Cell division and plant development from protoplasts of carrot cell suspension cultures

Summary Cell regeneration and sustained division have been observed in protoplasts from carrot cell suspension cultures. Carrot plants were produced from the protoplasts by embryogenesis.NRCC No. 12268.     

Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts

Summary Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.Supported by National Research Council of Canada, Grant A6304     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

Somatic embryogenesis in cell cultures of Glycine species

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

Cell proliferation and cell differentiation in tissue cultures of adult muco-cilliary epithelia.

Explants and monolayers from a variety of muco-ciliary epithelia were cultivated in vitro and the kinetics of their proliferation and differentiation described. New epithelial lining and epithelial-like monolayer sheets of cells formed in which the migration cells were all originally undifferentiated cycling stem cells. The divided and differentiated in ML growth into cell types characteristic of the tissue source: however, the control mechanisms which regulate cell division and cell differentiation in the tissues were lost outside the tissue framework. Cell division and cyto-differentiation in ML growths both in ciliated and in mucus-producing cells, were not always mutually exclusive.     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

Selection for high seed oil content in soybean families derived from plants regenerated from protoplasts and tissue cultures

Recurrent selection for high seed oil content was carried out with 2,008 progeny of 28 plants regenerated via embryogenesis, 95 via organogenesis and 25 from protoplasts via organogenesis from five different soybean cultivars. Two lines derived from plants regenerated from the cultivar Jack with small increases in seed oil content emerged after three selection cycles in the field but in both cases the protein content was decreased and the seed yield of one of the lines was also decreased. Apparently somaclonal variation for seed oil content can arise, but on the basis of the decreases in protein and yield found in this study, this small change is not useful for soybean improvement. Received: 22 July 2000 / Accepted: 28 July 2000     

Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue

A reproducible method for regeneration of plants from primary leaf tissue of 27 varieties of soybean (Glycine max), encompassing maturity groups 00 to VIII, has been developed. Progeny from seeds recovered from regenerated plants appear normal. Best regeneration was from leaf explants (2.1–4.0 mm) obtained from 5 day old seedlings. While 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was demonstrated to be essential for regeneration, addition of benzyladenine (BA) was found to enhance regeneration. Of the 6 other auxins tested, only picloram induced any regenerative response. Using identical volumes of medium and other conditions, regeneration could be obtained in 95 × 25 mm glass culture tubes but not in 60 × 15 mm Petri dishes.The regeneration of soybeans from primary leaf tissue was shown to be greatly enhanced by pyroglutamic acid (5-oxoproline). Stimulatory effects were attained if pyroglutamic acid was added directly to the medium or if it was formed in situ as a result of chemical transformation of glutamine during autoclaving. The active component produced by autoclaving glutamine was not a conjugate of glutamine with inorganic salts or another organic component of the medium. Filter-sterilized glutamine was shown to be inhibitory to regeneration.Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) basal media were compared to Gamborg B5 medium. All contained 0.1 mg/l 2,4,5-T, 40 mg/l adenine sulfate and 10 mM pyroglutamic acid. No regeneration occurred when MS medium was used. Growth and appearance of callus growing on SH and B5 media with the additives were similar. The incidence of regeneration among cultures growing on SH medium was only one third compared to cultures grown on B5 medium.     

Shoot regeneration from Glycine canescens tissue cultures

Shoots were reproducibly obtained from calli of both cotyledons and hypocotyls of Glycine canescens, a species related to soybean. A few shoots were also obtained from suspension cultures initiated from the callus tissues. Root formation was observed infrequently so no complete plants were recovered.     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos

Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [³H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [³H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [³H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [³H]thymidine, 63% incorporate [³H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule.     

Brassinolide affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida

Brassinosteroids are known to promote cell elongation in a wide range of plant species but their effect on cell division has not been as extensively studied. We examined the effect of brassinolide on the kinetics and final division frequencies of regenerating leaf mesophyll protoplasts of Petunia hybrida Vilm v. Comanche. Under optimal auxin and cytokinin conditions, 10–100 nM brassinolide accelerated the time of first cell division by 12 h but had little effect on the final division frequencies after 72–120 h of culture. One micromolar brassinolide showed the same acceleration of first cell division but inhibited the final division frequency by approximately 20%. Under sub-optimal auxin conditions, 10–100 nM brassinolide both accelerated the time of first cell division and dramatically increased the 72- to 120-h final division frequencies. Isolated protoplasts may provide a useful model system to investigate the molecular mechanisms of brassinosteroid action on cell proliferation. Received: 1 December 1997 / Revision received: 13 February 1998 / Accepted: 24 April 1998     

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45–50% were obtained in liquid medium and 55–60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1–2 mm in size) were obtained in 5–6 weeks, which upon transfer to MSB medium with 0.5 mg 1^–1 each of 2,4-D, BA, Kn and 500 mg 1^–1 CH produced compact green calli in 4–6 weeks. After 3–4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1^–1 each of BA, Kn, ZT, 0.1 mg 1^–1 NAA and 500 mg 1^–1 CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA₃ enhanced shoot regeneration up to 30%. Shoots of 0.5–1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1^–1 TH and 0.5 mg 1^–1 GA₃ for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2–3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1^–1 IBA or 0.5 mg 1^–1 NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.Abbreviations BA 6-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kn kinetin - MES 2[N-morpholino] ethane sulfonic acid - NAA naphthaleneacetic acid - TH thidiazuron - ZT zeatin     

Callus production from photoautotrophic soybean cell culture protoplasts

Summary Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - HT Heterotrophic - MES 2-(morpholino) ethane sulfonic acid - NAA Naphthaleneacetic acid - PA Photoautotrophic - PCM Protoplast culture medium     

Culture and regeneration of Populus leaf protoplasts isolated from non-seedling tissue

Leaf protoplasts of Populus alba L. × P. grandidentata Michx. (NC-5339) were isolted from shoot cultures of non-seedling origin and cultured through plant regeneration. Complete protoplast development was dependent on providing a stress-free culture environment which included eliminating ammonium, agar, exudate build-up, and light during the culture period. Contact with a solid surface appeared to stimulate development and thus the protoplasts were cultured in a liquid floating-disc system in which they adhered to the fibers of a polyester screen. Protoplasts exhibited a slow, staged development which resulted in cell division 6 weeks following protoplast isolation. The resulting colonies proliferated rapidly and rooted spontaneously. Shoot regeneration occurred when the protoplast-derived calli were exposed to thidiazuron, and such shoots could be readily rooted. This is the first report of reproducible plant regeneration from leaf protoplasts of non-seedling origin of a tree species.     

Cell polarity from cell division

Apical-basal polarity of epithelial cells is critical for their symmetric versus asymmetric division and commonly thought to be established in interphase. In a novel type of cell division termed "mirror-symmetric", apical cell constituents accumulate during M-phase at the cleavage furrow, resulting in epithelial daughter cells with opposite apical-basal polarity.