Callus formation and plant regeneration from mesophyll protoplasts of rape plants (Brassica napus L. cv. Zephyr)

P~ant Science Letters, 3 (1974) 265--271 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

CALLUS FORMATION AND PLANT REGENERATION FROM MESOPHYLL PROTOPL/L~TS OF RAPE PLANTS (BRAS,~TCA NAPUS L. CV. ZEPHYR)*

K.K. ~ R T H A * * , M.R. MICHAYLUK, K.N XAO, O.L. GAMBORG and F..CONSTABEL Prairie Regional Laboratory, National Research Council o f Canada, Saskatoon, Sask. STN OW9 (Canada) (Received March 25th, 1974)

SUMMARY

Protoplasts from mesophyll cells of rape plants (Brassica napu~ L. cv. Zephyr) were isolated by enzymatic removal of cell walls in an osmoticum consisting of sorbitol and mannitol. A two-step enzyme *,reatment involving 0.5 % each of Onozuka P1500 cellulase and Rhozyme H,~lS0 hemicellulase followed by 0.5 % e~ch of Driselase and hemicellulase at pH 6.2 resulted in the production of viable protoplasts in 80--90 % yield. The protoplasts were cultured in droplets of B5 medium in petri dishes at a light intensity of 100 lux and 26 °. They were able to regenerate cell walls. Cell division was apparent after 30 h of culturing and by 60 h up to 38 % of the protoplasts had dividea once. Cell clusters were formed within 15 days of culturing. The calli originating from cell clusters were induced to differentiate and to form complete plants.

INTRODUCTION

Plant protoplasts have been employed for the modification of cells by interspecific and intergeneric fusion and by transformation [ 1--7]. The ultimate objective of all these experiments is to grow somatic hybrids. The development of plants from somatic heterokaryons requires methods to regenerate cells from protoplasts and to induce sustained cell division. Callus has been obtained from mesophyll protoplasts of a few plant species only [ 8--12]. The isolation and culture conditions as well as procedures for inducing differentiation and plant development may vary considerably and have to be established for each species. So far, regeneration of complete plants from mesophyll protoplasts is limited to tobacco [9,13], petunia [14] ~nd cladodes of aspara* NRCC No. 14034. ** Guest Research Worker supported by the International.Development Research Centre, Ottawa, Canada. Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA3, gibberellic acid; MS, Murashige-Skoog; NAA, naphthaleneacetic acid.

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gus [15]. Emphasis has been placed on the investigation of the regeneration of plants from protoplasts of crop plants. Though prctoplasts from mesophyll cells of haploid rape (Brassica napua var. Oleifera cv. Canbra) have been isolated, cell wall regeneration and division were not obtained [ 16]. In this communication we report on the procedure for the isolation, culture, production of callus and regeneration of plants from mesophyll protoplasts of diploid Brassica napus L. cv. Zephyr. MATERIAL AND METHODS Leaf material was obtained from rape plants (Brassica napus L. cv. Zephyr) grown from seeds in vermiculite under greenhouse conditions and subjected to a total light intensity of 4500 lux (fluorescent lamps. 16/8 h photo period) throughout their growing period. The temperature and relative humidity averaged 19--21 ° and 40--45 % respectively. Nearly fully expanded leaves from one month old plants were ster~ized in 70 % ethanol for 60 sec and rinsed in 4 changes of sterile, distilled water. All subsequent operations were carried out aseptically m a laminar flow cabinet. The lower epidermis was removed in distilled water. The media employed for the isolation are summarized in Table I. The en zymes were desalted on Sephadex G-25 [ 17]. The pH of the dissolved enzyme mixtures was adjusted to 6.2 with 0.2 N KOH and sterilized through a 0.45 #m mfllipore f~ter. The first isolation medium (2 ml) was transferred to a 60 × 15 mm plastic petri dish to which approximately 150 mg of epidermisremoved leaf material had been added, sealed with Parafflm R, sheltered from light by wrapping the petri dishes with aluminum foil, and maintained at ambiant temperature (19--21 o). After 2 h of digestion, the leaf material was transferred to 2 ml of the second isolation medium. After 3 h, the enzyme-protoplasts mixture was passed through an 88/~ stainless steel sieve to remove undigested material. The protoplzsts were resuspended and washed 4 times by centrifugation (100 × g for 3 min) in 10 ml of culture medium (Table I) devoid of growth hormones, and resuspended in the culture medium containing the growth hormones. The protoplasts at a concentration of 2 . 0 - - 2 . 5 • 10S/ml were cultured in 60 × 15 mm petri dishes each containing 0.5 ml of a suspension of protoplasts in droplets of 50/~l [18]. The petri dishes were sealed with Parafflm R and incubated at 26 ° under diffuse light (100 lux, fluorescent lamps) in a humid cabinet. Fresh culture medium containing the same amount of growth hormones and 2 % sucrose instead of sorbitol and mannitol was added in drople~ (10 % v/v) at weekly intervals. After 15 days the cell clusters were transferred to agar plates of MS medium [ 19] containing vitamins as in B5 medium [20], 10 % coconut milk, 2.3 • 10-6M 2,4-D and 0.1% (w/v) vitamin-free casamino acids (Difco) at pH 5.7. When the size of the callus reached about 5--10 mm they were transferred to MS medium supplemented with different levels of BA or various combinations involving 266

TABLE I CONDITIONS FOR THE ISOLATION AND CULTURE OF RAPE MESOPHYLL PROTOPLASTS Isolation Enzyme Mixture I Enzyme Mixture II

0.5 0.5 0.5 0.5

% Onozuka P 1500a % hemicellulaseb % Driselasec % hemicellulase B5 d

Media Composition: (in g/lO0 ml) Sorbitol Mannitol D-Glucose D-Ribose N-Z-amine (Sheffield) CaCI2 • 2H20 CaH4(PO4)~ • H~O 2,4-D BA NAA pH Calculated osmolality

Culture

4.55 4.55

0.0875 0.0125

4.55 4.55 0.25 0.0125 0.0150 0.0875 2.3 • 10-6M

6.2 0.5

10"6M 10-6M 5.8 0.52

a All Japan Co. Ltd.; b Rohm and Haas of Canada; c Kyowa Hakko Kogyo Co. Ltd.; d Gamborg et al. (1968). The sucrose has been deleted from the medium.

BA and GA3 and cultured in a growth cabinet at a light intensity of 5000 lux (GRO-LUX Wide Spectrum), an alternating light and dark cycle of 20/4 h at 20°/15 ° respectively, and 70 % relative humidity. RESULTS

Only a few protoplasts were produced during incubation with Onozuka and hemicellulase. Protoplast production commenced 30 min after transferring into the second enzyme mixture, and was completed within 3 h by which time more than 90% of the cells had formed protoplasts (Fig.l). The chloroplasts were evenly distributed throughout the cytoplasm anc~ cytoplasmic streaming indicative of cyclosis was observed. The protoplasts isolated at pH 6.2 and 0.5 osmolality showed a better rate of survival than those produced at lower pH and at osmolality values of 0.45 and 0.55. Within 24 h more than 80 % of the protoplasts assumed an oval shape indicative of wall regeneration. Staining with Calcofluor White [ 21] showed that wall formation preceded cell division (Fig.3). The earliest mitosis occurred 267

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Fig. 1--5. Isolation, wall formation, division and cluster formation in protoplasts isolated from the leaf mesophyll cells of rape plants (Brassica napus L. cv. Zephyr). Fig. 1. Freshly isolated protoplasts at the end of a 3 h digestion period in the enzyme m i x t~re [× 525): Fig.2. First division of the cultured protoplast (x 1230). Fi~.3. S.~me as in Fig.2, but labelled with Calcofluor White to demonstrate the wall and O,-~ cross wall formation (x 1,.o0). Fig.4. Second cell division within 6--8 days of culturing (x 2250). Fig.5. Cell clusters obtained within 15 days of culturing and stained with Calcofluor White. Note the wall formation in all cells including the daughter ones (× 192).

after 30 h. In one e x p e r h n e n t 125 o u t of 1223 (10 %) protoplasts s h o w e d a first division within 36 h (Fig.2). A second c o u n t after 60 h s h o w e d t h a t 326 protoplasts o u t of 842 had divided (38.7 %). In m o s t experiments the first cell division required a culturing time of 4 8 - - 7 2 h. The second cell division (Fig.4) occurred after 6--8 days of culture. S u b s e q u e n t l y , the chloroplasts changed from greenish to either pale green or light brown. Clusters of cells were p r o d u c e d within 15 days in all experiments (Fig.5). After 28 days the calli were transferred to MS agar medium containing either BA, or BA and GA3. BA alone at 5 • 10-6M induced the calli to increase in mass and turn green, b u t no shoots were initiated even after pr~ionged periods of culture. In some cases r o o t f o r m a t i o n t o o k place. When GA~ was e m p l o y e d at 10-~M in c o n j u n c t i o n with BA at 5 • 10-~M green s h o o t buds were p r o d u c e d within 10 days. After 25 days the calli with buds were transferred to MS agar medium containing no h o r m o n e s and 7 days later root formation was observed. The s h o o t elongation occurred within 21 days and corn-

Fig.6 One-month-old plant, differentiated from callus of rape mesophyll protoplast~s.

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plete plants regenerated within I month (Fig.6). Calli supplied with BA at 10 -~, 5 - 10 -~, 10 -~ and 10-SM and GA3 at 10 -6, 5 - 10 -6 and 10-SM did not respond to the treatment and shoot differentiation did not occur. DISCUSSION

Our procedures permitted Lhe successful isolation and culture of protoplasts from rape mesophyll, and regeneration of complete plants. The physiological condition of the plant and the leaf material for protoplast isolation, and also the methods for isolation and culturing were critical for achieving cell regeneration and division. The leaves from plants grown at a higher temperature (26 °) and relative humidity (70 %) yielded only a few protoplasts and these failed to survive. The i~neficlal effects of slightly elevated pH on viability and subsequent division is similar to the results reported for bean mesophyll protoplasts [12]. The two-step enzyme treatment gave better surviving protoplasts possibly because the damaged cells resulting from peeling of the epidermis were removed by this step. The regeneration of plants from the callus was influenced by several factors. BA alone did not induce shoot formation although chlorophyll synthesis took place. BA alone induced plant regeneration from rape stem segments [22]. Apparently the origin and type of plant material influence organogenetic reactio~ BA and GAs appeared to have triggered bud initiation which was not observed with BA alone. However, the buds remained dormant as long as the growth regulators were present in the medium. Withdrawal of growth hormones allowed shoot elongation. GAs has been reported to stimulate vascularization and shoot development in callus [23,24 ] and meristem cultures [25,26]. In the present study a stimulatory effect of GAs on shoot formation was observed though shoot elongation was suppressed. It is difficdlt to define whether the suppression of shoot elongation is attributable to GA~ alone or the result of interaction of BA and GAs. Addition of organic substances like coconut milk, yeast extract or adenine or adenine sulfate to the medium containing either BA or BA and GAs did not improve shoot elongation. The root. formation in the callus and plants regenerated occurred without addition of auxins to the medium. Regeneratiori of plants from mesophyll protoplasts of diploid rape plants provides a means of using such protoplasts in somatic cell hybridization by fusion or transformation with the possibilities of hybrid plant regeneration. ACKNOWLEDGEMENTS The authors appreciated the generous gift of seeds of Brassica napus L. cv, Zephyr from Dr. Gary Stringham, Canada Department of Agriculture Research Station, Saskatoon. We wish to thank Mr. J.P. Shyluk, Mr. K. Pahl and Mr. D. Horn for technical assistance and Mr. A. Lutzko for preparing the photographic plate. 270

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