callus regeneration medium (CRM, Table 2) containing ... callus on CRM supplemented with kanamycin was exam- ..... vitrification of apple cultivars in vitro.
The Plant Journal (1994) 6(6), 949-956 TECHNICAL ADVANCE
Genetic transformation of the C4 plant, Flaveria bidentis Julie A. Chitty 1, Robert T, Furbank 1'2, Jerry S. Marshall 1, Zhenghua Chen 3 and William C. Taylor 1'2'* 1CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra 2601, Australia, 2Cooperative Research Centre for Plant Science, c/o Research School of Biological Sciences, Australian National University, G.P.O. Box 475, Canberra 2601, Australia, and 3Institute of Genetics, Chinese Academy of Sciences, Building 917, Datun Road, Deshengmenwai, Beijing, 100101 China Summary An efficient and rapid transformation system for the dicotyledonous C4 plant, Flaverla bidentis has been developed. The method involves Agrobacterium tumefaciens infection of explants followed by regeneration of shoots from kanamycin-resistsnt callus. Shoots first appear on callus 3-4 weeks after Agrobacterium infection, with plants in soil after another 12-15 weeks. Transformation has been verified by measuring the activity of the gusA and nptII transgenes and by genomic Southern blots to show the stable integration of T-DNA. Most regenerated plants show normal morphology, are self-fertile and transgenes show expected Mendelian segregations in the next generation. This system allows one to test a number of gone constructions rapidly and should accelerate progress in studies of the cell-specific expression of genes coding for C4 enzymes and the mechanisms regulating the activities of C4 enzymes. Introduction Photosynthesis in C4 plants involves a cooperative interaction between two cell types, mesophyll and bundle sheath. The mesophyll cells contain a specialized group of enzymes which constitute a biochemical 'pump', concentrating CO2 in the bundle sheath cells where Rubisco is located (reviewed Hatch, 1987). This biochemical compartmentation is achieved by the cell-specific expression of the nuclear gene encoding each enzyme (Langdale et al., 1988; Sheen and Bogorad, 1987). In principle this
Received 30 June 1994; accepted 25 August 1994. *For correspondence (fax + 61 6 2465000).
makes the C4 plant an excellent experimental system for the study of cell-specific gene regulation. Although the biochemical features of the C4 cycle are well defined, very little is known about how flux through the cycle is regulated, especially in the range of environmental conditions where C4 plants grow in nature. How each enzyme contributes to the physiological properties of C4 plants is also poorly understood. Further progress in our understanding of C4 photosynthesis and the associated cell-specific gene regulation is dependent on the development of an efficient and reproducible genetic transformation system. Although transformation of maize (Fromm eta/., 1990; Gordon-Kamm eta/., 1990; Rhodes et al., 1988) and sugarcane (Bower and Birch, 1992) has been reported, neither system is efficient enough to support the analysis of a large number of gene constructions over a reasonably short time period. The genus F/averia (Asteraceae-Flaveriinae) has several features making it attractive for molecular biological studies of C4 biochemistry and gene regulation. Some species are C4, others are C3 and some are physiologically intermediate, ranging from C3- to C4-1ike (Edwards and Ku, 1987; Ku et al., 1991). The more C4-1ike intermediate species show some differentiation of mesophyll and bundle sheath cells with relatively high levels of C4 enzymes (Ku et al., 1983). The ability to compare closely related species with different physiological properties and different programmes of gene regulation should be a helpful experimental approach. Martineau et al. (1989) reported the transformation of two C4-1ike intermediate species, Flaveria palmeri and F. brownii, suggesting that C4 Flaveria species might be good candidates for transformation. F. bidentis is a fully C4 species with a wide geographic distribution, probably native to South America (Powell, 1987). It is an annual weed which is self-compatible and easy to propagate sexually or by cuttings. We describe the development of an efficient transformation system for F. bidentis. Results and Discussion
Development of a shoot regeneration method As a first step in establishing a transformation system for a C4 Flaveria species, we attemp'(ed to develop a method for regenerating shoots from callus. Martineau et aL (1989) reported the transformation of two C4-1ike Flaveria species using Agrobactedum infection of mature leaf explants 949
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Julie A. Chitty et al.
followed by regeneration of transformed shoots from callus. Shoots were regenerated on Murashige and Skoog medium containing 4 mg 1-1 6-(7-~,-dimethylallylamino)purine (2iP) and 1 mg 1-1 indole acetic acid (IAA). Our attempts to adapt their methods to F. bidentis were unsuccessful. The primary difficulty encountered with F. bidentis was in developing conditions to promote shoot regeneration from callus, even with untransformed material. The most important factors proved to be explant source and hormone combination. We tested the ability of cotyledon, hypocotyl and root explants to grow callus and regenerate shoots on a range of media (Table 1). All explants were able to form friable callus but root and cotyledon callus tended to become brown. Plants in the Flaveria genus are known to contain high levels of phenolic compounds (Powell, 1987) which could be responsible for the observed browning through oxidation. However, the inclusion of the reducing agents ascorbate (200 mg 1-1) and citrate (100 mg 1-1) in culture media (basal medium is MS+B5, see Table 2) did not reduce browning. Shoots regenerated from hypocotyl callus on several cytokinin/ auxin combinations, but regeneration did not occur from root callus and was observed at only a low and variable frequency from cotyledon callus. Kinetin and IAA proved Table
to be the best hormone combination for hypocotyl explants. Pale yellow to green friable callus proliferated, especially at the cut edges and appeared to be less susceptible to browning. Shoots formed mainly from callus at the cut edges and were visible by 15-21 days in culture. Regeneration frequencies (the number of explants forming shoots per total number of explants) observed were commonly 60-80% and as high as 100%. A number of shoots suffered from vitrification (hyperhydricity). In F. bidentis vitreous leaves were thick, broad and translucent. The plantlets broke easily, grew poorly, were difficult to root in vitro and did not survive in soil. A number of factors have been implicated in causing vitreous shoots, including high ammoniacal nitrogen concentration (Daguin and Letouze, 1986), high cytokinin levels in the culture media (Phan, 1991), type and concentration of gelling agent (Debergh, 1983; Pasqualetto et aL, 1988) and in vitro ethylene production (Kevers et aL, 1984). The addition of activated charcoal and the ethylene inhibitors silver nitrate (5 mg 1-1) and silver thiosulphate (10 and 50 lig 1-1)to shoot culture media did not minimize the problem of vitreous shoots. Phloridzin, which may enhance lignin synthesis (Witrzens et al., 1988), was not helpful either. The use of half strength Murashige and Skoog macronutrients, the reduction of the total nitrogen
1. Shoot regeneration frequency Cotyledons
Growth regulators (mg 1-1) 4.0 2iP + 1.0 IAA 4.0 2iP + 1.0 NAA 4.0 2iP + 1.0 IBA 4.3 Kin + 1.0 IAA 4.0 BAP + 1.0 IAA
Hypocotyls
21 days
50 days
21 days
50 days
21days
50 days
0/100 0/100 0/100 0/100 0/100
1/100 1/100 0/60 5/100 0/100
0/50 0•50 0/20 39/50 0/50
6/50 0•50 0/20 39/50 10/50
0/50 0/50 0/20 0/50 0150
0/50 0/50 0/20 0/50 0/50
Shoot regeneration frequency: number of shoots regeneratingper number of explants.
Table
Roots
2. Tissue culture media
Component Salts Vitamins Sucrose (g I-I) Kinetin (mg 1-1) A d e n i n e (mg 1-1) IAA (mg 1-1) GA3 (mg I-I) PP 333 (mg 1-1) pH Gelling Agent (g 1-1)
GM
CRM
F M+S B5 30
F M+S B5 30
-
4.3
5.8 8.0 Agar
20 1.0 5.8 8.0 Agar
SPM1 0.8 M+S B5 30 0.5 5 0.05 0.05 0.5 6.0 2.8 Phytagel
SPM2
SPM3
0.8 M+S B5 30 0.2 0.0 0.05 0.2 6.0 2.8 Phytagel
0.8 M+S B5 30 -
6.0 2.8 Phytagel
GM, germination medium; CRM, callus regeneration medium; SPM, shoot proliferation medium; F, full strength; M+S, Murashige and Skoog medium; B5, Gamborg B5 medium; IAA, indole-3-acetic acid; GA3, gibberellic acid; PP 333, paclobutrazol.
Transformation of C4 Flaveria content of the medium and an increased concentration of agar also did not solve the problem. Although the occurrence of vitreous shoots was never entirely eliminated, several factors helped. The use of Phytagel (Sigma) at 0.28% (w/v) as a gelling agent together with a gradual reduction of hormones in shoot media and low humidity in tissue culture vessels promoted the growth of many shoots with normal morphology. The combination of the gibberellin GA 3 and the gibberellin synthesis inhibitor paclobutrazol (Davis and Curry, 1991), previously shown to have a synergistic effect on plant growth (Evans, 1969), also contributed to improved shoot growth. The response of the regeneration process to gelling agents changed at different stages. Shoots did not regenerate from callus on callus regeneration medium (CRM, Table 2) containing Phytagel, however once shoot regeneration began, Phytagel helped to minimize vitrification. Shoots were removed from callus 5-10 days after they first appeared and transferred to shoot proliferation medium 1 (SPM1, Table 2) and then sequentially to SPM2 and SPM3 at intervals of approximately 1-3 weeks, depending on the health and rate of development of the shoots. Transferring shoots from CRM directly to SPM3 (without hormones) was not successful. We observed that a gradual stepwise reduction of hormones and other media supplements was necessary to promote normal shoot morphology and growth. Shoots larger than 10 mm with normal morphology were transferred to hormone-free media (SPM3, Table 2) and readily grew roots. Shoots proliferated readily on these media, especially on SPM3, resulting in a number of individual plantlets from each of the regenerating calli. These plantlets were kept in culture until 25-40 mm tall, then transferred to a compost-perlite mix (1:1) in small pots in the glasshouse. Pots were covered with disposable clear plastic cups to maintain humidity for 1-2 weeks. The majority of regenerated plants had normal morphology. A small proportion exhibited abnormal growth. The most common abnormalities were three leaves instead of two at stem nodes and spiralled or flattened stems. These abnormalities appeared to be non-genetic because plants were fertile and produced normal progeny.
Transformation of explants With the establishment of a regeneration system for F. bidentis, the next step was to develop a method of transforming cells in explants. Kanamycin was tested for its effectiveness as a selective agent. The ability of untransformed cotyledon and hypocotyl explants to form callus on CRM supplemented with kanamycin was examined. After 3-4 weeks, untransformed explants had not formed callus and were bleached and dying on CRM containing 50, 100, 150 or 200 mg 1-1 kanamycin. In sub-
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sequent transformation experiments a selection level of 150 or 200 mg 1-1 kanamycin was maintained. In initial transformation experiments the binary vectors pBI121 (Jefferson et al., 1987) and pKIWI105 (Janssen and Gardner, 1989) were used in Agrobacterium strains LBA4404 (Hoekma et al., 1983) and AGL1 (Lazo et al., 1991). After co-cultivation with pKIWI105 in AGL1, transient ~-glucuronidase (GUS) expression was observed in both cotyledon and hypocotyl explants for several days (data not shown). The gusA gene in pKIWI105 has been shown to be expressed in plant cells but not in Agrobacterium (Janssen and Gardner, 1989). On medium containing 200 mg 1-1 kanamycin, callus growth on explants was less extensive than growth from control explants on kanamycin-minus medium. Many cells in kanamycin-selected callus showed GUS activity at all stages of proliferation. These observations showed that stably transformed callus was not difficult to obtain. Agrobacterium strains LBA4404 and AGL1 were initially used interchangeably. However, we observed more prolific growth and better health of calli from explants cocultivated with the AGL1 strain. AGL1 was also preferable because it was easier to wash off explants after co-cultivation than LBA4404 (which tended to clump). Therefore, the majority of transformation experiments were carried out using AGLI. There was, however, no suggestion that transformation should not work with other Agrobacterium strains or binary vectors. Agrobacterium overgrowth on callus and on regenerating shoots was an occasional problem in initial transformation experiments. We were able to prevent overgrowth after co-cultivation by supplementing media with 200 mg 1-1 of the antibiotic Timentin. At this concentration, Timentin, unlike Cefotaxime, did not affect growth or regeneration of transformed material.
Regeneration of transformed shoots A major problem was encountered in combining the regeneration system with transformation. The regeneration frequency of hypocotyl explants was significantly reduced to 0-5% after co-cultivation with Agrobacterium and selection on kanamycin medium. However, the addition of the cytokinin precursor, adenine, in the range of 10-30 mg 1-1 was observed to improve both frequency and consistency of shoot formation, with 20 mg 1-1 being the optimum level. Results from seven representative experiments (Table 3) illustrate the variability of shoot regeneration frequency between transformations, ranging from 5 to 47%. Shoot regeneration from hypocotyls proceeded best when explants were left undisturbed on CRM (Table 2) supplemented with 200 mg 1-1 Timentin and 200 mg 1-1 kanamycin for 4 weeks after co-cultivation. Very small shoots were often visible toward the end of this period.
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Julie A. Chitty et al. Table 3. Summary of transformation experiments
Binary construct
Selection (mg ml-I kanamycin)
Hypocotyl expiants formingshoots
Plants _Confirmed recovered transformantsa
pGA 470b
100 200
3/60 (5%) 0/60
39c
39/39
pGA 482
150
6/70 (8%)
29+
28/29
MCP3
150 200
24/100 (24%) 14/30 (47%)
2 12
2/2 11/12
pGA 470
150 200
7/123 (6%) 3/50 (6%)
7 2
pGA 470
150 200
21/80 (26%) 5/40 (12%)
24 10
MCP3
150 200
5190 (5%) 8/50 (16%)
8 5
8/8 4/5
pGA 470
150 200
7/135 (5%) 4/50 (8%)
2 0
1/1 0
2/3 Not tested 12/15 4/4
aTransformation was confirmed by NPT-II activity and/or GUS staining and Southern analysis. bThis experiment did not have adenine added to tissue culture media and was transformed using the bacterial strain LBA 4404. CSouthem analysis demonstrated that many plants were dedved from the same transformation event.
Callus was then transferred to fresh CRM plus 200 mg 1-1 Timentin (without kanamycin). Shoots which appeared on this medium were also found to be transformed. Putatively transformed shoots were transferred to SPM1, 2 and 3 and then to soil as for non-transformed shoots. Selection levels of 150 or 200 mg 1-1 kanamycin in culture media allowed transformed material to grow, and reduced the likelihood of 'escapes'. It was possible to re-select putatively transformed shoots at the stage when roots first appeared. After removal of roots, shoots showing normal morphology were transferred to SPM3 supplemented with 100 mg 1-1 kanamycin. Transformed shoots were able to grow roots on kanamycin within 2-3 weeks, but non-transformed shoots showed no significant root growth in this time period. Because we were able to assay shoots in culture for neomycin phosphotransferase (NPT-II) or GUS activity using small amounts of tissue and because we observed a very low incidence of escapes, kanamycin re-selection was not routinely applied as a method for screening transgenic shoots.
Verification of transformation Regenerated plants were shown to be transformed by monitoring the activity of transgenes and by showing the presence of T-DNA in genomic Southern blots. All transformation experiments used binary vectors carrying the
npt-II gene driven by the nopaline synthase (nos) promoter. This promoter provided adequate NPT-II activity for resistance to at least 200 mg 1-1 kanamycin in callus and 100 mg 1-1 in plants. NPT-II activity could be measured from as little as 2 mg of leaf tissue from putatively transformed shoots in culture. NPT-II assays were routinely carried out on these shoots or on larger plants in the glasshouse. Figure 1 shows an NPT-II dot blot assay of primary transformants, from a single transformation experiment, all containing the same binary construct. Between 13 and 15 mg of tissue were taken from the youngest fully expanded leaf, extracted and assayed as described in 'Experimental procedures'. Each assay contained 10-12 I~g protein. Samples labelled 2 and 3 are clones from the same transformation event as are samples 7 and 8. NPT-II activity varied from 1 x 103to 5 x 105 c.p.m. 14g-1 protein. NPT-II activity varied by less than 10% between plants from the same transformation event. The wide variation in NPT-II gene expression between individuals was not related to transgene copy number (data not shown) and was presumably due to positional effects of T-DNA insertion. In two experiments explants were transformed with the construct pMCP3 (Khan et al., 1994) containing the gusA gene driven by the cauliflower mosaic virus 35S promoter. Leaves from these putative transformants and from nontransformed plants were assayed histochemically for GUS
Transformation of C4 Flaveria activity. We observed intense blue staining in transformed leaf tissue (Figure 2). GUS expression was evident in both the bundle sheath and mesophyll cells. No GUS activity was detected in untransformed control plants. Plants transformed with pMCP3 were also analysed for bar gene expression by performing phosphinothricin acetyl transferase (PAT) assays on shoots in culture or plants from the glasshouse. PAT activity was detected in all plants tested. Further comparison of the expression of pMCP3 marker genes will be described elsewhere. The presence of T-DNA in regenerated plants was verified by genomic Southern blots. Figure 3 shows a blot of Hindlll digested DNA from seven transformants selected from four different experiments. The number of hybridizing bands indicates the number of transgenes in that plant as Hindlll does not cut within the npt-II DNA probe used or between the npt-II gene and the right border
Figure 1. Measurement of NPT-II activity in 10 regenerated plants from one transformation experiment, along with three untransformed plants (-) and kanamycin-resistant tobacco (+). Equal aliquots from NPT-II assays containing 10-12 ~g protein were spotted.
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of the inserted T-DNA. Kanamycin-resistant plants were found to be stably transformed with at least one copy of the npt-II gene. Two to six copies of the transgene were commonly observed. Tt progeny of plants shown in Figure 3 showed segregation of the npt-II gene as determined by NPT-II enzyme activity in seedling leaf material. The plant in Figure 3 (lane C) had one copy of the npt-II gene and its progeny showed the expected segregation of a single gene (Figure 4c). Twelve out of 45 plants had no NPT-II activity, 22 had intermediate levels of activity (one copy of the npt-II gene) and 11 had higher levels of activity (two gene copies). The plants in Figure 3 (lanes D and G) had two copies of the npt-II gene, but their progeny showed single gene segregation, due either to close linkage between the two copies or relatively low activity from one copy. The plant in Figure 3 (lane E) had two npt-II genes which are unlinked and show independent segregation in the T1 progeny. Only four out of 50 plants had no NPT-II activity, close to the expected 1/16 for two unlinked genes. Multiple shoots often arose from the same area of kanamycin-resistant callus. In many cases these shoots were derived from the same transformation event as shown by their identical Southern blot hybridization patterns. The average transformation experiment, starting with 50-150 explants, gave shoots from at least two and more commonly four to 10 separate transformation events. This transformation and regeneration method has proven to be reproducible and reasonably fast. We routinely obtain shoots from about 5% of kanamycinselected callus in 3-4 weeks. The average time from co-cultivation of explants with Agrobacterium to plants in soil is currently 15-20 weeks. To date, we have recovered over 130 confirmed transgenic plants with 12 different
Figure 2. Expression of the gusA gene driven by the 35S promoter was evident in most F. bidentis leaf cell types. The leaf section was incubated overnight at 37°C.
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Julie A. Chitty et al.
constructs. We anticipate rapid progress towards understanding the mechanisms of cell-specific gene regulation and the regulation of biochemical pathways in C4 plants. Experimental procedures Plant material
Figure 3. Integration of T-DNA in transgenic F. bidentis. Genomic DNA extracted from representative plants from four transformation experiments was digested with Hindlll, blotted and hybddized with a npt-II gene probe. Lanes A and B contain DNA from plants transformed with the pMCP3 binary vector; C, D, E, F and G were transformed with pGA470 or pGA482 binary vectors.
Our Flaveria bidentis plants were derived from field collections and the degree of genetic variability amongst them is unknown. Plants were grown to matudty in the glasshouse and allowed to self-fertilize. Seeds were collected and surface sterilized for 30 sec in 70% ethanol then 1% (w/v) sodium hypochlodte solution for 20 min, rinsed three times in sterile distilled water and placed on 90 x 25 mm petd dishes containing GM (Table 2). Dishes were sealed with Parafilm and maintained at 24°C in a 16 h light/8 h dark cycle to allow seed germination, or were stored at 4°C until required. Hypocotyls were excised from seedlings 7 - 8 days after germination when they were 3 - 6 mm in length and used as explants in regeneration and transformation experiments.
Figure 4. Distribution of NPT-II activities amongst T1 progeny of pdmary transformants. Phosphorimages of NPT-II dot blots were quantified by ImageQuant and protein concentration in each leaf extract determined. (a) progeny of plant in Figure 3 (lane D); (b) progeny of plant in Figure 3 (lane lane E); (c) progeny of plant in Figure 3 (lane C); (d) progeny of plant in Figure 3 (lane G).
Transformation of C4 Flaveria Tissue culture media and conditions
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Hybond N+. Filters were prehybddized, and probed in 50% formamide at 42°C, as described in Sambrook et al. (1989). Filters were probed with a 620 bp PCR product generated from the npt-II gene present in pGA470. This DNA fragment did not contain a Hindlll site. Filters were washed for 15 rain in 2 x SSC at 42°C, 15 min in 0.1 x SSC at 42°C and 60 min at 65°C in 0.1 x SSC, then exposed ovemight on a phosphodmager.
All culture media consisted of Murashige and Skoog (1962) macronutrients, micronutrients and iron salts and B5 medium vitamins (Gamborg eta/., 1968) (Table 2), pH was adjusted with 1 M KOH to pH 5.8---6.0. Gelling agents were 0.8% Sigma Pure Agar or 0.28% Sigma Phytagel. Hormones were added to media pdor to autoclaving at 121°C for 20 min. Antibiotics were added after autoclaving and cooling to 55°-65°C. Callus cultures were grown in Petd dishes sealed with Parafilm in foil-lined boxes. Regenerated shoot matedal was maintained in petd dishes sealed with medical Micropore tape which helped to reduce condensation in culture vessels by allowing improved air flow. All cultures were grown at 24°C under Philips Daylight 36 W fluorescent lights at a distance of 5-40 cm.
We thank Richard Brettell, Philip Laddn, Hart Schroeder and Therese Wardley-Richardson for advice, helpful discussions and encouragement. The skilled technical assistance of Jason Andrews and Michelle Couzens is gratefully acknowledged.
Bacterial strains and binary vectors
References
The disarmed Agrobactedum tumefaciens strains LBA4404 (Hoekma eta/., 1983) and AGL1 (Lazo eta/., 1991) were used in all transformation experiments. DNA constructs were based on the binary vectors pGA470 (An et al., 1985), pGA482 (An, 1986) or pMCP3 (Khan eta/., 1994). Agrobacterium strains were maintained in glycerol at -80°C or on LB agar plates plus appropdate selection at 4°C. Fresh cultures were grown overnight at 28°C in 10 ml LB plus MG salts without antibiotics. This AgrobacterJum suspension was diluted to approximately5>
