Time saving method for protoplast isolation, transformation and transient gene expression assay in barley
Barnabäs Jenes,MattiPuolimatka, Pedro Bittencourt and SeppoPulli Jenes,B.', Puolimatka, M.2,Bittencourt, P.1& Pulli, S.2 1994. Time saving
method for protoplastisolation,transformation and transient geneexpression assay in barley. AgriculturalScience inFinland 3: 199-205. (‘lnstitute for Plant Sciences, Agricultural Biotechnology Center, Gödöllö, P.0.80x 170,H-2100 Hun- gary and Agricultural Research Centre ofFinland, Institute of Crop and Soil Science, PlantBreeding Section, FIN-31600Jokioinen, Finland.)
This studywasconducted to establish arapidmethod forbarley(Hordeum vulgare L.) protoplastisolation toprovideaneasy-to-useprocedure for the transformation and primary investigation of new gene constructs by transient gene expression assays.Protoplasts weresuccessfully isolated from thechopped embryoand scutel-
lum parts of maturebarley seeds bydigestingthree hours withanenzyme mixture.
Isolatedprotoplasts were washed in W 5washing solution, sievedthrough plastic meshes and then cleanedon sucrosegradient.The suitabilityof thesedirectly from embryo-scutellum complexes derivedprotoplastsfor transient geneexpressionstudies wasdeterminedby transformingtheprotoplasts usingthe PEG (polyethylene gly- col) method. Plasmid pActl-Fcontaining the rice Actl promoter linked with the guscodingsequences and thenospolyadenylation signal wasused inthe transfor-
mation. After the PEG treatmentprotoplasts wereculturedonKPRculture medium and the transient gusexpressionwasassayed 24-36hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts withX-gluc.The results of this studyshow that theprotoplasts isolated directly from dissected maturebarley scutellum-embryo complexescould be used toinvestigatetransient geneexpressions in barley. Thisprocedure requires negligi- ble time priorthe transformation experiment and so canbe done in avery short timecomparedtotheprotoplast system basedon asuspensionculture.
Keywords: Hordeum vulgareL.,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 wellasin assaying the expression of recombinant geneconstructs 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 importantmonocot species and the first protoplast derived regenera- tion in cereals wasreported in rice (Oryza saliva L.) byFujimuraand his group (1985). Succesful protoplast-plant regeneration systems have also been reported in wheat (Vasiletal. 1990,Chang etal. 1991,Yang et al. 1993, Ahmed and SAgi 1993, Pauk et al. 1994),maize (Mörocz et al.
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1990)and barley (Jähne etal. 1991). Improve- mentsin protoplast culture and regeneration have significantly given a contribution to the genetic transformation ofmonocots. Inrice, the transfor- mation of protoplasts and the subsequent regen- eration of transgenic rice plants have become a routine procedure (Jenes etal. 1992). Protoplast transformation resulting in stable integration of foreign genes into thetarget cells has also been reported in other cereals such asrice (Shimamo-
toetal. 1989), maize (Möroczetal. 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, Jähneetal. 1991, Lazzeri etal. 1992). Junker etal. (1987) detected transient expression of NPT II (Neomycin phosphotransferase II)genein pro- toplasts derived from suspension culture andtrans- formed with PEG. The initiation and maintenance of fine suspensioncultures, aprerequisite forsuc- cessful protoplast isolation in barley aswell as in other species, is laboriuos and may take a con- siderably long time fromsome weeks to several months. Itcan therefore be a limiting factor in applying barley protoplasts to transient geneex- pression studies. In addition, a long lasting pre- culture phase before the transformation mayre- sult in undesirable somaclonal variation at the level of transgenicregenerants.Therefore theuse of protoplasts in transformation experiments would benifit from a procedure that would re- duce the time in culture prior to the genetrans- fer.
Diaz and Carbonero (1992) investigated tis- sue specific transient expression of the gus re- portergenein 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 tothe transformation of protoplasts de- rived from cell suspension but the time require- ment was negligible comparedto the suspension cultures.
Material and methods Plant material
Dry seeds of barley cultivars ‘Pohto’, ‘Kymppi’,
‘Prisma’ and Tgri’ wereprovided 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 distilledwater for seven times. After sterilization the seeds were imbibed in sterile distilledwater and incubated in 25°C for 24-36 hours. The imbibed embryo- scutellum complexeswereexcised from the seeds and chopped into small species with a scalpel (Fig. 1). The following three enzyme mixtures were originally used for the digestion ofproto- plasts:
1) JE enzyme mixture modified after Junkeret 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 Na2HP04, atpH 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% MgS04and 0.05%KH,P04,
3) LLE enzyme mixture modified after Luhrs and Lörz (1988) containing 1% Onozuka RS Cellulase, 0.5% MacerozymeRlO, 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
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cleaned by sieving the mixture through aplastic mesh with pores of 100 pm in diameter and cen- trifuged at800 rpm for 5 min. Protoplasts were resuspended in 2 ml
W 5
washing solution(Menc- zeletal. 1981)and the suspensionwasthen laid on thetop of0.6 M sucrose solution. After5 min of centrifugationat 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 (McElroyetal. 1991)waspro- vided by thecourtesy of Professor RayWu, Cor- nell University, Ithaca,NY. This plasmid includes the rice Actin 1 gene 5’ regulatory elements linked tothe gus gene coding sequences (synonym uidA, codes for (3-glucuronidase enzyme) from E. coli and thenospolyadenylation site from Agrobacte- rium
tumefaciens.
Transformation of protoplasts
The number of protoplasts suspended in the W
5
solutionwas estimated inaBuerker chamber and then divided into 1 x 106 aliquots in plastic Wassermann tubes. After centrifugation at 800 rpm for 5 min the supernatant wasremoved and the pellet was resuspended in 1 ml MgMa trans- formation buffer(Zhang etal. 1991). Twenty mg ofuncutpActl-F plasmid and 100 pg of Herring Testis DNA (SIGMA, ruptured by sonication) were added tothe 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 protoplastswere incubated with the PEG for 28 min. During this time the suspension wasgen- tly shaken by hand in every 5 min.
At the end of the incubation time the mixture was slowly diluted with
W 5 solution
upto 10 mlwithin 5 min, adding the
W 5 solution
drop bydrop 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 etal. 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 at25°C in dark until the GUS assay.
Detection of transient expression of gus 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 etal. 1986, Jefferson 1987)was added into each well. The plates were incubated at29°C for 12 hours before the visual counting of the cells show- ing transient expression wascarried out.
Results and discussion Protoplast isolation
Successful protoplast isolationwasachieved 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 whichwere 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 threeen- zyme mixtures applied toprotoplast isolation from em- bryo-scutellumcomplexesofbarley ‘Kymppi’.
Yield of protoplasts/ Enzymemixture
100 embryos 2.2 x106
1.2x10"
0.2x106 JE enzyme mixture1
KEenzyme mixture2 LEEenzyme mixture3
1modified after Junker et al. 1987.
2modified after Ahmed andSAgi 1993.
3modified after Luhrs and LOrz 1988.
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Table2,Average yields ofprotoplasts of the four different barley cultivars Kymppi, Prisma,Pohto and Igri. Diges- tionwasperformed bythe JE enzymemixture'.
Number ofprotoplasts/ Name of genotype
100embryos 2.2x I06 4.1x 106 2.5x 106 0.6x 106 Kymppi
Prisma Pohto Igri
1modified 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 106protoplasts per 100 dissect- ed embryo-scutellum complexes were obtained.
The protoplast yield seemedtobe dependent on the genotype and the enzyme mixture used (Table 2). Thegenotypesdiffered also in the size of released protoplasts. When using the JE en- zyme digestion, Pohto provided bigger protoplasts of50-80 |im in diameter than any of the other three cultivars (20-30(xm in diameter).
During the digestion of embryos from the ma- tureembryo-scutellum complexes it wasvisually estimated that about80% 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 celledaggregates.This ob- servation encouragesustowards ourfurther goals toobtain regenerated transgenic plants from these transformed protoplasts. It is well known from the earlier studies that the scutellar tissue in mono- cotspecies has a greatregeneration capacity (Fu-
jimuraetal. 1985).
Protoplast transformation and transient geneexpression
Protoplasts isolated directly from scutellum-em- bryo complexesweresuccesfully 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 thetrans- 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 cellswas estimated by visual examination. The frequency of the transformationevents that showed tran- sient expression of the gus genewasestimatedto 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 ofnew gene constructs. The establishment of a suspension- protoplastsystem in monocots is atimeconsum- ing process (Jähne etal. 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, ouraim was to setup arapid system for producing viable protoplasts suitable for transformation and transient geneex- pression studies. The embryo-scutellum derived protoplastsystem seemedtofullfil these require- ments. Only 24 hours was needed prior the pro- toplast isolation and transformation instead ofsev- eral months of subculturing. Thissystem was also suitable for obtaining 4.5% of the transformed cells showing transient gene expression.
The time needs of the protoplast isolation and transformationsystem 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,asprotoplast derivedregenerants havea single cell origin,one can expectgenetic 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 methodto isolate barley protoplasts directly from matureembryo-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-
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vide with the abilitytoproduce fertile transgenic plants of barley within much shorter time than through the suspension-protoplast-transgenic plant system.
Acknowledgements. The authors are gratefultothe Agri- cultural Research Centre ofFinland, Jokioinen, Finland, andAgricultural Biotechnology Centre, Gödöllö, Hunga- ry, and to the Ministry ofAgriculture and Forestry of Finland for providing the facilities and finance for this study.
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Fig. 1.Excised mature barley scutellum-embryo complex after24 hours of imbibitionin distilled,sterilized water.
Scalebar=I mm.(Photo:MattiPuolimatka)
Fig. 2. Barley protoplasts isolated from the mature scutel- lum-embryo complexes of ’Kymppi’. (Photo: Barnabäs Jenes)
Fig. 3.Celldivisionsinbarley protoplasts.(Photo; Bama bäs Jenes)
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Manuscriptreceived January1994 Agricultural ScienceinFinland3 (1994)
Research Note
SELOSTUS
Nopea menetelmä ohranprotoplastien eristämiseksi jasensoveltaminen geeninsiirtoon BarnabasJenes, MattiPuolimatka, Pedro Bittencourt jaSeppoPulli
Agricultural Biotechnology CenterjaMaatalouden tutkimuskeskus
Tutkimuksessa kehitettiin menetelmä,jossaprotoplasteja eristetäänsuoraan ohranjyvienalkion jasirkkakilvenso- lukosta ilman edeltävääsolukkoviljelyvaihetta. Jyvät pin- tasteriloitiin janiitä liuotettiin steriloidussa vedessävuo- rokausi. Tämänjälkeenalkionjasirkkakilven solukot eris- tettiin jyvistä ja pilkottiin pieniksi paloiksi. Protoplastien eristämiseksi kokeiltiin aluksi kolmea erilaistaentsyymi- liuosta, joista paras valittiin PEG (polyetyleeniglykoli)- menetelmällätehtyihin geeninsiirtokokeisiin.Siirtokokeissa käytetty rengasmainen plasmidi-DNA pActl-F sisälsirii- sin Acf/-säätelyjakson,Cics-geenin (koodaa P-glukuroni-
daasi-entsyymiä)jaAfav-lopetusjakson. PEG-käsittelynjäl- 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 prosentissatransformoiduista pro- toplasteista havaittiinGus-geenin ilmentymistä. Tulosten mukaan tämä nopea protoplastien eristystekniikkasovel- tuu erityisesti väliaikaisen geeni-ilmentymisen tutkimuk- seen, silläajansäästö protoplastien normaaliin suspensio- viljelmään verrattunaonhuomattava.
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