1 Dominant-negative inhibition of cytoplasmic

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Successful incorporation of the male sterility trait into F1 hybrid breeding requires tight sterility and ... The latter is usually achieved by crossing male-sterile plants with near-isogenic ... vitro cultured microspores are not able to develop into functional pollen grains in a medium ..... germination medium GK (C) (bar = 20 µm).
1 Dominant-negative inhibition of cytoplasmic glutamine synthetase in developing anthers and pollen leads to reversible male sterility

Alexandra Ribarits*,§, Mamun Ank*,‡,§, Shipeng Li†,§, Tatiana Resch*, Martijn Fiers†, Erwin Heberle-Bors*, Chun-Ming Liu† & Alisher Touraev*¶

*

Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department

of Plant Molecular Biology, Dr. Bohrgasse 9/4, 1030 Vienna, Austria. †Plant Research International, PO Box 16, 6700AA, Wageningen, The Netherlands. †Present address: Plant Biotechnology Division, I.F.R.B., A.E.R.E. Bangladesh Atomic Energy Commission, G.P.O. BOX NO. 3787, Dhaka – 1000, Bangladesh. §These authors contributed equally to the work. ¶To whom correspondence should be addressed: Alisher Touraev Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Plant Molecular Biology, Dr. Bohrgasse 9/4, A-1030 Vienna Tel: +43-1-4277-54681; Fax: +43-1-4277-9546; E-mail: [email protected]

Conflict of interest statement: No conflicts declared.

2 F1 hybrid seed production is one of the most important processes in plant breeding. F1 hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F1 hybrid seed technology based on metabolic engineering of glutamine in developing tobacco anthers and pollen. Dominant-negative mutant versions of tobacco glutamine synthetase were fused to the tapetum-specific TA29 or the microspore-specific NTM19 promoter, and transformed into tobacco resulting in male sterility. A homozygous population of male-sterile progeny was produced as doubled haploids. Fertility restoration was achieved by spraying plants with glutamine or by pollination with in vitro matured pollen. Tapetum-mediated sporophytic male sterility is of use in foliage crops whereas microspore-specific gametophytic male sterility can be applied to any field crop. In both types of male sterility no transgenic pollen is released into the environment.

glutamine synthetase | male sterility | microspore | Nicotiana tabacum L. | tapetum Abbreviations: DNM, dominant-negative mutation; GS, glutamine synthetase.

3 Today many important seed (corn, rape, soybean), vegetable (cucumber, tomato, pepper) and foliage crops (tobacco, forage grasses) are available as F1 hybrid cultivars. Two inbred lines are crossed to produce F1 hybrid seeds, and a reliable system of pollination control is mandatory to enforce cross-pollination (1). Genetic male sterility avoids the labor of manual emasculation and prevents pollen release to the environment (1, 2). The lack of a generic technology for reversible male sterility becomes a major barrier to exploit the advantages of F1 hybrid seed production in a wider range of plant species (1). Successful incorporation of the male sterility trait into F1 hybrid breeding requires tight sterility and efficient fertility restoration in order to maintain the male-sterile line (1). The latter is usually achieved by crossing male-sterile plants with near-isogenic male-fertile restorer lines. By contrast, no fertility restorer genes are required if male sterility is reversible (3-6). However, in all these systems the controlled restoration of fertility remains difficult, and incomplete male sterility, impracticable and inefficient methods for fertility restoration, or potential environmental risks limit their practical application (1). In addition, typically segregation occurs when a plant carrying a nuclear sterility gene is crossed with a near-isogenic male-fertile line. Plant reproduction involves precisely timed coordination of developmental and metabolic processes (7). This knowledge has been utilized in a number of attempts to obtain male-sterile plants by manipulating the developmental or metabolic pathways in anthers or pollen, or to deliver cytotoxic compounds to the tapetum or pollen grains (1, 8). Glutamine plays an essential role during pollen development, and glutamine synthetase (GS, E. C. 6.3.1.2) with its two iso-enzymatic forms, cytoplasmic GS1 and chloroplastic GS2, is tightly regulated during male reproductive development (9-11). Isolated and in

4 vitro cultured microspores are not able to develop into functional pollen grains in a medium lacking glutamine (12). Chemical inhibition of GS in developing rice anthers causes male sterility and depletes plants of crucial amino acids, including glutamine and glutamate (13, 14). We used a dominant-negative mutant (DNM) approach to inhibit GS activity specifically in the tapetum and in microspores of tobacco. Developing pollen aborted in transformed plants, resulting in 100% male sterility. Doubled haploid plants, which were 100% male-sterile, homozygous and, thus, non-segregating, were produced via microspore embryogenesis. Male-sterile plants could also be maintained by selfing with pollen grains rescued by in vitro pollen maturation, or by spraying flowering malesterile plants with glutamine.

Results Dominant-negative inhibition of GS in the tapetum and microspores of tobacco. Genome survey and RT-PCR analysis showed that there are at least five copies of GS1 and one copy of GS2 in the tobacco genome (data not shown). GS1 and GS2 cDNAs were isolated from Nicotiana tabacum cv Petit Havana SR1 (15) and confirmed by sequencing (data not shown). A DNM GS2 under control of the tapetum-specific TA29 promoter (16) (pTA29-∆GS2) was created by deleting the C-terminal 45 amino acids, which are critical for GS activity but not for sub-unit binding (17). Pollen fertility was not affected when this construct was transformed into Arabidopsis thaliana (data not shown). Therefore, in addition to the activity domain the N-terminal chloroplast targeting signal was deleted in the pTA29-∆GS2-L construct (Fig. 1A). The severe reduction of pollen fertility and very low seed set observed in Arabidopsis transformants (data not shown) showed that

5 deleting the chloroplast-targeting signal is essential to inactivate the enzyme. The DNM of GS1 was achieved by introducing point mutations in two critical sites identified in plants and E. coli (17, 18). To obtain maximum detrimental effects on GS activity and minimum interruption of sub-unit interaction, asparagine at position 56 was replaced by alanine, and arginine at position 291 by leucine, expected to result in severe structural changes. The mutated GS1 gene was fused to the tapetum-specific TA29 (16) or the microspore-specific NTM19 (19, 20) promoter (Fig. 1A). The constructs pTA29-∆GS2-L, pTA29-GS1A56L291 and pNTM19-GS1A56L291 were transformed into tobacco via Agrobacterium-mediated leaf disc transformation (21), and nineteen to fifty independent, kanamycin-resistant and PCR-positive transgenic plants were obtained per construct. Stable integration of the T-DNAs was confirmed by Southern blotting (Fig. 2), and aided by segregation analysis of seeds produced by cross-pollination on a kanamycincontaining medium to determine transgene copy number. As has been reported earlier (22), the Southern hybridization pattern revealed two common bands, most likely representing the endogenous GS1 and GS2 genes, in addition to the variable ones (Fig. 2). A 1:1 segregation ratio, indicative of a single insertion, was found in eight lines, whereas another ten lines carried two or more copies of the transgene (Table 1; Fig. 2). Western blots showed that GS1 and GS2 protein levels were lower in transgenic DNM GS anthers compared to the wild-type (Fig. 2), possibly caused by decreased protein stability or a co-suppression effect as in Petunia (23). However, the lower GS contents could also be the result of reduced tapetum and pollen biomass relative to the mass of other anther tissues as a consequence of the developmental defects in transgenic anthers.

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Fig. 1. Description of constructs and phenotypic alterations in tobacco transformants. (A) Schematic representation of pBINPLUS vectors used for transformation. (1) pTA29∆GS2-L. 45 amino acids from the N-terminal sequence and 59 amino acids from the Cterminal sequence were deleted. (2) pTA29-GS1A56L291. In GS1, arginine at position 291 (Ar) was replaced by leucine (L) and aspartate at position 56 (As) by alanine (Al), and fused to the tapetum-specific promoter TA29. (3) pNTM19-GS1A56L291. Mutated GS1 was fused to the microspore-specific promoter NTM19. L, left border; P, nopaline synthetase (nos) promoter; NPTII, neomycin phosphotransferase type II (nptII) conferring kanamycin-resistance; T, nos terminator; TA29, tapetum-specific promoter; NTM19, microspore-specific promoter; R, right border. (B) Phenotypic alterations in transgenic compared to wild-type tobacco flowers. Anthers in male-sterile plants turned olive to brown after first pollen mitosis stage (pTA29-GS1A56L291, L41), and became desiccated at the time of dehiscence. In most lines a small amount of aborted pollen was released (pTA29-∆GS2-L, L22). pNTM19-GS1A56L291 (L83), from left to right: fertile flower of a cross between a homozygous doubled haploid (DH) and a wild-type plant (DH×wt), restored fertile flower of a DH line after spraying with glutamine (DHr), malesterile flower of a homozygous DH plant (DHst), fertile flower of a wild-type plant (wt). (C) Anther development in wild-type (1-3) and three independent transgenic pTA29∆GS2-L lines (L8, L22, L32). Transverse sections (5 µm) were stained with toluidine blue, and examined by bright-field microscopy. The tapetum (►) appeared as a smooth layer in the anther locule at the microspore stage (1), started degenerating from the late uni-cellular microspore stage (2), and was fully degraded at the mature pollen stage, (3) accompanied by wall thickenings (►►) in the endothecium. In transgenic anthers at the late uni-cellular microspore stage (L8, L22, L32) degeneration of the tapetum (►) was clearly advanced, and wall thickenings (►►) were deposited precociously (Bars = 50 µm). (D) Cytology of microspores and pollen grains from transgenic pTA29-∆GS2-L (L12), pNTM19-GS1A56L291 (L11) and wild-type tobacco plants. Developing pollen grains were examined under the light microscope or stained with DAPI. At the tetrad stage (1) no differences were visible between transgenic and wild-type cells, at the microspore stage

7 (2) the number of viable microspores started to decrease in the transgenic compared to wild-type plants, and at the mature pollen stage (3) most of the pollen grains in the transformed plants were aborted (Bars = 20 µm).

Fig. 2. Molecular analysis of wild-type and male-sterile tobacco lines. (A) Southern blot analysis of wild-type and pTA29-∆GS2-L transformed plants. Genomic DNA (10 µg) was isolated from leaves of wild-type (wt) and four independent transgenic lines (22, 25, 29, 32). (B) Western blot of wild-type (wt), TA29-∆GS2-L (22, 25, 32) andTA29GS1A56L291 (11, 41, 45) plants. Total protein extracts (10 µg) were prepared from wildtype leaves (L) and anthers (A), and anthers of transgenic plants. GS polypeptides were detected using an antibody cross-reacting with both GS1 (38–40 kDa) and GS2 (45 kDa) isoenzymes. (C) Coomassie Blue-stained gel of the protein extracts used in (B) to show equal loading. The developmental defects are confined to male reproductive organs. Vegetative development of primary T0 transformants was like in wild-type plants. Five lines carrying pTA29-∆GS2-L and four lines harboring pTA29-GS1A56L291 (Table 1) were completely male-sterile. Eight more lines displayed highly reduced fertility (data not shown). After dehiscence, almost all pollen grains (91–99.5%) were found aborted and no single pollen was able to germinate in vitro (Table 1). Ten out of nineteen pNTM19GS1A56L291 lines exhibited 50% pollen viability (Table 1) as expected for single insertions of the transgene. Light and fluorescence microscopy demonstrated that pollen

8 started dying close to, or immediately after the first pollen mitosis stage (Fig. 1D). This result was independent of the construct and unexpected as the activity of both promoters sets in well before that stage (16, 19, 20). Tapetum-specific expression of the mutated GS1 or GS2 resulted in anthers which changed color from green to olive at about the first pollen mitosis stage, dried up prior to dehiscence, and released very small amounts of mostly aborted pollen grains (Fig. 1B). As expected, similar to rice panicles (13), we found slightly increased ammonia contents in anthers of pTA29-∆GS2-L plants compared to the wild-type (data not shown), which might explain the browning of anthers (Fig. 1B). The tapetum began to degenerate at the early microspore stage, accompanied by precocious deposition of wall thickenings in the endothecium (Fig. 1C). Wild-type and pNTM19-GS1A56L291 anthers looked similar until the dehiscence stage. However, the transgenic anthers clearly released less pollen (Fig. 1B). Cross-pollination of the male-sterile T0 plants with wild-type pollen led to normal seed set, showing that female fertility was not impaired (Table 1). Primary pNTM19GS1A56L291 plants set as many seeds as wild-type plants after self- and crosspollination (Table 1).

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Table 1. Pollen viability, in vitro germination, seed set and segregation ratios after crosses with wild-type or selfing in male-sterile T0, doubled haploid H1 and wild-type plants segregation seeds/ in vitro seed seed ratioa seed germination weight weight pod after fertility (g/seed (g/seed KanR:KanS (n=4restoration pod) pod) per 12) (%) selfing cross pTA29-∆GS2-L L8 14.0±0.9 0 0.06 0 n. d. n. d. 10:1 78.1±2.8 L12 16.2±1.1 0 0.11 0 n. d. n. d. 3:1 95.4±2.3 L22 5.4±0.3 0 0.07 0 8.4±1.6 120 3:1 80.4±6.7 L25 14.6±0.7 0 0.06 0 7.8±1.4 102 3:1 65.6±3.2 L32 7.8±0.6 0 0.07 0 10.1±2.4 151 1:1 91.2±2.1 pTA29L11 30.7±1.4 0 0 0.13 0 12.11±2.4 181 1:1 GS1A56L291 L14 24.5±5.6 0 0 0.09 0 n. d. n. d. 3:1 L41 30.9±2.7 0 0 0.08 0 0.3±0.1 0 3:1 L45 27.8±1.1 0 0 0.08 0 n. d. n. d. 3:1 pNTM19L3 75.0±4.2 52.3±4.4 46.5±2.9 0.17 0.13 n. d. n. d. 1:1 GS1A56L291 L8 73.2±2.6 52.6±0.6 46.0±2.7 0.17 0.14 n. d. n. d. 1:1 L39 75.0±0.4 50.7±0.7 46.5±2.2 0.16 0.17 n. d. n. d. 1:1 L61 76.7±0.7 49.6±2.5 43.7±2.7 0.15 0.14 n. d. n. d. 1:1 L64 74.0±2.3 50.2±1.5 46.4±3.7 0.17 0.15 n. d. n. d. 1:1 pNTM19L8 24.2±1.3 4.6±0.4 0 0.11 0 11.3±0.6 188 n. a. GS1A56L291 L39 23.9±1.6 4.9±0.2 0 0.09 0 10.8±1.4 129 n. a. doubled haploid L61 24.8±2.5 2.6±0.3 0 0.10 0 11.8±1.3 189 n. a. H1 L64 26.2±1.7 3.5±0.2 0 0.11 0 11.1±1.2 201 n. a. wild-type 96.2±2.1 95.5±1.3 95.3±0.1 0.17 0.18 n. a. >1200 n. a. a Segregation ratios were determined by plating seeds obtained after crosses between male-sterile and wild-type plants onto kanamycin-containing (50 mg/l) medium. n.d., not determined; n.a., not applicable, values are means ± s.d. construct

lines with constructs

viability of microspores in T0 (%)

viability of mature pollen in T0 (%)

in vitro germination of pollen in T0 (%)

10 Producing homozygous male-sterile plants via microspore embryogenesis. For complete sterility and to maintain the male-sterile lines as a non-segregating population the male sterility trait has to be homozygous. It is known that upon stress treatment isolated microspores become embryogenic and form haploid embryos when cultured in appropriate medium (24). Doubled haploid production is a rapid way to achieve complete homozygosity and is established in many important crops, including dicots (tobacco, pepper, rapeseed) and monocots (barley, wheat, corn) (25). Exploiting the finding that in all lines microspores were viable in sufficient numbers we regenerated doubled haploid lines homozygous for the transgenic male sterility trait via in vitro microspore embryogenesis (24). Microspores where isolated from anthers of heterozygous plants, reprogrammed towards embryogenesis by a starvation treatment and cultured in a medium adapted for embryo formation (26). Torpedo-shaped embryos (Fig. 3), formed after one month, were treated with colchicine for chromosome doubling, and selected on a kanamycin-containing medium. Completely male-sterile and homozygous doubled haploid H1 progeny was obtained from all lines (data not shown). Homozygosity is particularly crucial to achieve 100% male sterility in pNTM19-GS1A56L291 lines (Table 1). In a simulation of F1 hybrid seed production homozygous pNTM19-GS1A56L291 plants were used as female parents, and pollinated with wild-type pollen (male donor line). The T2 hybrid progeny was kanamycin-resistant, produced 50% viable, nontransgenic pollen, and self-pollination resulted in full seed set (Table 1).

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Fig. 3. Embryogenesis in microspores from TA29-GS1A56L291 (L11) and wild-type plants cultured in vitro. (A) Freshly isolated tobacco microspores, (B) globular embryos and (C) torpedo- and cotyledon-stage embryos after seven weeks of culture (bars = 20 µm, A; 100 µm, B and C).

Pollen fertility in the male-sterile lines can be restored by spraying plants with glutamine or by in vitro pollen maturation. Male sterility caused by chemical inhibition of GS in developing rice anthers can be overcome by exogenous application of glutamine (13). We likewise succeeded in restoring pollen fertility in DNM GS malesterile plants by spraying with glutamine onto the bottom of leaves twice a day throughout seven to ten days from the meiosis stage (Fig. 1b; Table 1). Tobacco microspores can be matured in vitro in medium supplemented with the necessary nutrient sources, including glutamine, and mature pollen may be used to pollinate flowers in vivo (26). Viable microspores were isolated from the male-sterile plants and, within six days, cultured in vitro to maturity in the presence of glutamine (Fig. 4). Both wild-type and transgenic in vitro matured pollen grains produced pollen tubes when transferred to appropriate germination medium (26). Pollination of receptive wild-type stigmas with an enriched suspension of viable, in vitro matured pollen resulted in fruit set, and segregation ratios of thus obtained seeds corresponded to those after conventional back-

12 crosses, proving that pollen of male-sterile plants can indeed be rescued. Self-pollination, by pipetting in vitro matured pollen onto stigmas of male-sterile plants, also resulted in seed set (data not shown). Hence, the application of glutamine onto male-sterile plants or in the culture medium to isolated microspores is sufficient to restore pollen viability, and indicates that a lack of glutamine leads to pollen abortion in the DNM GS transgenic plants.

Fig. 4. In vitro pollen maturation of isolated microspores from TA29-GS1A56L291 (L11), TA29-∆GS2-L (L12), and wild-type plants. After isolation, microspores (A) were cultured in medium T1, supplemented with glutamine. Starch accumulation was observed after six days of incubation (B). In vitro matured pollen grains germinated when incubated in germination medium GK (C) (bar = 20 µm).

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Discussion Dominant-negative inhibition of GS1 activity leads to male sterility, which is reversible by application of glutamine. Plant reproduction is a complex process requiring a precisely timed series of developmental steps and coordinated action of metabolic pathways (7). Consequently, male sterility can be engineered through disrupting the developmental or metabolic pathways in anthers or pollen, or delivering cytotoxic compounds to the tapetum or pollen grains (1, 8). We have produced 100% male-sterile plants by transforming modified GS1 or GS2 genes under control of the tapetum-specific TA29 or the microspore-specific NTM19 promoter into tobacco. Dominant-negative point mutations were introduced into GS1, whereas in GS2 the Nterminal sequence, targeting the protein to the chloroplasts, and the C-terminal functional domain were deleted to achieve a DNM effect. GS2-specific inhibition of enzyme activity did not result in sterility when only the functional domain was removed. However, deleting both domains suppressed pollen maturation, likely caused by the interaction of the DNM GS2 with the endogenous GS1. Hence, we concluded that GS1 is critical for tapetum and pollen development, while GS2 seems less essential. In all tobacco transformants, vegetative development remained unaltered compared to wildtype plants. However, nine lines transformed with tapetum-specific DNM GS were completely male-sterile. If the male sterility trait was induced by microspore-specific DNM GS, pollen viability was 50% reduced indicating insertion of an active foreign DNA in one locus. Pollen abortion commenced close to, or immediately after the first pollen mitosis stage independent of the construct. These results were in agreement with earlier findings that pollen maturation requires glutamine (12), and that the inhibition of

14 glutamine synthetase in anthers leads to male sterility (13). Doubled haploid plants were produced via microspore embryogenesis. These homozygous plants were male-sterile, showing that the trait was transmitted to the progeny, and could be used as inbred parental lines. Fertility of all male-sterile plants was restored by supplying glutamine either in the in vitro maturation medium or by spraying plants, indicating that indeed the lack of glutamine had caused male sterility.

Male sterility induced by metabolic engineering of glutamine offers important advantages. All currently available male sterility systems suffer from major drawbacks. Transgenic systems can be balanced in such a way that they provide tight sterility, no reduction of seed yield, no impairment of female fertility, and easy male fertility restoration (8). Cytoplasmic male sterility (CMS), often tried in F1-hybrid breeding, is only available in some crops, and the conversion of two potential parental inbred lines into a male-sterile and restoration line, respectively, is tedious (27). In addition, fertility restoration requires crossing the male-sterile line with a near-isogenic male-fertile line carrying an appropriate restorer gene (27). An interesting alternative to naturally occurring CMS is the recently reported engineered CMS via the chloroplast genome (6). However, chloroplast transformation has been established only in a few plant species (28), whereas nuclear transformation is possible in a number of plants, including economically important species. Also, fertility restoration by continuous illumination might prove inefficient. Another recently published approach uses the application of kinetin, a plant hormone, to restore fertility (5), which is likely to have unwanted side effects. Metabolic engineering of the carbohydrate supply (29) leads to pollen abortion

15 but fertility restoration has not been achieved. Two transformation events are necessary in the well-known barnase/barstar system (3), in which a sterility gene, i.e. barnase, is suppressed by expressing a restorer gene, i.e. barstar, in the same cells. In addition, herbicide treatment is necessary to eliminate segregating male-fertile plants. Usually, the sterility trait prevents the production of homozygous plants, which are required for maintenance breeding and F1 hybrid seed production. In most male sterility technologies, pollen abortion sets in early, and no viable microspores are produced. Early onset of pollen sterility has been considered desirable as it prevents unwanted self-pollination. If male fertility is affected late in male reproductive development, as caused by flavonoid deficiency the sterility might be leaky (30, 31). In contrast to these methods the technology described here targets pollen development in the stage just after the formation of microspores. This simultaneously ensures both tight sterility and the formation of viable microspores, which enabled us to isolate and culture them for microspore embryogenesis or in vitro maturation. In the breeding process, these two methods can be used to produce homozygous male-sterile plants, restore their fertility and to propagate them. Two different methods of fertility restoration and one new method of maintenance and propagation of male-sterile lines were made available. The novel F1 hybrid seed technology described here has further benefits. First, no transgenic pollen is released to the environment. This applies not only to the male-sterile lines, but also to F1 hybrids generated since all fertile pollen being produced is wild-type. Second, no toxic or potentially harmful substances need to be employed to induce male sterility or restore fertility. Glutamine is a regular metabolite, likely to be innocuous in an agricultural setting. Third, efficient fertility restoration by glutamine sprays or in vitro

16 maturation overcomes tedious back-crossing. Fourth, microspore embryogenesis provides fast access to completely homozygous material.

Fig. 5. Chart showing the generation and application of sporophytic (TA29) and gametophytic (NTM19) reversible male sterility induced by inactivation of glutamine synthetase. Transformation with pTA29-∆GS2-L or pTA29-GS1A56L291 yields heterozygous, completely male-sterile plants, whereas heterozygous pNTM19GS1A56L291 transformants release 50% non-transgenic pollen and, thus, are fertile. Homozygous, 100% male-sterile plants are produced as doubled haploids via microspore embryogenesis. These plants are used as female parents in a cross with a male-fertile inbred line. To maintain the homozygous male-sterile plants and to increase their number three methods are available: 1) self-pollination after spraying plants with glutamine, 2) pollination with in vitro matured pollen, 3) microspore embryogenesis.

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Finally, the two different modes of genetic transmission of the sterility trait allows to adapt the technology for F1 hybrid breeding in most species. Gametophytic male sterility using the constructs driven by the NTM19 promoter can be applied to seed crops, as 50% of the pollen in the resulting hybrids will be wild-type, fertile, and sufficient for full seed set (Fig. 5). Sporophytic male sterility using the TA29-driven constructs is of use for non-seed crops, as no fertile pollen is produced. For both types of male sterility, lines homozygous for the male sterility trait can be produced, and used as female parents in crosses with another male-fertile inbred line for F1 hybrid breeding. Providing proof of concept in tobacco, we feel confident that the presented environment-friendly F1 hybrid seed production technology can be adopted in many commercially important crops.

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Methods Plant material. Nicotiana tabacum L. cv Petit Havana SR1 (15) were grown in the greenhouse at 25 °C under 16 h daylight or in vitro on MS (32) medium at 25 °C for 4-6 weeks after surface sterilization of seed with 70% ethanol and 10% sodium hypochlorite (Fluka; v/v; 1% active chlorine).

Construction of plasmids with DNM in glutamine synthetase (GS) genes. Tobacco GS genes (GS1 and GS2) were isolated by RT-PCR from a tobacco cDNA library (Nicotiana tabacum cv Petit Havana SR1), and confirmed by sequencing analysis. The construct

pTA29-∆GS2-L was created by deletions of the C-terminal activity domain (amino acid 944-989) and the N-terminal chloroplast-targeting signal (amino acid 1-59) under control of the tapetum specific TA29 promoter (16). The DNM of GS1 was achieved by introducing point mutations in two critical sites (17, 18). Asparagine at position 56 was replaced by alanine, and arginine at position 291 by leucine. The mutated GS1 gene was fused to the tapetum-specific TA29 (16) or the microspore-specific NTM19 (19, 20) promoter, and cloned into the binary vector pBINPLUS. All constructs (Fig. 1A) were transformed into Agrobacterium tumefaciens (LBA4404) by electroporation.

Transformation. Agrobacterium-mediated leaf disc transformation was performed essentially as described (21). Transgenic shoots were selected on MS medium (32) containing 50 mg/l kanamycin and 500 mg/l cefotaxime sodium (Duchefa). Rooted plantlets were transferred to soil for further growth under the same conditions as described for wild-type plants in the greenhouse until flowering.

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Isolation of genomic DNA, PCR amplification, and Southern hybridization. Genomic DNA for PCR and Southern blotting was isolated from leaf tissues of kanamycin-resistant plants using the DNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions. The presence of the introduced constructs was assayed by PCR analyses using primers for the 3’-region of the TA29 promoter (5’GCGACATAGGCTCGAAGTATGCAC-3’)

or

the

NTM19

promoter

(5'-

GCTTCTAATTGATCAGACGAGGAC-3'), and specific for the introduced genes (5’GACCTGTGCTAGATCCATCATAGTT-3’

for

and

GS1

5’-

CTCAGAAGCATGCTTGACTGGCTTTG-3’ for GS2) under standard PCR conditions. Genomic DNA (10 µg) was digested for 12-16 h with NcoI and VspI (MBI Fermentas) using 2 U/µg DNA. Digested DNA was fractionated on 0.8% agarose gels, and transferred onto GeneScreenPlus membranes (PerkinElmer). Blots were probed with a 419-bp PCR product, generated by amplification with the primer pair 5’GGTACTAACGGAGAGGTTATGCCAGG-3’ CCTTTGCCTTGCTTCTCAGTGTCAGC-3’, and labeled with

and 32

5’-

P using a RadPrime

DNA Labeling System (Invitrogen). Hybridization was done at 65 °C in 0.5 M Na2PO4 (pH 7.2), 7% SDS. Protein isolation and immunoblotting. Frozen fresh young leaves (100 mg) and anthers collected from buds of 9–11 mm were ground in ice-cold protein extraction buffer (500 mM HEPES, 750 mM KCl, 100 mM MgCl2, 100 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 20,000 g for 40 min at 4 °C. 20 µg of protein per sample were run on 10% SDS-polyacrylamide gels, and blotted onto polyvinylidene

20 difluoride membranes (Immobilon-P; Millipore). Membranes were blocked overnight at 4 °C in PBS-T (0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 4 mM Na2HPO4, 0.05% Tween 20) containing 5% non-fat dried milk. Rabbit anti-glutamine synthetase (1:5,000) was used as the primary, and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma) as the secondary antibody. The reaction was visualized by fluorography using CDP-Star (Amersham Life Sciences) as a substrate.

Selection of male-sterile plants and segregation analysis of backcross-derived seeds. The primary T0 transgenic lines were self-pollinated, or backcrossed with pollen from wild-type tobacco plants. Seeds were germinated on MS medium containing 100 mg/l kanamycin to select for transformed T1 progeny, and copy numbers of DNM GS insertions were estimated by segregation rates.

Cytological analysis of male gametophyte development. The viability of pollen and the stage of pollen development were determined under a fluorescent microscope (LeitzDIAPLAN) using the FITC or UV filter channel after staining in fluoresceine diacetate solution (1 µg/µl; Sigma) or in 4’,6-diamidino-2-phenylindole (Partec) as described (26). Images were taken using Kodak Gold ISO400 or ISO800 films.

Histological analysis. Anthers collected at different developmental stages were fixed overnight in FAA (50% ethanol, 5.0% glacial acetic acid, 3.7% formaldehyde; v/v), and kept in 70% ethanol until further processing. Fixed samples were embedded using the Kulzer Technovit 7100 Kit (Serva). Sections of 5 µm were stained in 1% toluidine blue

21 (w/v) for 10 min, and photographed under an Olympus BX50 microscope with a Nikon D100 digital camera.

In vitro pollen germination. In vivo matured pollen grains were collected from open tobacco flowers, germinated in medium GK for 3-6 h at 25 ºC in the dark (26), and pollen tube growth and germination frequencies were observed under the light microscope.

Ammonia extraction and quantification. Protein was isolated from anthers 9-11 mm in size as described for immunoblotting, and the phenol hypochlorite assay (Berthelot) reaction was used to determine ammonia contents as described (33).

Production of homozygous T1 male-sterile plants via microspore embryogenesis. Homozygous T1 doubled haploid male-sterile lines were produced essentially as described (26). Microspores were isolated from anthers aseptically excised from 11-mmbuds, cultured in starvation medium B at 33 °C for 6 d, and transferred to embryogenesis medium AT3 resulting in the formation of embryos. After 6-8 weeks, chromosome doubling was performed by incubating embryos in AT3 medium supplemented with 0.1% (w/v) colchicine for 6-8 h at 25 °C in the dark. Diploidized embryos were transferred onto kanamycin-containing MS medium (100 mg/l) for selection of transgenic plants. Flow cytometry was performed as described (34).

Fertility restoration of male-sterile lines by spraying. Glutamine (0.01-0.05 mM) was dissolved in water, filter-sterilized, and sprayed twice a day throughout 7-10 d onto

22 leaves and inflorescences of young greenhouse grown plants starting from the meiosis stage. In vitro pollen germination frequencies were determined as described (26). Transgenic and wild-type plants were manually self- and cross-pollinated with rescued pollen, and seeds were subjected to segregation analysis on kanamycin-containing (100 mg/l) medium.

Fertility restoration by in vitro pollen maturation. Microspores at the late uni-cellular stage were isolated from male-sterile lines and matured for 6 d as described (26). After gradient centrifugation in 60% Percoll (Amersham, UK) and B medium with 1 M mannitol to remove dead pollen, the mature pollen grains were re-suspended in germination medium GK. 3 µl of suspension (3,500 – 5,000 grains/µl) were applied to stigmas of emasculated flowers protected with paper bags. Seeds were collected after 3-4 weeks, and subjected to segregation analysis on kanamycin-containing medium.

We are grateful to Dr. Robert Dirks (Rijk Zwaan, NL) for helpful advice, Dr. Ken Kasha (University of Guelph, CA) and Dr. Brian Forster (SCRI, UK) for critical reading of the manuscript, and Dr. Ralf Buchner (Department of Ultrastructure Research and Palynology, University of Vienna) for assistance with histological images and access to facilities. This work was supported by the European Commission funded HybTech project (QLK5-CT-1999-30902), and by a fellowship of the Austrian Exchange Service (ÖAD) to M.A.

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