Maataloustieteellinen Aikakauskirja Vol. 63: 371—378, 1991 Protoplast culture and plant regeneration of different agronomically important Brassica species and varieties JANOS PAUK*, SANDOR FEKETE*, JUHA VILKKI** and SEPPO PULLI** * Cereal Research Institute, POB. 391, H-6701 Szeged, Hungary ** Agricultural Research Centre, Institute of Plant Breeding, SF-31600 Jokioinen, Finland Abstract. Protoplast cultures were prepared from 6-day-old hypocotyls of six spring, sev- en winter cultivars of Brassica napus L. and one line of Brassica campeslris L. The molarity of enzyme solution was raised to 0,714 M mannitol resulting in well manipulable, cytoplasm dense protoplasts. In the protoplast purification procedure densitygradient centrifugationwas used to minimize physical damage of protoplasts. Three different protoplast culture systems —(1) liquid, (2) 2nd day embedded, (3) directly embedded in low melting agarose were compared. The two different protoplast embedding techniques resulted in the same efficiency of cell division as the liquid culture method and over this fact the colony browning was avoided. Using protoplast agarose-embedding and culture techniques, healthy calli were obtained for plant regeneration experiments. Incorporation of silver nitrate into the regeneration medium improved the efficiency of plant regeneration in responsive genotypes and the regeneration was induced in three non- responsive (without silver nitrate) genotypes, too. The supplement of silver nitrate in regener- ation medium was especially advantageous in plant regeneration of B. campeslris. Out of fourteen commercial cultivars of Brassica napus and B. campeslris, there is only one recalcitrant genotype in obtaining plantlets from protoplast-derived calli. Index words: protoplast isolation, protoplast culture, silver nitrate, plant regeneration, Brassica napus L, Brassica campeslris L. Introduction Since several of the most widespread methods of introducing foreign genes into plant cells rely on protoplasts, the establish- ment of a reproducible protoplast-plant sys- tem is of a crucial importance in any plant spe- cies of biotechnological interest. The first successful! protoplast isolationand plant regeneration experiments have been per- formed by Wenzel (1973). The Brassica spe- cies vary considerably in their ability to 371 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=U8O8IfEsqgLzTEib.C-ZFi2k_0F23pzDueLCV3A.48TnVGoaIagUjK7Z6Ijmge-DcOpeIRZMjM9JCGRSCFSyKyOssAO6OG60w46DUvlx-EML0E_TBXLfZkzw0NqJMQuxiI5kHp-EWuzQvaaDAytznZQnSQWt75gNoYU_7VrhQLTy6M6ilzjLSuxtYCr_Wv2FxNf5mm-ebO2GLV9LaAiqmM_wS3mLcezMzL7KWiQt2meT9LnqJB4J-aMAW54HTUv6skK0_pe1531pgEDyzyLYGRGLo4gPliZ7htWCclROj2sq0FDzK-UOM9w-M2VG-e3_PYcUvKHgJ2lXLWNWMU4UnXPZqTPWYX5jRrWv regenerate plant from protoplast. While the Brassica napus and the Brassica oleracea (Sidney et al. 1983, Robertson et al. 1984) generally give good results in protoplast re- generation experiments (Kartha et al. 1974, Li et al. 1982, Glimelius 1984), in case of turnip rape (B. campestris, B. rapa) the plant regeneration from protoplast derived calli re- mained a sporadic event (Glimelius 1984, Ulrich et al. 1980). The aim of the present study was to develop a largely genotype independent, efficient plant regeneration procedure for B. napus L. and B. campestris L. with agronomical impor- tance. Material and methods To extend the protoplast-plant system to different genotypes of agronomic importance various spring and winter type varieties were included in the study. Spring cultivars of Bras- sica napus L. spp. oleifera cv. Hanna, Kun- to, Olga, Omega, Varma, Westar and winter cultivars Arabella, Belinda, Danubia, Dar- mor, Santana, Ujfertodi, Viking were tested. One Brassica campestrisL. ssp. oleifera breed- ing line Jo. 4001 was used, too (Table 2). The data were calculated from 2 or 3 in- dependent experiments. For protoplast isolation, the seeds were sur- face sterilized with 75 % alcohol for I—21 —2 minutes, then immersed in 2 % NaO’Cl with some drops of Tween 80 and continually shaken for 20 minutes followed by a thorough wash in autoclaved sterile distilled water. Sterilized seeds were germinated on a hormon- free MS medium (Murashige and Skoog 1962) for 5—6 days in darkness at 28°C. The 5—6 cm long hypocotyls were cut into small pieces of about 1.0 mm and treated for 17—18 hours (generally overnight) with 1 % CellulaseR-10 and Macerozyme R-10 enzymes dissolved in K 3 (Nagy and Maliga 1976) medium. The osmotic pressure of the enzyme mixture was raised to 0.714 M using mannitol as osmotic stabilizer. This treatment resulted in a great number of comparatively small pro- toplasts with dense cytoplasm. The filtration of the isolated protoplasts was omitted to min- imize physical damage. The enzyme mixture together with the protoplast and the hypocotyl debris were diluted with W 5 salt solution (Menczel et al. 1981) to 5—6 times the origi- nal volume and centrifuged for 5 minutes at 120 g. The supernatant was discarded and the protoplasts, mixed with debris, were suspend- ed in 0,6 M sucrose and W 5 was layered on the top, then centrifugated at 120 rpm for 5 minutes. The floating protoplasts were collect- ed by Pasteur pipette and washed twice with W 5 solution. The isolated protoplasts were cultured in 0,4 M K-75 medium (Kao and Michayluk 1975) modified by Glimelius (1984), at 28°C in dark. Protoplasts were cul- tured at a density of 6 X 104 in three differ- ent culture systems: (1) liquid medium, (2) embedded on the 2nd day from liquid into agarose and (3) embedded into agarose direct- ly after isolation. The embeddedcultures were solidified by 0,8 % low melting agarose (Sigma type VII). After about 10 days the liq- uid and agarose embedded cultures were fed with liquid medium with reduced osmoticum (3 % sucrose). The cell aggregates were ready to plate from the beginning of the third week. The liquid cultures were plated on agar solid- ified regeneration medium and the agarose blocks were floated in liquid medium before plating. The hormones were decreased to quater strength in the nutrient and floating medium as described by Vamling and Glime- lius (1990). The plant regeneration was carried out on K 3 medium supplemented with 0.5 mg/1 BAP, 0,5 mg/1 Zeatin and 0,1 mg/1 lAA (Nagy and Maliga 1976, Vamling and Glimelius 1990). To increase the efficiency of plant regenera- tion 5 mg/1 AgNG 3 was added to the re- generation medium. Regenerants were grown up with space isolation in different greenhouse chambers. Results In each variety the protoplasts (Fig. la) 372 were isolated from hypocotyl. The relatively high osmotic pressure (0,714 M) results in small, cytoplasm-dense protoplasts. Small, cytoplasm-rich (Fig. lb) protoplasts were well manipulable during isolation procedure and proved to be excellent for different culture procedures. The culture methods applied resulted in difference in initiation of cell division at 24- hour age (Table 1). Protoplasts cultured in liq- Fig. I. Development of Brassica protoplast-callus system using agarose embedding technique with ’Varma’ cv. a. Freshly-isolated protoplasts collected from the interphase ofgradient centrifugation. Bar = 40 pm. b. Cytoplasm- rich protoplast embedded in agarose solidified medium. Bar = 40 pm. c. Freshly divided protoplast embedded into agarose from liquid medium on the second day of culturing. Bar = 40 pm. d. Second cell division within 4 days of culturing. Bar = 40 pm. e. Cell clusters obtained within 40 days of culturing. Bar = 0,1 mm. f. Protoplast-derived colonies of ’Varma’ cultivar in agarose-solidified medium and agarose blocks floated in liquid medium. Magnifica- tion = 2x. 373 Table I. Percentage of dividingcells using three different culture methods in protoplast cultures of three B. napus cv. The numbers of division (in percent) were counted after 24-, 48- and 96 hours. Genotype Culture Percentage of dividing method* cells after 24 48 96 hours hours hours liquid 1,1 18 48 emb. 2nd d. 1,0 14 46 emb. direct 0 16 47 Olga liquid 2,3 12 51 Varma emb. 2nd d. 2,4 14 53 emb. direct 0,1 10 49 liquid 0,7 9 44 Arabella emb. 2nd d. 0,9 7 42 emb. direct 0 7 45 * See in Materials and methods uid medium were able to undergo divisions from the end of the Ist day depending on cul- ture methods. Protoplasts embedded directly after isolation into agarose were capable to divide from the 2nd day of culturing showing visible divisions at 48th hour (Fig. 1c). The agarose technique seems to be a hindering fac- tor in the starting of cell division, but this hin- dering effect disappeared on the fourth day of culturing. There was no significant differ- ence in percentage of dividing cells obtained from different varieties using different culture techniques on the fourth day of culturing (Ta- ble 1). Each variety produced about the same percentage of divisions on the fourth day (in 96 hours) of subculturing. Second divisions (Fig. Id) were observed from the third day of subculturing. Among the varieties, Varma was the most responsive in the efficiency of cell division at the end of the 4th day of culture, but in com- mencement of microcolony development there was no substantial difference between culture methods regarding the percentage of devel- oped colonies. Observing of cell division after 96 hours of culturing was easier in agarose embedded cul- tures because the well-spread cells, protoplast- derived colonies in agarose were in fixed po- sitions while in liquid medium the cells were aggregated spontaneously and it was difficult to distinguish between division-derived micro- colonies and spontaneous cell groups. After about ten days in cultures small calli or micro colonies (Fig. le) had developed in both culture systems. There was an essential difference between liquid culture technique and agarose solidified culture. In the liquid cultures a brown precipitation started to de- velop around microcalli from the second week. This phenomenon, observed by other authors as well (Schenck and Hoffman 1979, Glimelius 1984), was detrimental to the sub- sequent culture work, inhibiting or completely stopping the growth. This harmful phenome- non was not observed in agarose solidifiedcul- tures (Fig. If) indicating more favourable out- come when using agarose-embedding in Bras- sica protoplast culture. Except for one variety Ujfertddi —, ev- ery variety in both Brassica napus and cam- pestris formed a higher number of calli for plant regeneration experiments (Table 2). At the end of 2 nd and 3rd week, the number of formed microcalli varied, depending on in- dividual cultures and genotypes, but no essen- tial differences were found between cultivars. In most cases white calli with green or greenish morphogene structures (Fig. 2, indi- cated by arrows) were obtained on the regener- ation medium but the efficiency of plant regeneration remained low (Table 3). Initial- ly, only in ten of fourteen Brassica genotypes plant regeneration was successful. The re- generants (Fig. 3) were obtained from green organogenetic centres on the surface of white calli, and generally one or two shoots were ob- tained from one callus (Fig. 3). Consequent- ly, to test its effect on regeneration, AgNQ3 was added to the regeneration medium. In the presence of 5 mg/I AgN0 3 the regeneration efficiency was substantially increased includ- ing multiple shoots (Fig. 4 and 5) in the posi- tive cases. Out of the fourteen Brassica geno- types studied, thirteen gave plantlets with varying numbers of regenerants (Table 3). This modified regeneration procedure re- 374 375 Table 2. Summary of results in protoplast isolation, culture, callus induction, plant regeneration and source of geno- types used in investigations. Species, variety and Life form Protoplast Plant regene- source ~ ' ; I ' ; " ~ ration isolation culture derived calli B. napus Olga S spring > Varma SF » > Westar CAN » > Kunto S » > Hanna S » > Omega S » > •> Viking DK winter > Arabella D » > Darmor F » > Santana D » > Danubia H » > > Belinda D » > Ujfertddi H » > B. campestris Jo. 4001 SF spring > •> > without silver nitrate in regeneration medium ■> with silver nitrate in regeneration medium generation medium supplemented with AgNG3 has a particularly advantageous effect on regeneration of B. campestris which has been Table 3. The effect of silvernitrate (AgNO,) on plant re- generation from protoplastderived calli of B. napus and B. campestris varieties. Name of genotype % of shoot regenerating and life form calli —AgNO, + AgNO, B. napus Olga *s. 24 41 Varma s. 31 39 Westar s. 19 33 Kunto s. 11 29 Hanna s. 9 19 Omega s. 0 4 Viking **w. 9 23 Arabella w. 10 17 Darmor w. 4 14 Santana w. 6 10 Danubia w. 0 8 Belinda w. 2 6 Ujfertddi w. 0 0 B. campestris Jo. 4001 s. 0 22 • s = spring, ** w = winter Regeneration without AgNOj(—) with AgN03( + ) known to respond poorly to in vitro manipu- lation. The regenerated plantlets were transferred to K 3 medium without AgN0 3 which al- lowed the plantlets to develop rapidly. Discussion The molarity of enzyme solution was raised from 0,4 M sucrose suggested by Glimelius (1984) and Vamling and Glimelius (1990) to Fig. 2. Protoplast-derived calli with morphogene struc- ture on their surface (arrows) on plant regeneration me- dium without silver nitrate. 0,714 M mannitol. We thus used higher molarity as Kohlenbach et al. (1982) who used 0,55 M and 0,65 M mannitol for leaf and stem embryo protoplast isolation, respective- ly. Eapen et al. (1989) obtained good results using 0,6 M mannitol in enzyme solution in mesophyll protoplast isolation of Brassica juncea. The cytoplasm dense smallprotoplasts proved to be well manipulable during isola- tion procedure. They will be an essential ad- vantage in direct protoplast transformation experiments where protoplasts are manipulat- ed washing etc. several times. The agarose embedding culture technique developed by Shillito et al. (1983) has proven to be a successful method in Brassica also. Our results confirmed the applicability of the embedded protoplast culture technique (directly or on the 2nd day of subculturing) which produced the same division results as the liquid culture method (basic culture meth- od) published for Brassica protoplast culture (Bidney et al. 1983, Glimelius 1984, Jourdan et al. 1989, Loudon et al. 1989). This tech- nique has been already used for 2—3- week old colony embedding by Vamling and Gli- melius (1990) resulting in a good culture practice, but our results (early embedding af- ter isolation) show other advantages over this culture technique, too. The colony browning, Fig. S. Typical rapeseed white callus with one shoot on regeneration medium without silver nitrate. Fig. 5. One shootlet on surface of green callus develo- ping on AgNOi supplemented regeneration medium. Fig. 4. Well differentiated green protoplast-derived cal- lus with multiple shoot primordia (arrows) on regenera- tion medium supplemented with 5 mg/1 AgN03 . 376 widely reported to be a feature of Brassica protoplast culture (Xu et al. 1982, Glimelius 1984, Loudon et al. 1989) was prevented and healthy calli were induced for plant regenera- tion experiments. One of the noticeable features of this study is the wide range of responses observed (Ta- ble 2). These results show, in accordance with other studies (Jourdan and Earle 1989, Lou- don et al. 1989) that various Brassica varie- ties of great economic importance can be suc- cessfully regenerated from protoplasts. There was only one (Ujfertodi) out of the fourteen varieties tested that failed to produce plant- lets. Incorporation of silver nitrate (5 mg/1) into the regeneration medium improved the effi- ciency of plant regeneration. With the in- troduction of AgNOj three of the examined varieties became responsive in regeneration ex- periments (Table 3) and regeneration was more efficient in each variety. There is only one recalcitrant genotype Ujfertodi in our experiments (Table 2 and 3). In agreement with our results, the positive effect of AgNQ 3 on the induction of plant regeneration has been recently published by other authors as well (Lentini et al. 1988, Songstad et al. 1988) in Brassica and in other crop plants (Purnhauser et al. 1987). Our results support the findings of Purn- hauser et al. (1987) and Williams et al. (1990) that AgNQ 3 has the most favourable effect on plant regeneration when applied at a later stage of callus development (when mor- phogene zones had already been formed) rath- er than right from thebeginning of callus in- duction. Acknowledgements. The authors are thankful to Miss Outi Manninen, Mrs. Maija-Liisa Penttila, Mrs. lldiko D. Bartok, for their skilful assistance and Mr. B. Dusha and Mr. B. Kdrmdn in preparing the photographic ma- terial. They also wish to thank Mr. L. Nagy for kindly supplying one part of seed material. This work was sup- ported by J. Pauk’s 6-month fellowship in Finland. References Sidney, D.L., Shepard, J.F., Kaleikau, E. 1983. Re- generation of plants from mesophylle protoplasts of Brassica oleracea. Protoplasma 117: 89—92. Eapen, S., Abraham, V., Gerdemann, M., Schieder, O. 1989. Direct somatic embryogenesis, plant regenera- tion and evaluation of plants obtained from mesophyll protoplasts of Brassica juncea. Annals of Botany 63, 369—372. Glimelius, K. 1984. High growth rate and regeneration capacity ofhypocotyl protoplast in some Brassicaceae. Physiol. Plant. 61: 38—44. Jourdan, P.S. Earle, E.-D. 1989. Genotypic varia- bility in the frequency of plant regeneration from leaf protoplasts of four Brassica spp. and of Raphanus salivas. J. Amer. Soc. Hort. Sci. 114 (2): 343—349. Kao, K.N. Michayluk, M.R. 1975. Nutritional re- quirements for growth of Vida hajaslana cells and protoplasts at very low population density in liquid media. Planta 126: 105—110. Kartha, K.K. Michayluk, M.R. Kao, K.N. Gamborg, O.L. Constable, F. 1974. Callus for- mation and plant regeneration from mesophyll pro- toplast of rape plants (Brassica napuscv. Zephir) Plant Sci. Lett. 3: 265—271. Kohlenbach, H.W., Wenzel, G., Hoffmann, F. 1982. Regeneration of Brassica napus plantlets in cultures from isolated protoplasts of haploid stem embryos as compared with leaf protoplasts. Z. Pflanzenphysiol. Bd. 105. S. 131—142. Lentini, Z. Mursell, M.A. Mutschler, M.A. Earle, E.D. 1988. Ethylen generation and reversal of ethylene effects during development in vitro of rapid- cycling Brassica campestris L. Plant Sci. 54: 75—81. Li, L.C. Kohlenbach, H.W. 1982. Somatic embryo- genesis in quite a direct way in cultures of mesophyll protoplasts of Brassica napus L. Plant Cell Rep. 1: 209—211. Loudon, P.T. Nelson, R.S. Ingram, D.S. 1989. Studies of protoplast culture and plant regeneration from commercial and rapid-cycling Brassica species. Plant Cell Tiss. Org. Cult. 19: 213—224. Murashige, T. Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cul- tures. Physiol. Plantarum 15: 473—497. Nagy, J. Maliga, P. 1976. Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana silveslris. Z. Pflanzenphysiol. 78: 453—455. Purnhauser, L. Medoyesi, L. Czak6, M. Dix, 377 P.J. Marton, L. 1987. Stimulation of shoot regeneration in Triticum aeslivum and Nicotiana plumbaginifolia Viv. tissue culture using the ethylen inhibitor AgNG3 Plant Cell Rep. 6: I—4. Robertson, D. Earle, E.D. Mutschler, M.A. 1984. Enhanced rates ofplant regeneration from pro- toplasts of cultivar Green Comet Broccoli Brassica oleracea. Plant Physiol. 75: 134. Schenk, H.R., Hoffmann, F. 1979. Callus and root regeneration from mesophyll protoplasts of basic Brassica species: B. campestris, B. oleracea and B. nigrra. Z. Pflanzenziichtg. 82: 354—360. Shillito, R.D., Paszkowski, J., Potrikus, I. 1983. Agarose plating and bead type technique enable and stimulate development of protoplast-derived colonies in a number of plant. Songstad, D.D. Duncan, D.R. Widholm, J. M. 1988. Effect of 1-aminocyclopropane-l-carboxylic acid, silver nitrate and norbornadiene on plant re- generation. Plant Cell Rep. 7: 262—265. Ulrich, T.H. Chowdhury, J.B. Wildholm, J.M. 1980. Callus and root formation from mesophyll pro- toplasts of Brassica rapa. Plant Sci. Lett. 19; 347 —354. Vamling, K., Glimelius, K. 1990. Regeneration of plants from protoplasts of oilseed Brassica crops. In: Bajaj (ed.); Biotechnology in Agriculture and Forestry. Vol. 10. 385—417. Springer-Verlag, Heidelberg. Wenzel, G. 1973. Isolation of leaf protoplasts from haploid plants of petunia, rape and rye. Z. Pflanzen- ziich. 69: 58—61. Williams, J. Pink, D.A.C. Biddington, N.L. 1990. Effect of silver nitrate on long-term culture and regeneration of callus from Brassica oleracea var. gem- mifera. Plant Cell Tissue Org. Cult. 21: 61—66. Xu, Z.H., Davey, M.R., Cocking, E.C. 1982. Plant regeneration from root protoplasts of Brassica. Plant Sci. Lett. 24: 117—121. Ms received June 9, 1991 SELOSTUS Maataloudelle tarkeiden Brassica-lajien protoplastien viljely ja kasvien regenerointi Janos Pauk*, Sandor Fekete*, Juha Vilkki** and Seppo Pulli** • Cereal Research Institute, Pob. 391, H-6701 Szeged, Hungary ** Agricultural Research Centre, Institute of Plant Breeding, SF-31600 Jokioinen, Finland Tutkimuksessa eristettiin protoplasteja kolmentoista rapsilajikkeen (Brassica napus) ja yhdenkevatrypsilinjan (B. campestris) 6 vrk:n ikaisista hypokotyyleista. Protoplastien eristyksessa kaytettiin tavanomaista kor- keampimolaarista entsyymiliuosta (0.714 M mannitoli). Main saatiin pienia, kestavia, runsaasti solulimaa sisal- tavia protoplasteja. Protoplastien vaurioitumisen valtta- miseksi eristyksessa kaytettiin tiheysgradienttisentrifugoin- tia. Tutkimuksessa verrattiin kolmea eri protoplastivilje- lymenetelmaa: 1)nesteviljely, 2) maljaus agaroosiin toi- sena viljelypaivana, 3) suora maljaus agaroosiin. Agaroo- siviljelmissa solut jakautuivat yhta aktiivisesti kuin nes- teviljelyssa, lisaksi valtyttiin solukolonioiden ruskettumi- selta. Hopeanitraatin (AgNG3) kaytto regeneraatioalustassa (5 mg/1) lisasi kalluksesta muodostuvien versojen maa- raa niilla 10 lajikkeella, joilla havaittiin vahaista regene- raatiota jo ilman hopeanitraattia. Kolmella lajikkeella re- generaatio onnistui ainoastaan silloin kuin regeneraatio- alustassa oli hopeanitraattia. Erityisen suotuisa vaikutus hopeanitraattilisayksella oli kevatrypsin regeneraatioon. 378