Plant Cell Tissue and Organ Culture 47:111-118, 1997. 111 (~) 1997 Kluwer Academic Publishers. Printed in the Netherlands.

An improved method for direct gene transfer and subsequent regeneration of Arabidopsis thaliana Landsberg erecta and two marker lines

M. Torres 1, J. Siemens, M. Meixner & M. D. Sacristtin* Institut fiir Angewandte Genetik, Freie Universitgit Berlin Albrecht-Thaer-Weg 6, 14195 Berlin, Germany (* requests for offprints)(lpresent address: Universidad San Francisco de Quito, Campus Cumbayd, Via Interocedmica y Jardines del Este, Ecuador)

Received 6 March 1995; accepted in revised form 13 September 1996

Key words: PEG-mediated transformation, protoplasts, transgene inheritance

Abstract

A protocol for PEG-mediated transformation of protoplasts is described for A. thaliana ecotype Landsberg erecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 gg plasmid DNA and 28 gg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5-3.2% and absolute transformation frequencies of 1-2 x 10 -4, were obtained. Shoot regeneration frequencies of 40-60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.

Abbreviations: GUS -/3-glucuronidase; HPT - hygromycin phosphotransferase; La-er - Landsberg erecta; PEG - polyethylene glycol, MES - 2(N-morpholino)-ethanesulfonic acid; PE - plating efficiency

Introduct ion

Arabidopsis thaliana has become a widely used plant in molecular biology studies because of its special char- acteristics such as small genome size, short generation time, presence of extensive genetic maps and availabil- ity of many mutants. With the establishment of plant regeneration protocols from different types ofexplants, callus or protoplasts for various Arabidopsis thaliana ecotypes (cf. Morris and Altmann, 1994), transfor- mation procedures based principally on Agrobacteri- um tumefaciens have been developed (Feldmann and Marks, 1987; Valvekens et al., 1988; Schmidt and Willmitzer, 1988; Bechtold et al., 1993). Direct gene transfer to protoplasts of A. thaliana has also been reported(Damm et al., 1989; Karesch et al., 1991; Mathur et al., 1995), and these techniques enabled tran- sient gene expression studies in Arabidopsis, which are useful for understanding regulatory mechanisms

involved in gene expression (Hoffman et al., 1994; Abel and Theologis, 1994; Mathur et al., 1995). With- in Arabidopsis, the ecotype Landsberg erecta (La-er) has been widely used in classic and molecular genet- ic experiments, and is the origin of different mutant and marker lines (Koornneef et al., 1987; Hauge et al., 1993). Although this ecotype has been considered to be recalcitrant for protoplast in vitro culture, two plant regeneration protocols from protoplasts have recent- ly been described for La-er (Siemens et al., 1993; Park and Wernicke, 1993). Here, we report an effi- cient protocol for direct gene transfer to protoplasts of A. thaliana ecotype La-er and two marker lines which derive from it, M4 and M10. The regeneration of fer- tile transgenic plants, the transmission and segregation of the marker gene in their progeny are also reported.

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Materials and methods

Plant material

Arabidopsis thaliana ecotype La-er and the two marker lines derived from La-er, M4 (long hypocotyl) and M 10 (reduced petals, large sepals), were obtained from the AIS Seed Stock Center, Frankfurt.

Plant culture conditions and protoplast isolation

Plants grown from surface-sterilized seeds were culti- vated in AM medium (Damm and Willmitzer, 1988). Protoplasts isolation from leaves of 3-4 weeks old plants was performed according to Siemens et al., (1993).

Plasmid DNA

Transformation experiments were performed using either the 4.5 kb plasmid pGL2, which carries the hygromycin phosphotransferase (HPT) gene (Gritz and Davis, 1983), or pRT103gus, a plasmid derived from pRT104 (Trpfer et al., 1987), which contains the/3- glucuronidase (GUS) gene. Both genes are controlled by the 35S promoter of the cauliflower mosaic virus.

Transformation protocol

Freshly isolated protoplasts were centrifuged and resuspended in transformation buffer (15 mM MgC12, 0.5 M mannitol, 2 mM MES, pH 5.6) at a density of 1.8-2.0 x 106 protoplasts/ml, and distributed in 0.3 ml aliquots in 2.0 ml Eppendorf tubes. Subsequent- ly, circular plasmid DNA (7-50 p-g) and carrier DNA (calf thymus DNA, 14-100 p.g) were added to each aliquot. After 5-10 rain of incubation, 0.32 ml PEG solution (26%, 17%, 15% or 13% (w/v) PEG 6000, 65 mM Ca(NO3)2, 0.26 M mannitol, pH 7-9) was mixed with the protoplast-DNA suspensions in the Eppendorf tubes. Control samples were treated in the same way but either the plasmid DNA, or both plasmid and car- rier DNA were omitted. After an incubation of 20-30 min, the transformation suspensions were transferred to 12 ml centrifuge tubes, carefully diluted stepwise with 10 ml W5-mannitol solution (170 mM mannitol, 125 mM CaCL2, 14 mM NaC1, 5 mM glucose, pH 5.6) and then centrifuged for 7 min at 140 x g. Sub- sequently, the protoplast pellets were washed in 3 ml 0.5 M mannitol and 3 ml seawater and were again cen-

trifuged for 5 min at 140 × g. Finally, the protoplasts were resuspended in 0.5 M mannitol.

Protoplast culture, selection of transformed cell lines and plant regeneration

The protoplasts were embedded in alginate disks (Siemens et al., 1993), and cultivated for the first 20- 24 days in NT-medium (Gleba and Hoffmann, 1978) in the dark; the medium was renewed every 10-12 days. After this period, the NT-medium was replaced by MII-medium (Datum and Willmitzer, 1988) and the cultures were transferred to dim light (2-3 Ixmol m -z s - i , 16-h light, 20-25 °C). In transformation experi- ments with the plasmid pGL2, selection was applied on the 10th day of culture using 10 mg 1-1 hygromycin B. Ten days later, the selection continued with 20 mg 1-1 hygromycin B. After 8 weeks of culture, protoplast- derived colonies were counted and transformation fre- quencies determined. When resistant colonies reached 1 mm in size (after 60-80 days of culture), they were picked out from the alginate matrix and transferred to shoot regeneration media N2 (Chuong et al., 1987, modified by Sacrist~in et al., 1989). Shoots 2-3 mm long were detached from calluses and further culti- vated on AM-medium. To promote root development, shoots were placed on AMI (AM-medium containing 1 mg 1-1indole-3-butyrate). Rooted shoots were trans- ferred to soil.

Analysis of plant genomic DNA

Genomic DNA from plants or calluses was isolated according to Rogers and Bendich (1985), but using only a 2% CTAB-extraction. After digestion with Eco RV or BamHI, the fragments were separated by elec- trophoresis in 0.8% agarose gels, alkaline vacuum blotted to a nylon membrane (Hybond N-Filter, Amer- sham) and hybridized to the 32p-labelled 1.1 kb BamHI fragment of pGL2, which covers the coding region of the HPT gene.

fl-glucuronidase enzyme assay

Protoplasts treated with pRT103gus were incubated in NT-medium for 2 days at 23 °C in the dark. Thereafter they were centrifuged at 140 x g for 5 min and the pellets were resuspended in extraction buffer (50 mM phosphate buffer pH 7, 10 mM /3-mercaptoethanol, 10 mM EDTA, 0.1% v/v Triton X-100), transferred to an Eppendorf tube and subjected to three cycles of

freezing in liquid nitrogen and thawing at room tem- perature to lyse the protoplasts and release the GUS enzyme. After centrifugation at 15800 × g for 5 min, the supernatant was assayed in replicate for protein content (Bradford, 1976) and for GUS activity by flu- orimetry as described by Jefferson (1987).

Progeny analysis

Seeds harvested from self-fertilized transformants (ST1 and ST2 progeny) were germinated on AM- medium + 20 mg 1-1 hygromycin B. After 3 weeks, resistant seedlings could be clearly distinguished from sensitive ones.

Results and discuss ion

Protoplast transformation and regeneration

Plating efficiencies (PE) of 1.5-1.8% and shoot regen- eration frequencies of 40-60% were achieved for La- er, M4 and M 10, employing NT-medium for micro- colony culture and N2-medium for shoot regeneration (Torres, 1994)• Higher PEs have been reported for La-er by previously selecting protoplasts subpopula- tions obtained through a Percoll gradient (Park and Wernicke, 1993), and for other ecotypes by using opti- mized plant culture conditions (Masson and Paskows- ki, 1992) or conditioned root cultures (Mathur et al. 1995). Applying the protocol of Park and Wernicke (1993) to La-er, M4, and M10 without separation of protoplasts PEs of 1.4-2.3% and shoot regeneration frequencies of 11- 18% were obtained (Torres, 1994). The transformation conditions established by Damm and Willmitzer (1991) for the ecotype C24 did not work efficiently with our lines. Transient gene expres- sion studies were undertaken to determine plasmid and PEG-concentrations suitable for transformation. The results are shown in Figure 1.

Transient gene expression decreased with lower PEG concentrations, these decreases could be compen- sated by simultaneously increasing the plasmid con- centration. Mathur et al. (1995) obtained similar tran- sient GUS-activity for the ecotype Columbia using 40 gg plasmid per 1.5 x 106 protoplasts and 26% PEG. For stable transformation, however, such high PEG- concentrations were not appropriate because they led to a drastic reduction of the plating efficiency (data not shown). Table 1 shows the results achieved using two different plasmid DNA concentrations and differ-

113

L a n d s b e r g erecia

! 4 5 ~ [ i pEG 26%1w~) ! 4o I- I~rr-cis~("~')l • } 3st I U r ~ " " ~ ' l t -

,

0 I i i i I 7Ng 14/ag 20/ag 2Sl~g 50~tg

amount plssmid D N A / 600,000 protoplasts

6o

55

-I ,01

40 !3, • 30

2O

15

M 4

_ i iU rEG l~-/, (,,~) I

L k[ [ 7gg 14$ag 20lag 2gpg 5011g

amount plasmid DIqA / 600,000 protoplasts

7o

65 !°° 55

50 !4, 40

3O 25

:~ 2o w 15

t~ 5 0

7btg 14tZg 20Hg 2g~tg 50tlg

amount plasmid D N A / 6 0 0 , 0 0 0 protoplssts

Figure 1. Transient GUS activity in La-er (a), M4 (b) and MI0 (c) after transformation with different plasmid DNA (pRT103gus) and PEG concentrations. The 2:1 carrier to plasmid DNA proportion was always maintained. Data are mean values taken from 4 independent experiments.

ent PEG concentrations. The relative transformation frequencies increased in all cases with the higher plas- mid DNA concentration, independently of the PEG concentration used, under the applied selection con-

114

~tb/e 1. Relative transformation frequencies (% SD) estimated after 8 weeks of culture using two different pGL2 plasmid concentrations (amounts of DNA per 6 x 105 protoplasts) and different PEG-concentrations. Selection began with 10 mg 1-1, and after 10 days continued with 20 mg I - l hygromycin B. Data are mean values taken from 5 independent experiments, with at least 10 replicates per experiment.

La-er M4 M10

PEG % (w/v) 7 gg plasmid 14 lag plasmid 7 lag plasmid 14 lag plasmid 7 lag plasmid 14 lag plasmid

26% 1.4 -I- 0.2 1.8 4- 0.3 0.9 -t- 0.2 1.44- 0.4 0.6 4- 0.2 1.24- 0.3

17% 1.5-t-0.2 2 .44-0 .2 1.14-0.3 2.04-0.2 0 .94-0.3 1 .74-0 .4

15% 1.94-0.3 3.1:t::0.4 1.34-0.1 2.74-0.2 1.14-0.1 2 .54-0 .2

13% 2.04- 0.2 3.24- 0.l 1.5 4- 0.I 2.94- 0.1 1.3 4- 0.2 2.54- 0.3

ditions. The more suitable transformation parameters were showen to be: 14 lag plasmid DNA, 28 ~tg carri- er DNA per 6 x 105 protoplasts and 15% (w/v) PEG, concentrations that enabled both plant regeneration and efficient transformation. Mean relative transformation frequencies of 3.1%, 2.7% and 2.5%, and mean abso- lute transformation frequencies of 2 x 10 -4, 2 x 10 -4 and 1 x 10 -4 were obtained for La-er, M4 and M10, respectively (Table 1).

Co-transformation experiments using the plasmids pGL2 and pRT103gus (15 lag each and 50 lag carrier DNA per 6 x 105 protoplasts) were carried out. Cal- luses which grew under hygromycin-selection condi- tions were subsequently tested for GUS activity. Co- transformation frequencies of 50 %, 30 % and 22 % were found for La-er, M4 and M10, respectively.

Plant regeneration was achieved by transferring macrocolonies developed under selection conditions (60 days) to non-selective shoot regeneration medi- um. On average, 40-60% of the hygromycin-resistant calluses regenerated shoots in all three lines analyzed. Calluses derived from different transformation exper- iments varied in their shoot regeneration capacity. The shoot regeneration rate of La-er reached 90% in some experiments, and was in general higher than that of the marker lines. Sixty to 80% of the shoots developed into fertile transgenic plants. The transfer of rooted shoots to soil was an important prerequisite for obtaining good plant development and seed yield.

Proof of integration of the HPT gene in the plant genome

Genomic DNA isolated from plants or calluses derived from hygromycin-resistant colonies was digested with Eco RV or Barn HI and subjected to Southern-blot anal- ysis using the 1.1 kb Bam HI-fragment from the pGL2 plasmid as a molecular probe. Because there is only

one single Eco RV restriction site in the plasmid pGL2 (within the promoter sequence of the HPT gene), diges- tion of the genomic DNA with this restriction enzyme should prove the integration of the HPT gene into the plant genome. The presence of a single hybridizing band at 4.5 kb would indicate vector contamination, and bands larger or smaller than 4.5 kb would prove integration. In all samples analyzed, more or less com- plex integration patterns could be detected. The marker lines showed frequently multi-copy integration of the hygromycin gene, whereas in the ecotype La-er the number of integrated copies was usually lower (see Table 2 and Figure 2).

When genomic DNA was digested with Bam HI, the presence of the full-length HPT coding sequence could be detected since this restriction enzyme cleaves the 1.1 kb pGL2-fragment corresponding to the coding region of this gene. This band was detected in all sam- ples tested. As shown in Figure 2, different patterns were found depending on the transformant analyzed. Among other possible explanations, a multi-band pat- tern could indicate that not all HPT genes were present in the plant genome as full-length gene copies, or that one of the Barn HI cleavage sites of this gene was lost. This would explain the additional bands above 1.1 kb (Figure 2). Rearrangements or other modifications in the plasmid, prior to or during integration, would also lead to such complex patterns.

Inheritance of the hygromycin resistance

For progeny analysis, seeds harvested from self- fertilized plants regenerated from hygromycin- resistant colonies were tested under selection condi- tions (20 mg 1-1 hygromycin B) in order to study the transmission of the antibiotic resistance in the ST1 (offspring derived from a self-fertilized primary trans- formant). Seeds germinated with rates of 80-100%.

Table 2. Number of integrated HPT gene copies, estimated from the number of bands detected by Southern-blot hybridizations using the 1.1 kb Barn HI-fragment from pGL2 as probe, and genomic DNA from transformants digested with Eco RV.

Number of integrated HPT genes Line No. of transformants 1 copy 2-5 copies >5 copies

analysed

La-er 30 5 22 3 M4 24 3 12 9 M10 10 0 4 6

115

E B U E B U E B U C C Z. 1 2 3 4 5 6 7 8 9 10 11 12

23.1kb

9.4kb

6.6kb

4.3kb

2.3kb 2.0kb

1.1kb

0.5kb

Figure 2. Southern-blot analysis of pGL2 transformants. DNA samples were digested with Eco RV (E) or Barn HI (B), and undigested DNA was also loaded (U). Hybridization was performed with the 1.1 kb Bran HI-pGL2 fragment. Lanes 1 - 3: La-er, lanes 4 - 6: M10, lanes 7-9: M4. Untransformed (C) L. erecta and M4 DNA were loaded in lanes 10 and 11, respectively. Lane 12 represents ,X DNA-Hind IlL

When the hygromycin-resistant STl-plants were visu- ally evaluated, no change in their phenotype with respect to the original material could be detected. The

phenotypic markers of M4 (long hypocotyl) and M10 (reduced petals, large sepals) were still present. The segregation of hygromycin resistance in the ST1 proge- ny from 22 independent transformants of La-er, M4 and M10 is presented in Table 3. An attempt was made to correlate the segregation patterns with the number of bands detected in the Southern-blot analysis of the pri- mary transformants. In most cases, the observed segre- gation corresponded to the presence of 1 or 2 functional

copies of the HPT gene. However, these segregations could not always be correlated with the number of bands detected in Southern-blot hybridizations, sug- gesting that not all integrated HPT genes copies were active or that more than one copy was integrated in the same locus. No segregation at all was observed for the transformants T37, T50, T53, T64 and T78, all ST1 seedlings being hygromycin-resistant. These transformants always had a multi-copy integration of the HPT gene, and the reason for the absence of seg- regation could be either that the number of analyzed plants was too low, or that gene copies had been inte-

116

Table 3. Segregation of hygromycin resistancein the progeny of 22 primary trans- formants from La-er, M4 and M10.

Landsberg erecta

Number of seedlings Trans- Resistant Sensitive Presumed X 2 HPT formant segregation detected bands 1

T3 438 20 15:1 2.75 #2 4 T5 96 40 3:1 1.40 # 2 T6 690 210 3:1 1.33 # 2 T10 378 34 15:1 2.81 # 2 TI2 137 15 15:1 3.39 # 2 TI5 112 28 3:1 1.86 # 1 T16 307 86 3:1 2.02 # 1 T24 112 12 15:1 2.48 # 2

M 4

T30 97 23 3:1 2.17 # 5 T32 116 10 15:1 0.65 # 9 T37 270 0 (63:1) 3 3 T43 336 29 15:1 1.78 # 9 T46 352 102 3:1 1.54 # 1 T49 143 35 3:1 2.69 # 2 T50 139 0 (255:1) 4 T53 241 0 (4095:1) 6

M 10

T62 135 15 15:1 3.60 # 4 T64 132 0 (255:1) 4 T67 170 46 3:1 1.57 # 7 T69 183 6 15:1 3.04 # 9 T76 240 10 15:1 2.14 # 6 T78 112 0 (4095:1) 6

i Number of HPT bands detected by Southern-blot analysis in the respective primary transformant. z # accordance with presumed ratio (p > 0.05) 3 Segregation pattems in parenthesis represent patterns expected according to the number of bands detected by Southern-blot analysis.

grated in both h o m o l o g o u s c h r o m o s o m e s o f one pair.

In addit ion, also changes in p lo idy level cannot be

exc luded , a l though no plant showed any m o r p h o l o g i -

cal trait which cou ld indicate polyploidy, and r andom

samples cy to log ica l ly ana lyzed a lways revea led the

diploid c h r o m o s o m e number (data not shown). The

segregat ion o f h y g r o m y c i n res is tance in the ST2 gen-

erat ion was s tudied in the p rogeny of 30 ST1 plants

de r ived f rom 3 independen t t ransformants (Table 4).

H y g r o m y c i n resis tance was a lways t ransmit ted to the

ST2 generat ion, but also in this genera t ion unexpec t -

ed segrega t ion patterns were observed. For instance,

in the ST2 progeny f rom T15 ( S T I : I func t iona l H P T

copy) , depending on the ST1 mothe r plant, a 3:1 segre-

gat ion (ST1 he te rozygous for HPT) wou ld be expec ted

or all p rogeny should be resis tant (ST1 homozygous ) .

For the ST2 progenies f r o m T10 and T69 (ST1:2 func-

t ional H P T copies), no segrega t ion (all ST2 plants

resistant), 15:1 or 3:1 segrega t ion pat terns wou ld be

expected . The devia t ions f r o m these expec ta t ions sug-

gest that there were modi f ica t ions in the express ion of

the t ransgene over generat ions .

Lack of correlat ion be tween the segrega t ion data

and the results o f the Southern-b lo t analysis has

117

Table 4. Segregation of the hygromycin resistance in the proge- ny of hygromycin-resistant ST1 plants.

ST2 from T15 (La-er)

Plant Resistant Sensitive Expected X 2

No. seedlings seedlings segregation

1 50 15 3:1 0.12

2 26 24 3:1 14.08 *l 3 38 22 3:1 4.34 4 53 13 3:1 0.98

5 45 18 3:1 0.42 6 64 11 3:1 4.26 * 7 48 11 3:1 1.26

8 58 17 3:1 0.21 9 56 11 3:1 2.62

10 52 16 3:1 0.07

ST2 from T10 (La-er)

1 32 18 3:1 3.33 2 44 32 3:1 11.85 *

3 45 23 3:1 2.82 4 30 20 3:1 5.23 * 5 53 18 3:1 0.004

6 61 6 15:1 0.84

7 53 24 3:1 1.24 8 44 4 15:1 0.55

9 47 32 3:1 9.73 * 10 52 15 3:1 0.24

ST2 from T69 (M10)

1 51 18 3:1 0.04 2 28 16 3:1 3_02 3 43 6 15:1 3.18 4 44 26 3:1 5.87

5 37 8 3:1 1.24 6 37 18 3:1 1.74 7 45 6 15:1 2.66 8 40 6 3:1 3.5

9 58 21 3:1 0.09 10 52 12 3:1 1.33

i • Statistical evidence (p < 0.05) of deviation from expected ratio.

also b e e n o b s e r v e d b o t h afterAgrobacterium-mediated t r a n s f o r m a t i o n a n d af te r d i rec t gene t rans fe r in A.

thaliana ( F e l d m a n n and Marks , 1987; S c h m i d t and

Wil lmi tzer , 1988; D a m m et al., 1989; Sche id et al.,

1991; Ki lby et al., 1992). To da te no r egene ra t i o n pro-

tocol for p ro top l a s t s has b e e n app l i cab le to all e co types

o f A. thaliana. O u r i m p r o v e d pro toco l of fers a s imp le

m e t h o d for r e g e n e r a t i o n and t r ans fo rma t ion o f mes -

ophyl l p ro top las t s o f A. thaliana e c o t y p e La-er , a n d

the m a r k e r l ines M 4 an d M 1 0 . Th i s is a p r e r e q u i s i t e

for somat i c h y b r i d i z a t i o n and c o m p l e m e n t a t i o n s tud ies

at the p ro top l a s t level . In the p r e s e n t r e sea rch , s t ab le

t r a n s f o r m a t i o n by d i rec t gene t r ans fe r of m u t a n t l ines

has b e e n a c h i e v e d for the first t ime.

118

Acknowledgements

We would like to thank Annette N6h and Hannelore Lehmann for technical assistance. This work was supported by a scholarship from the Katholischer Akademischer Auslander-Dienst (M.Torres).

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