Plant Cell, 7issue and Organ Culture 28: 255-260, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts

K. C. Katsirdakis 1'2 & K. A. Roubelakis-Angelakis ~ 1Department of Biology, University of Crete, and 2Institute of Molecular Biology and Biotechnology, P.O. Box 1470, 711 10 Heraklio, Greece

Received 9 November 1990; accepted in revised form 17 October 1991

Key words: culture medium, grapevine, protoplast culture, protein synthesis

Abstract

Studies were undertaken to determine optimum conditions for grapevine protoplast culture. Highest viability was obtained at 25 ° in the dark, and at initial pH values of 5.2 to 7.2, in the presence of 1 or 2% (w/v) sucrose and 0.6 or 0.7M mannitol or osmolality between 729 and 930mOsmolkg -~. Optimum plant growth regulators were 6-BAP/NAA at 2.3 x 10 6/10-15 × 10 -6 M, respectively. 35S- Methionine incorporation into de novo synthesized proteins for protoplasts of Nicotiana tabacum cv Xanthi (a readily regenerating species) and for the recalcitrant grapevine was studied. In tobacco the rate of protein synthesis showed 3 maxima which coincided with the distinct stages of naked protoplasts, cell wall reconstitution and induction of cell division. In grapevine protoplasts the first peak exceeded the second one, whereas no peak corresponding to the induction of cell division was observed.

Introduction

Isolated protoplasts and in particular leaf proto- plasts offer an excellent experimental system for molecular, biochemical, and physiological studies and can also serve as a tool for produc- tion of genetically transformed plants. Grapevine leaf protoplasts have been isolated and cultured from several Vitis vinifera cultivars [2, 5, 13-15, 23, 27, 30] and from Vitis rotundifolia cv Summit [14], but only in few cases cell divisions were reported [13-15, 23, 27]. The difficulties in ob- taining regeneration can have many facets. Dur- ing the isolation of protoplasts various stresses occur that may modify the physiological prop- erties and decrease their viability [20]. Also, protoplasts are very sensitive to various culture conditions, such as the composition of the medium and the conditions during culture de- pending upon what species or cultivar is used [8].

With grapevine the growth regulator require- ment are somewhat cultivar dependent; optimum

cell division was obtained with 2,4-D and BA in Koshu Sanjaku protoplasts [23], whereas NAA was slightly more effective than 2,4-D in grape pericarp protoplasts [25] and Dattier protoplasts divided only when NAA and kinetin were used [6]. However, Cabernet Sauvignon and V. rotun- difolia cv Summit protoplasts divided and de- veloped microcalluses in the presence of 2.2/ 4.5 ~M 6-BAP/2,4-D, respectively [14]. In con- trast, Sultanina protoplasts did not respond to various combinations of 2,4-D and 6-BAP [27]. In addition, the concentration of osmotic adjuv- ant appeared critical for protoplast wall regene- ration and cell division [23, 25].

In a previous communication, the optimum conditions for the isolation of leaf protoplasts from axenic shoot cultures of Vitis vinifera L. cv. Sultanina were established and some culture con- ditions were examined [27]. Also, the physiologi- cal functioning of the protoplast membrane was tested by studying the uptake characteristics of labeled sugars and amino acids [22, 26, 28].

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In this communication, further results are pre- sented on a modified culture medium (Grapevine Cell Wall Regenerating, GCWR) and optimiza- tion of some of the conditions which promote sustained protoplast viability and enhance cell elongation of Vitis vinifera L. cv. Sultanina leaf protoplasts. Also, the incorporation of labeled methionine, as a test of the physiological status of protoplasts, was examined. The results were compared to those obtained with protoplasts from tobacco (Nicotiana tabacum cv Xanthi), which is a good plant regenerating system.

Materials and methods

Plant material

Heat-treated Vitis vinifera L. CVo Sultanina meristem-derived plants were sequentially mi- cropropagated and cultured in vivo in Roubelakis hormone-free medium [21] at 25°C, 16 h photoperiod and total energy of 1,500 ixWcm -2 provided by cool-white fluores- cent lamps. Leaves from the middle part of the young shoots were used for protoplast isolation.

Protoplast isolation

Protoplasts from Vitis vinifera L. CVo Sultanina leaf tissues were isolated as described previously [27]. The purified protoplasts were suspended in a small volume of culture medium (see below) and placed into 2ml of the defined culture medium in 10 × 15 mm sterile plastic Petri dishes at a density of approx. 2 x 105 protoplasts/ml. In addition, protoplasts were isolated from seed- lings of tobacco (Nicotiana tabacum cv Xanthi), grown in a controlled-temperature glasshouse by applying the same procedure.

Culture media

The starting culture solution contained Murashige & Skoog (MS) salts [18], and the following in mg 1-1 , biotin 0.1, myo-inositol 100, nicotinic acid 1, pyridoxine-HC1 1, thiamine-HCl 10, Ca-pantothenic acid 1, and cellobiose 250. To this basic medium, mannitol (0.3 to 0.8 M), sucrose (1 or 2% (w/v)), 6-BAP (0.7 to 4.5 x

10 -6 M) and NAA (2.5 to 15 x 10 -6 M) were added. The combination which showed promis- ing results was 2.3 x 1 0 - 6 M 6-BAP and 10- 15 × 10 -6 M NAA. The osmolality of the media was determined with a Gonotek cryoscopic Os- momat 030 Osmometer.

Stains

Viability was assessed using fluorescein diacetate (FDA) [29] and the results were expressed as the percentage viability, based on counts of ten microscopic fields.

Calcofluor white, a very sensitive stain for cellulose, was also used to confirm the removal of cell walls from freshly isolated protoplasts and to monitor the reformation of walls by the plated protoplasts with time [10]. The percentage of cell wall regenerating protoplasts was calculated as the number of protoplasts fluorescing blue per total number of viable protoplasts, based on counts of ten microscopic fields.

Radioactive labeling of de novo synthesized proteins

Protoplasts were labeled by adding 21xCi of 35S- methionine (1070 Ci/mmol specific activity) to 2 ml culture medium containing 6 × 105 proto- plasts as a pulse for a period of 3 h after 0, 0.5, 1, 2, 5 and 7 days of culture.

Extraction of proteins

At the end of the labeling period, the protoplasts were recovered by centrifugation at 100 g for 2 rain, and the pellet was frozen at -80°C until used. Proteins from each pellet were extracted for 20 rain at 4°C in an extraction buffer de- scribed by Lowry [16], and centrifuged at 19,000 g for 5 min. The resulted supernatant was treated with 10% (v/v) trichloroacetic acid (TCA) and proteins were allowed to precipitate at 4°C for 15 min. Following centrifugation at 19,000 for 15 rain, the pellet was dissolved in 100 Ixl of a reagent consisting of 2 g NaOH, 10 g N a z C O 3 , 0.1 g Na-K-tartrate/500ml HzO [16]. A 50 txl aliquot was used to count radioactivity by liquid scintillation spectrometry and a 50 Ixl aliquot was used to determine the protein con- tent by the method of Lowry [16].

Results

Osmotic requirement

An initial study was peformed to determine the opt imum concentration of mannitol, as os- moticum, when sucrose was used as carbon source in culture medium (Table 1). Highest survival rates were observed at 0.5 to 0 .7M mannitol in combination with either 1% or 2% sucrose. Therefore , osmotic pressure values below 729 and above 930mOsmol /kg were not suitable for grapevine Sultanina protoplast culture.

Plant growth regulators

Protoplasts from grapevine leaves cv Sultanina did not divide in the presence of 2,4-D and their viability was very low (data not shown). The combined effect of N A A at 2.5-15 x 10 -6 M and 6-BAP at 0.7-4.5 x 10 -6 M on protoplast viabili- ty and elongation was examined. The elongation was determined as the percentage of viable pro- toplasts that were elongating 12 days after isola- tion. The highest viability and elongation were observed at 2 . 3 x 1 0 - 6 M 6-BAP and 10- 15 × 10 -~ M NAA (Fig. 1A, B).

Table 1. Effect of the concentration of mannitol and osmotic pressure on the viability of grapevine leaf protoplasts after an 8-day culture period in an MS basal medium.

Sucrose, % Mannitol, M mOsmol/kg % viability

1 0.3 513 11 g 1 0.4 613 20 defg 1 0.5 729 32 bc 1 0.6 868 39 ab 1 0.7 920 43 a l (1.8 1028 21 def 2 0.3 537 15 fg 2 0.4 642 26 cde 2 0.5 760 30 bcd 2 0.6 887 44 a 2 0.7 930 43 a 2 0.8 1050 18 efg

All data are the mean values from 4 independent experi- ments. Means are separated in column by Duncan's multiple range test; the same letters in a column designate non significant difference at p = 0.05.

257

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C o n c e n t r a t i o n o f 6-BAP, X10 -6M Fig. 1. Effect of growth regulators on % viability (A) and on % elongation (B) of Vitis vinifera L. cv Sultanina leaf protoplasts after 12 days of culture in GCWR medium.

Effect of temperature

The survival rates of protoplasts at 5°C and 25°C were bet ter than those at 37°C. At the end of 8-day culture period, the survival rates were 38 + 2, 46_+ 2, and 13-+ 3%, at 5, 25 and 37°C, respectively.

Cell wall regeneration medium

After the systematic study of environmental con- ditions, osmotic requirement and plant growth

258

regulators ratio, the improved GCWR culture medium has the macro- and micro- elements of MS [18], the vitamins of Barbier & Bessis [3], cellobiose concentration of 250 mg 1-1 (as sub- strate for cellulase), mannitol 0.6 M, sucrose 2% (w/v), NAA 15 × 10 -6M and 6-BAP 2.3 × 10 -6 M, pH 6.2. Results in Fig. 2A indicate that the percentage viability in this medium was sig- nificantly greater than in basal MS medium sup- plemented with vitamins, growth regulators and

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Days of c u l t u r e Fig. 2. Percentage of viable (A) and cell wall regenerating (B) Vitis vinifera L. cv Sultanina leaf protoplasts cultured in GCWR medium (11) and in basal MS medium supplemented with vitamins, growth regulators and osmoticum of Barbier & Bessis [3] ( e ) .

osmoticum of Barbier & Bessis [3]. As it is shown in Fig. 2B grapevine protoplasts began to regenerate their cell walls; after 2 days of culture more than 80% of the viable protoplasts showed complete fluorescence. Recognizable cytological phases during protoplast culture were followed. Expansion of protoplasts was observed after 2 to 4 days of culture and after 4 to 6 days the shape of protoplasts changed from spherical to ellip- soidal.

De novo protein synthesis

The rate of labeled methionine incorporation into insoluble TCA proteins was studied during the first 8 days of culture of grapevine proto- plasts in comparison to tobacco protoplasts in the same GCWR medium (Fig. 3). In grapevine protoplasts, the incorporation of the added 35S- methionine showed a distinct peak after 12 h of culture and a second peak on the 4th day. Incor- poration decreased thereafter. In tobacco proto- plasts, three distinct peaks were observed; one after 6 to 9 h, one after 2 days and another one 5 days following the isolation of protoplasts (Fig. 3).

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Days of c u l t u r e Fig. 3. Incorporation of 35S-methionine into the TCA-insolu- ble protein fraction of grapevine (line A) and tobacco (line B) leaf protoplasts cultured in the same (GCWR) medium.

Discussion

The factors which seem to contribute to the plant regenerating potential of plant protoplasts are related to the donor plants and tissues, the con- ditions of isolation, the composition of culture medium and the conditions of culture [12]. Gen- erally, high light intensity inhibits protoplast growth when applied from the beginning of the culture [1]. The inhibition by high light intensity is not clearly understood but it might be related to the rapid bleaching of chloroplasts. Grapevine protoplasts showed higher viability rates in the dark than in the light [27].

The temperature usually employed for the cul- ture of protoplasts ranges between 22°C and 28°C, but Miihlback & Thiele [19] reported that chilling of freshly isolated tomato mesophyll pro- toplasts at 7°C enhanced the division frequency. This effect has been ascribed to the excretion of some factor(s) by the protoplasts into the medium. Our results also, indicated high viabili- ty of grapevine protoplasts at 5°C compared to 25 ° .

Usually, the pH used for protoplast culture is in the range of 5.5 to 5.8. Higher pH values have been reported to significantly enhance cell divi- sion of pea [11], cowpea [4], and asparagus [17] protoplast-derived cells. The survival rate of grapevine protoplasts was higher at initial pH 5.4 to 7.0 compared to lower pHs. The pH changes of the medium during culture indicated that leaf protoplasts readily acidify the medium. This fact would indicate a high capacity of the protoplasts to excrete into the medium anions rather than cations. The observed changes of pH during culture (data not shown), may be related to ammonium and nitrate utilization, or solute ab- sorption by the mechanism of cotransport with H +, and this fact ensures the normal physiologi- cal status of protoplasts, while alkalinization was observed in the degradation phase [7].

Lee & Wetzstein [14] reported that grapevine protoplasts divided in the presence of 2.2 IxM 6-BAP and 4.5 ~M 2,4-D. Shimizu [23] found that some grapevine leaf protoplasts divided in the presence of 2,4-D at 0.1-2.0mg ! -~ and BA at 0.1-2.0mgl -~, while other auxins and cyto- kinins, in various combinations, were not effec- tive in promoting cell division [27]. Apparently,

259

the hormone requirement is probably cultivar dependent, as Skene [25] reported that NAA was slightly more effective than 2,4-D in reduc- ing the lag phase prior to division, and Brezeanu & Rosu [6] obtained cell division only when NAA and kinetin were used. In addition, 2,4-D/ 6-BAP at ratios from 0.1 to 5 had no effect in increasing survival rates of Sultanina protoplasts [27]. The results obtained in this study show that optimal auxin and cytokinin concentrations for Sultanina leaf protoplasts were 15 × 10- ~' M NAA and 2.3 x 10 6 M 6-BAP. It appears that auxin is required for cell wall formation in cul- tured protoplasts (Fig. 1).

The studies of the physiological functioning of plasmalemma of cultured grapevine protoplasts, e.g. uptake of sugars and amino acids, have shown that they indeed have a high uptake capacity [22, 28]. The study of the de n o v o

protein synthesis by these protoplasts could give some information on the metabolic competence of these recalcitrant protoplasts which could be further more useful if compared to that of re- generating protoplasts, e.g. tobacco. Three peaks of protein synthesis were identified in tobacco and two in grapevine protoplasts (Fig. 3). The first one could be due to shock protein synthesis [9] and was of comparable magnitude in both species. The second peak coincided with the cytologically identified stage of cell wall syn- thesis, which occurs approximately the 2nd and 4th day of culture in tobacco and grapevine protoplasts, respectively [31]. The 5th day of culture, which coincides with cell division, the rate of protein synthesis in tobacco protoplasts showed a 2-fold increase; in contrast, the rate of protein synthesis in grapevine protoplasts de- clined constantly after the second peak. Before being able to suggest whether this peak of pro- tein synthesis is due to cell division-related pro- tein synthesis further work is required.

In summary, results from this report suggest that the GCWR medium is suitable for sustain- ing high viability rates, for grapevine protoplasts, during the first 8-12 days of culture, a period particularly suitable for basic morphological and physiological studies. It also promotes cell wall regeneration and cell elongation, events which are prerequisite for subsequent cell division, and it increases the percentage of dividing cells.

260

Acknowledgements

The authors thank Mr. C. Siminis for useful discussions and help during the labeling work, and Mrs S. Makrygiannakis-Toufexis for techni- cal and typing assistance.

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