small pieces of agar containing protoplasts on to the top of a fresh medium containing lower amounts (0.2 M) of sorbitol. The protoplasts continued to divide to form cell colonies, and finally masses of callus after 4–5 weeks. In a liquid medium (B5) the protoplasts reacted similarly, they regenerated walls within 2 days and after 2 weeks anthocyanin containing callus was formed. In both media embryo-formation occurred after 6 weeks of culture.By combining the polyethylene glycol and the high pH fusion techniques intergeneric fusion was achieved between carrot protoplasts and mesophyll protoplasts from haploidNicotiana tabacum cv. “Badischer Burley”. The fused products could be readily identified and isolated by using the difference in colour as the visual markers. Conditions for the culture of these fused protoplasts are being worked out.

Factors affecting high-frequency embryo formation inab initio pollen cultures ofNicotiana

SummaryConsistent high-frequency embryo formation, up to 30% of the cultured pollen, was possible when embryogenic pollen were selectively isolated from the gametophytic pollen on centrifugation, at 12–15 °C, in Percoll diluted by a low level of sucrose (4%). Higher level of sucrose in separation medium was inhibitory for embryogenesis. The embryogenic pollen were to be from the buds, petal length 2.4 ± 0.1 cm, of plants in early stage of flowering and induced to flower at 15 °C. The pollen from buds of plants in late stage of flowering gave a variable response. Prior to isolation of pollen, these buds were to be given an additional cold treatment at 10°C for 10 days.There was no response on mere water-sucrose but the nutrient requirements of the pollen for embryogenesis were very simple, filtersterilized mineral-sucrose (2%) medium at pH 6.8. Incubation of the cultures at 24°C than at 28°C resulted in high-frequency embryogenesis. Initiation of embryo formation, at high-frequency, was possible at low level of iron (2×10−5M Fe-EDTA) than customarily employed (10−4M) but for further development of embryos higher levels of iron were required. Pollen embryogenesis was also possible on either nitrate or ammonium as sole source of nitrogen. Increased level of sucrose (4%), growth regulators (cytokinins, gibberellin), monochromatic light (red or blue) and incubation in dark did not improve the response.An appreciable frequency of embryogenesis, up to 8% of the pollen, was also possible by taking pollen from buds of plants flowering at 24°C provided the buds were given a cold treatment at 10 °C for 10 days, prior to isolation of pollen.