Time saving method for protoplast isolation, transformation and transient gene expression assay in barley Barnabäs Jenes, Matti Puolimatka, Pedro Bittencourt and Seppo Pulli Jenes, B.', Puolimatka, M. 2 , Bittencourt, P. 1 & Pulli, S. 2 1994. Time saving method for protoplast isolation, transformation and transient gene expression assay in barley. Agricultural Science in Finland 3: 199-205. (‘lnstitute for Plant Sciences, Agricultural Biotechnology Center, Gödöllö, P.0.80x 170, H-2100 Hun- gary and Agricultural Research Centre of Finland, Institute of Crop and Soil Science, Plant Breeding Section, FIN-31600 Jokioinen, Finland.) This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutel- lum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W 5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene gly- col) method. Plasmid pActl-F containing the rice Actl promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transfor- mation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligi- ble time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture. Key words: Hordeum vulgare L., plasmid, gene transfer, (I-glucuronidase Introduction During the last decade much interest has been focused on the culture of plant protoplasts and their potential in producing transgenic plants as well as in assaying the expression of recombinant gene constructs in transformed cells. The use of protoplasts provides the advantages of easier up- take of foreign DNA by the target cell in the absence of the cell wall and also single cell ori- gin of regenerants thus avoiding chimerism in transformants. Considerable progress has been made in protoplast culture of important monocot species and the first protoplast derived regenera- tion in cereals was reported in rice (Oryza saliva L.) by Fujimura and his group (1985). Succesful protoplast-plant regeneration systems have also been reported in wheat (Vasil et al. 1990, Chang et al. 1991, Yang et al. 1993, Ahmed and SAgi 1993, Pauk et al. 1994), maize (Mörocz et al. 199 Agricultural Science in Finland 3 (1994)Research Note https://www.c-info.fi/en/info/?token=XLFuR93EFmJw2E_X.bmKM1pjy-oyFc5761tKV9g.8Q_fDWjvcvXTWUjwDBHXmFfxFuXNY_O1rrS0QRR6MmJkzNcbyPq8PJGlhmXRtb_rfB_opJTs7ZoLSACeYCVBRSPndJjyvU6g_Va8lrCeTFDMQFE7wlOSFuyfJgUQD5_DjPp5Z8pErXmADz2SeCP9d6jT5wp7ZcS-GoF6oK9VMg12djXpeDbeVAqYUXz6EiHnl6NVdtU7cRvIlUDKu6FFdhBoSyWqCX9aa9-Rcd_PjHjvloH3feIAtDLpF8l-SU9Rgu9z6AMk5yh8rS4hpp04MtRxCbaJwW52fPjONzF2XHdC61XwfOia7SfRhHp5K_fJMJKb09V0-CUIxE7asjPU Research Note 1990) and barley (Jähne et al. 1991). Improve- ments in protoplast culture and regeneration have significantly given a contribution to the genetic transformation of monocots. In rice, the transfor- mation of protoplasts and the subsequent regen- eration of transgenic rice plants have become a routine procedure (Jenes et al. 1992). Protoplast transformation resulting in stable integration of foreign genes into the target cells has also been reported in other cereals such as rice (Shimamo- to et al. 1989), maize (Mörocz et al. 1990) and barley (Lazzeri and Lörz 1990, Lazzeri et al. 1992). In barley, most of the reported studies in pro- toplast isolation and transformation have so far been based on suspension derived protoplasts (Luhrs and Lörz 1988, Lazzeri and Lörz 1990, Jähne et al. 1991, Lazzeri et al. 1992). Junker et al. (1987) detected transient expression of NPT II (Neomycin phosphotransferase II) gene in pro- toplasts derivedfrom suspension culture and trans- formed with PEG. The initiation and maintenance of fine suspension cultures, a prerequisite for suc- cessful protoplast isolation in barley as well as in other species, is laboriuos and may take a con- siderably long time from some weeks to several months. It can therefore be a limiting factor in applying barley protoplasts to transient gene ex- pression studies. In addition, a long lasting pre- culture phase before the transformation may re- sult in undesirable somaclonal variation at the level of transgenic regenerants. Therefore the use of protoplasts in transformation experiments would benifit from a procedure that would re- duce the time in culture prior to the gene trans- fer. Diaz and Carbonero (1992) investigated tis- sue specific transient expression of the gus re- porter gene in transformed barley protoplasts iso- lated from developing endosperm. There are re- ports on the isolation of barley aleurone proto- plasts for transient expression studies (Skriver et al. 1991). This isolation procedure, however, requires very specific skills. In this paper we in- troduce a quick method to isolate barley proto- plasts directly from dissected mature scutellum- embryo complexes and the use of such proto- plasts in transient gene expression studies. This isolation method was found to be equivalent in efficiency to the transformation of protoplasts de- rived from cell suspension but the time require- ment was negligible compared to the suspension cultures. Material and methods Plant material Dry seeds of barley cultivars ‘Pohto’, ‘Kymppi’, ‘Prisma’ and Tgri’ were provided by the Institute of Plant Breeding, Agricultural Research Centre ofFinland, Jokioinen. Protoplast isolation Dry seeds of barley were surface sterilized in 0.01% solution of mercuric chloride and then washed and rinsed with sterile distilled water for seven times. After sterilization the seeds were imbibed in sterile distilled water and incubated in 25°C for 24-36 hours. The imbibed embryo- scutellum complexes were excised from the seeds and chopped into small species with a scalpel (Fig. 1). The following three enzyme mixtures were originally used for the digestion of proto- plasts: 1) JE enzyme mixture modified after Junker et al. (1987) containing 1.0% Onozuka RS Cel- lulase, 0.5% Macerozyme R 10 (Serva), 0.05% Pectolyase Y23 (Seishin), 5 mM CaCl,, 0.5 mM Na 2 HP04, at pH 5.8, 2) KE enzyme mixture modified after Ahmed and Sägi (1993) containing 2% Onozuka RS Cel- lulase, 0.5% Driselase, 0.1% Pectolyase Y-23, 6.5% Glycerol, 1.0% CaCl„ 0.1% MgS04 and 0.05% KH,P0 4, 3) LLE enzyme mixture modified after Luhrs and Lörz (1988) containing 1% Onozuka RS Cellulase, 0.5% Macerozyme RlO, 0.1% Pec- tolyase Y-23 and 0.1% Casein hydrolysate. The protoplasts were kept in the enzyme at 25°C for 3 to 6 hours. The protoplast were then 200 Agricultural Science in Finland 3 (1994) Agricultural Science in Finland 3 (1994) cleaned by sieving the mixture through a plastic mesh with pores of 100 pm in diameter and cen- trifuged at 800 rpm for 5 min. Protoplasts were resuspended in 2 ml W 5 washing solution (Menc- zel et al. 1981) and the suspension was then laid on the top of 0.6 M sucrose solution. After 5 min of centrifugation at 800 rpm the protoplasts were collected from the interphase. The cleaning was finished by washing the protoplasts twice with W 5 solution. Plasmids Plasmid pActl-F (McElroy et al. 1991) was pro- vided by the courtesy of Professor Ray Wu, Cor- nell University, Ithaca, NY. This plasmid includes therice Actin 1 gene 5’ regulatory elements linked to the gus gene coding sequences (synonym uidA, codes for (3-glucuronidase enzyme) from E. coli and the nos polyadenylation site from Agrobacte- rium tumefaciens. Transformation of protoplasts The number of protoplasts suspended in the W5 solution was estimated in a Buerker chamber and then divided into 1 x 106 aliquots in plastic Wassermann tubes. After centrifugation at 800 rpm for 5 min the supernatant was removed and the pellet was resuspended in 1 ml MgMa trans- formation buffer (Zhang et al. 1991). Twenty mg of uncut pActl-F plasmid and 100 pg of Herring Testis DNA (SIGMA, ruptured by sonication) were added to the protoplast suspension. After 5 min of gentle handvortexing 1 ml of 30% solu- tion of PEG (Polyethylen Glykol, fw 3450, SIG- MA) solution was added (Zhang et al. 1991). The protoplasts were incubated with the PEG for 28 min. During this time the suspension was gen- tly shaken by hand in every 5 min. At the end of the incubation time the mixture was slowly diluted with W 5 solution up to 10 ml within 5 min, adding the W 5 solution drop by drop and mixing. The diluted mixture then was centrifuged and the protoplasts washed twice in W 5 solution. Culture of protoplasts The washed and pelleted protoplasts were resus- pended in 1 ml of KPR protoplast culture medi- um (Thompson et al. 1986) and placed into a 24 well sterile plate (CORNING) so that 250 pi of suspension was transferred into each well. The plate was sealed with PARAFILM and incubated at 25°C in dark until the GUS assay. Detection of transient expression ofgus gene Samples of control and transformed protoplast cultures were placed into a 96 well sterile plate (CORNING), 100 pi of culture into each well. Forty pi of X-Gluc staining solution (Jefferson et al. 1986, Jefferson 1987) was added into each well. The plates were incubated at 29°C for 12 hours before the visual counting of the cells show- ing transient expression was carried out. Results and discussion Protoplast isolation Successful protoplast isolation was achieved from the mature scutellum-embryo complexes of each of the four genotypes used in this study (Fig. 2). In our first experiments three different digestion enzyme mixtures were tested, each of which were modified from the original references. The re- sults showed the superiority of the JE enzyme mixture in protoplast yielding (Table 1). Conse- Table 1. Differencies in the effectiveness of the three en- zyme mixtures applied to protoplast isolation from em- bryo-scutellum complexes of barley ‘Kymppi’. Yield of protoplasts /Enzyme mixture 100 embryos 2.2 x 106 1.2 x 10" 0.2 x 106 JE enzyme mixture 1 KE enzyme mixture2 LEE enzyme mixture 3 1 modified after Junker et al. 1987. 2 modified after Ahmed and SAgi 1993. 3 modified after Luhrs and LOrz 1988. 201 Research Note Agricultural Science in Finland 3 (1994) Table 2, Average yields of protoplasts of the four different barley cultivars Kymppi, Prisma, Pohto and Igri. Diges- tion was performed by the JE enzyme mixture'. Number of protoplasts /Name of genotype 100 embryos 2.2 x I06 4.1 x 106 2.5 x 106 0.6 x 106 Kymppi Prisma Pohto Igri 1 modified after Junker et al. 1987. quently, this enzyme mixture was chosen for the later experiments. The four barley genotypes tested in this study showed differences in protoplast yielding and characteristics. In the digestion experiments as much as x 106 protoplasts per 100 dissect- ed embryo-scutellum complexes were obtained. The protoplast yield seemed to be dependent on the genotype and the enzyme mixture used (Table 2). The genotypes differed also in the size of released protoplasts. When using the JE en- zyme digestion, Pohto provided bigger protoplasts of 50-80 |im in diameter than any of the other three cultivars (20-30 (xm in diameter). During the digestion of embryos from the ma- ture embryo-scutellum complexes it was visually estimated that about 80% of the protoplasts were released from the scutellar tissue. These proto- plasts started cell division 3-5 days after isola- tion and transformation (Fig. 3) and continued further forming 20-30 celled aggregates. This ob- servation encourages us towards our further goals to obtain regenerated transgenic plants from these transformed protoplasts. It is well known from the earlier studies that the scutellar tissue in mono- cot species has a great regeneration capacity (Fu- jimura et al. 1985). Protoplast transformation and transient gene expression Protoplasts isolated directly from scutellum-em- bryo complexes were succesfully transformed with the PEG method showing transient expression of the reporter gene under the control of the rice Actinl promoter, similarly to the results in rice transformation experiments (McElroy et al. 1991).This became evident after treating the trans- formed cells with X-gluc. Some of the cells showed the typical blue colour resulting from the reaction between the enzyme and its substrate (Fig 4.). The frequency of transformed cells was estimated by visual examination. The frequency of the transformation events that showed tran- sient expression of the gus gene was estimated to be in some cases up to 6% of the isolated and transformed protoplasts. In plant molecular biology research cell sus- pension is the most common source of barley protoplasts used in the investigation of new gene constructs. The establishment of a suspension- protoplast system in monocots is a time consum- ing process (Jähne et al. 1991) taking three to six months of culture until the first protoplasts can be digested and transformed (Junker et al. 1987). To reduce this time, our aim was to set up a rapid system for producing viable protoplasts suitable for transformation and transient gene ex- pression studies. The embryo-scutellum derived protoplast system seemed to fullfil these require- ments. Only 24 hours was needed prior the pro- toplast isolation and transformation instead of sev- eral months of subculturing. This system was also suitable for obtaining 4.5% of the transformed cells showing transient gene expression. The time needs of the protoplast isolation and transformation system presented in this paper is comparable to the transformation with particle bombardment but it still has all the advantages of the protoplast system. For example, as protoplast derived regenerants have a single cell origin, one can expect genetic uniformity in the whole plant after regeneration from transformed protoplast which is not always the case after regeneration from the bombarded material. Further refinary of this method to isolate barley protoplasts directly from mature embryo-scutellum complexes is need- ed to improve the yield of protoplasts and the induction level of cell divisions. A working and repeatable protoplast-plant regeneration system based on a quick protoplast isolation could pro- 202 Research Note Research Note vide with the ability to produce fertile transgenic plants of barley within much shorter time than through the suspension-protoplast-transgenic plant system. Acknowledgements. The authors are grateful to the Agri- cultural Research Centre of Finland, Jokioinen, Finland, and Agricultural Biotechnology Centre, Gödöllö, Hunga- ry, and to the Ministry of Agriculture and Forestry of Finland for providing the facilities and finance for this study. References Ahmed, Z. K. & SÄGI, F. 1993. Culture of and fertile plant regeneration from regenerable embryogenic sus- pension cell-derived protoplasts ofwheat (Triticum aes- tivum L.). Plant Cell Reports 12: 175-179. Chang, Y.-F., Wang, W.Ch., Warfield, C.Y., Nguyen, H.T. & Wong, J.R. 1991. Plant regeneration from pro- toplasts isolated from long-term cultures of wheat (Triti- cum aestivum L.). Plant Cell Reports 9: 611-614. Fig. 1. Excised mature barley scutellum-embryo complex after 24 hours of imbibition in distilled, sterilized water. Scale bar = I mm. (Photo: Matti Puolimatka) Fig. 2. Barley protoplasts isolated from the mature scutel- lum-embryo complexes of ’Kymppi’. (Photo: Barnabäs Jenes) Fig. 3. Cell divisions in barley protoplasts. (Photo; Bama bäs Jenes) Fig. 4. Blue colour shows transient Gus gene activity in the transformed protoplasts of barley. (Photo: Barnabäs Jenes) 203 Agricultural Science in Finland 3 (1994) Research Note Diaz, I. & Carbonero, P. 1992. Isolation of protoplasts from developing barley endosperm: a tool for transient expression studies. Plant Cell Reports 10: 595-598. Fujimura, T., Sakurai, M., Akagi, H., Negishi, T. & Hi- rose, A. 1985. Regeneration of rice plants from proto- plasts. Plant Tissue Culture Letters 2: 74-75. Jähne,A., Lazzeri, P. A. & Lörz, H. 1991. Regeneration of fertile plants from protoplasts derived from embry- ogenic cell suspension of barley ( Hordeum vulgare L.). Plant Cell Reports 10: 1-6. Jefferson, R. A. 1987.Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular and Bi- ological Reports 5: 387-405. -, Burgess, S. M. & Hirsh, D. 1986. p-glucuronidase from Escherichia coli as a gene fusion marker. Pro- ceedings of the National Academy of Sciences. USA. 83:8447-8451. Jenes, 8., Moore, H., Zhang, W., Cao, J. & Wu, R. 1992. Techniques for gene transfer. In: Kung, S.-D. & Wu, R. (eds.). Transgenic Plants, Vol. I. Academic Press, Inc., San Diego, California, p. 125-147. Junker, 8., Zimny, J., Luhrs, R. & Lörz, H. 1987. Tran- sient gene expression of chimeric genes in dividing and non-dividing cereal protoplasts after PEG-induced DNA uptake. Plant Cell Reports 6: 329-332. Lazzeri, P. A., Brettschneider, R., Luhrs, R. & Lörz, H, 1992. Stable transformation of barley via PEG-in- duced direct DNA uptake into protoplasts. Theoretical and Applied Genetics 81: 437-444. - & Lörz, H. 1990. Regenerate suspension and proto- plast cultures of barley and stable transformation via DNA uptake into protoplasts. In; Lycett, G. W. & Gri- erson, D. (eds.). Genetic engineering of crop plants. Butterworth. London, p. 231-238, Luhrs, R. & Lörz, H. 1988. Initiation of morphogenic cell suspension and protoplast cultures of barley (Hor- deum vulgare L.). Planta 175: 71-81. McElroy, D„ Blowers, A., Jenes, B. & Wu, R. 1991. Construction of rice actin-1 (Actl-) based expression vectors for use in monocot transformation. Molecular and General Genetics 231; 150-160. Menczel, L., Nagy, F., Kiss, ZS. R. & Maliga, P. 1981. Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum tobacco + Nicoliana knightiana: correlation of resistance to Nicoliana tabacum plas- tids. Theoretical and Applied Genetics 59: 191-195. Mörocz, S., Donn, G., Németh, J. & Dudits, D. 1990. An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture. Theoretical Applied Genetics 80: 721-726. Pauk, J., Kertesz, Z., Jenes, 8., Purnhauser, L., Man- ninen, 0., Pulu., Barabas, Z. & Dudits, D. 1994. Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture. Plant cell, tissue and organ culture. In press. Shimamoto, K., Terada, R., Izawa, T. & Fujimoto, H. 1989. Fertile transgenic rice plants regenerated from tranformed protoplasts. Nature 338: 274-277. Skriver, K., Olsen, S.L., Rogers, J.C, & Mundy, J, 1991. cw-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proceedings of the Nation- al Academy of Sciences. USA. Vol 88: 7266-7270. Thompson, J. A., Abdullah, R. & Cocking, E. C. 1986. Protoplast culture of rice (Oryza saliva L.) using me- dia solidified with agarose. Plant Science 47: 123- 133. Vasil, V., Redway, F. & Vasil, I.K. 1990. Regeneration of plants from embryogenic suspension culture proto- plasts of wheat (Triticum aestivum L.).8i0/Technology 8: 429-134. Yang, Y.M., He, D.D. & Scott, K.J. 1993. Plant regener- ation from protoplasts of durum wheat (Triticum aesti- vum Desf., cv. D6962). Plant Cell Reports 12: 320- 323. Zhang, W., McElroy, D. & Wu, R. 1991. Analysis of rice Actl 5’ region activity in transgenic rice plants. Plant Cell 3: 1155-1165. Manuscript received January 1994 204 Agricultural Science in Finland 3 (1994) Research Note SELOSTUS Nopea menetelmä ohran protoplastien eristämiseksi ja sen soveltaminen geeninsiirtoon Barnabas Jenes, Matti Puolimatka, Pedro Bittencourt ja Seppo Pulli Agricultural Biotechnology Center ja Maatalouden tutkimuskeskus Tutkimuksessa kehitettiin menetelmä, jossa protoplasteja eristetään suoraan ohran jyvien alkion ja sirkkakilven so- lukosta ilman edeltävää solukkoviljelyvaihetta. Jyvät pin- tasteriloitiin ja niitä liuotettiin steriloidussa vedessä vuo- rokausi. Tämän jälkeen alkion ja sirkkakilven solukot eris- tettiin jyvistä ja pilkottiin pieniksi paloiksi. Protoplastien eristämiseksi kokeiltiin aluksi kolmea erilaista entsyymi- liuosta, joista paras valittiin PEG (polyetyleeniglykoli)- menetelmällä tehtyihin geeninsiirtokokeisiin. Siirtokokeissa käytetty rengasmainen plasmidi-DNA pActl-F sisälsi rii- sin Acf/-säätelyjakson, Cics-geenin (koodaa P-glukuroni- daasi-entsyymiä) ja Afav-lopetusjakson. PEG-käsittelyn jäl- keen protoplasteja kasvatettiin KPR-kasvatusalustalla. Transienttinen (väliaikainen) Gu.v-geenin ilmentyminen määritettiin vuorokauden kuluttua käsittelemällä transfor- moituja protoplasteja X-gluc-entsyymisubstraatilla. Enim- millään noin kuudessa prosentissa transformoiduista pro- toplasteista havaittiin Gus-geenin ilmentymistä. Tulosten mukaan tämä nopea protoplastien eristystekniikka sovel- tuu erityisesti väliaikaisen geeni-ilmentymisen tutkimuk- seen, sillä ajansäästö protoplastien normaaliin suspensio- viljelmään verrattuna on huomattava. 205 Agricultural Science in Finland 3 (1994)