High efficiency transformation of peppermint (Mentha × piperita L.) with Agrobacterium tumefaciens

Plant Science

ELSEVIER

136 (1998)

101

108

High efficiency transformation of peppermint (Mentha x piperita L.) with Agrobacterium tumefaciens F. Diemer a**, F. Jullien a, 0. Faure a, S. Moja ‘, M. Colson ‘, E. Matthys-Rochon J.C. Caissard a

Received

9 March 1998; received

in revised

form

1 I May 1998: accepted

26 May

“,

1998

Abstract

Transgenic peppermint (Men#~a x piprritu L.) plants were obtained by using Agrohuctrriw~ tltr,lelircien.s-mediated gene transfer. The effects of the coculture period and of the Agrobucterium strain were tested. 10% transformed plants were regenerated by leaf disk culture after inoculation with strain EHAlO5/MOG harbouring /I-glucuronidase and neomycin phosphotransferase II genes. with a coculture period of 5 days. Rooting of regenerated plants was achieved on selective medium with 150 mg/l kanamycin. The presence of transgenes in DNA was shown through PCR and Southern blot hybridization and transgene product activity via histoenzymatic GUS test and leaf callus assay. Transgenic plants were successfully acclimatized in the greenhouse. 10 1998 Elsevier Science Ireland Ltd. All rights reserved. Kgwwds: MU&U phosphotransferase

x pipe&u II

L.; Agrobucterium

rwmfuciens:

.Ahhrrriutions: BAP. h-benzylaminopurine; CTAB, hexadecyltrimethylammonium bromide; 2.4-D. 2.4-dichlorophenoxyacetic acid: GUS. /J-glucuronidase: GUS INT, /i-glucuronidase gene with intron: IBA, indole-3-butyric acid; Kan’. kanamycin resistant: MS, Murashige and Skoog: NAA,

Genetic

transformation:

j’-glucuronidase:

Neomycin

1. Introduction Peppermint matic

plant,

(Mmthct vegetatively

x piperitcl propagated,

L.) is an aroproducing

naphtalenacetic acid; NPTII, neomycin phosphotransferase II: PCR. polymerase chain reaction: rpm, rotation per min: TDZ, thidiazuron.

essential oils of great economical interest for their medicinal. culinary and fragrance properties. The

* Corresponding author. Tel.: + 33-4-7748 1583; Fax: 4-7725 I8 17; E-mail: [email protected]

major constituents found in peppermint oil are monoterpenes, which are Cl0 molecules all struc-

Ol68-9452:98:$19.00

SC“1998 Elsevier Science

PII: SOl68-9452(98)00107-l

Ireland

+ 33.

Ltd. All rights

reserved.

turally derived from the condensation of two isoprene units [l]. Since the beginning of the 197Os, monoterpene biosynthesis has been largely studied and many enzymes of the pathway have been characterized. Modification of the activity of these enzymes [2] could be of great interest both for commercial use and as a suitable tool for studying regulation mechanisms controlling major endproducts accumulation. For instance, overproduction of geranyl diphosphate or limonene should inform on rate-limiting reactions in the accumulation of compounds such as menthone or menthol. Furthermore, the modification of oil composition could also be attempted by the decrease of undesirable products, like menthofuran. Unfortunately, such modifications can not be obtained by classical selection as peppermint is sterile, even if a few attempts to modify essential oil production were performed by irradiation mutagenesis of rhizomes [3]. Nevertheless, progress in biotechnologies offer an alternative promising way to improve mint genotypes by gene transfer. To date, a few cDNA encoding enzymes of the pathway, such as 4S-limonene synthase [4], linalool synthase [5], and myrcene synthase [6] have been cloned. Therefore, the effects of antisense constructs and constitutive or organ-specific expression of selected transgenes in peppermint could be studied, if a genetic transformation method was available. Up to now, a few papers related to mint transformation have been published. However, most of these transformation experiments were performed using wild strains of Agrobucterium tun~eficiens. First attempts [7] with the virulent strains ChrlIB and B6 of A. tunzejkirn.s failed to obtain transformed calli. Later. using the wild strains T37 and C58. Spencer et al. [8,9] obtained transformed shooty teratoma of M. x piperitu L. and M. x citrutu. However. this procedure gave plants harbouring the wild T-DNA genes and was unsuitable for the transfer of other genes. Berry et al. [lo] confirmed the susceptibility of peppermint to wild strain A281, but they did not report plant regeneration. Using disarmed Agrobacterium strains, transient GUS expression was obtained by Caissard et al. [l 11, and recently transgenic plants containing GUS and NPTII genes were regenerated by Niu et al. [12], but with a transformation rate of l’%).

A reliable leaf disk regeneration method for peppermint has been lately reported [13]. Using this method, we report a procedure for successful regeneration of transgenic peppermint expressing the GUS and NPTII reporter genes leading to a transformation efficiency of 10%.

2. Materials and methods

2.1. Plunt muterid

und regeneration

nlediiunl

Plants of M. x piperitu L. cv. Black Mitcham 38 micropropagated in vitro [l l] were used as source of explants. Leaf disks, 4 mm in diameter, were excised with a cork borer and cultured on callus formation medium, for 2 weeks at 25 _+ 2°C in the dark. This medium was constituted of MS salts [ 141, Morel and Wetmore [I 51 vitamins, 0.03 M sucrose, 0.3 M mannitol, 2 PM IBA, 2 PM BAP and solidified with 0.15% phytagel (pH 5.8). Explants were then transferred onto caulogenesis medium, at 25 f 3’C under a 16/8 h photoperiod (6 pmoljm’ s). Subculturing was performed every 2 weeks. Caulogenesis medium was constituted of 112 MS salts, Morel and Wetmore vitamins, 0.09 M sucrose, 9 yM BAP, 0.5 PM NAA, 0.5 FM TDZ and solidified with 0.15% phytagel (pH 5.8). _ 1.5 cm high, were transRegenerated shoots, ferred in jars onto the same medium without growth regulators to allow both rooting and shoot elongation, at 25 i: 2’C under a 16i8 h photoperiod (94 pmol/m’ s). 2.2. Agrobucteriunl

struins und rectors

A total of five different A. tumejticiens binary vectors harbouring the GUS INT and NPTII were used. They were LBA4404/Gl, genes C%pMP90/Gl, GV2260/Gl, AGLl/BGl and EHAlOS/MOG. The LBA4404/Gl, C58pMP901 Gl and GV2260/Gl strains contain the p35S GUS INT plasmid [16], they are respectively derived from LBA4404 [17], C58pMP90 [IS], and GV2260 [19]. The strain AGLl/BGl was derived from AGLl [20] and contained the pB + GIN plasmid (provided by L. Jouanin, INRA Versailles, France) where the GUS INT is near the right

F. Dirnw

et al.

Plrolt Srietrw

border of the T-DNA. The EHAlO5/MOG was derived from the strain EHA105 and contained the pMOG410 plasmid [21]. .?.3. Coculticution

urzd phrlt

r~gmrration

Bacteria were grown overnight in LB medium (Sigma, St. Louis, MO, L3522) with the appropriantibiotics ate and 50 uM acetosyringone (Aldrich, Germany, D13 440-6) at 100 rpm on a rotary shaker. Bacterial suspensions (OD,,,, = 0.5) were rinsed three times in liquid callogenesis medium by centrifugation (15 min, 1600 x g). Leaf disks were immersed in a lo-fold dilution of this suspension and shaked at 170 rpm on a rotary shaker for 30 min. They were then dried on sterile filter paper before being placed onto callus formation medium. After 2 or 5 days of coculture, explants were rinsed three times for 10 min in liquid callus formation medium with 400 mg/l Augmentin (Smithkline Beecham. France), under shaking at 170 rpm. Leaf disks were then dried as before and cultured onto regeneration medium, supplemented with 400 mg/l Augmentin and 50 mgl kanamycin monosulfate (Sigma, USA, K4378). Regenerated shoots were rooted in the presence of 150 mg;l kanamycin.

GUS assay was carried out according to the method of Jefferson et al. [22]. Tissues were incubated overnight in 1 mM 5-bromo-4-chloro-3-indolyl-/?-D-glucuronide. 0.5 mM potassium hexacyanoferrate (III). 0.5 mM potassium hexacyanoferrate (II), 10 mM NazEDTA and 50 mM sodium phosphate buffer. GUS positive spots were observed under a stereomicroscope.

Leaf disks were taken from several leaves of each transformed plant. They were cultured in darkness for 2 weeks in the presence of 50 m&/l kanamycin onto a medium triggering callus formation. This medium contained MS salts, Morel and Wetmore vitamins, 0.06 M sucrose, 4.5 uM 2,4-D, and was solidified with 0.7% agar (pH 5.8).

lib

(1998) 101 108

2.6. DNA

103

c~struction

High molecular weight DNA was isolated from 2 g of leaves of transformed and control plants according to the CTAB method [23] with the following composition of the extraction buffer: 100 mM Tris-HCl pH 7.5. 0.7 mM NaCl, 10 mM Na,EDTA pH 8, 1% 2-mercaptoethanol and 2% CTAB.

PCR was performed on a Perkin Eilmer thermocycler with DNA extracted from putative transformants and control plants. The primers used for amplification of a 469 pb fragment of the NPTII gene were 5’-CAAGATGGATTGCACGCAGGTTC-3’ and 5’-TCCAGATCATCCTGATCGACAAG-3’. and those used for amplification of a 1062 pb fragment of the GUS gene were 5’TAGAAACCCCAACCCGTGAAATC-3’ and 5’CGACCAAAGCCAGTAAAGTAGAA-3’. For each PCR. 100 ng of plant DNA and 10 ng of the corresponding plasmid for control were used. Samples were heated to 94°C for 3 min, followed by 35 cycles of 94°C (30 s). 60°C (30 s), 72°C ( 1 min 15 s) and then 72°C for 5 min. Amplified products were detected by ultraviolet light fluorescence (3 12 nm) after electrophoresis on 1.5% agarose gels and staining with ethidium bromide.

A total of 10 ug of DNA from control and transformed plants were digested with HirrdIII or EcoRI and separated on 0.8% agarose gels. DNAs were blotted to nylon membrane (HybondN + , Amersham) under alkaline conditions following standard procedures [34]. A 1062 pb fragment. obtained from PCR with GUS primers, was used as a probe after labelling with “P-dCTP by random priming. Filters were prehybridized for 4 h at 60°C in 4 x SSC (3 M sodium chloride, 300 mM sodium citrate), 1% SDS, 1 x Denhardt’s reagent (0.02% ficoll, 0.02’% bovine serum albumin. 0.02% polyvinylpyrrolidone) and 50 ug:ml salmon sperm DNA. Hybridization was carried out for 20 h in prehybridization conditions. Fil-

104

F. Diemrr

Table I Effect of coculture Coculture

period

period

on the formation

(days)

Agrohucwriutt~

et ul.

‘Phi

of peppermint strains

Sciencr

Number

LBA4404iGI GV?260,/GI EHAIO5/MOG

0 (0) 0 (0) 2 (1)

5

LBA4404/GI GV226O/GI EHAIO5;MOG

0 (0) 72 (36) YO (45)

explants

were inoculated

per condition,

results

ters were washed with 2 x SSC, 0.1% SDS, and besides with the same washing solution diluted x 20.

3. Results and discussion 3.1. Chdturr

period und Agrobacterium

101 -108

Kan’ calli

2

~’Two hundred

136 (1998)

strains

Comparisons were made between 2 and 5 days of coculture with three Agrobactrrium strains (LBA4404/GI, GV2260/GI and EHA105/MOG). The results (Table 1) showed that a lenghtening of the coculture period increased the percentage of kanamycin resistant (Kan’) calli obtained after 3 months of culture. This reached a maximum of 45% Kan’ calli for the strain EHA105. Thus, despite the loss of some explants due to the more significant growth of Agrobucteriunl, a 5 days coculture period was a critical step to allow transformation events. Similar results have been already reported with Forsvthiu and Cucumis [25,26]. Using a 5-day coculture, five strains of Agrobacteriunl were compared for their ability to mediate the production of Kan’ plants. In this experiment (Table 2) we obtained Kan’ rooted shoots (with frequencies between 2 and 10%) after inoculation with the strains C58pM90/GI, AGL 1/ BGI, GV2260/GI and EHA105/MOG, while the strain LBA4404/GI was inefficient. Comparisons of C58pM90/GI, AGLl/BGI and GV2260/GI strains showed no significant difference, while EHA105/MOG gave a significantly higher per-

of explants

were scored

producing

Kan’ calli.’ (corresponding

after 3 months

‘%)

of culture.

centage of Kan’ plants (10%). In fact, EHAlOSI MOG is known to be an hypervirulent strain, which also allows high transformation frequency on tobacco leaf disks [21]. Kan’ In this transformation protocol, organogenic calli were obtained within 228 months of culture (Fig. lA), and shoots were rooted on kanamycin selection pressure within 1 month. Plants were successfully acclimatized (Fig. 1B) and further analysed for gene expression and integration. 3.2. GiYS uttd NPTII

genes espression

A total of 20 putative transformed plantlets obtained with the strain EHAlOS/MOG were studied. The rooting of regenerated plants on selection medium with 150 mg,/l kanamycin showed the expression of NPTII gene while untransformed control plants were not able to root. Each plant was tested for GUS gene expression when transferred to rooting medium. From the 20 plants analyzed, 16 were GUS positive. GUS activity was observed in different kinds of organs and tissues, but especially in vessels of stems and leaves (Fig. IC). Four plants showed no GUS expression. although they grew well on selection medium. These 20% negative results could be explained either by the silencing of the GUS gene or by an incomplete T-DNA transfer, as already reported [27729]. They could also be due to a problem with the GUS assay as reviewed by Taylor [30].

Table 2 Effect of d,grohactrrium --

strains

on the formation

of peppermint

Number

LBA4404;Gl CSpMPYOGI AGLI ;BGI GV226O’GI EHA 105.MOG

200 210 210 200 200

3.3. GUS and NPTII

shoots

producing

Kan’

of culture, values followed by different comparison followed by a G test.

letters

of explants

Number

of explants

rooted

shoots”

(corresponding

‘!i,) .._~.._

0’ (0) 4b (2) 4b (2) Sb (3) 20” (IO)

____ ,’ Results here scored after 9 months using a x’-square

rooted

__~__

._(,~r”hn(,rc,ril~/?l strains

probohllity

Kan’

global

genes integration

Among the 20 selected plants, four were chosen: three of them were GUS positive and the last one was GUS negative. These four plants were analysed using PCR and Southern blot hybridization. In all these plants, a 0.5 kb and a 1 kb fragments. corresponding respectively to the region between the NPTII primers and the region between the GUS primers (Fig. 2A), were amplified by PCR (Fig. 2B). The plasmid p35S GUS INT was used as positive control and non transformed plants as negative control. So, plants 1 --3 were GUS and PCR positives but plant 4 was GUS negative and PCR positive. Further investigations on gene integration were then performed by Southern blot hybridization (Fig. 2C). DNAs were digested either by HindtlI or EcoRI and hybridized with the GUS probe shown in Fig. 2A. Hybridization patterns with EwRI digests enabled us to estimate the number of GUS gene copies, while hybridization with Hirrdlll digests was used to check transgene integrity. In both cases, the probe did not hybridize with the total genomic DNA from the control plants. but it hybridized with DNA from the four putative transformed plants, showing that they had integrated the GUS gene. Results from EcoRI digestion (Fig. 2C, left panel) showed that plants I, 2 and 4 probably beared only one copy of the transgene. On the contrary in plant 3, at least four copies were detected. Analysis of hybridization patterns after Hind111 digestion (Fig. 2C, right panel) showed the cxpccted 2.7 kb GUS fragment, in plants 1, 2 and 3.

__.___are significantly

different

at 0.05 level of

indicating that these plants had integrated at least one intact copy of the transgene. This result is in accordance with the positive histoenzymatic test performed on these plants. It must be emphasized that for plant 3, among the several integrated copies, at least two of them seemed to be truncated, as indicated by their longer size. Concerning plant 4, the GUS fragment was longer than the expected one. This result could be explained by a suppression of a Hind111 site. Moreover, the PCR being positive and the GUS assay negative would seem to indicate that a deletion appeared near the right border of the T-DNA. leading to the suppression of gene expression. The occurrence of such a deletion is more unusual than near the left border, but had already been reported [29], sometimes with a high level (e.g. 20’%, in [27]). 3.4. Chimneric

status of transfbrmrd

plunts

Leaf callus assay was used to evaluate the chimaerism of the transformed plants (Fig. ID). Disks were cut off from leaves inserted at different levels along the stem, and cultured on callus formation medium containing kanamycin. The four transgenic plants tested regenerated calli, from all explants cultured. On the contrary. callus formation did not occur from non transformed control. This result indicated that transformed plants were probably not chimaeric. In fact. it is likely that the strong selection pressure applied with I50 mg,,l kanamycin during rooting limits the occurrence of a high percentage of chimaeric plants. Peppermint being a sterile hybrid, the inheritance of transgenes was not studied by analysing

106

from a peppermint leaf disk ( x 1.9). (B) Acclimatized transgenic peppermint plant ( x 0.7). CC) Fig. I. (A) Bud regeneration _ - _ ^ .. Histoenzymatic observation of GUS gene expression in leaves and stem of transgemc peppermmt ( x 3). (D) Leat callus assay on selection medium with 50 mg/l kanamycin ( x 0.4). Formation of callus from leaf disks of transformed peppermint plant (up). Control with non transformed plant (down).

gene segregation in the progeny. However, we present strong evidence of peppermint transformation with GUS and NPTII reporter genes. Our transformation method has several advantages comparing to the transformation protocol proposed by Niu et al. [12]. First, we used a leaf disk method which permits a great regeneration rate. Moreover, the use of a high selection pressure

with 50- 150 mg/l kanamycin instead of 15-50 mg/l decreases the occurrence of escapes and reduces the number of chimaeric plants, as shown by the leaf callus assay. Finally, this new protocol greatly improved the transformation efficiency up to 10% and will allow to introduce genes of interest such as cDNA encoding enzymes of the monoterpene pathway.

107

Acknowledgements

HrndIII EcoRI

Thanks are due to P. Heizmann (ENS Lyon, France) for his assistance with PCR and Southern blot hybridization. The authors are grateful to L. Jouanin (INRA Versailles. France), for kindly providing Agrohucteriunl twm~fircic-ws strains C58pMP901’GL GV2360/Gl. AGLl !BGI and LBA4404:GI. They also thank Mogen International N.V. (The Netherlands) for giving the strain EHAIOS!MOG.

HzndIII 2.7Kb

.

l

References [‘I D.J. [:I

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3

4

5

6

7

8

9

10

Kb 10

2.5 Fig. 2. (A) Restriction map of the plasmid pMOG410 (from Mogen International N.V.. The Netherlands). N and N’ are primers for amplification of a 0.47 kb fragment of the NPTIl gene. G and G’ are primers for amplification of a I kb fragment of the GUS gene. (B) PCR analysis of regenerated peppermint transgenic plants with detection of the NPTIl dene (above) and detection of the GUS gene (below). Lane I. Kb DNA ladder (Stratagene. USA). Lane 2. control without DNA. Lane 3. positive control with plasmid. Lane 4. negative control with non transformed plant. Lanes 5-8, transformed plants I-4 with EHAlO5:MOG. (C) Southern blot hybridization of peppermint transgenic plants. Probe was expected to hybridize to a 2.7 kb GUS fragment. Lanes I 5. digestion by E,oRI. Lanes 6 IO. digestion by HbzdlII. Lanes I-4 and 7 IO. transformed plants I 4 with EHAIOS’MOG. Lane 5 and 6. control with non transformed plant.

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