Rapid separation of the plastid,mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena

Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-m aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 l microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.Abbreviations BSA bovine serum albumin - Cyt c cytochrome c - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenol pyruvic acid - RuBP ribulose-1.5-bis-phosphate     

Auxin-binding by particulate fractions from tobacco leaf protoplasts

In vitro binding of 1-naphthaleneacetic acid (NAA) to particulate fractions from tobacco leaf protoplasts was studied. In freshly isolated protoplasts no specific binding could be detected, whereas it was present in particulate fractions from tobacco leaves. It is concluded that the NAA-binding-sites are probably located at the external face of the plasma membrane; they are destroyed during protoplast isolation by proteolytic enzymes in the cellulase and macerozyme preparations. After culturing the protoplasts for 3–4 d, the first cell divisions were observed and at the same time specific NAA-binding became detectable. The affinity constant for NAA was approx. 2·10⁶ mol^-1 and the number of binding sites increased during further culture.Abbreviations MES 4-morpholinoethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.     

A method for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves

A method is described for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves of two higher plant species. Sodium dodecyl sulfate (1%) and 25 mM magnesium ions are required to inhibit ribonuclease action during RNA purification by phenol deproteinization. The ethanol-precipitated RNA product, including 23s chloroplast ribosomal RNA, is completely stable during electrophoresis in the absence of magnesium ions, even in the presence of EDTA. The $\text{in}$$\text{vivo}$ mole fraction of chloroplast ribosomes relative to cytoplasmic ribosomes is estimated. Bentonite is shown to cause preferential losses of chloroplast RNA during extraction.     

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-¹⁴C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Isolation of intact plastids from protoplasts from castor bean endosperm

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose “Onozuka” R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

Plant regeneration from leaf protoplasts of Solanum torvum

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10⁴ per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1–2 weeks and 4 weeks later p-calli were 1–3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CPA p-chlorophenoxyacetic acid - IAA indoleacetic acid - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthaleneacetic acid - 2ip 6-dimethylallyamino purine Michigan Agricultural Experiment Station Journal Article No. 12167     

Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts

Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h.     

High-yield isolation of chloroplast DNA from Hordeum vulcare barley protoplasts

A fraction of intact chloroplasts free of other cell components in isolated from barley leaf chloroplasts. Instead of mechanical desintegration of plant tissue, the method described includes the cellulysine treatment, thus increasing the yeild of chloroplast DNA. The mean content of DNA per barley chloroplast is found to be 1.10(-4) g. Base composition of barley chloroplast DNA is 39.8 mol.% of G+C. The treatment of chloroplast DNA with restriction endonuclease EcoRI results in the appearance of 17-19 bands under agarose gel electrophoresis.     

Somatic embryogenesis from Lycopersicon peruvianum leaf mesophyll protoplasts

Summary One to five percent of Lycopersicon peruvianum (L.) Mill. leaf mesophyll protoplasts undergo cell division and concomitant organization to form embryogenic-like structures when cultured in Murashige and Skoog medium (1962) containing 3% sucrose, 9% mannitol, 1.0 mg/l kinetin (K) and 1.0 mg/l naphthalene acetic acid (NAA) at pH 5.6–5.8 (medium A). These embryogenic structures, after passing through developmental stages similar to those observed in zygotic embryogeny, are capable of forming shoots on hormone-free medium A. In medium B, wherein 0.5 mg/l of 2,4-dichlorophenoxyacetic (2,4-D) replaced the hormones (K and NAA), embryogenic structures did not develop. However, callus originating in medium B retained morphogenetic capacity as was evidenced by subsequent shoot regeneration when they were transferred to medium A with K and NAA replaced by 1.0 mg/l zeatin (Z). The potential value of incorporating this regeneration trait into Lycopersicon species and cultivated lines for use in tissue culture programs is discussed.Michigan Agricultural Experiment Station Journal No. 9676     

Isolation of intact mesophyll cells and their protoplasts from higher plants

Eighteen herbaceous species of angiosperms were tested for theisolation of mesophyll cells and their protoplasts by applyingenzymatic procedures originally developed for tobacco. Intactmesophyll cells and their protoplasts were obtained from a varietyof plants, indicating the general applicability of these procedures. (Received May 17, 1969; )     

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.     

Labeling and isolation of plasma membranes from corn leaf protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [¹²⁵I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg²⁺-dependent, K⁺-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.     

Regeneration from leaf protoplasts of Arabidopsis thaliana ecotype estland

Protoplasts (2 x 10(7)/g fresh wt) were isolated from leaves of A. thaliana ecotype estland, with a viability of more than 90%. Protoplasts cultured in calcium alginate beads or layers showed division while culture in liquid or agarose beads failed to elicit any division. Effect of culture density showed highest frequency of division occurring at 5 x 10(5) while no division was seen when cultured at a density of 5 x 10(4). Culture in MS medium resulted in higher division frequency and better sustenance of microcolonies as compared to B5 medium. Under optimized conditions, macrocolonies were formed at a frequency of 1.8%. Shoot regeneration was seen in 50% of microcalli transferred to shoot induction medium for regeneration. Shoots were rooted and plantlets transferred to pots. The plants produced flowers and were fertile.