Plant Cell Reports

Plant Cell Reports (1994) 14:94-97

,9 Springet-Verlag 1994

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants Chung-Jui Tsai 1, Gopi K. Podila 2, and Vincent L. Chiang 1 i School of Forestry and Wood Products, Michigan Technological University, Houghton, Michigan 49931, USA 2 Department of Biological Sciences, Michigan Technological University, Houghton, Michigan 49931, USA Received 22 November 1993/Revised version received 24 June 1994

SUMMARY AgrobacteHum-mediated gene transformation of Populus tremuloides

Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with AgrobacteHum turnefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ft-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZy was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.

Abbreviations. AMV RNA4: Alfalfa mosaic virus RNA4; BA: 6-benzyladenine; CaMV: cauliflower mosaic virus; 2,4-D: 2,4-dichlorophenoxyacetic acid; EDTA: ethylenediaminetetraacetic acid; FAA: formalin-acetic acid-alcohol; GUS: 8-glucuronidase; NAA: l-naphthylacetic acid; NPT I1: neomycin phosphotransferase II; PCR: polymerase chain reaction; SDS sodium dodecyl sulphate; TE: Tris-CI/EDTA ; TDZ: N-phenyl-N'-l,2,3-thiadiazol-5-yl-urea (thidiazuron); WPM: woody plant medium (Lloyd and McCown 1980); X-GLUC: 5-bromo-4chloro-3-indolyl-t~-glucuronic acid INTRODUCTION Genetic engineering of forest tree species to conform to desired traits has shifted the emphasis in forest tree improvement away from the traditional breeding programs during the past decade. Although research on genetic engineering of forest trees has been vigorous, the progress has been slow due either to a lack of an efficient gene transfer mechanism or an in vitro culture system for plant regeneration. So far, transformation and regeneration systems have only been developed for a few tree species such as Douglas-fir (Dandekar et al. 1987), walnut (McGranahan et al. 1988; 1990), sweetgum (Chen 1991; Sullivan and Lagrimini 1993), European larch (Huang et al. 1991), yellow poplar (Wilde et al. 1992), and Populus spp. (Minoeha et al. 1986; Parsons et al. 1986; Fillatti et al. 1987; Pythoud et al. 1987; De Block 1990; Brasileiro et al. 1991; 1992; Howe et al. 1991; Klopfenstein et al. 1991; Leple et al. 1992; Correspondence to." C.-J. Tsai

Communicated by J. Widholm

Nilsson et al. 1992;). It is not surprising that, since Populus is known as a natural host for Agrobacterium tumefaciens (Parsons et al. 1986; Leple et al. 1992), various species of the genus Populus have been studied extensively for their genetic transformation via AgrobacteHum and their capability to regenerate in in vitro cultures. In fact, for forestry species, the first Agrobacterium-mediated transformation of an agronomically useful gene which confers tolerance to herbicide glyphosate and whole plant regeneration from the transformed cells were established for Populus (P. alba x P. grandidentata) (Fillatti et al. 1987; Riemenschneider et al. 1988). Since then, hybrid poplar has become a model system for genetic engineering of forest tree species (Ostry and Ward 1991). Of Populus species, quaking aspen is one of the most commonly used species for woodpulp production in North America due to its good fiber properties, fast growth, and world-wide distribution. In addition to these characteristics desired for a pulpwood species, it would be economically beneficial if, through genetic engineering, a new trait can be introduced into quaking aspen to improve the efficiency of pulping of this species. Based on our study (Dwivedi et al. 1994), it is plausible to regulate the expression of a lignin-pathway specific gene, O-methyltransferase (Bugos et al. 1991), through antisense technology in order to genetically manipulate the quality and quantity of lignin in plants for ease of pulping. In order to achieve this genetic engineering of lignin biosynthesis in quaking aspen, a transformation and regeneration system needs to be established for this species, since the available protocols for transformation and regeneration of model hybrid poplar are inadequate for quaking aspen, The only Agrobacterium-mediated transformation and regeneration in in v#ro culture for quaking aspen using hypocotyl and leaf segments of in vitro plants derived from seedlings was first reported in a conference proceedings by Minocha et al. (1986). However, no supporting data or figures were available in this report for comparison_ Furthermore, using the protocol of Nob and Minocha (1986) and Minocha et al. (1986) for organogenesis, we were unable to regenerate whole plant from either leaf segments excised directly from greenhouse plants or from leaf segments of in vitro plants derived from greenhouse-grown plants. The continual availability of greenhouse plants through vegetative propagation and the ease of maintaining these plants are the obvious advantages of using greenhouse plants over the use of in vitro plants derived from seedlings. However, the difficulty of shoot regeneration from greenhouse plants has been one of the main reasons that in vitro plants from seedlings are the preferred explant materials. We have circumvented the difficulty of whole plant regeneration from greenhouse-grown plants through the introduction of TDZ into the in vitro culture system as a plant growth regulator. Furthermore, this organogenesis system also allows an efficient whole plant regeneration from cuttings of field-grown mature quaking aspen and hybrid poplar, which represents a potential means of producing transgenic aspen and hybrid poplar of valuable genotypes. Thus, this effective TDZ-

95 assisted organogenesis for greenhouse- as well as field-grown plants would represent a new and important addition to the transformation and regeneration systems for forest tree species. Using this organogenesis system, we present, in this paper, a protocol for the Agrobacterium-mediated transformation of chimeric NPT II and GUS genes for greenhouse-grown quaking aspen and regeneration of whole plants from transformed callus. Conclusive transformation and expression of these genes in transgenic quaking aspen are presented. This protocol is currently adopted for generating transgenic quaking aspen with altered lignin biosynthesis. MATERIALS AND METHODS Plant Materials: Young leaves from cuttings of aspen (Populus tremuloides Michx. clone 271) grown in the greenhouse of School of Forestry and Wood Products, MTU, were used. Explants were surface sterilized in 20% commercial bleach for 10 minutes followed by rinsing three times with sterile double-distilled water. Leaves from cuttings of field-grown aspen and hybrid poplar were treated the same way as above. Culture Media and Culture Conditions: WPM (Lloyd and McCown 1980) supplemented with 2% sucrose was used as a basal medium and 650 mg/L calcium gluconate and 500 mg/L MES were added as pH buffers as described by De Block (1990). The combination of BA and 2,4-D at the concentrations of 0.5 and 1 mg/L, respectively, was used to induce callus while 0.5 mg/L TDZ was added for shoot regeneration. Cefotaxime (300 mg/L, Claforan, Hoechst) was used in both callus induction and shoot regeneration media to kill ~lgrobacterium. Whenever necessary, cefotaxime (300 mg/L) was also added in the hormone-free medium for elongation. For selecting transformed tissue, 40 mg/L of kanamycin was used at callus induction stage, whereas 100 mg/L was used for shoot regeneration, elongation, and rooting stages. All media were adjusted to pH 5.5 prior to the addition of 0.75% Difco Bacto Agar and autoclaved at 121~ and 15 psi for 20 min. Filter sterilized antibiotics were added after autoclaving. The cultures were maintained at 23+1 ~ in a growth chamber with 16 h photoperiods (160 p,E x m -2 x-S -1) except for callus induction, which was maintained in the dark. Plant Transformation: The binary vector BinSynGus containing double 35S promoter/AMV RNA4/untranslated region-GUS/NOS gene fusion with the NOS/NPT II/NOS gene cassette (obtained from Dr. Raju S.S. Datla) was mobilized into Agrobacterium tumefaciens strain C58 by freeze-thaw method (Holstein et al. 1978). Leaf disks cut from young leaves along the midrib using a corkborer (7 mm in diameter) were precultured on callus induction medium for 2 days to screen for healthy leaf disks which were then inoculated with an overnight-grown agrobacterial suspension for 2 hours on a shaker. After infection, explants were blotted dry with sterile Whatman NO. 1 filter papers and cocultivated on callus induction medium for 2 days. Selection of Transformed Cells and Shoot Regeneration: After cocultivation, explants were washed in sterile distilled water containing 300 mg/L cefotaxime to decontaminate, blotted dry and transferred onto the callus induction medium containing 40 mg/L kanamycin for selection of transformed cells. The kanamycinresistant tissues were then subcultured on fresh media every 2 weeks. Callus formed on selection medium was separated from the explant and subeultured periodically for further proliferation. When callus clumps reached to 3 lima in diameter or bigger, they were transferred to shoot regeneration medium containing 100 mg/L kanamycin. Adventitious shoots were then transferred to hormone-free elongation medium containing 100 mg/L kanamycin. Shoots of 2-3 cm in length were separated and planted in hormone-free rooting medium containing 100 mg/L kanamycin. Plantlets so derived were subjected to PCR and Southern analyses and GUS expression assay. Transgenic plants, when confirmed, were transplanted into soil medium (vermiculite:peatmoss:perlite = l: 1:1) and grown in the greenhouse. PCR Analysis: The PCR analysis is based on a modified method of Edwards et al. (1991) using Perkin Elmer/Cetus (Norwalk, CT)

AmpliTaq Recombinant Taq DNA polymerase. NPT II and GUS primers (Blake et al. 1991) bordering a 780 bp fragment of NPT H and 1097 bp fragment of GUS genes, were used for PCR. The template DNA for PCR was extracted from a single leaf excised from control untransformed and transgenic plants. The leaf was first ground in liquid nitrogen in a microcentrifuge tube and homogenized in 400 #L extraction buffer (200 #M Tris-HCl pH 7.5, 250 #M NaCI, 25 tzM EDTA, 0.5% SDS) for 30 seconds and followed by phenol/chloroform and chloroform extractions. After precipitation in ethanol, DNA was pelleted and dissolved in 20 t~L TE buffer. PCR reactions (final volume = 50 /aL) were performed using 5 I~L of template DNA. Samples were heated to 95~ for 4 min, followed by 35 cycles of 95~ for 45 seconds, 55~ for 30 seconds, and 73~ for 2.5 minutes with a final extension step of 73~ for 5 minutes in a thermal cycler (Perkin Elmer/Cetus 480). Amplified DNA fragments were electrophoresed on a 0.8% agarose gel and visualized by staining with ethidium bromide. Southern Analysis: Total cellular DNA was isolated from young aspen leaves according to Aitchitt et al. (1993). DNA (10 ~g) was digested with 2 units/gg DNA of EcoR I, Hind III, Pst I, or EcoR I + Hind III at 37 ~ overnight and was fractionated on a 0.8% agarose gel and blotted to nylon membrane. The blot was probed with randomly primed 32p-labelled probe of either NPT II or GUS DNA fragment from BinSynGus vector. Hybridization and washing of the blot were carried out at high stringency and the blot was autoradiographed according to Bugos et al. (1991). Histochemieal GUS Assay: Leaves, roots and stems from regenerated and kanamycin-resistant plants were analyzed for GUS gene expression in situ with X-GLUC according to Jefferson et al. (1987). Plant materials were stained with 1 mM X-GLUC in 100 mM phosphate buffer (pH7.0) containing 10 mM EDTA (pH 7.0), 0.5 mM potassium ferrocyanide, 0.5 rnM potassium ferricyanide, and 0.1% Triton X-100 at 37~ overnight. After staining, plant tissues were soaked in hot 70% ethanol to clear chlorophyll and were subsequently fixed in FAA for one day, followed by dehydration in t-butanolethanol series and embedded in paraffin. After removal of paraffin, tissue sections of 15 #m in thickness were photographed with a Nikon Optiphot light microscope. RESULTS AND DISCUSSION It is known that whole plant regeneration from greenhouse-grown plant materials is more difficult than that from in vitro plant materials or embryonic materials including mature and immature embryo, cotyledon, hypocotyl, and seedling. In our preliminary study, shoot regeneration could not be achieved from callus derived from greenhouse aspen leaves when 1 mg/L BA (Noh and Minocha 1986) or BA + NAA was used as the common growth regulator. However, shoot regeneration was successful when these common regulators were replaced by TDZ in the concentration range of 0.05 to 1 mg/L. At high concentrations of TDZ, however, swelling or vitrification of shoots and, in some cases, inhibition of shoot elongation (Nilsson et al. 1992) occurred. To avoid these adverse effects, shoot regeneration was conducted on medium containing 0.5 mg/L TDZ and shoots were transferred onto elongation medium immediately after their formation. In this way we have always been able to achieve satisfactory shoot regeneration and elongation from leaves of greenhouse-grown aspen as well as from cuttings of field-grown mature quaking aspen and hybrid poplar. In order to select positive transformants conferring to kanamycin resistance, leaf explants from non-transformed in vitro aspen were tested for their tolerance to kanamycin. These explants were maintained for one or two months on callus induction medium containing 0, 20, 40, 60, 80, or 100 mg/L kanamycin. For both oneand two-month growth periods, the growth of callus was inhibited in the presence of kanamycin and the yields of callus decreased significantly at the concentration of 20 mg/L kanamycin but decreased to almost a constant level at concentrations higher than 40 mg/L kanamycin. However, even at a concentration of 100 mg/L kanamycin, leaf disks still remained green in color, but at levels of 50 or 100 mg/L kanamycin, shoot regeneration and root formation were completely inhibited. We then decided to use a concentration of 40 mg/L kanamycin at the callus induction stage for primary selection of

96 the putative transformants. A concentration of 100 mg/L kanamyein was used for selecting transformants in subsequent shoot regeneration, elongation, and rooting stages to prevent possible escapes. After selection through a two-day preculture, healthy leaf disks were inoculated and cocultivated with A. tumefaciens C 58 hosting BinSynGus vector. When these explants were transferred to callus induction medium containing kanamycin, callus formation occurred at the wound sites of leaf disks after about 2 weeks. The callus formed on the selection medium was further proliferated on selection medium. Shoots were regenerated about 4 weeks after callus was transferred to regeneration medium (Fig. 1A). As soon as the shoots were

Fig. 1. Production of transgenie plants from leaf disks of quaking aspen inoculated with Agrobacteriurn tumefaciens C58/BinSynGns. A: Adventitious shoots differentiating from the surface of subcultured callus at one month after subculture ( b ~ = 1 ram); B: Transgenic planflets forming roots within 2 weeks in kanamycin-containing rooting medium (picture was taken 6 weeks after subculture). regenerated they were then transferred to kanamyein-eontaining hormone-flee medium to promote elongation. At this stage, some shoots did not grow and started browning. Green and healthy shoots elongated to 2-3 cm in length were excised and planted separately in rooting medium containing kanamycin for further selection. The efficient uptake of kanamycin by shoots during their rooting stage provide the most effective selection for positive transformants. During rooting stage, shoots that were apparently escaped from previous selections developed reddish leaves with black spots and necrosis in both shoot and leaves within 2 weeks and eventually died. Regenerated plants that survived the selection at rooting stage formed roots (Fig. 1B) within 2 weeks in fresh rooting medium and were analyzed for transformation based on in situ GUS activity assay and putative transgenic plants, once confirmed, were transplanted into soil and grown in the greenhouse for further molecular genetic analysis. The effectiveness of this transformation and regeneration system for aspen was indicated by a production of about fifty individual regenerated and kanamyein-resistant plantlets from every leaf disk. Young leaves of twenty randomly selected putative transgenic plants grown 2 to 3 months in the greenhouse were excised for total cellular DNA isolation for PCR and genomic Southern analyses to verify the integration of GUS and NPT II genes in nuclear genome of these plants. PCR analysis revealed that 1097 bp GUS and 780 bp NPT II DNA fragments were amplified from genomic DNA of all putative transgenic plants (data not shown). However no GUS and NPT II PCR products were seen for the control non-transformed plants. These PCR analyses indicated transformation of GUS and NPT II genes into these putative transgenic plants. To further confirm the integration of these genes into the nuclear genome of transgenic plants, total cellular DNA was subjected to Southern hybridization analysis. The results from the analysis of three~utative transgenic plants are shown in Fig. 2. Figure 2A shows that ~P-labelled NFF II probe hybridized to various DNA fragments of transgenic plants (lanes 2 to 7) and non-digested BinSynGns plasmid (lane 1), but no hybridization was seen for control non-transformed aspen (lanes 8 & 9), indicating positive integration of NPT II gene into the genome of transgenic plants. Furthermore, different pattern of multiple hybridization was observed for these three transgenic plants. For instance, NFF 11 probe hybridized to three (Fig. 2A, lane 2), two 0ane 4) and one (lane 6) DNA fragments of genomie DNA restricted with EcoR I for these three transgenie plants, respectively. Similarly, different pattern of multiple hybridization was also observed for these three transgenic plants of which genomie DNA was digested with Hind III (Fig. 2A, lanes 3, 5, and 7). This hybridization of NPT II probe to multiple DNA fragments indicates a random integration and multiple insertions of NPT II gene in genome of transgenic plants,

Fig. 2. Southern analysis of integration profiles of GUS and NPT II genes in nuclear genome of transgenic aspen plants. Panel A: Southern hybridization of DNA with NPT II gene probe: Lane 1, nondigested BinSynGus plasmid DNA (positive control); Lanes 2, 4, and 6, nuclear DNA (EcoR I digested) from transgenic plants a, b, and c, respectively; Lanes 3, 5, and 7, nuclear DNA (Hind III digested) from transgenie plants a, b, and c, respectively; Lanes 8 and 9, nontransformed control aspen nuclear DNA digested with EcoR I and Hind III, respectively. Arrow head indicates 13 kb linearized BinSynGUS plasmid which hybridized to t h e NPT II probe. B: Southern hybridization of DNA with GUS gene probe: Lanes I, 3, and 5, nuclear DNA (EcoR I digested) from transgenic plants a, b, and c, respectively; Lanes 2, 4, and 6, nuclear DNA (Hind III digested) from transgenic plants a, b, and c, respectively. C: Southern hybridization of DNA with GUS gene probe: Lanes 1, 2, and 3, nuclear DNA (double digested with EeoR I and Hind III) from transgenic plants a, b, and c, respectively. Hybridizing DNA fragment of about 3 kb corresponds to the size of the GUS gene cassette (double CaMV 35S promoter/AMV RNA4/GUS/NOS). The the positions of DNA standard markers are indicated by arrows. which is typical for Agrobacterium-mediated gene transformation of plants (Horsch et al. 1988). Random integration and multiple insertions of GUS gane in genome of these three transgenic plants were also observed, as shown in Fig. 2B. When nuclear DNA from transgenic plants was digested with Pst I and probed with 32p-labelled NPT II DNA in a Southern blot, a hybridizing DNA fragment was found at about 2 kb for these three transgenic aspen, which is the expected size for NPT II gene-containing insert (NOS/NPT II/NOS) (data not shown). Likewise, Southern analysis with ~'P-labelled GUS DNA on three transgenic aspen genomic DNA double digested with EcoR I and Hind III revealed a strongly hybridizing DNA fragment at about 3 kb (Fig. 2C) -- an expected size for the GUS gene cassette (double CaMV 35S promoter/AMV RNA4/GUS/NOS). The above results clearly provide evidence that GUS and NPT II genes were integrated in the genome of transgenic aspen, confirming the validity of this Agrobacterium-mediated gene transformation and regeneration of the transgenic plant systems for quaking aspen. In addition to the PCR and genomic Southern hybridization analyses,

in situ GUS activity assay with X-GLUC (Fig. 3) was also conducted

Fig. 3. Histochemical analysis of GUS gene expression in transgenic plants, Tissues were first stained with X-GLUC and embedded in paraffin for sectioning (15 ~m). A: Leaf paradermal section (bar = 100 /zm); B: Stem cross section (bar = 100 /~m); C: Longitudinal section of developing root (bar = 25 #m).

97 for control and transgenic plants to further confirm the integration and expression of T-DNA in the genome of these plants. Initially, a single intact leaf from each of one hundred and three transgenic plants were assayed. Of all these transgenic plants assayed, only six plants gave negative response to GUS staining. PCR analysis also revealed that no GUS or NPT II gene fragment was amplified from the genomic DNA of these plants which gave negative GUS staining (data not shown). GUS activity assay was then carried out for stems and roots of transgenic plants of which leaves gave positive GUS staining. As shown in Fig. 3, an intense blue staining was observed for tissues of leaf (Fig. 3A), stem (Fig. 3B) and root (Fig. 3C) of transgenie plants, but no staining was seen for control non-transformed plants (data not shown), confirming positive transformation and expression of T-DNA in these transgenic plants. We have established a transformation and regeneration system for quaking aspen using leaf explants excised directly from greenhouse, and this protocol should be applicable to in vitro plants as well as field-grown mature trees. One of the potential applications of this system is the genetic manipulation of lignin biosynthesis in quaking aspen to improve the pulping efficiency. However, the progress on cloning lignin-pathway specific genes has by far exceeded the pace of developing gene transformation and regeneration systems for tree species. Together with the available ]ignin-specificgenes, the system we have presented here for one of the most important pulpwood species, quaking aspen, would represent a timely addition to the alternatives to conserving energy and materials for wood pulping and bleaching processes. Acknowledgements. We are grateful to Dr. Raju S.S. Datla for providing BinSynGus vector and to NOR-AM Chemical Co., Wilmintong, DE. for TDZ. This research was supported by a grant (#92-37301-7598) from the Plant Genetic Mechanism and Molecular Biology Program of the USDA National Research Initiative Competitive Grants Program to VLC and GKP and from CPBR-DOE Energy from Biomass Program to GKP and VLC. REFERENCES Aitchitt M, Ainsworth CC, Thangavelu M (1993) Plant Mol Biol Reporter 11(4):317-319 Blake NK, Ditterline RL, Stout RG (1991) Crop Sci 31:1686-1688 Brasileiro ACM, Leple JC, Muzzin J, Ounnoughi D, Michel MF, Jouanin L (1991) Plant Mol Biol 17:441-452 Brasileiro ACM, Tourneur C, Leple JC, Combes V, Jouanin L (1992) Transgenic Res 1:133-141 Bugos RC, Chiang VL, Campbell WH (1991) Plant Mol Biol 17: 1203-1215 Chen ZZ (1991) Nodular culture and Agrobacterium-mediated transformation for transgenic plant production in Liquidambar styraciflua L. (sweetgum). PhD thesis, North Carolina State University, Raleigh, North Carolina Dandekar AK, Gupta PK, Durzan DJ, Knauf V (1987) Bio/Technology 5:587-590 De Block M (1990) Plant Physiol 93:1110-1116 Dwivedi UN, Campbell WH, Yu J, Datla RSS, Bugos RC, Chiang VL, Podila GK (1994) Plant Mol. Biol. (in press) Edwards K, Johnstone C, Thompson C (1991) Nucleic Acids Res 19 (6): 1349-1352 Fillatti JJ, Sellmer J, McCown B, Haissig B, Comai L (1987) Mol Gen Genet 206:192-199 Holstein M, De Wacek D, Depicker A, Messers E, Von Montagu M, Schell J (1978) Mol Gen Genet 163:181-187 Horsch RB, Fraiey RT, Rogers SG, Klee I-IJ, Fry J, Hinchee MAW, Shah L (1988) Iowa State J. Res. 62:487-502 Howe GT, Strauss SH, Goldfarb B (1991) In: Woody Plant Biotechnology. Ahuja MR (ed) Plenum Press, New York, pp 283-294 Huang Y, Diner AM, Karnosky DF (1991) In vitro Cell Dev Biol 4:201-207 Jefferson R (1987) Plant. Mol. Biol. Rep. 5:387-405 Klopfenstein NB, Shi NQ, Kernan A, McNabb Jr HS, Hail RB, Hart ER, Thornburg RW (1991) Can J For Res 21:1321-1328

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