Received 8 February 2002; accepted in revised form 14 March 2003. Key words: Cactaceae, pigments, plastids, tissue culture, transformation, ultrastructure.
Plant Cell, Tissue and Organ Culture 75: 117–123, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Morphology and ultrastructure of Mammillaria gracillis (Cactaceae) in in vitro culture ˇ ´ 3 & M. Krsnik-Rasol 1 D. Poljuha 1, *, B. Balen 1 , A. Bauer 2 , N. Ljubesic 1
Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 ˇ ˇ 6, 10000 Zagreb, Croatia; 3 Ruder Zagreb, Croatia; 2 Clinical Hospital Dubrava, Avenija Gojka Suska ˇ ´ Institute, POB 1016, 10001 Zagreb, Croatia ( * requests for offprints; Fax: ⫹385 -1 -482 -6260; EBoskovic mail: danijela@ zg.biol.pmf.hr) Received 8 February 2002; accepted in revised form 14 March 2003
Key words: Cactaceae, pigments, plastids, tissue culture, transformation, ultrastructure
Abstract Morphogenetic status of cactus Mammillaria gracillis Pfeiff. tissue culture was studied by light and electron microscopy. In vitro propagated shoots spontaneously developed callus. This callus regenerated normal and hyperhydric shoots without exogenous hormones. Tumour tissue induced by wild or rooty strains of Agrobacterium tumefaciens never expressed any morphogenetic potential. Light microscopy showed cellular characteristics of morphologically different tissues. Ultrastructural studies revealed changes in plastids: secondary dedifferentiation of mature chloroplasts, thylakoid swelling and disruption, phytoferritin accumulation, plastid elongation and increase in size. Changes in chlorophyll and carotenoid content were in accordance with degradation of the thylakoid system. Plastids were confirmed as very sensitive organelles to an artificial hyperhydric environment as well as to Agrobacteria-mediated cell transformation.
Introduction There are few reports on in vitro culture of cacti (Bhau, 1999; Mangolin et al., 1999; Llamoca-Zarate et al., 1999a, b). As plants with crassulacean acid metabolism (CAM), cacti are highly affected by artificial environments in tissue culture. CAM plants grown ex vitro incorporate CO 2 into organic acids at night and close their stomata during the day to reduce water loss. The CO 2 uptake by in vitro-cultured cacti happens continuously in the light and dark, consequently the growth of cacti can be considerably accelerated by in vitro culture (Malda et al., 1999). The high relative humidity and rich nutrient medium can alter tissue growth even in the absence of exogenous growth regulators (Elias-Rocha et al., 1998). In vitro propagated Mammillaria gracillis shoots developed callus without any exogenous growth regulator (Krsnik-Rasol and Balen, 2001). This habituated (hormone independent) callus regenerated
morphologically normal and hyperhydric shoots. Mammillaria was susceptible to tumour transformation by the Agrobacteria-Ti-plasmid-system. Such an easy switch from the organised to unorganised way of growth makes Mammillaria gracillis in vitro culture a suitable model to study plant development. There are relatively few references related to ultrastructural changes caused by transformation and artificial con` ditions in tissue culture (Crevecoeur et al., 1992; Lorkovic´ et al., 1993). In vitro propagated cactus shoots, callus, tumours and regenerants were compared with regard to their protein, glycoprotein, isoperoxidase and isoesterase patterns (Krsnik-Rasol and Balen, 2001; Balen et al., 2002, 2003 in press). In the present study, a morphology and ultrastructure of in vitro Mammillaria tissues were investigated with the intention to connect the structural and biochemical data. Attention was paid to plastids as markers of cell differentiation / dedifferentiation and to chlorophyll and carotenoid content.
118 Materials and methods
were established by one-way ANOVA followed by Duncan’s test (Duncan, 1955).
Plant material Mammillaria gracillis plants were cultivated in vitro, at 24 ⬚C, on a solid MS (Murashige and Skoog, 1962) nutrient medium containing 9 g l ⫺1 of agar and 30 g l ⫺1 sucrose without any growth regulator at 16 / 8-hlight / night-photoperiod (light intensity 90 mol s ⫺1 m ⫺2 ). Spontaneously formed callus was subcultivated every 3 weeks on the hormone-free MS medium (Krsnik-Rasol and Balen, 2001). Tumours were induced by Agrobacterium tumefaciens, a wild strain B6S3 (tumour line TW) and a rooty mutant GV3101 (tumour line TR) (KrsnikRasol and Balen, 2001). Light microscopy Semi-thin sections of material fixed for ultrastructural studies were stained with 2% toluidine blue and examined under a Zeiss Axiovert 35 light microscope. Handmade sections of fresh tissue were used for light microscopy. Electron microscopy Plant material was fixed in 1% glutaraldehyde in cacodylate buffer (pH 7.2) and postfixed in 1% OsO 4 . After dehydration in a graded ethanol series, the material was embedded in araldite. Ultrathin sections, 50–100 nm thick, obtained with Reichert Om U3 ultramycrotome, using diamond knives, were stained in uranyl acetate and lead cytrate and examined with the Zeiss EM 10A transmission electron microscope. Chlorophyll and carotenoid content Photosynthetic pigments were extracted from six replicates of each tissue sample in 100% acetone. The absorbance of the extracts was measured at 470, 644.8 and 661.6 nm using an ATI Unicam UV/ VIS Spectrometer UV4. Chlorophyll and carotenoid content were quantitatively determined using extinction coefficients provided by Lichtenthaler (1987). Statistics Statistical differences between Mammillaria tissues regarding data of content of photosynthetic pigments
Results In vitro culture of Mammillaria gracillis In the culture on hormone-free MS medium, M. gracillis shoots developed abundant callus masses (Figure 1A). These were detached from the plants and subcultivated on the MS medium without any growth regulator. The callus had a snowy surface and rather compact yellowish or light green inner portion (Figure 1B). Regeneration of normal and hyperhydric shoots occurred simultaneously from the same callus. Transformed tissue, derived from two primary crown gall tumours, one induced by a wild type of Agrobacterium tumefaciens B6S3 (TW line) and the other induced by a rooty strain of A. tumefaciens GV3101 (TR line), never expressed any organogenic potential. The TW tissue was yellowish to orange–brown while the TR was green to yellowish (Figure 1C,D). Light microscopy observations Semithin sections through a cactus stem (Figure 2A) showed a single-layered epidermis with stomata. Parenchyma cells were highly vacuolated with plastids located in the cytoplasm close to their cell walls. The tissue was rather loose and intercellular spaces were well developed. Sections through areoles at the bases of lignified spines revealed areas with small dividing cells and vascular bundles (Figure 2B). Callus was composed of vacuolated, isodiametric, thin walled cells and randomly dispersed groups of smaller dividing cells (meristemoids) (Figure 2C). On the friable snowy surface of the callus, cells were larger and more elongated (Figure 2D). Tumour line TW consisted of closely packed elongated cells of various sizes; division centres were spread throughout the tissue (Figure 2E), tracheid-like cells were frequent. Tumour line TR consisted of larger and mostly round-shaped cells (Figure 2F). Electron microscopy Parenchyma cells of in vitro grown cactus shoots had large nuclei, with deep invaginations of the nuclear envelope (Figure 3A). Plastids varied in size and shape from oval proplastid-like type (up to 2 m in
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Figure 1. In vitro culture of Mammillaria gracillis. (A) Cactus plant with spontaneously formed callus, (B) subculture of hormone independent callus with regenerated shoots, (C) tumorous callus induced by Agrobacterium tumefaciens, B6S3 (line TW), (D) tumorous callus induced by A. tumefaciens, GV3101 (line TR). Bar⫽0.5 cm.
diameter) to elongated chloroplasts (length up to 10 m) with well-developed thylakoid system. Plastids often accumulated phytoferritin in the stroma (Figure 3B). Starch grains were occasionally present in some plastids. Cells from lateral areoles were smaller than parenchyma cells and contained chloroamyloplasts with large starch grains and thylakoids (grana and stroma) on the periphery (Figure 3C). Plastids in callus cells were giant (up to 20 m in diameter), frequently vacuolized, with a lot of unorganized thylakoid membranes (figure not shown). In the vacuol-
ated TW tumour cells, proplastid-like plastids with few dilated thylakoids, phytoferritin and small plastoglobuli were observed (Figure 3D). Some tumour cells had chloroamyloplasts with layers of single thylakoids arranged on the organelle periphery and small aggregations of plastoglobuli (Figure 3E). The most obvious changes in plastid morphology were noticed in the TR tumour cells. These plastids had dense stroma and an extensive system of grana with dilated thylakoids arranged in layers around the large starch grain in the plastid centre (Figure 3F).
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Figure 2. Light micrographs of M. gracillis stem and callus sections. (A) Transverse section through a stem, (B) areole with spines, (C) parenchyma like callus cells and a group of smaller dividing cells, (D) elongated cells from the callus surface, (E) closely packed tumour cells (line TW), (F) tumour cells (line TR). Abbreviations used: dc – division centre; e – epidermis; me – meristemoid; pc – parenchyma cell; s – spine; st – stomata; vb – vascular bundle; tc – tracheid-like cell. Bar⫽30 m.
Chlorophyll and carotenoid content Chlorophyll and carotenoid contents of Mammillaria tissues are shown in Table 1. Chlorophyll a, b and total chlorophyll concentrations were lower in callus extracts than in the extracts of shoots. Both tumour lines had significantly lower concentrations of total chlorophyll, and of chlorophyll a and b with a re-
markable decrease in the content of chlorophyll a. The chlorophyll a /b ratio was lower in the callus than in the shoot; it was extremely low in both tumour lines. Carotenoid content of callus and tumour tissue was significantly reduced in comparison to cactus shoot. Chlorophyll / carotenoid ratios were not statistically different for cactus shoot, callus and both tumour lines.
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Figure 3. Plastid ultrastructures of M. gracillis cultivated in vitro. (A) part of shoot cell with large lobed nucleus, proplastids and mitochondria, (B) elongated chloroplast of the same cell type, (C) chloroamyloplasts with developed thylakoid system and large starch grains in cells from lateral areoles, (D) proplastid-like plastids with few dilated thylakoids and phytoferritin in a tumour cell (line TW), (E) layers of thylakoids on the periphery of plastid (line TR), (F) an extensive system of grana with dilated thylakoids on plastid periphery noticed in tumour cells (line TR). ca – chloroamyloplast; ch – chloroplast; dt – dilated thylakoids; f – phytoferritin; m – mitochondria; n – nucleus; pp – proplastid; s – starch. Bar⫽1 m.
Discussion Spontaneous formation of hormone independent callus with regeneration potential was most likely caused by hyperhydric stress in the culture. The lower agar
concentration and the higher MS salt concentration stimulated callus growth at the base of cactus shoots (Krsnik-Rasol and Balen, 2001). This habituated callus bears some resemblance to crown gall tissue, which is also hormone independent. Habituation has
122 Table 1. Chlorophyll and carotenoid content (mg g ⫺1 f.w.) of Mammillaria tissues
Total chlorophyll Chlorophyll a Chlorophyll b Chloro a /b ratio Total carotenoids Chloro / car. ratio
Shoot
Callus
Tumour line TW
Tumour line TR
0.139⫾0.005a 0.095⫾0.005a 0.044⫾0.040a 2.242⫾0.271a 0.025⫾0.002a 5.647⫾0.416a
0.043⫾0.002b 0.025⫾0.001b 0.019⫾0.001b 1.493⫾0.171b 0.008⫾0.001b 5.415⫾0.289a
0.016⫾0.001c 0.007⫾0.001c 0.009⫾0.001c 0.720⫾0.070c 0.004⫾0.0003c 4.455⫾0.348a
0.022⫾0.004c 0.009⫾0.001c 0.012⫾0.002bc 0.780⫾0.035c 0.004⫾0.0005c 5.444⫾0.729a
The content of photosynthetic pigments was given as the mean value of six replicates of each tissue line⫾standard errors. According to Duncan’s New Multiple Range Test significantly different values ( p⬍0.05) are marked with different letters.
been considered a step of neoplastic progression that can be initiated in in vitro cultures (Gaspar, 1995). This process implicates a progressive reduction of cell-to-cell adhesion (Liners et al., 1994), many mor` phological abnormalities (Crevecoeur et al., 1992), biochemical deviations (Le Dily et al., 1993), and irreversible loss of organogenic totipotency at the terminal stage. Very sensitive plant cell organelles reacting to unfavourable influences are plastids. Damage may occur on mature plastids, causing secondary misbalances in the cell, or affecting plastid development and differentiation. Our results confirmed that plastids react very sensitively to the artificial environment in the culture as well as to cell transformation after Agrobacteria infection. Underdeveloped chloroplasts with reduced thylakoid systems, noticed in shoot cells, are most likely the result of cultivation in the rich nutrient medium where photosynthetic activity was reduced (Birchem et al., 1981). In the same culture conditions, callus cells contained giant plastids with reduced grana, wavy swollen thylakoids and dilated luminal spaces. Similar plastid morphology has been described in water-stressed wheat (Freeman and Duysen, 1975). Caredda et al. (1999) reported disruptions which affected the plastid size, the feature of plastid envelopes, thylakoid and grana organization, as well as starch accumulation in the barley callus. It has also been reported that the exclusive regeneration of albino plantlets in the spring cultivar Cork of barley from the anther culture may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions (Caredda et al., 2000). Plastid morphology in TW tumour cells revealed a dedifferentiation of already mature chloroplasts toward proplastids. The process is usual in cells cultivated in vitro (Sjolund and Weier, 1971) and occurs also during anaplasia or
callus formation. Phytoferritin particles found in plastids could be a product of thylakoid disintegration ˇ ´ 1976). Circular arrangement of thylakoid (Ljubesic, membranes is most likely a consequence of thylakoid stack disruption or of blocking of thylakoid aggregation. Similar changes in plastid ultrastructure have been found after treatment with some herbicides (Srivastava et al., 1971). Giant and multilobed nuclei, vacuoles in the nucleoli and highly vacuolated cells in tumour tissue were also observed in habituated sugarbeet callus ` (Crevecoeur et al., 1992). Pigment analysis was in accordance with ultrastructural observations. Significantly lower concentrations of total chlorophyll, chlorophyll a and b in callus and both tumour lines than in the cactus shoot confirms thylakoid degradation in these lines. Total chlorophyll content was highly affected by transition from organised to unorganised growth. On the basis of the observation that gibberellin and auxins stimulate growth at the same time as they inhibit chlorophyll formation in tissue culture (Kirk and TilneyBassett, 1978) we suppose that endogenous hormones of unorganized habituated and tumour tissues inhibit greening and promote rapid growth. Chlorophyll deficiency, accompanied with lower peroxidase activity, could be related to abnormal porphyrin metabolism as has been already reported ` for habituated sugar beet callus (Crevecoeur et al., ` et al., 1992). 1987; Hagege
Acknowledgements We would like to thank Dr. Gordana Rusak for helpful discussions, Dr. Marin Greenwood for critical reading ˇ of the manuscript and Karmela Suvak for her technical assistance.
123 References ˇ ´ J & Krsnik-Rasol M (2002) Protein and Balen B, Milosevic glycoprotein patterns related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture. Food Technol. Biotech. 40: 275– 280 Balen B, Krsnik-Rasol M & Simeon-Rudolf V (2003) Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture. J. Plant Physiol. (in press) Bhau BS (1999) Regeneration of Coryphantha elephantides (Lem.) Lem. (Cactaceae) from root explants. Sci. Horticult. 81: 337– 344 Birchem R, Sommer HE & Brown CL (1981) Comparison of plastids of Pinus pallustris Mill. and Pinus elliottii Engelm. in callus tissue culture. Pflanzenphysiol 102: 101–107 ´ Caredda S, Devaux P, Sangwan RS & Clement C (1999) Differential development of plastids during microspore embryogenesis in barley. Protoplasma 208: 248–256 Caredda S, Doncoeur C, Devaux P, Sangwan RS & Clement C (2000) Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.). Sex Plant Reprod. 13: 95–104 ` Crevecoeur M, Kevers C, Greppin H & Gaspar T (1987) A comparative biochemical and cytological characterization of normal and habituated sugarbeet calli. Biol. Plant 29: 1–6 ` ` D, Catesson AM, Greppin H & Gaspar T Crevecoeur M, Hagege (1992) Ultrastructural characteristics of cells from normal and habituated sugar beet calli. Plant Physiol. Biochem. 30: 87–95 Duncan DB (1955) Multiple range and multiple F-tests. Biometrics 11: 1–42 Elias-Rocha MA, Santos-Diaz MD & Arredondo-Gomez A (1998) Propagation of Mammillaria candida (Cactaceae) by tissue culture techniques. Haseltonia 6: 96–101 Freeman TP & Duysen ME (1975) The effect of imposed water stress on the development and ultrastructure of wheat chloroplasts. Protoplasma 83: 131–145 Gaspar T (1995) The concept of cancer in in vitro plant cultures and the implication of habituation to hormones and hyperhydricity. Plant Tiss. Cult. Biotech. 1: 126–136 ` Hagege D, Werck-Reichhart D, Schmitt P & Gaspar T (1992) Deficiency in tetrapyrrole-containing compounds in a non-organogenic habituated sugarbeet cell line. Plant Physiol. Biochem. 30: 649–654
Kirk JTO & Tilney-Bassett RAE (1978) The Plastids: Their Chemistry, Structure, Growth and Inheritance. Elsevier / NorthHolland Biomedical Press, Amsterdam Krsnik-Rasol M & Balen B (2001) Electrophoretic protein patterns and peroxidase activity related to morphogenesis in Mammillaria gracillis tissue culture. Acta Bot. Croatica 60: 219–226 Llamoca-Zarate RM, Studart-Guimaraes C, Landsmann J & Campos FAP (1999a) Establishment of callus and cell suspension cultures of Opuntia ficus-indica. Plant Cell Tiss. Org. Cult. 58: 155–157 Llamoca-Zarate RM, Aguiar LF, Landsmann J & Campos FAP (1999b) Whole plant regeneration of Opuntia ficus-indica Mill. (Cactaceae). J. Appl. Bot. 73: 83–85 Le Dily FJ, Billard JP, Gaspar T & Huault C (1993) Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet. Physiol. Plant 88: 129–134 Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth. Enzym. 148: 350–382 Liners F, Gaspar T & Van Cutsem P (1994) Acetyl- and methylesterification of pectins of friable and compact sugar-beet calli: consequences for intercellular adhesion. Planta 192: 545–556 Lorkovic´ Z, Muraja-Fras J, Krsnik-Rasol M & Wrischer M (1993) Ultrastructural and biochemical changes in potato tuber cells related to tumourigenesis. Plant Physiol. Biochem. 31: 633–638 ˇ ´ N (1976) Phytoferritin in plastids of blackberry leaves. Ljubesic Acta Bot. Croatica 35: 51–55 Malda G, Backhaus RA & Martin C (1999) Alterations in growth and crassulacean acid matabolism (CAM) activity of in vitro cultured cactus. Plant Cell Tiss. Org. Cult. 58: 1–9 Mangolin CA, Ottoboni LMM & Machado MFPS (1999) Twodimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins. Electrophoresis 20: 626–629 Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15: 473–479 Sjolund RD & Weier TE (1971) An ultrastructural study of chloroplast structure and dedifferentiation in tissue cultures of Streptanthus tortuosus (Cruciferae). Am. J. Bot. 58: 172–181 Srivastava LM, Vesk M & Singh AP (1971) Effect of chloramphenicol on membrane transformation in plastids. Can. J. Bot. 49: 587–593
