PEG-mediated transformation of leaf protoplasts of Solanum tuberosum L. cultivars

transcript

Plant Cell, Tissue and Organ Culture 27: 105-114, 1991. 1991 Kluwer Academic Publishers. Printed in the Netherlands.

A. Feh6r, K. Felf61di 1, J. Preiszner & D. Dudits Institute of Plant Physiology and Institute of Genetics, Biological Research Center of Hungarian Academy of Sciences, H-6701, P.O.B. 521, Szeged, Hungary. (1present address: Cereal Research Institute, H-6701, P.O.B. 391, Szeged, Hungary)

Received 18 September 1990; accepted in revised form 11 June 1991

Key words: direct gene transfer, kanamycin-resistance, PEG-treatment, potato protoplasts

Abstract

As an alternative to Agrobacterium-mediated gene transfer, direct transformation of potato (Solanum tuberosum L.) cultivars was achieved by using the Mg2+/PEG protocol (Negrutiu et al. 1987) to stimulate DNA uptake into leaf protoplasts. The frequency of kanamycin-resistant clones varied between 1-12% from experiment to experiment independently of the genotype used. A deleterious effect of heat shock treatment (45°C for 5min.) has been found on colony formation during optimization of the transformation procedure. The Mg 2+ ion concentration (15-35 mM), the denaturation (100 ° C, 10 min. right before the treatment) and the form of the plasmid DNA (linear or circular) had no significant effect on the efficacy of the transformation. Application of denatured calf thymus DNA as carrier molecules (at concentration of 50/~g m1-1, however, resulted in a significant decrease in the number of the resistant cell colonies). Sixty to 80 percent of the kanamycin-selected callus tissues regenerated shoots. The presence and expression of the introduced neomycin phosphotransferase neo gene in regenerants with normal morphology and tetraploid chromosome number was proved by biological tests based on rooting and callus formation in the presence of the antibiotic and by NPT enzyme activity assay. Southern analysis revealed the integration of the introduced DNA molecules carrying the neo marker gene, into the genome of potato.

Introduction

Potato (Solanum tuberosum L.) is one of the important crop plants amenable to basic cell and tissue culture techniques. Fertile plants can be regenerated in vitro from different explants and from leaf protoplasts (e.g. Shepard & Totten 1977; Haberlach et al. 1985; Feh6r et al. 1989). Plant regeneration is a basic prerequisite of suc- cessful genetic manipulations resulting in transgenic potato plants.

Moreover potato is sensitive to Agrobacterium infection and nowadays several procedures have been developed for Agrobacterium-mediated

transformation of this plant species (e.g. Sheer- man & Bevan 1988; Stiekema et al. 1988; De Block 1988; Tavazza et al. 1988; Visser et al. 1989).

Direct transfer of DNA into plant protoplasts represents an alternative way to introduce foreign genes into plants. Direct transformation may have advantages as compared to Agrobac- terium-mediated transformation in attempts for 'shotgun' experiments using plant genomic DNA or gene expression libraries to transform plant protoplasts. For these approaches high transformation frequencies are needed.

There are several methods based on chemical

(Krens et al. 1982; Paszkowski et al. 1984; Hain et al. 1985; Negrutiu et al. 1987) or electrical treatments (Fromm et al. 1986, Bates et al. 1988) or a combination of both (Shillito et al. 1985) to promote DNA uptake by plant protoplasts. A simple and efficient procedure applying a high concentration of polyethylene glycol in the presence of Mg 2+ ions was described by Negrutiu et al. (1987) for transformation of Nicotiana protoplasts. Because of the simplicity and efficacy of this protocol, it has been successfully adapted to several other plant species including Arabidopsis thaliana (Damm et al. 1989), Brassica nigra and Vigna aconitifolia (K6hler et al. 1989), Helianthus annuus (Moyne et al. 1989), Zea mays (Maas & Werr 1989) and Medicago varia (Feh6r & Gy6rgyey, unpub- lished).

Recently a few reports have been published about successes in direct protoplast transformation experiments with potato (Knapp et al. 1988; Masson et al. 1989; Jones et al. 1989). Here we describe a comprehensive study to optimize DNA uptake into leaf protoplasts of different tetraploid potato cultivars (Gracia, Desir6e, Boro). The high efficiency transformation method based on MgZ+/PEG treatment was demonstrated in several experiments by formation of several hundreds of kanamycin-resistant microcolonies that could be regenerated into transgenic potato plants expressing the neomycin phosphotransferase marker gene.

Materials and methods

Plant material, cell and tissue culture techniques

In vitro shoot cultures of tetraploid Solanum tuberosum cultivars (Gracia, Desir6e, Boro) were maintained on MSP medium (Maliga 1984) at 20/15°C day/night temperature and 12-h fluorescent light (50-120/zmol m -2 s- l) . Proto- plasts were isolated from leaf tissues of 4-6- weeks-old preconditioned (2 days 24°C plus one day 4°C in dark) plants (Haberlach et al. 1985, Feh6r et al. 1989). The incubation medium con- tained 1% Cellulase (Onozuka R-10) and 0.5% Macerozyme (Onozuka) dissolved in V-KM culture medium (Bokelmann & Roest 1983) sup-

plemented with 0.42 M sucrose as osmotic stabil- izer. Incubation was performed overnight at 24°C in dark. The same medium was used for the culture of the protoplasts but with glucose (0.45 M) instead of sucrose. The 'agarose-bead' type culture (Shillito et al. 1983) system was used for stepwise decreasing the osmotic pressure and replacing the glucose with sucrose in the reservoir medium (Feh6r et al. 1989). Re- generation of shoots from the protoplast derived cell colonies was achieved through the use of the series of the media ('C', 'D', 'E' media) developed by Shepard (1980).

The development of primary callus tissues on leaf pieces was induced on MS (Murashige & Skoog 1962) medium supplemented with 27/zM naphthaleneacetic acid and 0.44/zM ben- zyladenine.

Transformation of potato leaf protoplasts

Protoplast transformation experiments were carried out closely following the protocol described by Negrutiu et al. (1987), using the MaMg and PEG-CMS solutions, but for protoplast washing a buffered mannitol/CaCl 2 solution (190raM mannitol, 100 mM CaCI 2 2H20 , 0.5% MES, pH 5.6) was used instead of the W5 solution. Poly- ethylene glycol (Mw: 4000 or 6000) was purchased from Merck. Unless stated otherwise 15 mM MgCI 2 was present in the transformation buffer. Heat shock (45°C, 5 min.) was applied after adding the DNA and before the PEG- treatment.

Plasmid and carrier DNA

The plasmid pGA 471 (An et al. 1985) developed as a binary vector for Agrobacterium- mediated transformation of plants was used at a final concentration of 10/xg ml 1. This vector (15.6 kb) carries the neo gene under control of the nopaline synthase promoter between the T- DNA borders. Plasmid DNA was purified by CsCI density gradient centrifugation. Lineariza- tion of the plasmid molecules was achieved by Eco R1 digestion (see: An et al. 1985).

As carrier DNA, calf thymus DNA (Sigma) was added to the protoplast suspension at a final concentration of 50/~g ml 1.

Denaturation of the plasmid and/or the carrier DNA was accomplished by 10 min. boiling and subsequent cooling on ice immediately before the transformation.

Scoring viability and transformation efficiency

Division of the agarose embedded protoplasts was investigated at the end of the second week of culture. Division frequency (%) was determined as the number of cells accomplishing at least one cell division as compared to the number of the treated protoplasts. Altogether at least 600 cells were investigated per treatment in several randomly chosen areas. The frequency of colony formation (%) was estimated as the percentage of the treated protoplasts that could form green microcolonies after 6-7 weeks of culture. Relative transformation frequency (RTF value, %) was calculated as the ratio between the number of the selected kanamycin-resistant green calluses and of the colonies grown from protoplasts in cultures without selection.

Selection of the kanamycin resistant microcalluses

After transformation treatment the protoplasts were divided into three petri dishes and cultured at a density of 5 × 10 4 protoplasts/ml (5 ml per dish). Cells were embedded in agarose (0.6% Seaplaque, FMC) dispersing them as uniformly as possible. Two or three days later the agarose was cut into four equal blocks and transferred into liquid reservoir medium. At the end of the second week of culture, one of the four agarose blocks was transferred from each petri dish rep- resenting one treatment into a new dish and this culture was used to determine the colony formation capacity, while the others were subjected to selection pressure (100mg 1-1 kanamycin in the reservoir medium).

Treatments with carrier DNA only as well as with plasmid and carrier DNA but without PEG, always were included in the experiments as con- trols.

Analysis of the transgenic plants

The activity of the neomycin phosphotransferase

(NPT-II) enzyme was determined in some of the putatively transformed calluses and in the regenerated plants using a dot assay (Platt & Yang 1987) and a gel assay (Reiss et al. 1984), respectively. Plant DNA was isolated according to Rogers & Bendich (1988). Restriction enzymes were purchased from New England Biolabs and used according to the manufacturers instructions. DNA fragments were electrophoretically separated in 0.8% agarose gel (approximately 10/~g DNA/lane) and transferred onto nylon mem- brane (Hybond N, Amersham) by vacuum blot- ting followed by UV fixation. Hybridization took place overnight at 65°C in a solution containing 0.25% dried skimmed milk, 3 × SSC, 0.1% SDS. The 0.92 kb Pst I. fragment of pNeo (Pharmacia) carrying the neo gene coding region was labeled to a specific activity around 8 x 108cpm/~g DNA by random priming. The filter was washed in 2 × SSC, 0.1% SDS for 2 x 10min. at room temperature and in 0.1 × SSC, 0.1% SDS for 3 x 30 min. at 65°C. The filter was exposed to X-Omat AR diagnostic film (Kodak) at -80°C for three days using one amplifying screen (Cronex Lightning Plus, Dupont).

In order to determine the chromosome number, shoot tips of greenhouse plants were rooted in soil. The root tips were prefixed in 0.002 M 8-hydroxyquinoline, fixed in Carnoy solution and stained with carbolfuxin.

Results and discussion

Protoplast treatment and survival

Potato leaf protoplasts are frequently considered to be sensitive to polyethylene glycol treatment, which is why electromanipulations are often pre- ferred in somatic hybridization (e.g. Puite et al. 1986; de Vries et al. 1987; Fish et al. 1988) and genetic transformation (e.g. Masson et al. 1989; Jones et al. 1989) experiments with potato. We found, however, that the protoplast transformation protocol described for Nicotiana species by Negrutiu et al. (1987) using a high final concentration of PEG (20%) to promote DNA uptake can be successfully used in the case of potato protoplasts as well.

According to our observations, around 60% of the isolated potato protoplasts could tolerate the PEG treatment in comparison to the untreated control protoplast population. Careful handling of the protoplasts during the treatment by gradu- al mixing of the PEG with the protoplast suspension was found to be very important to avoid extreme protoplast damage. Neither the molecular weight nor deionization of the PEG solution (Kao & Saleem 1986) influenced significantly the survival of the potato leaf protoplasts (data not shown). The PEG treatment promoted division of the protoplasts that survived the procedure. Division frequencies of the surviving cells were 63.4-+3.8% in the treated cultures and only 47.5---7.2% in the control ones. The treated protoplasts often started to divide earlier and formed larger cell colonies at the end of the second week of the culture as compared to the untreated ones. The overall division frequency based on the total number of the treated protoplasts and the colony formation frequency, how-

Diviaion Colony formation

PEG ~ Heat ~ Heat+PEG

Fig. 1. Effect of heat shock (45°C for 5 min.) and PEG- treatment on the division frequency and colony forming capacity of potato (cv. Gracia) leaf protoplasts. Data derived from three independent experiments. Untreated control cultures were considered to present 100% division and colony formation frequencies.

ever, decreased by 24% and 16%, respectively, by the treatment in comparison to the control cultures (Fig. 1).

Heat shock (45°C, 5min.) proved to be deleterious for potato protoplasts. The heat treatment resulted in a 50% decrease in the division frequency (Fig. 1), which was decreased another 30% by adding the PEG solution. Colony formation was low and unpredictable following a com- bined heat /PEG effect (Fig. 1).

The Mg 2+ concentration in the transformation buffer in the range that was used by Negrutiu et al. (1987) for Nicotiana protoplasts (15-35 mM) had no effect on protoplast survival. Higher concentrations, however, caused a strong pre- cipitation of the potato protoplasts by PEG finally resulting in low viability (data not shown).

Selection of the resistant cell colonies

Protoplasts were cultured in 'agarose-beads' sur- rounded by liquid medium (Shillito et al. 1983), which enabled a continuous and uniform selection of the cells. Kanamycin (100mg 1-1) was added to the medium at the end of the second week of culture. Two weeks later agarose blocks were smashed carefully and the colonies were transferred onto agar-solidified medium (Shep- ard's medium 'C', 1980) supplemented with the same amount of kanamycin. The green kanamycin-resistant calluses could be easily recog- nized in two weeks following the transfer (Fig. 2a). Control plates without kanamycin always were included in the experiments. Untreated protoplasts were never able to form colonies under these conditions.

To test the transformation origin of the out- growing calluses, ten of them were chosen randomly to investigate the expression of the neo gene by using a simple dot assay (Platt & Yang 1987; Fig. 2b). All of the investigated clones gave a positive signal.

Efficiency of the transformation

The few reports on the stable direct transformation of potato protoplasts were limited to use of diploid clones (Knapp et al. 1988; Masson et al. 1989). Efficient direct transformation of the protoplasts of tetraploid potato cultivars could be of

Fig. 2. Selection of the putative transformant cell colonies on kanamycin-containing medium. (a) Colonies originating from treatments in the absence (pl - ) as well as in the presence (pl +) of the transforming DNA were plated onto 100 mg l- kanamycin-containing (KM ÷) and control (KM-) media. Kanamycin-resistant colonies could be observed only in cultures derived from treatments of the protoplasts in the presence of the plasmid DNA. (b) Neomycin phosphotransferase enzyme activities of ten selected colonies were detected by a dot assay (a -kanamycin-resistant Solanum bre- videns cell line as positive control; b -non- t rans fo rmed potato protoclone as negative control; c - t - ten independent kanamycin-resistant calluses).

interest to breeders for transfer of monogenic characters into potato cultivars via 'shotgun' transformation experiments preserving their heterozygosity at the tetraploid level with simul- taneous maintenance of their agronomic value.

In our experiments three potato cultivars (Gracia, Desiree, Boro) exhibiting good plant regeneration capacity from leaf protoplasts (Feh6r et al. 1989) were involved. All of them could be transformed with high efficacy (1-12% RTF; Table 1), which is at least as high as was reported for diploid genotypes after electroporation (Masson et al. 1989) and one order of magnitude higher than in the case of the reported PEG-mediated transformation (Knapp et al. 1988). The transformation frequency within this range, however, varied from experiment to experiment depending on the quality of the isolated protoplasts. Independent treatments using the protoplasts deriving from the same isolation always resulted in similar transformation frequencies. In our experiment correlation between the cultivars used and the transformation frequencies could not be determined, although the significant role of the genotype in the transformation process has been suggested in several cases (e.g. Kohler et al. 1987; Tyagi et al. 1989; Damm et al. 1989).

Since a number of factors, including the genotype used, may affect the efficiency of the direct protoplast transformation (e.g. Shillito et al. 1985; Negrutiu et al. 1987; Damm et al. 1989), there was a need to optimize the parame-

Table I. Efficiency of direct transformation of leaf protoplasts of potato (Solanurn tuberosum L.) cultivars.

Cultivar Exp.~ Treated Colony RTF (%)3 protoplasts formation (%) 2

Gracia A 1.0 × 1 0 6 1.61 4.1 B 9.5 × 105 1.18 12.1 C 1.5 x 106 0.37 2.3

Desiree A 7.5 x 105 0.76 2.0 B 7.0 × l0 s 1.26 0.9

Boro A 1.0 × 1 0 6 0.30 3.0

Independent protoplast isolations and transformations under standard conditions: 10 #g ml 1 linearized pGA 471 (An et al. 1985) plasmid and 50/~g m1-1 calf thymus DNA; 20% PEG~000 and 15 mM MgCl2; 1 × 1 0 6 protoplasts/ml; according to Negrutiu et al. (1987). 2 Percentage of the treated protoplasts forming green cell colonies determined 6 weeks after the treatment. 3 Relative transformation frequency: percentage of the colony-forming protoplasts showing resistance against 100mg 1-1 kanamycin applied from the third week of culture (see Materials and methods).

ters for every plant species to be transformed by these methods. We found, however, that the basic protocol described by Negrutiu et al. (1987) for Nicotiana species gave the best results for potato protoplasts as well, and any change in some of the tested parameters had negative or no effects on the transformation frequency.

For example, heat shock as we described above proved to be deleterious for potato protoplasts similarly to Nicotiana plumbaginifolia (Negrutiu et al. 1987) and Arabidopsis thaliana (Damm et al. 1989) protoplasts, although it was reported to promote plasmid uptake into Nicotiana tabacum (Shillito et al. 1985; Negrutiu et al. 1987) and Oryza sativa (Zhang et al. 1988) protoplasts.

The Mg > concentration of the transformation buffer with a narrow range (15-35 mM) had no effect on the transformation efficiency, at least in the case of the potato cultivar Gracia (Table 2), but it was observed to affect the frequency of the transformed cells of Nicotiana (Negrutiu et al. 1987) and Brassica (Golz et al. 1988) species.

The form of the plasmid molecules (linear or circular) did not change the efficacy of the transformation in our hands as in the case of Arabidopsis (Damm et al. 1989), although linear plasmid molecules are usually considered to be better vectors for stable protoplast transformation (Shillito et al. 1985; Negrutiu et al. 1987).

The highly efficient Agrobacterium-mediated transformation of plants proceeds via introduction of single-stranded DNA into the cells (Stachel et al. 1986). We investigated the effect of the denaturation of the plasmid and/or the carrier DNA prior to direct protoplast transformation on the transformation frequency (Table 3).

We found, in agreement with the results of Furner et al. (1989) that the denaturation of the plasmid molecules had no influence on the transformation frequency, probably due to the rapid generation of double-stranded molecules within the cells. Interestingly, however, we observed that the denaturation of the carrier DNA decreased the frequency of transformed colonies by

Table 2. Effect of the Mg 2+ concentration in the transformation buffer on the transformation efficiency. Transformation of potato (cv. Gracia) leaf protoplasts was carried out under standard conditions (see Materials and methods), only the Mg 2÷ concentration of the transformation buffer was changed.

Mg 2* conc. Treated Colony RTF (%) 3 (mM) protoplasts formation (O~)2 151 1 × 106 2.4 0.0 15 1 × 106 1.6 4.1 25 1 x 106 1.75 4.9 35 1 × 106 1.9 4.75

Control treatment in the absence of the plasmid DNA. -' and 3 as in the Table 1.

Table 3. Effect of plasmid and/or carrier DNA denaturation prior to protoplast transformation on the frequency of resistant cell colonies. Transformation of potato (cv. Desir6e) leaf protoplasts was carried out under standard conditions (see Materials and methods) with 10/xg mt -1 plasmid D N A and 50 ~g ml l carrier DNA.

Treatment 1 of Treated Treated Colony RTF (%)3 plasmid D N A carrier protoplasts formation (%)2

no - 7 x 105 2.4 0.0 - - 7 x 105 1.3 1.2 + - 7 x 105 1.2 0.9 - + 7 x lO s 1.2 0.3 + + 7 x 105 1.45 0.4

1 Plasmid and /o r carrier D N A molecules were denatured by 10 min. boiling and subsequent cooling on ice right before adding to the protoplasts (+ denatured; - not denatured; no = not added). 2 and 3 as in Table 1.

about 50%, irrespective of whether plasmid D N A was denatured or not. A possible explana- tion of this feature can be the supersaturat ion of the cells with single-stranded D N A molecules prevent ing the rapid enzymatic second strand format ion and finally resulting in increased nu- clease sensitivity.

Regeneration and characterization of transgenic potato plants

Around 60 -80% of the selected kanamycin-resistant cell colonies regenera ted shoots. Regenera-

tion was carried out under non-selective conditions, but ever~¢ shoot was rooted in the presence of 100mg 1 - - kanamycin. This concentrat ion complete ly inhibited root format ion of the non- t ransformed control shoots. Expression of the kanamycin-resis tance trait in the t ransformants was fur ther suppor ted by pr imary callus formation on leaf strips on kanamycin-containing med ium (Fig. 3c). All shoots regenerated f rom the selected calluses proved to be resistant and it was confirmed by detecting the activity of the N P T enzyme in several regenerated plants (Fig. 3a,b). Significant variat ion was detected, how-

Fig. 3. Regenerated transgenic potato plants expressing the neo marker gene. (a) One of the transgenic plants. (b) The activity of the neomycin phosphotransferase enzyme in some of the regenerated plants (lanes: 1 -positive control as in Fig. 1; b - empty lane; c-f-independent kanamycin-resistant regenerants; g-untransformed control potato protoclone) (c) Primary callus formation on leaf strips on transgenic potato lines in the presence of kanamycin (a control in the presence and b in the absence of kanamycin (100 mg l-~), c-g leaf strips of independent transformants on kanamycin-containing medium).

ever , in the level of n e o gene expression among the t ransformants (Fig. 3b). Randomly selected plants grown in soil in the greenhouse exhibited normal morphology. The presence of the n e o

gene in three of these plants was also proved by Southern analysis.

Following direct protoplast t ransformation the integration of one to three (Negrutiu et al. 1987; Christou et al. 1987; D a m m et al. 1989; Kartzke et al. 1990) or several (Paszkowski et al. 1984; Shillito et al. 1985; Potrykus et al. 1985; Czer- nilofsky et al. 1986; Schocher et al. 1986; Christ- ou et al. 1987; D a m m et al. 1989; Tyagi et al. 1989) copies of the introduced gene was observed. Integrat ions took place at one (Paszkow- ski et al. 1984; Potrykus et al. 1985; Mouras et al. 1987; Kar tzke et al. 1990) or more (Hain et al. 1985; Riggs & Bates 1986; Tyagi et al. 1989, Kar tzke et al. 1990) physical locations within the plant genome. Truncation, tandemizat ion or re- a r rangement of the foreign gene copies were frequently proved (Hain et al. 1985; Potrykus et al. 1985; Riggs & Bates 1986; Czernilofsky et al. 1986).

The restriction enzyme Eco RI cut only at one position of p G A 471 and was used to linearize the plasmid before t ransformation. That is why it was not expected to cleave within the integrated D N A sequences. Therefore , it was possible to de termine the number of sites at which plasmid D N A was integrated by digestion of the plant D N A with this enzyme. Hybridization patterns indicated the integration of one gene copy in the case of two t ransformants and four copies in the case of the third one (Fig. 4) at different posi- tions of the pota to genome. It was confirmed by dot blot analysis (data not shown). Fragments hybridizing with the n e o gene coding sequence are smaller than the total plasmid (15 .6kb) re- vealing truncation.

C h r o m o s o m e counts revealed the tetraploid nature of most of the t ransformants but some octoploids also occurred. In the case of diploid pota to genotypes mainly tetraploid transformants were observed following PEG-media ted protoplas t t ransformation (Knapp et al. 1988; Masson et al. 1989). It seems, however, that the reason could be the spontaneous tetraploidiza- tion of the diploid protoplasts during regenera-

Fig. 4. Southern analysis of some of the transgenic greenhouse-grown plants. Total DNA was isolated by the CTAB method (Rogers & Bendich 1988), digested by Eco RI and the fragments were separated in 0.8% agarose (10/xg in 0.8% per lane). The 920 bp Pst I fragment of pNeo (Phar- macia) was labelled with 32p ATP to high specific activity (8 × 108 cpm/.Lg 1) by random priming and used as a probe. Lanes: a - c three independent transgenic paints; d non-transformed potato plant; e 100 pg pNeo fragment. Pst I fragments of lambda phage DNA we used as molecular weight stan- dards.

tion independent of the t ransformation procedure itself.

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