Plant Cell Reports
Plant Cell Reports (1996) 15:929-933
A grobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants Annemieke Vergauwe 1, Ronny Cammaert a, Dirk Vandenberghe 1, Christiane Genetello M a r c Van Montagu 2, and Elfride Van den Eeckhout 1
2, Dirk Inze 2
1 Laboratorium voor Farmaceutische Biotechnologie, Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium z Laboratorium voor Genetica, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Received 21 October 1995/Revised version received 5 January 1996 - Communicated by W. Barz
S u m m a r y . A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N 6benzyladenine after transformation with the C58C1 Rift (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a ~-glucuronidase-positive response. Abbreviations: 2,4-D = 2,4-dichlorophenoxyacetic acid; BA = Nr-benzyladenine; GM = germination medium; GMVIT = germination medium with vitamins; GUS = ~-glucuronidase; Kin = kinetin (N6-furfurylaminopurine); NAA = ~-naphthaleneacetic acid; NPT II = neomycin phosphotransferase II; PCR = Polymerase Chain Reaction; T-DNA = transfer-DNA; X-glucuronide = 5-bromo4-chloro-3-indolyl [~-D-glucuronide
Introduction Many attempts have already been made to increase the production of the secondary metabolite and antimalarial compound artemisinin in the Chinese medicinal herb Artemisia annua L. (Asteraceae). Artemisinin is mainly concentrated in the aerial parts of the plant, but low yields are obtained (Liersch et al. 1986; Vandenberghe et al. 1995). The production of artemisinin by means of cell or tissue cultures has not been successful (Nair et al. 1986; Brown 1994). It would therefore be economically interesting to produce transgenic plants of A. annua which ensure a constant high production of artemisinin by overexpressing a key enzyme in the biosynthetic pathway of artemisinin or Correspondence to: E. Van den Eeckhout
by inhibiting an enzyme of another pathway, competitive for the precursors. This paper describes the first Agrobacterium tumefaciens-mediated transformation of A. annua plants and the recovery of transgenic plants. Shooty teratomas of A. annua were already established by infecting stem tissue with a wild-type A. tumefaciens nopaline strain for studying secondary metabolism, but no specified protocol was given (Paniego et al. 1993). Recently there have been some attempts made to transform A. annua plants, using A. rhizogenes (Weathers et al. 1994; Jaziri et al. 1995). In all cases transformed hairy roots were obtained, but no transgenic plants. Up to now no successful procedure to regenerate A. annua plants in a sufficient short time to be applied in an Agrobacterium tumefaciens-mediated transformation procedure has been established.
Materials and methods Agrobacterium strains. The following Agrobacterium grffms were used: C58C1 ~ (pGV2260) and C58C1 Rff (pMP90), both harbonng the binary vector pTJK 136 (Kapila and Angenon, persoaal communication). ThepTJK136 plasmid contains a ~-glucttrc~idase (gus) gene with intron, derived from the original gus intron cassette from p35SGUSINT (V~mc.~aeytet al. 1990), and the neomycin phosphotransferase FI (npt ]2) geae drives by the 35S and nos promotors, respectively. The intron in the gus gene assttres only plant-specific GUS expression (Vanc~meyt et al. 1990). The bacteria were grown on YEB medium (Vervliet et al. 1975) suvplemeated with the following selective antibiotics: for the C58C1 (pGV2260) (pTJK136) strain: spectinomycin (100 gg/ml), streptomycin (100 gghnl)~ad ampicillin (100 gg/ml) ~ad for the C58Cl Ri~ (pMP90) (pTJK136) strain: spectinomycin (100 gg/ml), streptomycin (100 gg/ml) and g~atamycin (40 gg/ml). Prior to infection the Agrobacteria were incubated overnight at 28~ in agitated liquid YEB medium. Plant material and plant growth. Seeds from West-Virginian and exYugoslavimaArtemisia annua strains were surface sterilized by stinhng in 5% NaOC1 during 20 minutes after immersing in 70% (v/v) ethanol for
930 The same transformation proceditre was repeated, using 750 mg/L vancomyein (Vaneocin| Lilly, Giessen, Germany) instead of cefotaxime.
2 minutes. The seeds were rinsed several times with sterile distilled water until the pH was 7 or lower. They were finally germinated under sterile conditions in 90 x 20 m m Pari dishes containing one quarter Mttrashige
The vancomycin concentration was reduced to 500 mg/L after five weeks
and Skoog salts (Murashige and Skoog 1962), supplemented with 2% (w/v) sucrose and solidified with 0.8% (w/v) agar. The pH was adjusted to
andfurther gradually reduced to 400, 300, 200 and 100 mg/L. Only after five months was this antibiotic totally omitted. Obtained calluses were
5.9 with 1 M KOI-I before the addition of agar. The medium was autoclaved at 1 bar for 20 rain. This meddumais further referred to as the
removed fromthe e x p ~ t s and cultured separately on the same medium for farther proliferation. Healthy shoots were cut off at the base and planted in rooting medium containing 0.1 mg/L NAA- Newly regenerated plantlets were fiagher treated as described for the regeneration procedure.
Crmllk3~on Medium (GM). Germination started within two or three days. Plants were cultivated in an experimental greenhouse using I-Ig- and Navapour lamps 16 hours a day and at a temperature of 22~ and a relative humidity of 40%. After two to three weeks seedlings were transferred to preserving jars containing the same medium.
Regeneration o f Artemisia annua plants. The basal medium for callus, shoot and root induction consisted of Murashige and Skoog salts (M~ashige and Skoog 1962) containing 2% (w/v) sucrose supplemented withthe following vitamins: 1 mg/L thiamine, 100 mg/L inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxin. The medium was solidified with 0.8% (w/v) ag~. ThepHwas adjustedto 5.6 with 1 M KOH prior to the addition of agar. The media were autoclaved at 1 bar for 20 rain. The vitamins were addedto a 50~ solution after autoclaving, This medium is timber referred to as GMVIT. Various hormone concentrations were added to the medium to induce callus and shocts on leaf, stem and root explants. Leaves were eta transversely intotwo segments and cultivated upside-down on the m e d i u ~ Stems were cut crossardse in pieces of ca. 0.5 em leagth and cultivated separately, while roots were stacked together and cut into segments of c& i em length and cldtivated in packets of five to ten segments. The Pexi dishes were sealed with Urgo Pore Tape (Laboratoires Urgo, France) and cultivated in the experimental greenhouse. In that way the cultures were kept under a 16 h light/8 h dark c2r All explants and callus were tratasferred to flesh medium weekly during the first month. Afterwards subctfltures were made every two weeks. Larger paris of callus were separated from the rest of the explant and cultivated separately on the medium for further growth and eventual shoot induction. Induced shoos of
Plant DNA isolation. D N A extracts were made from kanamycin resistant callus andtrmsgeincplant leaves according to DeUaporta et al. (1983) and acceding to Stacey and Isaac (1994). The same extraO,Jon was repeated for control untransformed callus and plants.
Polymerase chain reaction (PCR) analysis. The PCR-alyproach to show the transformation of A. annua plants and callus grown on selection medium, was based on the amplification of a 206 bp fragment of the 35S promotor of the gus gene integrated in the plant genome and c~ the demonstration of the absence of a 282 bp fragmmt situated outside the T-DNA on the pTJK 136 plasmid. The first polymerase chain reaction to amplify a part of the 35S promotor was performed on 1 gl of template DNA in a reaction volume of 50 gl with 1.2 units of T aq D N A polymerase. The PCR buffer consisted of 10 m M Tris-HC1 ptI 8.3, 25 mMKC1 and 0.01% geLatine. MgC12 was added m concentrations ranging from 3 to 7 raM. The different nucleotides were added in concentrations of 0.2 m M each. A emcentr~lien of 150rig of each primer (5'-ATGTCACATCAATCCACTTGCTITG-3' and 5'-TTGCCCAGCTATCTGTCACTTCATC-3') was maintained. Samples were sutxnitted to 30 cycles of 94~ for 30 s, 55~ for 30 s, and 72~ for 30 s with a final exte~tsion step of 72~ for 5 min in a thermal cycler (Cetus 480/Perkin Elmer, Foster City, California). The second polymerase chain reaction was performed with 200 ng of each of the following primers: 5'-TCGTGGCTGGCTCGAAGATA-3' and 5'-TTGTACGGCTCCGCAGTGGA-3' in lx
rooting medium. Different combinations of hormones were tested for their
PCR-reaction buffer inclusive of 1.5 m M MgCI2 (Boehringer, lviannlieim, Gmaa~y) using 0.2 m M of each deoxyribonueleotide and 1.2 units of Taq
root inducing capacity on shoots obtained from the differeat tested shoot
polymemse. Amplification conditions were for 30 cycles at 94~ for 1 min,
inducing media. Fully regenerated plants were transported to GM medium
50~ for 1 rain, and 72~ for 1 rain with a final extension step of 72~ for 5 rain. Amplified DNA fragments were electrophoresed on a 2% agarose gel and visualizedby staining with ~hidium bromide. The same procedure was performed on control tmtransformed callus and plants.
about l em height were separated from the basal callus and planted in
in preserving jars. One month later the plants were pored in soil.
TransformaO'on andselection. Leaf, stem and root explants from 12 to 18 weeks old in vitro cttltured plants were floated in 90 x 20 m m Pari dishes m 10 ml of liquid GMVIT medium supplemented with NAA 0.05 mg/L and BA 0.5 mg/L. To each PeWi dish, 25 ~tl of a late logAgrobacterium culture was added- These plates were cultivated in the experimental greeali~se dmingtwo days. After two days the leaf, stem and root explants were washed with the same medium containing 500 mg/L cefotaxime (Claforan| tIoechst AG, Frankfurt, Germany) for five minutes in order to destroy theAgrobacteria. Then they were blotted on sterile filter paper and incubated on solid mediurn with the same hormone conemtrations and in the presence of 500 mg/L cef~axime. Half of the media was supplemented with 20 mg/L kanamycin to select the transformants, based on the incorporation mad expression of the kanamycin resSst~ce encoding npt 11 gene. The media without l~aamycin were considered as controls. Again the Petri dishes were sealed with Urgo Pore Tape and cultured in the experimental greenhouse. The plant explants were transferred to fresh medium weddy daring the first month. Afterwards subcultures were made every two weeks. After eight weeks the concentration of cefotaxime was reducedto 250 mg/L. The cefotaxime was totally omitted after 12 weeks.
DNA sequencing. The product amplified by the first PCR reaction was purified using the WizardT M Preps DNA thxrification System (Promega, Madisen, Wisconsin). The DNA was eluted from the minicolumn using distilled water. DNA samples were prepared according to the PRISMT M Ready Reaction DyeDeoxyT M Terminator Cycle Sequeacing Kit (Perkin Elmer) by usingthe primer 5'-TTGCCCAGCTATCTGTCACTT CAT C-3'. The sequence was elucidated on an ABI 373A DNA sequencer (Perkin Elmer).
~-glucuronidase (GUS) assay. Callus cultivated on selectien medium and leaves, stems and roots from regmerated kanamycin resistant plants were analyzed for gus gene e ~ e s s i o n with X-glucuronide according to Jefferson (1987). Coloring was observed visually or microscopically.
Analysis of artemisinin and bioprecursors. Callus and regenerated plants were analyzed for their content of artemisinin and the bioprecursors artemnum B and mtemisitmeby HPLC with reductive electrochemical and
UV detectionalter solidphase extracticf~accordingto Vandenbe~gheet al. (1995). The synthetic artonisinin derivative artemether was used as internal~tmdard./nsteadofu,smgI ml of the tcgzl 6 ml toluene extract,this extract was evaporatedto dryness madtaken up in 1 ml toluene prior to performing the solid phase extractiaa. This led to a sixfold increase in sensitivityof the mahod.
Based on previous reports on more or less organized tissue cultures (Nair et aL 1986; Whipkey et al. 1992; Basile et al. 1993), we decided to test hormone combinations as described in Table 1. Whereas some authors limited their experiments to only leaf or stem explants, we expanded our experiments to leaf, stem and root explants. Table 1. Different combinations of growth regulators trod their callus inducing capacity~on leaf, stem and root explants NAA (mg/L)
2,4-]) BA (rag/L) ( m g / L )
0.02 0.05 0.05 1.0 1.0 -
0.1 0.5 1.0
0.1 1.0 0.1 0.1 10
Callus on expl~ats Leaf S t e m Root + +++ + +++ +++ + +
+ +++ + +++ +++ + ~ +
++ +++ ++ +++ +++ ++ +
between the color, and thus the chlorophyll content, of the callus and its capacity to regenerate. It was favorable to culture the callus under a light/dark cycle, since complete darkness led easily to browning of the callus, in accordance with the observations by Nair et al. (1986) and by Paniego et al. (1993). Some of the media described in Table 1 led to the formation of roots after two weeks. Root induction at such an early stage is undesirable, but those media were tested further on their root inducing capacity on induced shoots (Table 2). When the calluses were separated from the explants and further cultured on medium with high cytokinin concentrations such as B A 1.0 mg/L and Kin 1.0 mg/L shoot formation was rare. Even when callus from a medium other than the N A A 0.05 mg/L and B A 0.5 mg/L medium was transferred to a medium containing a combination of N A A 0.05 mg/L and B A 0.5 mg/L, few shoots were formed. It seems therefore best to induce shoots directly from explants, than from induced callus. The callus and shoot inducing capacity was in general higher for the West-Virginian strain than for the exYugoslavian strain. Therefore all further experiments were conducted on the West-Virginian strain. Table 2. Influence of various hormone combinationson the rooting of shoots generated on a medium containing NAA 0.05 mg/L and BA 0.5 mg/L NAA (nag/L)
~Capacityis e:qrressedas + = low, ++ =medium, +++ =high,-=no callus F r o m all the tested hormone combinations the combination of N A A 0.05 mg/L and B A 0.5 mg/L revealed the best results. Within three weeks all root explants, 98% of the stem explants (n=171) and 95% o f the leaf explants (n=351) had developed callus. After five weeks 55% of the stem explants and 85% of the leaf explants gave rise to shoots. However the shoot regeneration continued further and one explant led to the formation of clusters of several shoots, so that the number of regenerated shoots was higher than the original number o f leaf explants. This led to a total number of regenerated shoots that was 122% of the number o f leaf explants and 66% of the number of stem explants. Roots never gave rise to shoots, independent of the incubation time. The shoot inducing capacities o f N A A 0.02 mg/L c o m b i n e d with B A 0.1 mg/L and N A A 0.05 mg/L combined with B A 1.0 mg/L were inferior to that of N A A 0.05 mg/L combined with B A 0.5 mg/L. Callus cultures from leaf and stem explants established by the use of 2,4-D were friable white or yellowish and did not lead to shoot production, while the green and more compact callus cultures established b y use o f N A A led to shoot formation. This is in agreement with the results found by Paniego et al. (1993). W e suppose there is a relationship
0.1 1.0 0.5 1.0
0.1 0.1 0.1
Results after 3 weeks No roots Normal fine roots Thick roots Roots and callus Thick roots and callus
Shoots obtained from medium containing N A A 0.05 mg/L combined with B A 0.5 mg/L were transferred to different rooting media. The results are summarized in Table 2. Most shoots had rooted within three weeks. With the addition of N A A 0.1 mg/L 99% (n=125) shoots developed roots. The regenerated A. annua plants were not different in appearance from the normally grown A. annua plants. All the regenerated plants survived after transfer to preserving jars and potting in soil. Previously no successful procedure to regenerate A. annua plants in a short time had been established. Nair et al. (1986) reported the regeneration ofA. annua plants starting from leafexplants by using a hormone combination of the auxin N A A (0.05 mg/L) and the cytokinin B A (0.1 to 0.2 mg/L). In about 60 days distinct shoots were evident in this procedure, but the rooting o f the shoots took three months. Comparison with our regeneration procedure leads to the conclusion that a higher concentration of the cytokinin B A is necessary for a rapid induction of shoots. However when the B A concentration is brought to 1.0 mg/L the same results are obtained as for 0.1 mg/L BA.
932 A concentration of 10 mg/L BA without the addition of an auxin was toxic for leaf and stem explants. This unhealthy influence of a high BA concentration was previously reported by Brown (1994).
9 Norrn~ Jn~tro grown plants
Fig. 1. C~t~as (% DW = % dry weight) of artemisinin and its precursors arteannuin B and artemisiteae in leaves of 3 months oldnormal in vitro gown plants and regenerated plaats (n=l 0 for each group of plants). The bars indicate the st,~dard deviation (SD).
Artemisinin contents in leaves of regenerated plants, compared with concentrations found in normal in vitro grown plants, are represented in Figure 1. Remarkable is the higher content of artemisinin in plants regenerated from leaf explants in comparison with normal grown plants of the same age. He et al. (1983) reported an artemisinin content of 0.92% in an A. annua plant regenerated from a 0.56% artemisinin containing plant. No artemisinin was detectable in callus. The lack of artemisinin or related compounds in non-transformed callus is consistent with other reports (He et al. 1983; Nair et al. 1986; Martinez and Staba 1988; Brown 1994). However Nair et al. (1986) found artemisinin in the culture fluid from liquid suspension cultures of callus cells, which means that the compound probably leaches into the medium. Generally the presence of artemisinin in various A. annua plant tissue cultures is more than 500 times lower than in the plant (Martinez and Staba 1988), which is consistent with the trace amounts we found in three months old transformed callus obtained from leaf explants.
Transformation procedure Two possibilities were considered for the decontamination of the Agrobaeteria after infection of the explants. Using cefotaxime as decontaminating antibiotic resulted in a retardation in callus formation (eight weeks for leaf explants) and an inhibition of the shoot inducing capacity. The other tested antibiotic, vancomycin, was not toxic for
the plant material. Besides, (transgenic) callus is produced after two weeks and within five weeks the production of transgenic shoots is started. However, this antibiotic has two disadvantages: it is rather expensive and the activity against the Gram-negative Agrobaeteria is rather weak. This weak activity led to bacterial overgrowth in many Petri dishes. Therefore further research is being performed to replace vancomycin by another more potent, but not toxic antibiotic for the plant material. The efficiency of the shoot induction is summarized in Table 3. Considering the different kind of explants, the use of leaf explants seems best for the production of transgenic A. annua plants. Root explants give most callus in the shortest time, but do not produce shoots. Stem explants are less potent in producing callus and shoots than leaf explants. Ten weeks after the transformation with the C58C1 (pGV2260) (pTJK136) strain 65% of the transgenic shoots were already obtained, The shoots grown on a medium containing NAA 0.1 mg/L (with an additional 250 mg/L vancomycin concentration for shoots formed within the first 2 months) developed roots in 90% of the cases. After transferring these plants to GM medium in preserving jars 92% survived. From the 24 plants potted in soil only four plants died. Of the shoots grown on the control medium 97% (n=168) rooted on NAA 0.01 mg/L. The survival rate in GM medium and afterwards in soil was 100%. The kanamycin resistant shoots grown after transformation with the C58C1 ~ (pMP90) (pTJK136) strain were very small and died on the rooting medium. Table 3. Shoot production after infection with the Agrobacterium tumefaciens strains C58C1 Ri~(pGV2260) (pTJK136) and C58C1 Ri~ (pMP90) (pTJK136) after 7 months by using vancomyciu as decontaminating antibiotic Leaf explants
Analysis of the PCR product by electrophoresis The results for callus and regenerants are presented in Figure 2. PCR analysis revealed that 75% of the putative transgenic plants contained the 206 bp fragment in their genomic DNA, without giving the 282 bp fragment of the outer part of the plasmid. The identity of the 206 bp PCR product was confirmed by DNA sequencing. An important observation was that the PCR was influenced by the method used for the DNA extraction, however a DNA extraction according to Stacey and Isaac (1994) led to successful PCR reactions in all cases. Best results were obtained with a 3 mM MgC12 concentration for the amplification of the 206 bp fragment.
206bp 1.Okb "
Considering the results obtained by the double PCR approach, DNA sequencing and the positive GUS assays obtained w i t h t h e g u s g e n e w i t h intron, c o m b i n e d w i t h the fact that the plants were grown on selection medium, we m a y c o n c l u d e t h a t w e h a v e s u c c e e d e d in e s t a b l i s h i n g the first t r a n s f o r m e d A r t e m i s i a a n n u a plants. T h e d e s c r i b e d procedure will allow u s to i n f l u e n c e t h e m e t a b o l i c p a t h w a y o f a r t e m i s i n i n b y e i t h e r o v e r e x p r e s s i n g or i n h i b i t i n g enzymes in order to o v e r p r o d u c e this v a l u a b l e antimalarial drug. Acknowledgements. The authors thank G. Angeaan and J. Kapila for providing the Agrobacteria strains and Raimundo Villarroel and Nancy Ten-ynfor preparing the PCR primers (Laboratory of Genetics, University of Ghent, Belginm). Dr. D.L. Klaym~ (Walter Reed Army In~Jtute of Research, Washington, DC) is gratefully acknowledged for providing Artemisia annua seeds and purified artemisinin, artemiffaene ~ad arteannuin B. This research was supported by the National Fund for Scientific Research, Grant no. 9.0034.93. A_ Vergauwe is a Research Aspirmat of the National Fund for Scientific Research (Belgium).
Fig. 2A-B. Polymerase chain rea~ions to show the integration of the new
genes in the plant genome. A S e ~ a t of the linear map of plasmid pTJK136, in which the positions of the PCR products are indicated. LB = left border, liB = right border. B In a 2% agarose gel 10 ~tl of the PCRDNA samplewas loaded. Lines 1 and 8:100 Base-Pair Ladder (Pharmacia Bioteda,Uppsah, Sweden). The 206 bp fragment of the 35S promotor was found in callus grown on selective medium after disinfection with cefotaxime (Lane 2) and vancomycin (Lane 3) and in plants grown on selectionmedinm (L~e 5). Control untr~asformed callus and plants did not possessthis fragment (Lanes 4 and 6). PCR on the pure pTJK136 plasmid was performed as positive control (Lane 7). The 282 bp fragment of the pTJK 136 plasmid was not found in the same callus or plants (Results not
The use of PCR combined with D N A sequencing instead of Southern blotting for the characterization of transgenic plants has the advantage that plants can be detected for their p r e s e n c e o f t h e n e w l y i n s e r t e d g e n e s i n a n earlier stage. L e s s D N A is n e e d e d , w h i c h m e a n s less p l a n t material.
~-glucuronidase (GUS) assay In 90% (n=40) of the eases the calluses produced on selection medium were found to be GUS-positive, independent of the kind of antibiotic used for the destruction of the Agrobacteria. From the transformed plants (identified by PCR) 94% were found to be GUSpositive. The expression of the gus gene was found in the leaves, as well as in the stems and roots.
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