Time saving method for protoplast isolation , transformation and transient gene expression assay in barley

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 Hungary and Agricultural Research Centre of Finland, Institute of Crop and Soil Science, Plant Breeding Section, FIN-31600 Jokioinen, Finland.)


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 uptake 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 regeneration 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.  1990) and barley (Jähne et al. 1991).Improvements 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 (Shimamoto 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 derived from 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 protoplasts for transient expression studies (Skriver  et al. 1991).This isolation procedure, however, requires very specific skills.In this paper we introduce 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 requirement was negligible compared to the suspension cultures.

Plant material
Dry seeds of barley cultivars 'Pohto', 'Kymppi', 'Prisma' and Tgri' were provided by the Institute of Plant Breeding, Agricultural Research Centre of Finland, Jokioinen.
The protoplasts were kept in the enzyme at 25°C for 3 to 6 hours.The protoplast were then 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 provided by the courtesy of Professor Ray Wu, Cor- nell University, Ithaca, NY.This plasmid includes the rice 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 Agrobacterium tumefaciens.

Transformation of protoplasts
The number of protoplasts suspended in the W 5 solution was estimated in a Buerker chamber and then divided into 1 x 10 6 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 gently 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 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  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.

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 enzyme mixtures applied to protoplast isolation from em- bryo-scutellum complexes of barley 'Kymppi'.
The four barley genotypes tested in this study showed differences in protoplast yielding and characteristics.In the digestion experiments as much as x 10 6 protoplasts per 100 dissected 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 observation 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 (Fujimura 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 transient 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 suspensionprotoplast 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 requirements.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-vide with the ability to produce fertile transgenic plants of barley within much shorter time than through the suspension-protoplast-transgenic plant system.

Table 2 ,
Average yields of protoplasts of the four different barley cultivars Kymppi, Prisma, Pohto and Igri.Digestion was performed by the JE enzyme mixture'.