Plant Cell Reports
Plant Cell Reports (1992) 11:20-24
(c~Springer-Verlagt 992
Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Lilium longiflorum Leonne M . van der Leede-Plegt, Bernadette C. E. van de Ven, Raoul J. Bino, Theo P. M. van der Salm, and Arjen 3. van Tunen DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, 6700 AA Wageningen, The Netherlands Received September 26, 1991/Revised version received December 2, 1991 - Communicated by I. Potrykus
SUMMARY As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the 13-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flowerspecific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollenspecific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2' mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species. Abbreviations. ADHI: Alcohol Dehydrogenase 1; A. tumefaciens: Agrobacterium tumefaciens; CaMV: Cauliflower MosaicVirus; ChiA : Chalcone Flavanone lsomerase A; L. tongiflorum: Lilium longiflorum; N. glutinosa: Nicotiana glutinosa; Nos: Nopaline Synthase; N. tabacum: Nicotiana tabacum.
INTRODUCTION Broad application of genetic manipulation techniques in monocot plants is still seriously hindered by the lack of transformation and regeneration procedures of Offprint requests to: L. M. van der Leede-Plegt
these often commercially important crop species. Agrobacterium-mediated gene transfer has only been successful for a small number of monocot plant species, probably due to the limited host-range of this bacterium. Only in one case transgenic plants have been obtained after transformation of asparagus spears with Agrobacterium tumefaciens (Bytebier et al. 1987). Direct gene transfer via PEG- and electroporationmediated DNA uptake by protoplasts has resulted in the generation of stably transformed tissues of Lolium multiflorum (Potrykus et al. 1985), Zea mays (Fromm et al. 1986), Saccharum spp. (Chen et al. 1987) and Oryza sativa (Yang et al. 1988; Shimamoto et al. 1989). Using these gene transfer systems full proof of a transformation event has only been demonstrated for two Japonica rice varieties (Zhang and Wu 1988) and one Indica rice variety (Datta et aL 1990 a,b). A new method for direct DNA transfer into plants has been developed by Klein et al. (1987). The procedure is based on the introduction of genetic material by bombardment of cells or tissues with high velocity microprojectiles using a particle delivery system. Stably transformed maize plants have recently been obtained using this method (Spencer et al. 1990). We are developing an alternative approach to obtain a transformation procedure for the recalcitrant plant species Lilium longiflorurn using the male gametophyte as a transformation vector. Twell et al. (1989a) confirmed the introduction of foreign DNA in tobacco pollen by particle bombardment using GUS as a marker. The development of our alternative transformation procedure requires the availability of a promoter active in Lilium longifiorum pollen. Therefore four .different chimeric genes consisting of various promoter fragments fused to the GUS reporter gene were tested for transient gene expression in pollen. Mature pollen grains from N. glutinosa and L longiflorum were bombarded and assayed for GUS activity.
21 MATERIALS AND METHODS
Plant material Nicotiana tabacum cv. Petit Havanna SR1 and Lycopersicon esculentum MSK93 (Koornneef et aL 1987) were transformed with Agrobacterium tumefaciens strain LBA4404-pBI121 (Jefferson et aL 1987) according to the leaf disc method of Horsch et aL (1985). L. esculentum MSK93 was also transformed with A. tumefaciens strain LBA4404-pCPO1.2'GUS (Dr. B. Visser, CPRO, pers. comm.). Petunia hybrida was transformed with A. tumefaciens LBA4404MIP138 (PA2-GUS construct: Van Tunen et aL 1990). Transgenic and untransformed control plants were grown to maturity in green~ houses at a 24~176 temperature regime under daylight conditions. N. glutinosa plants, as a source of pollen for bombardment treatments, were grown in a 17-18~ growthchamber (55% Relative Humidity, Light intensity 22 W/m 2 for 16hr/24hr). L. longiflorum cv. Gelria plants were induced to flower simultaneously by storing bulbs at &C before planting. Plasmids The binary plasmid pBI121 has been described previously (Jefferson et al. 1987). pCAL1Gc (Dr. V. Walbot, Stanford University USA) consists of the GUS gene fused to a 450 bp CaMV 35S promoter combined with a 670 bp ADH1 intron in the untranslated leader and the nos terminator in a pUC8 background. The total size of this plasmid (refered to in the text as 35S(adh1)-GUS) is 5.8 kb. In pCPO1.2'GUS (refered to in the text as TR2'-GUS) the GUS gene is fused to the 2' dual mannopine synthase gene promoter from A. tumefaciens (Velten et al. 1984) and a 35S terminator. This 11.8 kb plasmid was constructed by replacement of the cDNA-Ag7 terminator fragment from a PCV708 background (Koncz et aL 1990) by a GUS-35S terminator fragment (Dr. B. Visser CPRO, personal communication). The 5.8 kb plasmid pLAT52-7 (refered to as LAT52-GUS) contains a chimeric gene consisting of a 600 bp promoter fragment of the flower-specific LAT52 gene from tomato (Twell et aL 1989b) fused to the GUS reporter gene and a nos terminator (Twell et al. 1989a). For the construction of pMIP138 (refered to in the text as chiA PA2-GUS) the 35S promoter of pBI121 was replaced by a 440 bp pollen-specific chalcone flavanone isomerase chiA PA2 promoter originally isolated from petunia (van Tunen et al. 1989; 1990). GUS assays
Preparation of plant extracts and the fluorometric assay of GUS activity were carried out as described by Jefferson et aL (1987). Protein concentrations were measured according to Bradford (1976). TR2'- and CaMV 35S-driven GUS activity in the anthers of transgenic plants prior to anthesis was histochemically localized by addition of 2 mM X-gluc (5-bromo-4-chloro-3-indolyl 13-Dglucuronide; Research Organics Inc., Cleveland, Ohio) in 50 mM sodium-phosphate buffer (NaPi), pH 7.5 to fresh cross-sections. Histochemical analysis of the transient expression in pollen was performed by addition of 2 mM X-gluc in 150 mM NaPi, pH 7.5 with 12.5% sucrose to the bombarded pollen on a nylon membrane. These conditions prevented endogenous GUS-like activity in the pollen grains (Plegt and Bino 1989). Particle b o m b a r d m e n t
N. gtutinosa and L. longiflorum pollen were collected from mature dehiscent flowers. After rehydration approximately 70,000 pollen grains of N. glutinosa (10 mg) and L. longiflorum (40 rag) were placed in the middle of agar plates (solified MS25% medium) which were overlayed with a filter paper and a nylon membrane (Gene ScreenTM). Bombardment was performed with a DuPont PDS1000 apparatus at 28.5 inchHg within 25 minutes after placing the pollen onto the agar plate. The distance from stopper plate to target cells amounted 12.8 cm. DNA was precipitated on 1.6 t~m thungsten particles by the addition of successively 2.5 tzl DNA (1 t~g//zl), 25 ,ul 2.5 M CaCI2 and 10 #1 0.1 M spermidine to 1.5 mg (25~1 volume) particles. After 10 minutes of incubation, 45 tzl of the supernatant
was removed and 2 t~l of the remainig particle suspension was used per bombardment. In each experiment 4 plates with either N. glutinosa pollen or L. longiflorurn pollen were bombarded with the four different chimeric GUS genes. Three sep:rrate experiments have been performed. As a control 1.6 t~m particles were used without adhering DNA. After bombardment the plates were incubated at 18~ for 16 hours. 80% to 90% of the N. glutinosa and L. longiflorum pollen was viable. After bombardment and a 16 hour incubation period 50% of the N. glutinosa and 75% of the L. longiflorum pollen was still viable as was determined by fluorescein diacetate accumulation (Heslop-Harrisonand Heslop-Harrison 1970). Subsequently the nylon membrane carrying the pollen was excised and placed on top of a Whatmann 4 MM filter. X-gluc was applied to the membrane. Extra buffer was added underneath the membrane to prevent pollen from drying out during microscopical analysis. In this way each membrane was analyzed for blue staining pollen grains after several time periods . Bombarded N. glutinosa pollen was germinated under rotation (30 rpm) of the pollen in BK medium (Brewbaker and Kwack 1963) including 15% sucrose for 8 hours.
RESULTS AND DISCUSSION Activity of various promoters in pollen of transgenic plants containing promoter-GUS gene constructs Before our research on pollen transformation was started an inventory study was carried out in order to identify promoters active in pollen grains. Therefore transgenic plants containing chimeric genes consisting of the LAT52, chiA PA2, CaMV 35S and TR2' promoter fused to the GUS reporter gene (see Fig. I) were analyzed for expression in the male gametophyte. Table 1 summarizes the data. For the activity of the LAT52 promoter in the male gametophyte we refer to a study by Twell et al. (1989a). They showed that this promoter is highly active in pollen of transgenic N. tabacum plants (83,800 pmol 4-MU/min.mg protein). For mature pollen of a Petunia hybrida plant transformed with chiA PA2-GUS, we observed a reporter enzyme activity of 1100 pmol 4-MU/min.mg protein. This is similar to data reported previously (Van Tunen et aL 1990). Mature N. tabacum pollen, pooled from eleven independent chiA PA2-GUS transformants, contained somewhat higher levels of the GUS reporter enzyme (3180 pmol 4-MU/min.mg protein). The level of GUS expression driven by the CaMV35S Table 1. GUS-activity driven by various promoters in pollen of
transformed dicot plants.
Promoter
Plantspecies
GUS -'~ activity
Reference
LAT52
tobacco
83800
Twell et aL 1989a
ChiA PA2 petunia petunia tobacco
1100 670 3180
CaMV 35S tobacco tobacco
25 63
TR2'
tobacco
3970
this study van Tunen et aL 1990 this study this study Twell et al 1989a this study in pmol 4-MU/min.mg protein
22 N. glutinosa (N) and L. longiflorum (L) driven by various promoters after particle bombardment. GUS activity is shown as the number of blue pollen per 100,000 viable pollen.
Table 2. Transient GUS expression in pollen grains of
CaMV time of GUS staining (hrs)
LAT52
ChiA
35S
TR2'
no DNA
PA2
N
L
N
L
N
L
N
L
t=2.5
expl exp2 exp3
1678 867 2173
0 0 0
0 0 0
0 0 0
0 4 0
0 0 0
0 0 0
0 0 0
0 0 0
t=5
expl exp2 exp3
2333 1200 2530
0 0 0
0 0 0
0 0 0
2 7 0
0 0 2
5 2 0
0 0 0
0 0 0
t=10
expl exp2 exp3
2684 1346 2607
0 0 0
0 0 0
0 0 0
5 8 4
0 2 6
5 2 0
0 0 0
0 0 0
t=24
expl exp2 exp3
3276 1850 3209
0 0 0
0 0 0
0 0 0
14 8 4
4 9 12
12 2 0
0 0 0
0 0 0
t=50
expl exp2 exp3
3482 1942 3287
0 0 0
0 0 0
0 0 0
14 13 12
6 20 25
12 4 0
0 0 0
0 0 0
promoter was low in mature pollen of six transgenic N. tabacum plants (25 pmol 4-MU/min.mg protein). This result is in line with experiments in which no indigo-blue precipitation was histochemically detected in pollen of ten independent transgenic 35S-GUS N. tabacum plants and four L. esculentum transformants examined (data not shown). A low level of GUS activity was also found by Twell et al. (1989a) implicating that the CaMV35S promoter is only active at a low level in mature pollen. TR2'-driven GUS activity has been assayed histochemically in anthers derived from four independent transgenic TR2'-GUS L. esculentum and fourteen TR2'-GUS N. tabacum plants. In both species the TR2' promoter was active in mature pollen (see also Fig. 2). The GUS activity driven by the TR2' promoter in pollen pooled from the transgenic N. tabacum plants was 3970 pmol 4MU/min.mg protein. The presence of TR2'-driven GUS activity in pollen of L. esculenturn and N. tabacum is in contrast with an absence of TR2'-driven luciferase synthesis in N. tabacum pollen (Langridge et aL 1989). Here, measuring luciferase as a reporter in pollen extracts, the TR2' promoter was found to be activated only after pollen germination. Apart from pollen germination, both wounding and external addition of growth regulators are known to activate the TR dual promoter (Langridge et al. 1989, Saito et al. 1991). As none of these treatments has been applied in our experiments it remains unclear why TRY-driven GUS activity was present in nongerminating pollen whereas TR2'-driven luciferase activity was not. In the sporophytic anther tissues from the transgenic TR2'-GUS L. esculentum and N. tabacum plants the TR promoter was also found to be active. Here an intense indigo-blue precipitation was observed in the stomium (Fig. 2). No indigo-blue precipitation was observed in sporophytic and gametophytic tissues of control plants at the same
conditions. From the results in transgenic plants we conclude that the LAT52, chiA PA2 and TR2' promoters are and the CaMV 35S promoter is not or only at a low level active in mature N. tabacum pollen.
Promoter activity in Nicotiana glutinosa pollen after particle bombardment The activity of the LAT52, chiA PA2, TR2' and CaMV 35S promoters has been tested in pollen of N. glutinosa after particle bombardment. The transient GUS expression in pollen grains of N. glutinosa (a plant species producing high numbers of viable pollen grains) driven by these various promoters is shown in Table 2. In a bombardment experiment with the LAT52-GUS gene construct a high number of blue N. glutinosa pollen was observed (Fig. 3A and 3B). Within 10 minutes of X-gluc incubation the first blue pollen grains were detected. The time curve of the histochemical analysis resembles a logarithm with 1.5% and 2.9% indigo-blue pollen after respectively 2.5 and 50 hours of X-gluc staining. A logarithmic time course of the histochemical GUS analysis has also been shown recently for the ADH1 promoter in bombarded coleoptiles of Zea mays (Reggiardo et al. 1991). Bombardment with the chiA PA2-GUS and TR2'-GUS gene construct lead to the generation of blue N. glutinosa pollen .The first indigo-blue pollen grains were detected after 2.5 to 5 hours incubation in Xgluc (Table 2). The amount of blue N. glutinosa pollen after introduction of the chiA PA2- or TR2'-GUS genes was significantly less than found for the LAT52GUS gene and reached a maximum of 0.014% after 50 hours of X-gluc incubation.Bombardment with the 35S(adhl)-GUS construct did not give rise to blue N.
23
Fig 1. Chimeric genes used for particle bombardment of N. glutinosa pollen and L. longiflorum pollen. A: pLAT52-7 ; B: pMIP138; C: pCPO1.2'GUS; D: pCAL1Gc. Fig 2. Anther section from a transgenic TR2'-GUS L. esculentum plant. Indigo-blue precipitation in stomium and mature pollen, e= endothecium, I=loculus, p=pollen, s=stomium, v=vascular sheet. Fig 3. A. Overall picture of a nylon membrane showing LAT52-driven GUS expression in N. glutinosa pollen.; B. LAT52-driven GUS expression in N. glutb~osa pollen; C. TR2'-driven GUS expression in L. longiflorum pollen; D. TR2'-driven GUS expression in germinated N. glutinosa pollen.
glutinosa pollen (Table 2). As a control the same construct was introduced into N. glutinosa leaf tissue. Here the CaMV 35S promoter was highly active as demonstrated by the high amount of blue coloured spots (data not shown). These observations are in line with the data obtained for the activity of the CaMV 35S promoter in transgenic N. tabacum plants where no activity was detected in pollen in a histochemical assay and only a very low GUS activity was measured with the fluorimetric assay. Summarizing the data we conclude that the expression level of the promoters in N. glutinosa pollen reflects the activity levels found in N. tabacum pollen from transgenic plants: high for the LAT52 promoter, intermediate for the TR2' promoter, lower for the chiA PA2 promoter and no or a very low level of expression for the CaMV 35S promoter. Apparently the pollen bombardment system provides a way to compare the relative strengths of different promoters.
Promoter activity in Lilium longiflorum pollen after particle bombardment In order to assay the activity of the LAT52, chiA PA2, CaMV 35S and TR2' promoter in pollen of a recalcitrant monocot plant species, bombardment experiments were performed on pollen of L. longiflorum using again promoter-GUS constructs. The results obtained are summarized in Table 2 and differ from the data obtained for N. glutinosa pollen. In N. glutinosa pollen the LAT52 promoter was highly active. However this promoter was not active in pollen of the monocot L. longiflorum. The chiA PA2 promoter was also silent in L. longiflorum pollen. Therefore it can be concluded that these two promoters are differentially active in pollen grains from a monocot and dicot plant species and that factors activating the chiA PA2 and LAT52 promoters in N. glutinosa are absent in the lily pollen. This may reflect a different developmental
24 programme between the male gametophytes of these plant species. No GUS activity was demonstrated in L. longiflorum pollen after bombardment with the 35S(adh1)-GUS gene construct. Combined with the data described above we conclude from this that the regulatory factors necessary for the functioning of the CaMV 35S promoter seem to be absent or present at a low level in pollen of the monocot and dicot plant species used. In contrast, the TR2' promoter was clearly active in both L. longiflorum and Nicotiana pollen (Fig. 3C). This reveals a new property of this promoter namely that it is active in the generative stage of N. glutinosa and L. longiflorum. These results combined with recent findings such as the expression of this promoter in vegetative tissues of transgenic rice plants (Meijer et al. 1991) and the monocot tulip (Wilmink et al. 1991) suggests that the TR2' promoter is a promoter active in vegetative and sporogenous tissues of both dicot and monocot plants.
Germination of bombarded pollen Addition of a germination medium after bombardment of N. glutinosa pollen with TR2'-GUS and subsequent X-gluc staining produced pollen with extended tubes showing GUS expression (Fig. 3D). From the pollen expressing the GUS gene 81% was able to germinate. In the same population, pollen without GUS expression showed a similar in vitro germination percentage (78%). From this we conclude that pollen vigour does not seem to be affected by the bombardment procedure. Bombarded L. longiflorum pollen, expressing the same chimeric gene was also able to germinate (data not shown). Therefore future experiments will be directed towards pollination with bombarded pollen and subsequent screening of the progeny for a transformation event. ACKNOWLEDGEMENTS We thank Dr. S. McCormick (Plant Gene Expression Centre, LA, USA) and Dr. V. Walbot (Stanford University, USA) for kindly providing some of the gene constructs; Drs. 3.3.M. Dons and G.C. Angenent for critical reading of the manuscript and stimulating discussions and Mr. 3. Franken for technical support. Part of this work was funded by the Innovation Funding for Plant Breeding Research (Inpla) and by the Urgency Programme for Research on Diseases and Breeding of Flower Bulbs. REFERENCES MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254. Brewbaker JL. Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. of Bot. 50: 859-865. Bytebier B, Deboeck F, De Greve H, Van Montagu M (1987) TDNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis. Proc. Natl. Am. Sc. USA 84: 5345 -5349. Chen WH, Gartland KMA, Davey MR, Sotak R, Gartland JS, Mulligan BJ, Power JB, Cocking EC (1987) Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. Plant Cell Rep. 6: 297-301. Datta SK, Datta K, Potrykus I (1990a) Fertile Indica rice plants Bradford
regenerated from protoplasts isolated from microspore derived suspensions. Plant Cell Rep. 9: 253-256. Datta SK, Peterhans A, Datta K, Potrykus I (1990b) Genetically engineered fertile Indica-rice recovered from protoplasts. Bio/Technology 8: 736-740. Fromm M, Taylor LP, Walbot V (1986) Stable transformation of maize after electroporation. Nature 319: 791-793. Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Tech. 45: 115-120. Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eicholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transfering genes into plants. Science 227: 1229-1231. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Bglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70-73. Koncz C, Mayerhofer R, Koncz-Kalman Z, Nawrath C, Reiss B, Redei GP, Schell J (1990) Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. EMBO J. 9: 1337-1346. Koornneet M, Hanhart CJ, Martinelli L (1987) A genetic analysis of cell culture traits in tomato. Theor. Appl. Genet. 74: 633-641. Langridge WHR, Fitzgerald KJ, Koncz C, Shell J, Szalay AA (1989) Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc. Natl. Am. Sc. USA 86: 3219-3223. Meijer EGM, Schilperoort RA, Rueb S, Van Os-Ruygrok PE, Hensgens LAM (1991) Transgenic rice cell lines and plants: expression of transfered chimeric genes. Plant Mol. Biol. 16: 807820. Plegt LM, Bino RJ (1989) 13-Glucuronidase activity during development of the male gametophyte from transgenic and nontransgenic plants. Mol. Gen. Genet. 216: 321-327. Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer to cells of a gramineous monocot. Mol. Gen. Genet. 199: 167-177. Reggiardo MI, Arana JL, Orsaria LM, Permingeat HR, Spitteler MA, Vallejos RH (1991) Transient transformation of maize tissues by microparticle bombardment. Plant Sc. 75: 237-243. Saito K, Yamazaki M, Kaneko H, Murakoshi I, Fukuda Y, Van Montagu M (1991) Tissue-specific and stress-enhancing expression of the TR promoter for mannopine synthase in transgenic medical plants. Planta 184: 40-46. S h i m a m o t o K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274-276. Spencer TM, Gordon-Kamm WJ, Daines RJ, Start WG, Lemaux PG (1990) Bialaphos selection of stable transformants from maize cell culture. Theor. AppL Oenet. 79: 625-631. Twell D, Klein TH, Fromm ME, McCormick S (1989a) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol. 91: 1270-1274. Tweli D, Wing R, Yamaguchi J, McCormick S (1989b) Isolation and expression of an anther-specific gene from tomato. Mol. Gen. Genet. 217: 240-245. Van Tunen AJ, Hartman SA, Mur LA, Mol JNM (1989) Regulation of chalcone flavanone isomerase (CHI) gene expression in Petunia hybrida: the use of alternative promoters in corolla, anthers and pollen. Plant Mol. Biol. 12: 539-551. Van Tunen AJ, Mur LA, Brouns GS, Rienstra JD, Koes RE, Mol JNM (1990) Pollen-and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene. Plant Cell 2: 393401. Velten J, Velten J, Hain R, Schell J (1984) Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J. 3: 2391-2406. Wilmink A, Van de Ven B, Dons JJM (1991) Expression of the GUS-gene in the monocot Tulip after introduction byAgrobacterium and particle bombardment (submitted). Yang H, Zhang HM, Davey MR, Mulligan BJ, Cocking EC (1988) Production of kanamycin resistant rice tissues following DNA uptake into protoplasts. Plant Cell Rep. 7: 421-425. Zhang W, Wu R (1988) Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor. Appl. Genct. 76: 835-840.
Plant Cell Reports (1992) 11:20-24
(c~Springer-Verlagt 992
Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Lilium longiflorum Leonne M . van der Leede-Plegt, Bernadette C. E. van de Ven, Raoul J. Bino, Theo P. M. van der Salm, and Arjen 3. van Tunen DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, 6700 AA Wageningen, The Netherlands Received September 26, 1991/Revised version received December 2, 1991 - Communicated by I. Potrykus
SUMMARY As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the 13-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flowerspecific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollenspecific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2' mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species. Abbreviations. ADHI: Alcohol Dehydrogenase 1; A. tumefaciens: Agrobacterium tumefaciens; CaMV: Cauliflower MosaicVirus; ChiA : Chalcone Flavanone lsomerase A; L. tongiflorum: Lilium longiflorum; N. glutinosa: Nicotiana glutinosa; Nos: Nopaline Synthase; N. tabacum: Nicotiana tabacum.
INTRODUCTION Broad application of genetic manipulation techniques in monocot plants is still seriously hindered by the lack of transformation and regeneration procedures of Offprint requests to: L. M. van der Leede-Plegt
these often commercially important crop species. Agrobacterium-mediated gene transfer has only been successful for a small number of monocot plant species, probably due to the limited host-range of this bacterium. Only in one case transgenic plants have been obtained after transformation of asparagus spears with Agrobacterium tumefaciens (Bytebier et al. 1987). Direct gene transfer via PEG- and electroporationmediated DNA uptake by protoplasts has resulted in the generation of stably transformed tissues of Lolium multiflorum (Potrykus et al. 1985), Zea mays (Fromm et al. 1986), Saccharum spp. (Chen et al. 1987) and Oryza sativa (Yang et al. 1988; Shimamoto et al. 1989). Using these gene transfer systems full proof of a transformation event has only been demonstrated for two Japonica rice varieties (Zhang and Wu 1988) and one Indica rice variety (Datta et aL 1990 a,b). A new method for direct DNA transfer into plants has been developed by Klein et al. (1987). The procedure is based on the introduction of genetic material by bombardment of cells or tissues with high velocity microprojectiles using a particle delivery system. Stably transformed maize plants have recently been obtained using this method (Spencer et al. 1990). We are developing an alternative approach to obtain a transformation procedure for the recalcitrant plant species Lilium longiflorurn using the male gametophyte as a transformation vector. Twell et al. (1989a) confirmed the introduction of foreign DNA in tobacco pollen by particle bombardment using GUS as a marker. The development of our alternative transformation procedure requires the availability of a promoter active in Lilium longifiorum pollen. Therefore four .different chimeric genes consisting of various promoter fragments fused to the GUS reporter gene were tested for transient gene expression in pollen. Mature pollen grains from N. glutinosa and L longiflorum were bombarded and assayed for GUS activity.
21 MATERIALS AND METHODS
Plant material Nicotiana tabacum cv. Petit Havanna SR1 and Lycopersicon esculentum MSK93 (Koornneef et aL 1987) were transformed with Agrobacterium tumefaciens strain LBA4404-pBI121 (Jefferson et aL 1987) according to the leaf disc method of Horsch et aL (1985). L. esculentum MSK93 was also transformed with A. tumefaciens strain LBA4404-pCPO1.2'GUS (Dr. B. Visser, CPRO, pers. comm.). Petunia hybrida was transformed with A. tumefaciens LBA4404MIP138 (PA2-GUS construct: Van Tunen et aL 1990). Transgenic and untransformed control plants were grown to maturity in green~ houses at a 24~176 temperature regime under daylight conditions. N. glutinosa plants, as a source of pollen for bombardment treatments, were grown in a 17-18~ growthchamber (55% Relative Humidity, Light intensity 22 W/m 2 for 16hr/24hr). L. longiflorum cv. Gelria plants were induced to flower simultaneously by storing bulbs at &C before planting. Plasmids The binary plasmid pBI121 has been described previously (Jefferson et al. 1987). pCAL1Gc (Dr. V. Walbot, Stanford University USA) consists of the GUS gene fused to a 450 bp CaMV 35S promoter combined with a 670 bp ADH1 intron in the untranslated leader and the nos terminator in a pUC8 background. The total size of this plasmid (refered to in the text as 35S(adh1)-GUS) is 5.8 kb. In pCPO1.2'GUS (refered to in the text as TR2'-GUS) the GUS gene is fused to the 2' dual mannopine synthase gene promoter from A. tumefaciens (Velten et al. 1984) and a 35S terminator. This 11.8 kb plasmid was constructed by replacement of the cDNA-Ag7 terminator fragment from a PCV708 background (Koncz et aL 1990) by a GUS-35S terminator fragment (Dr. B. Visser CPRO, personal communication). The 5.8 kb plasmid pLAT52-7 (refered to as LAT52-GUS) contains a chimeric gene consisting of a 600 bp promoter fragment of the flower-specific LAT52 gene from tomato (Twell et aL 1989b) fused to the GUS reporter gene and a nos terminator (Twell et al. 1989a). For the construction of pMIP138 (refered to in the text as chiA PA2-GUS) the 35S promoter of pBI121 was replaced by a 440 bp pollen-specific chalcone flavanone isomerase chiA PA2 promoter originally isolated from petunia (van Tunen et al. 1989; 1990). GUS assays
Preparation of plant extracts and the fluorometric assay of GUS activity were carried out as described by Jefferson et aL (1987). Protein concentrations were measured according to Bradford (1976). TR2'- and CaMV 35S-driven GUS activity in the anthers of transgenic plants prior to anthesis was histochemically localized by addition of 2 mM X-gluc (5-bromo-4-chloro-3-indolyl 13-Dglucuronide; Research Organics Inc., Cleveland, Ohio) in 50 mM sodium-phosphate buffer (NaPi), pH 7.5 to fresh cross-sections. Histochemical analysis of the transient expression in pollen was performed by addition of 2 mM X-gluc in 150 mM NaPi, pH 7.5 with 12.5% sucrose to the bombarded pollen on a nylon membrane. These conditions prevented endogenous GUS-like activity in the pollen grains (Plegt and Bino 1989). Particle b o m b a r d m e n t
N. gtutinosa and L. longiflorum pollen were collected from mature dehiscent flowers. After rehydration approximately 70,000 pollen grains of N. glutinosa (10 mg) and L. longiflorum (40 rag) were placed in the middle of agar plates (solified MS25% medium) which were overlayed with a filter paper and a nylon membrane (Gene ScreenTM). Bombardment was performed with a DuPont PDS1000 apparatus at 28.5 inchHg within 25 minutes after placing the pollen onto the agar plate. The distance from stopper plate to target cells amounted 12.8 cm. DNA was precipitated on 1.6 t~m thungsten particles by the addition of successively 2.5 tzl DNA (1 t~g//zl), 25 ,ul 2.5 M CaCI2 and 10 #1 0.1 M spermidine to 1.5 mg (25~1 volume) particles. After 10 minutes of incubation, 45 tzl of the supernatant
was removed and 2 t~l of the remainig particle suspension was used per bombardment. In each experiment 4 plates with either N. glutinosa pollen or L. longiflorurn pollen were bombarded with the four different chimeric GUS genes. Three sep:rrate experiments have been performed. As a control 1.6 t~m particles were used without adhering DNA. After bombardment the plates were incubated at 18~ for 16 hours. 80% to 90% of the N. glutinosa and L. longiflorum pollen was viable. After bombardment and a 16 hour incubation period 50% of the N. glutinosa and 75% of the L. longiflorum pollen was still viable as was determined by fluorescein diacetate accumulation (Heslop-Harrisonand Heslop-Harrison 1970). Subsequently the nylon membrane carrying the pollen was excised and placed on top of a Whatmann 4 MM filter. X-gluc was applied to the membrane. Extra buffer was added underneath the membrane to prevent pollen from drying out during microscopical analysis. In this way each membrane was analyzed for blue staining pollen grains after several time periods . Bombarded N. glutinosa pollen was germinated under rotation (30 rpm) of the pollen in BK medium (Brewbaker and Kwack 1963) including 15% sucrose for 8 hours.
RESULTS AND DISCUSSION Activity of various promoters in pollen of transgenic plants containing promoter-GUS gene constructs Before our research on pollen transformation was started an inventory study was carried out in order to identify promoters active in pollen grains. Therefore transgenic plants containing chimeric genes consisting of the LAT52, chiA PA2, CaMV 35S and TR2' promoter fused to the GUS reporter gene (see Fig. I) were analyzed for expression in the male gametophyte. Table 1 summarizes the data. For the activity of the LAT52 promoter in the male gametophyte we refer to a study by Twell et al. (1989a). They showed that this promoter is highly active in pollen of transgenic N. tabacum plants (83,800 pmol 4-MU/min.mg protein). For mature pollen of a Petunia hybrida plant transformed with chiA PA2-GUS, we observed a reporter enzyme activity of 1100 pmol 4-MU/min.mg protein. This is similar to data reported previously (Van Tunen et aL 1990). Mature N. tabacum pollen, pooled from eleven independent chiA PA2-GUS transformants, contained somewhat higher levels of the GUS reporter enzyme (3180 pmol 4-MU/min.mg protein). The level of GUS expression driven by the CaMV35S Table 1. GUS-activity driven by various promoters in pollen of
transformed dicot plants.
Promoter
Plantspecies
GUS -'~ activity
Reference
LAT52
tobacco
83800
Twell et aL 1989a
ChiA PA2 petunia petunia tobacco
1100 670 3180
CaMV 35S tobacco tobacco
25 63
TR2'
tobacco
3970
this study van Tunen et aL 1990 this study this study Twell et al 1989a this study in pmol 4-MU/min.mg protein
22 N. glutinosa (N) and L. longiflorum (L) driven by various promoters after particle bombardment. GUS activity is shown as the number of blue pollen per 100,000 viable pollen.
Table 2. Transient GUS expression in pollen grains of
CaMV time of GUS staining (hrs)
LAT52
ChiA
35S
TR2'
no DNA
PA2
N
L
N
L
N
L
N
L
t=2.5
expl exp2 exp3
1678 867 2173
0 0 0
0 0 0
0 0 0
0 4 0
0 0 0
0 0 0
0 0 0
0 0 0
t=5
expl exp2 exp3
2333 1200 2530
0 0 0
0 0 0
0 0 0
2 7 0
0 0 2
5 2 0
0 0 0
0 0 0
t=10
expl exp2 exp3
2684 1346 2607
0 0 0
0 0 0
0 0 0
5 8 4
0 2 6
5 2 0
0 0 0
0 0 0
t=24
expl exp2 exp3
3276 1850 3209
0 0 0
0 0 0
0 0 0
14 8 4
4 9 12
12 2 0
0 0 0
0 0 0
t=50
expl exp2 exp3
3482 1942 3287
0 0 0
0 0 0
0 0 0
14 13 12
6 20 25
12 4 0
0 0 0
0 0 0
promoter was low in mature pollen of six transgenic N. tabacum plants (25 pmol 4-MU/min.mg protein). This result is in line with experiments in which no indigo-blue precipitation was histochemically detected in pollen of ten independent transgenic 35S-GUS N. tabacum plants and four L. esculentum transformants examined (data not shown). A low level of GUS activity was also found by Twell et al. (1989a) implicating that the CaMV35S promoter is only active at a low level in mature pollen. TR2'-driven GUS activity has been assayed histochemically in anthers derived from four independent transgenic TR2'-GUS L. esculentum and fourteen TR2'-GUS N. tabacum plants. In both species the TR2' promoter was active in mature pollen (see also Fig. 2). The GUS activity driven by the TR2' promoter in pollen pooled from the transgenic N. tabacum plants was 3970 pmol 4MU/min.mg protein. The presence of TR2'-driven GUS activity in pollen of L. esculenturn and N. tabacum is in contrast with an absence of TR2'-driven luciferase synthesis in N. tabacum pollen (Langridge et aL 1989). Here, measuring luciferase as a reporter in pollen extracts, the TR2' promoter was found to be activated only after pollen germination. Apart from pollen germination, both wounding and external addition of growth regulators are known to activate the TR dual promoter (Langridge et al. 1989, Saito et al. 1991). As none of these treatments has been applied in our experiments it remains unclear why TRY-driven GUS activity was present in nongerminating pollen whereas TR2'-driven luciferase activity was not. In the sporophytic anther tissues from the transgenic TR2'-GUS L. esculentum and N. tabacum plants the TR promoter was also found to be active. Here an intense indigo-blue precipitation was observed in the stomium (Fig. 2). No indigo-blue precipitation was observed in sporophytic and gametophytic tissues of control plants at the same
conditions. From the results in transgenic plants we conclude that the LAT52, chiA PA2 and TR2' promoters are and the CaMV 35S promoter is not or only at a low level active in mature N. tabacum pollen.
Promoter activity in Nicotiana glutinosa pollen after particle bombardment The activity of the LAT52, chiA PA2, TR2' and CaMV 35S promoters has been tested in pollen of N. glutinosa after particle bombardment. The transient GUS expression in pollen grains of N. glutinosa (a plant species producing high numbers of viable pollen grains) driven by these various promoters is shown in Table 2. In a bombardment experiment with the LAT52-GUS gene construct a high number of blue N. glutinosa pollen was observed (Fig. 3A and 3B). Within 10 minutes of X-gluc incubation the first blue pollen grains were detected. The time curve of the histochemical analysis resembles a logarithm with 1.5% and 2.9% indigo-blue pollen after respectively 2.5 and 50 hours of X-gluc staining. A logarithmic time course of the histochemical GUS analysis has also been shown recently for the ADH1 promoter in bombarded coleoptiles of Zea mays (Reggiardo et al. 1991). Bombardment with the chiA PA2-GUS and TR2'-GUS gene construct lead to the generation of blue N. glutinosa pollen .The first indigo-blue pollen grains were detected after 2.5 to 5 hours incubation in Xgluc (Table 2). The amount of blue N. glutinosa pollen after introduction of the chiA PA2- or TR2'-GUS genes was significantly less than found for the LAT52GUS gene and reached a maximum of 0.014% after 50 hours of X-gluc incubation.Bombardment with the 35S(adhl)-GUS construct did not give rise to blue N.
23
Fig 1. Chimeric genes used for particle bombardment of N. glutinosa pollen and L. longiflorum pollen. A: pLAT52-7 ; B: pMIP138; C: pCPO1.2'GUS; D: pCAL1Gc. Fig 2. Anther section from a transgenic TR2'-GUS L. esculentum plant. Indigo-blue precipitation in stomium and mature pollen, e= endothecium, I=loculus, p=pollen, s=stomium, v=vascular sheet. Fig 3. A. Overall picture of a nylon membrane showing LAT52-driven GUS expression in N. glutinosa pollen.; B. LAT52-driven GUS expression in N. glutb~osa pollen; C. TR2'-driven GUS expression in L. longiflorum pollen; D. TR2'-driven GUS expression in germinated N. glutinosa pollen.
glutinosa pollen (Table 2). As a control the same construct was introduced into N. glutinosa leaf tissue. Here the CaMV 35S promoter was highly active as demonstrated by the high amount of blue coloured spots (data not shown). These observations are in line with the data obtained for the activity of the CaMV 35S promoter in transgenic N. tabacum plants where no activity was detected in pollen in a histochemical assay and only a very low GUS activity was measured with the fluorimetric assay. Summarizing the data we conclude that the expression level of the promoters in N. glutinosa pollen reflects the activity levels found in N. tabacum pollen from transgenic plants: high for the LAT52 promoter, intermediate for the TR2' promoter, lower for the chiA PA2 promoter and no or a very low level of expression for the CaMV 35S promoter. Apparently the pollen bombardment system provides a way to compare the relative strengths of different promoters.
Promoter activity in Lilium longiflorum pollen after particle bombardment In order to assay the activity of the LAT52, chiA PA2, CaMV 35S and TR2' promoter in pollen of a recalcitrant monocot plant species, bombardment experiments were performed on pollen of L. longiflorum using again promoter-GUS constructs. The results obtained are summarized in Table 2 and differ from the data obtained for N. glutinosa pollen. In N. glutinosa pollen the LAT52 promoter was highly active. However this promoter was not active in pollen of the monocot L. longiflorum. The chiA PA2 promoter was also silent in L. longiflorum pollen. Therefore it can be concluded that these two promoters are differentially active in pollen grains from a monocot and dicot plant species and that factors activating the chiA PA2 and LAT52 promoters in N. glutinosa are absent in the lily pollen. This may reflect a different developmental
24 programme between the male gametophytes of these plant species. No GUS activity was demonstrated in L. longiflorum pollen after bombardment with the 35S(adh1)-GUS gene construct. Combined with the data described above we conclude from this that the regulatory factors necessary for the functioning of the CaMV 35S promoter seem to be absent or present at a low level in pollen of the monocot and dicot plant species used. In contrast, the TR2' promoter was clearly active in both L. longiflorum and Nicotiana pollen (Fig. 3C). This reveals a new property of this promoter namely that it is active in the generative stage of N. glutinosa and L. longiflorum. These results combined with recent findings such as the expression of this promoter in vegetative tissues of transgenic rice plants (Meijer et al. 1991) and the monocot tulip (Wilmink et al. 1991) suggests that the TR2' promoter is a promoter active in vegetative and sporogenous tissues of both dicot and monocot plants.
Germination of bombarded pollen Addition of a germination medium after bombardment of N. glutinosa pollen with TR2'-GUS and subsequent X-gluc staining produced pollen with extended tubes showing GUS expression (Fig. 3D). From the pollen expressing the GUS gene 81% was able to germinate. In the same population, pollen without GUS expression showed a similar in vitro germination percentage (78%). From this we conclude that pollen vigour does not seem to be affected by the bombardment procedure. Bombarded L. longiflorum pollen, expressing the same chimeric gene was also able to germinate (data not shown). Therefore future experiments will be directed towards pollination with bombarded pollen and subsequent screening of the progeny for a transformation event. ACKNOWLEDGEMENTS We thank Dr. S. McCormick (Plant Gene Expression Centre, LA, USA) and Dr. V. Walbot (Stanford University, USA) for kindly providing some of the gene constructs; Drs. 3.3.M. Dons and G.C. Angenent for critical reading of the manuscript and stimulating discussions and Mr. 3. Franken for technical support. Part of this work was funded by the Innovation Funding for Plant Breeding Research (Inpla) and by the Urgency Programme for Research on Diseases and Breeding of Flower Bulbs. REFERENCES MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254. Brewbaker JL. Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. of Bot. 50: 859-865. Bytebier B, Deboeck F, De Greve H, Van Montagu M (1987) TDNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis. Proc. Natl. Am. Sc. USA 84: 5345 -5349. Chen WH, Gartland KMA, Davey MR, Sotak R, Gartland JS, Mulligan BJ, Power JB, Cocking EC (1987) Transformation of sugarcane protoplasts by direct uptake of a selectable chimeric gene. Plant Cell Rep. 6: 297-301. Datta SK, Datta K, Potrykus I (1990a) Fertile Indica rice plants Bradford
regenerated from protoplasts isolated from microspore derived suspensions. Plant Cell Rep. 9: 253-256. Datta SK, Peterhans A, Datta K, Potrykus I (1990b) Genetically engineered fertile Indica-rice recovered from protoplasts. Bio/Technology 8: 736-740. Fromm M, Taylor LP, Walbot V (1986) Stable transformation of maize after electroporation. Nature 319: 791-793. Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Tech. 45: 115-120. Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eicholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transfering genes into plants. Science 227: 1229-1231. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Bglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70-73. Koncz C, Mayerhofer R, Koncz-Kalman Z, Nawrath C, Reiss B, Redei GP, Schell J (1990) Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. EMBO J. 9: 1337-1346. Koornneet M, Hanhart CJ, Martinelli L (1987) A genetic analysis of cell culture traits in tomato. Theor. Appl. Genet. 74: 633-641. Langridge WHR, Fitzgerald KJ, Koncz C, Shell J, Szalay AA (1989) Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc. Natl. Am. Sc. USA 86: 3219-3223. Meijer EGM, Schilperoort RA, Rueb S, Van Os-Ruygrok PE, Hensgens LAM (1991) Transgenic rice cell lines and plants: expression of transfered chimeric genes. Plant Mol. Biol. 16: 807820. Plegt LM, Bino RJ (1989) 13-Glucuronidase activity during development of the male gametophyte from transgenic and nontransgenic plants. Mol. Gen. Genet. 216: 321-327. Potrykus I, Saul MW, Petruska J, Paszkowski J, Shillito RD (1985) Direct gene transfer to cells of a gramineous monocot. Mol. Gen. Genet. 199: 167-177. Reggiardo MI, Arana JL, Orsaria LM, Permingeat HR, Spitteler MA, Vallejos RH (1991) Transient transformation of maize tissues by microparticle bombardment. Plant Sc. 75: 237-243. Saito K, Yamazaki M, Kaneko H, Murakoshi I, Fukuda Y, Van Montagu M (1991) Tissue-specific and stress-enhancing expression of the TR promoter for mannopine synthase in transgenic medical plants. Planta 184: 40-46. S h i m a m o t o K, Terada R, Izawa T, Fujimoto H (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274-276. Spencer TM, Gordon-Kamm WJ, Daines RJ, Start WG, Lemaux PG (1990) Bialaphos selection of stable transformants from maize cell culture. Theor. AppL Oenet. 79: 625-631. Twell D, Klein TH, Fromm ME, McCormick S (1989a) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol. 91: 1270-1274. Tweli D, Wing R, Yamaguchi J, McCormick S (1989b) Isolation and expression of an anther-specific gene from tomato. Mol. Gen. Genet. 217: 240-245. Van Tunen AJ, Hartman SA, Mur LA, Mol JNM (1989) Regulation of chalcone flavanone isomerase (CHI) gene expression in Petunia hybrida: the use of alternative promoters in corolla, anthers and pollen. Plant Mol. Biol. 12: 539-551. Van Tunen AJ, Mur LA, Brouns GS, Rienstra JD, Koes RE, Mol JNM (1990) Pollen-and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene. Plant Cell 2: 393401. Velten J, Velten J, Hain R, Schell J (1984) Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J. 3: 2391-2406. Wilmink A, Van de Ven B, Dons JJM (1991) Expression of the GUS-gene in the monocot Tulip after introduction byAgrobacterium and particle bombardment (submitted). Yang H, Zhang HM, Davey MR, Mulligan BJ, Cocking EC (1988) Production of kanamycin resistant rice tissues following DNA uptake into protoplasts. Plant Cell Rep. 7: 421-425. Zhang W, Wu R (1988) Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants. Theor. Appl. Genct. 76: 835-840.
