Plant regeneration from patchouli protoplasts encapsulated in alginate beads

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×10³ protoplasts per 25 l bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10^-6 M NAA and 10^-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - f. wt. fresh weight - GC gas chromatography - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self-renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse-derived NSCs were cultured in three-dimensional calcium alginate beads (Ca-Alg-Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca-Alg-Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, beta-tubulin, and GFAP. The results show that the 2-mm diameter Ca-Alg-Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 x 10(5) cells x mL-1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca-Alg-Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca-Alg-Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca-Alg-Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.     

Nurse culture of low numbers of Medicago and Nicotiana protoplasts using calcium alginate beads

Protoplasts of two species, lucerne and tobacco, were cultured in semi-solid droplets of calcium alginate as a means of nurse culturing very low numbers of protoplasts. It was shown that increasing autoclave times decreased the gelling capacity of the alginic acid. A convenient measure of viscosity is described to allow appropriate adjustment of the alginate solution. Tobacco protoplasts are shown to be more sensitive to higher alginate concentrations than lucerne, however beads with a final alginate concentration of approximately 1.5% were suitable for both species. Agitation of the beads in liquid medium was needed for optimum division frequencies. The volume of liquid medium affected the culture response. Interestingly, the local cell density (bead cell density) was shown to be more influential than the total cell density. Nurse beads with higher densities of protoplasts of the same species were visually marked with activated charcoal. Experiments were performed to determine whether nursing was effective with calcium alginate encapsulation and to what extent the cell densities could be lowered. When there were no nurse beads, divisions effectively ceased at 10⁴ per ml with lucerne and 10³ per ml with tobacco. In the presence of nurse beads, protoplasts in the test beads grew at high frequency down to the lowest densities tested, namely 50 per ml for tobacco. With these methods transformed lucerne protoplasts from electroporation experiments and somatic hybrids have been recovered and plants regenerated with much greater efficiency that was hitherto possible.     

Proteases of Melilotus alba mesophyll protoplasts

Proteases from mesophyll protoplasts of Melilotus alba were identified by standard proteolytic assays and separated using different chromatographic techniques. Their characterization also included their subcellular location. Besides the evidence for the multiplicity of the proteolytic enzymes, two protease sets were distinguished endopeptidases, which are exclusively vacuolar, and aminopeptidases, which are widely distributed throughout the cell. Cytosol-located enzymes were tested as substrates of the two sets of proteases, by studying comparatively the time-course changes of enzyme activities during incubation in total protoplast extracts, or in cytosol fractions devoid of vacuolar proteases. The degradation of phosphoenolpyruvate-carboxylase protein, a typical cytosolic enzyme, in the presence of purified amino-and endopeptidases, was also estimated by immunoprecipitation studies. Only the vacuolar endopeptidases are effective in the degradation of cytosolic enzymes. Hydrolytic enzyme activities mostly of vacuolar origin were very stable during incubation in total protoplast extracts. These proteins therefore appear to be particularly resistant to proteolytic attack. The results indicate that, in plants, the effective proteolytic system acting on cytosolic enzymes seems to be vacuole-located, and that the selectivity in protein degradation may be imposed by the susceptibility of the protein being degraded and by its transfer into the vacuoles.Abbreviations Leu-pNA leucine-p-nitroanilide - lys-p-NA lysine-p-nitroanilide - pCMB p-chloromercuribenzoic acid - PEPCase phosphoenolpyruvate carboxylase - PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Electro-fusion of mesophyll protoplasts ofAvena sativa

In order to assay the viability of electrically fused mesophyll protoplasts ofAvena sativa a technique was developed to determine adenylate levels in single protoplasts and fusion products. The results demonstrate that the intracellular ATP/ADP ratios are identical before and after fusion (values between 1.4 and 1.8) and that the time of the rounding up process is directly related to the ATP level of the hybrid. This was shown by the manipulation of the intracellular ATP/ADP ratio in the light using different effectors. Hybrids with an ATP/ADP ratio of 2.3 needed 54 s to round up completely; in the presence of antimycin (inhibition of both oxidative and light-dependent cyclic electron flow: ATP/ADP=1.1) or dibromothymoquinone (plastoquinone antagonist: ATP/ADP=1.0) the time for rounding up was slightly increased (64 s and 76 s respectively), whereas after preincubation with antimycin, dichlorophenyldimethylurea (inhibition of oxidative and light-dependent electron flow) or uncouplers (ATP/ADP=0.19–0.32) this process needed 128–153s for completion. These results are discussed in relation to the viability of electrically induced fusion products and to energy-dependent events involved in the process of fusion.     

A transient expression system in soybean mesophyll protoplasts reveals the formation of cytoplasmic GmCRY1 photobody-like structures

Soybean(Glycine max(L.) Merr.), grown for its plant oils and proteins, is one of the most important crops throughout the world.Generating stable and heritable transgenic soybeans is relatively inefficient; therefore, there is an urgent need for a simple and high-efficient transient transformation method by which to enable the investigation of gene functions in soybeans, which will facilitate the elucidation and improvement of the molecular mechanisms regulating the associated agronomic traits. We established a system of transient expression in soybean mesophyll protoplasts and obtained a high level of protoplast transfection efficiency(up to 83.5%). The subcellular activity of the protoplasts was well preserved, as demonstrated by the dynamic formation of GmCRY nucleus photobodies(NPs) and/or cytoplasmic photobody-like structures(CPs) in response to blue light.In addition, we showed that GmCRY1b CPs colocalized with GmCOP1b, a co-ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1(COP1), which provided new insight into the potential roles of GmCRY1s in the cytoplasm.     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Plantlets from mesophyll protoplasts of Solanum xanthocarpum

Young leaves of Solanum xanthocarpum from axenic shoot cultures released viable protoplasts when treated with appropriate enzymes. The protoplasts on culture in modified Murashige and Skoog (1962) medium supplemented with 2,4-dichlorophenoxy-acetic acid (0.5 mg/l), naphtha leneacetic acid (1 mg/l), kinetin (1 mg/l) and organic nutrients of KM (Kao and Michayluk 1975) regenerated to form callus tissue as a result of repeated divisions. Protoplast-derived calli differentiated into shoots on MS medium enriched with kinetin (0.5 mg/l) and rooting could be initiated by transferring the shoot-buds to basal medium.     

DNA fiber autoradiography of tobacco mesophyll protoplasts

DNA fiber autoradiography was successfully applied to protoplasts isolated from tobacco leaf mesophyll. After an incubation period of 37 h, DNA began to be labeled with ³H-thymidine. These cells, with regenerated thin cell walls, were then effectively disrupted in a lytic solution containing sodium dodecyl sulfate. DNA fiber autoradiography confirmed that the mean replicon size of this plant is approximately 60 in good agreement with the values reported previously in animal cells.     

Culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads

Yeast immobilization offers operational advantages such as high cell concentration, and some drawbacks related to cell leaking and restricted mass transfer inside particles. The influence of bead size, chitosan, bead charge, volume of liquid media, and the use of corncob hydrolyzates and vinasses as culture medium were analyzed on xylitol production by Debaryomyces hansenii immobilized in alginate beads. The results showed a profuse growth of free cells, accounting 75–95% of total biomass, but electron micrographs revealed the generation of a dense biofilm with hyphal morphology at the bead surface and a very low intraparticular growth. Xylitol production was not affected by the size of particle; however chitosan had a negative effect. The use of corn cob as carbon source and twofold diluted vinasses as economic nutrients incremented xylitol concentration to 13.7 g L^?1 (Y_P/S = 0.56 g g^?1; Q_P = 0.29 g L^?1 h^?1). The best conditions corresponded to high bead charges and intermediate liquid volumes (44 g Na-alginate and 110 mL liquid medium). These results showed the feasibility of employing these cheap substrates, reflected the importance of the microaerobical conditions, and pointed to the favorable effect of cell immobilization on the metabolism of xylitol production.     

Vacuole development in cultured evacuolated oat mesophyll protoplasts

Oat leaf mesophyll protoplasts were evacuolated and shown to develop acidic vacuoles when cultured for 3 d. Vacuole development was followed by cell wall formation. Developing vacuoles, stained with acridine orange, took the form of a tubular network when viewed by confocal laser scanning microscopy. The tubules expanded and fused to form a series of interconnected vacuoles. When thin sectioned material was examined by transmission electron microscopy, the tubular network appeared as a number of small, expanding vesicles. The vacuolar H⁺-ATPase, H⁺-PPase and a membrane integral protein of 23 kDa (VM23) were shown, by Western blotting, to be removed from protoplasts following evacuolation. After 5 d culture the H⁺-ATPase and H⁺-PPase, but not VM23, were detectable in microsomal fractions.This study describes, for the first time, successful vacuole regeneration in a monocotyledenous plant. This regeneration follows a similar pattern to that seen in non-cereal protoplasts.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Plasmalemma fine structure in isolated tobacco mesophyll protoplasts

Summary Tobacco mesophyll protoplasts have been examined by electron microscopy during isolation procedures and after 24 hours culture in a medium known to support cell wall regeneration. During isolation the plasmalemma shows little structural differentiation apart from the formation of small vacuoles in the cytoplasm. After 24 hours of culture, several types of activity are seen at the plasmalemma surface. Microtubules, profiles of endoplasmic reticulum, electron dense granules and coated vesicles are associated with the inner surface of the membrane. External to the plasmalemma fibrillar structures occur, both as extensive networks and as individual fibrils apparently associated with the membrane itself. Techniques and criteria for electron microscopy are presented, and the results discussed in terms of plasmalemma function and the regeneration of the cell wall.     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Rapid plant regeneration from Nicotiana mesophyll protoplasts

A procedure is described for rapid plant regeneration from tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Six to seven days after protoplast isolation, colonies are placed on double filter feeder plates that consist of a strong regeneration medium containing 7.5 mg/l 6-(γ,γ-dimethylallylamino)-purine (2iP) and 0.1 mg/l p-chlorophenoxyacetic acid (pCPA). Complete plants are regenerated in about 5 weeks after transfer to a rooting medium (hormone-free Murashige and Skoog (MS) medium). However, upon remaining on shoot regeneration medium, 50–75 shoots are regenerated from single colonies derived from individual protoplasts. This procedure may reduce the amount of somaclonal variation (as measured by ploidy level) which is usually expressed in plants obtained by conventional regeneration techniques.     

Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts

Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated. The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds. Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks. These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h). Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes). Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H₄ gene. They seem to be induced in protoplasts by a synergistic effect of wounding and maceration. Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium.     

Plant regeneration from mesophyll protoplasts in Isatis indigotica

Protoplasts of an accession of Isatis indigotica Fort. were isolated from mesophyll tissue by enzymatic digestion and cultured using a feeder cell system. Shoot regeneration efficiency was 100% via organogenesis among 627 isolated calluses within 30–37 days. Among these shoot initiating calluses, 162 (22.6%) developed normal shoots with multiple (2–5) shoots per callus. The remaining calluses developed only vitreous shoots. High concentration (5 μM) of indole-3-butyric acid had a positive effect on rooting compared to low concentration (0.5 μM) of indole-3-butyric acid and α-naphthalene-acetic acid. The average rooting efficiency of regenerated shoots of two experiments was higher on LS medium with 5 μM indole-3-butyric acid than on LS medium without growth regulators. Twenty-nine plantlets, with 2–3 expanded leaves and roots were potted in soil and 22 developed normally to maturity in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Subcellular localization of acid proteinase in barley mesophyll protoplasts

Chloroplasts prepared from lysed protoplasts of barley mesophyll contain 2–8% of the total acid proteinase activity. This residual activity is not associated with intact chloroplasts isolated by means of density gradient centrifugation. Vacuoles isolated from lysed protoplasts contain 80–85% of the total acid proteinase activity, indicating that the enzyme(s) which is presumably responsible for the degradation of chloroplastic proteins is located largely in the central vacuoles of mesophyll cells.