Procedure for the regeneration of plants from cell suspension protoplasts of tetraploid potato (Solanum tuberosum L.) cv. Desirée

Plant Science, 58 (1988) 2 2 3 - 230 Elsevier Scientific Publishers Ireland Ltd.

223

PROCEDURE FOR THE REGENERATION OF PLANTS FROM CELL SUSPENSION P R O T O P L A S T S O F T E T R A P L O I D P O T A T O (SOLANUM TUBEROSUM L.) CV. DESIRi~E

R A F F A E L A TAVAZZA a, RICARD0 J. ORDAS b and GIORGIO ANCORA"

°ENEA, Divisione Ingegneria Genetica, C.R.E. Casaccia~ 1-00060Rome fltalyJ and bLaboratory of Plant Physiology, Faculty of Biology, Oviedo (Spainj (Received July 9th, 1988) (Revision received July 11th, 1988) (Accepted July l l t h , 1988)

A routine procedure for the isolation, culture and plant regeneration of a large number of protoplasts from suspension cultures of Solanum tuberosum cv. Desir~e has been developed. An optimal protoplast yield of up to 800/0 was obtained by gently shaking at 30 °C overnight the cell suspension with an enzyme mixture {3°/0 cellulase R10, 10/0 macerozyme R10 and 10/0 pectinase in 0.3 M Mannitol). The protoplasts cultured at a density of 7-10 ~ prot./ml in a modified MS liquid medium supplemented with naphthaleneacetic acid (NAA) (2 rag/l), 6-benzylaminopurine (BAP) (0.5 rag/l), 2,4-dichlorophenoxyaeetic acid (2,4-D) (2 mg/1) and 0.4 M Mannitol regenerated cell wall within 2 days and they underwent the first division within 3 days. The first regenerated shoot was obtained approximately 3 - 4 months after protoplast isolation. The critical factors seem to be associated with the quality of the culture and the enzyme composition used for protoplast isolation rather than with specific culture conditions for the protoplasts.

Key words: Solarium tuberosum L.; protoplast; regeneration; suspension culture; potato

Introduction

Protoplasts with a high viability and ability to regenerate into plants are potentially interesting materials, especially for the somatic hybridization, transformation and selection studies. The first prerequisite for the application of these studies in the improvement of economically important plants is the standardization of the isolation and cultivation procedures, including the selection of suitable starting materials. Besides, large numbers of protoplasts with both high plating and plant regeneration efficiencies are often required. In the case of potato, protoplast culture and regeneration procedures have been developed from mesoAbbreviations: BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; MS, Murashige and Skoog's (1962) medium; NAA, naphthaleneacetic acid; PCV, packed cell volume; UM, Uchimiya and Murashige (1974) medium.

phyll [1--4] or from apical root [5] of various tetraploid commercial cultivars, while no data concerning the regeneration of plants from cell suspension protoplasts have been reported. In an earlier communication, we reported the regeneration of protoplasts isolated from mesophyll of a commercial potato cultivar Desir~e [6]. The regeneration of plants from this cultivar has been carried out successfully. However, the use of leaf mesophyll tissue, as a source of protoplasts, can be very poor when the isolation of protoplasts in large quantities is required. In addition, protoplasts from cell suspension lacking chlorophyll are advantageous in somatic hybridization experiments with mesophyll protoplasts because heterokaryons can be easily recognized. For these reasons, we have decided to use as a starting material suspension cultures derived from leaf tissue of the cv. Desir~e. The main objective of this investigation is to develop an efficient method for the large scale

0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

224

isolation of cell suspension protoplasts of cv. Desir6e which can be induced to regenerate into plants, and at the same time, serve as phenotypic markers for the identification of 'somatic hybrid cells'. Materials and methods

Plant material

Virus free stocks of potato line cv. Desir6e were used for callus induction. In vitro explants were maintained on MS [7] basal medium, supplemented with 1% w/v sucrose and 0.8% w/v agar. The cultures were incubated in a growth chamber at 19°C with a daylength of 16 h under 400 lux light intensity. L e a f callus and cell s u s p e n s i o n

Young leaves collected from 3--4-week-old shoot cultures were induced to form callus on an agar solidified UM medium [8] supplemented with 0.25 mg/1 kinetin and various concentration of 2,4-D (0.1, 0.5, 1, 2 and 5 mg]l). The pH of the media was adjusted to 5.6-5.8 by the addition of 0.1 N HC1 or 0.1 N NaOH. Calli were grown in darkness and in light (2000 lux) at 26 °C. To evaluate the effect of different 2,4-D concentrations on the growth of the calli, 1 g of induced callus was inoculated on fresh medium in each subculture. Calli were subcultured on fresh medium every four weeks. After 3 subcultures 5 g of fast growing callus were placed into liquid medium of the same composition. The suspensions were grown in 250 ml Erlenmeyer flasks containing 50 ml medium. They were maintained on a rotary shaker (120 rev./min) at 26°C with a daylength of 16 h under 2000 lux light intensity. After 10 days of growth, the cells were dispersed; at this time, large cell aggregates were recovered through 500 pm and 125 pm mesh filters respectively. Cells settling on the filter with a pore size of 125 ~m were resuspended in 50 ml of a fresh medium (4.7 _+ 0.9.105 cell/ml) and grown in the same conditions gave rise to a fast growing, fine suspension culture composed of single cells and small cell aggregates. Subculturing was performed every two weeks

by transferring 10 ml of suspension culture to 40 ml fresh medium. P r o t o p l a s t isolation and culture

For protoplast isolation, 4-, 6- and 10-day-old cell cultures were used. Two millilitres packed cell volume (PCV) of each suspension was suspended in 10 ml of enzyme for digestion. Two enzyme compositions were examined (Table I). The mixture was incubated overnight at 30 °C in the dark on a rotary shaker at 30 rev./min. Following incubation, the protoplasts were separated from cell debris by filtration through two filters with a pore size of 88 and 38 pro, respectively. The protoplasts were collected by low speed centrifugation (70 x g, 5 rain), followed by washing two times with 10 ml of washing medium (0.32 M NaCI) applying centrifugation (70 x g, 3 rain). After washing, the protoplasts were suspended in liquid culture medium at a density of 7.104 protoplasts/ml. The protoplast culture medium was MS [7] in which NH4NO 3 was replaced by casein hydrolysate (1000 mg/1) and to which 0.4 M mannitol was added. The concentrations of nicotinamide, pyridoxine--HCl and t h i a m i n e - H C l were respectively increased to 5 mg/1, 5 rag/1

Table I. Compositionof enzyme solution for protoplast isolation frompotato suspensioncells.

Enzymesolution I

II

Enzyme f%wlvJ

OnozukaR-10 Pectinase MacerozymeR-10

3 2

3 1 1

408

408

MES

5

5

Osmotica fMJ Mannitol

0.3

0.3

pH

5.5

5.5

Mineral (mMJ

CaCl- H20 Buffer froM)

225 and 1 rag/1 respectively and sucrose concentration increased to 2%. Two millilitres of protoplast suspension was plated in 5 cm petri dishes and incubated in a growth cabinet at 26 °C under continuous light (1000 lux). Cell suspension protoplasts were cultured following the culture procedures used for mesophyll protoplasts [6]. Shoots were regenerated using the procedure of Bokelmann and Roest [3].

and nylon filtered (53 pm) before FCM analysis. All fiow-cytometric analyses were carried out by a BD FACS 400 equipped with an argon laser tuned at 488 nm and run at 200 mW power output; the filter set included a BP 535/15 for the green channel, a 570 dicroic mirror and a LP620 for the red channel; the protoplasts were analysed at a flow rate of 500/sec. Data analysis were performed with an 'FDASIII' dedicated computer.

Viability Viability of the isolated protoplasts was assessed by FCM using the fluorescent dye rhodamine 123 (RH 123) (EXT 485 nm; EMS 530 nm). RH 123 (Sigma) was dissolved in absolute ethanol at a concentration of 5 rag/1 and the solution was stored at - 20 °C. The protoplasts were stained with RH 123 at the concentration of 5.0 pg/ml and incubated with the fluorochrome for 40 rain. After centrifugation (100 x g, 3 min), the samples were resuspended into fresh culture medium

Results and discussion The growth responses, texture, colour and friability of the callus obtained were influenced by the different media. When the calli were transferred from a medium containing high auxin (2,4-D 5 rag/l) to low auxin concentration (2,4-D 0.1 mg/l) or to auxin free medium, a sudden decrease in growth was observed and cell division was strictly inhibited (Fig. 1). After the second subculture, the calli turned brownish, a

Fresh weig)ht, /no

j

• ~SUeCUL~JRE

10.0

7.5

5.0

2.5

0.0 0.1

0.5

1.0

1.5

2,0

2.5

3.0

3,5

4.0

4.5

5.0

2,4-D Concentration (rag/I) Fig. 1. Influence of different concentrations of 2,4-D on the increase of fresh weight of potato calli cv. Desir~e. The initial weight of the callus in each subculture was I g . . , Subculture 1; [~, Subculture 2.

~

~J ~1d

crq

i -I

o

~-~

?!

Cr~

227 Table II. Effect of enzyme solution and time after subculture on the yield of protoplasts isolated from potato suspension culture. Age of cell cultures (days)

Yield of protoplasts ( x 106/ml PCV) Enzyme solution

4 6 10

I

II

1.35 1.60 0.20

2.88 3.00 0.77

phenomenon already observed by Bajaj and Dionne [9]. There were non-significant differences in the growth of calli under light and dark conditions.

However, in the latter condition, calli contained highly vacuolised cells. As the growth and dissociation of the callus in suspension was better in the medium containing 5 mg/1 of 2,4-D, it was used for all subsequent experiments. The physiological state of the starting cell culture appeared to be very important for the yield and viability of the isolated protoplasts. As already observed by Opatrny et al. [10], a special problem is represented by the isolation of protoplasts from older, highly vacuolised cells whose cell walls were fairly resistant to digestive enzyme. Experiments were performed to determine the optimum time interval after subculturing the cell suspension for protoplast isolation. The highest efficiency

Fig. 4. Viability of potato protoplasts stained with Rhodamine 123 performed by FCM analysis. The tridimensional cytc~ gram contains 3 • 104 protoplasts. It shows t h a t nearly 90°/0 of the protoplasts show both enzymatic activity (green fluorescence) and ceil membrane integrity (iarger scatter signals). X = forward iight scatter (signais derived from the iight scattered at small angles (0.5-2.0 o) from an incident laser beam; the scatter intensity is proportionai to the cell volume). Y = relative green fluorescent intensity from RH 123. Z = number of cells.

228

Fig. 5.

A cell cluster after three weeksof culture.

of intact protoplasts was obtained from fast growing cell suspensions, 4 - 6 days after they had been subcultured in fresh medium (Figs. 2 and 3). Cultures containing clusters of small spherical cytoplasmic cells (about 20 cells) gave rise to dividing protoplasts while the oldest produced very large sized protoplasts that usually did not regenerate cell wall [10]. The yield of protoplasts (Table II) was also influIII. Desir~e).

Table

enced by the enzyme composition. Enzyme solution II gave the highest yield of protoplasts, 3.0. 106/ml PCV. Enzyme solution I, which did not contain pectinase, was less effective. In fact, the cell wall was not completely digested, and after enzyme treatment, 10-20o/o of the protoplasts were still surrounded by cell wall. This indicates that the addition of pectinase stimulates not only the dispersal of cell aggregates but also the process

Platingefficiencyand percentage of shoot regeneration in suspensionculture-derivedprotoplasts of potato (cv.

Protoplast yield per ml PCV (n)

Dividingcells after 7 days (%)

Plating efficiency after 30 days (%)

Regenerating calli (%)

3 x 106

40

1 + 0.3

46.5 _

5.5

Shoots/callus (n) 6--8

229

6

Fig. 6

Regeneratingcalli on agar-solidified regeneration medium, 3 months after protoplasts isolation.

of protoplast isolation and the overall output. Protoplasts isolated from both enzyme mixtures were equally viable (80--900/0 depending on the age of the culture) and were intact. Protoplast viability was evaluated by FCM analysis of the fluorescent emission from the cationic dye RH 123 as related to the scattered light diffracted by the intact cells [11] (Fig. 4). RH 123 is a specific probe for active mitochondria where the cationic dye is attracted inside by the relatively high negative electric potential across the mitochondrial membrane [12]. The filtration through a fine 38 ~m filter encouraged the collection of relatively highly cytoplasmic protoplasts while discarding the large and vacuolated protoplasts which were normally obtained from the older cells. The first cell division occurred after 2 - 3 days of culture, while the frequency of cell division was

estimated around 400/0 after a week of culture. To avoid protoplast aggregation, fresh culture medium was added 3 days after isolation. This dilution enabled the formation of white colonies and helped their separation. After 2--3 weeks of culture, when most of the dividing cells formed cell aggregates, the cultures were softened by diluting with an equal volume of a (40°C) culture medium, containing only 5 rag/1 NAA as hormonal component and supplemented with 0.40/0 agar (Fig. 5). The plated cells were then cultured under the same conditions as before. Minicalli, large enough to be picked up, were transferred to shoot regeneration medium. The first shoot formation was obtained after culturing 10 days on shoot regeneration medium containing zeatin (1 rag/l) and NAA (0.01 rag/l) (Fig. 6). Shoot regeneration fre-

230 q u e n c y w a s a r o u n d 5 0 % ( T a b l e III). T h e w h o l e process from protoplast isolation to plantlet formation took 3--4 months, This process was considerably faster with respect to the one used for the regeneration of plants from m e s o p h y l l p r o t o p l a s t s , in w h i c h c a s e 6 m o n t h s were required. Most calli consistently produced 6--8 shoots. Regenerated shoots were successf u l l y e x c i s e d f r o m t h e c a l l u s a n d r o o t e d in a g a r s o l i d i f i e d M S m e d i u m c o n t a i n i n g lO/o s u c r o s e . These data indicate that protoplasts can be successfully isolated and regenerated from s u s p e n s i o n c u l t u r e s of S o l a n u m t u b e r o s u m cv. Desirde. As a result, this procedure can he used in s o m a t i c h y b r i d i z a t i o n s t u d i e s .

erosum cv. Marls Bard). Plant Sci. Lett., 23 (1981) 8 1 -

3 4

5 6

7

Acknowledgements

8

T h e a u t h o r s w i s h t o t h a n k D r . M. P e z z o t t i f o r his v a l u a b l e s u g g e s t i o n s in s u s p e n s i o n cult u r e s e s t a b l i s h m e n t , D r . S. L u c r e t t i a n d D r . F . Moretti for flow cytometric analysis and Dr. H u r r y d e o U n m o l e f o r h i s h e l p in m a n u s c r i p t p r e p a r a t i o n . A c k n o w l e d g e m e n t is a l s o d u e t o F I C Y T of A s t u r i a s (Spain) f o r p r o v i d i n g a research fellowship to R.J.O.

9 10

11

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