JXB Advance Access published online on January 19, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl271
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers
Instituto para la Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia Camino de Vera s/n, edificio I-4, E-46022 Valencia, Spain
* To whom correspondence should be addressed. E-mail: seguisim{at}btc.upv.es
Received 12 September 2006; Revised 15 November 2006 Accepted 16 November 2006
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Abstract |
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In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.
Key words: Androgenesis, anther culture, electron microscopy, haploid embryogenesis, Lycopersicon esculentum, microspore culture, morphogenesis, organogenesis, plant regeneration, Solanum lycopersicum
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Introduction |
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In higher plants, the developmental fate of the male gametophyte or its precursors can be experimentally deviated towards haploid androgenic development. By means of in vitro procedures, haploid and doubled-haploid plants can be regenerated. This process presents diverse advantages in both basic and applied research (Touraev et al., 2001; Maluszynski et al., 2003), the most important one being related to plant breeding. A doubled-haploid plant constitutes a pure line (100% homozygous), and can be obtained in just one in vitro generation. The inherent saving in costs and time have promoted the use of doubled-haploid technology in breeding programmes, replacing classical selfing selection techniques in those species where the method is well implemented. Examples of such species include rapeseed, barley, maize, rice, asparagus, pepper, aubergine, or wheat (reviewed in Thomas et al., 2003).
Tomato (Solanum lycopersicum L.) is the most important vegetable crop in the world in terms of both production and harvested area (FAOSTAT, 2005). However, despite the enormous economic importance of tomato all over the world, doubled-haploid technology is still far from being routinely applied in tomato breeding programmes, mostly due to the lack of knowledge about androgenesis induction in this species. Experimental induction of androgenesis from cultured anthers was first attempted in tomato (Sharp and Dougall, 1971; Gresshoff and Doy, 1972) soon after the discovery of the process by Guha and Maheshwari (1964). In these last 35 years, little progress has been made; results are somewhat controversial and there is not a standardized method to obtain doubled-haploids in tomato. Nearly all the research has focused on in vitro anther culture, mostly reporting the induction of callus (Dao and Shamina, 1978; Jaramillo and Summers, 1990, 1991) and the regeneration of roots (Sharp and Dougall, 1971; Gresshoff and Doy, 1972; Levenko et al., 1977; Gulshan and Sharma, 1981) or apical shoots (Ma et al., 1999). Regeneration of plants has also been reported from anther cultures of S. lycopersicum (Zamir et al., 1980; Brasileiro et al., 1999) and S. lycopersicumxS. peruvianum interspecific hybrids (Cappadocia and Ramulu, 1980), but a somatic origin for the regenerants could not be excluded. Only four studies have previously reported haploid or doubled-haploid regenerated plants (Gresshoff and Doy, 1972; Ziv et al., 1984; Zagorska et al., 2004; Seguí-Simarro and Nuez, 2005). Thus, it seems clear that tomato is a strongly recalcitrant species, very little is known, and much work still needs to be devoted to obtain satisfactory results.
In this regard, one of the critical issues to resolve is the determination of the right stage during anther development for androgenesis induction. In nearly all species, this stage revolves around the first pollen mitosis (Touraev et al., 2001). In tomato, this matter has been subject to an open debate for years. Initial studies postulated meiocytes as the stage inducible through anther culture (Gresshoff and Doy, 1972). In 1978 a report was published stating that callus can be generated from anthers carrying young microspores just released from the tetrad, whereas embryoids can be obtained from vacuolate microspores or young, bicellular pollen grains (Dao and Shamina, 1978). In parallel, some reports proposed the optimal stage to be the unicellular microspore (Levenko et al., 1977; Gulshan and Sharma, 1981; Varghese and Gulshan, 1986), whereas some others pointed back to the meiocyte (Gresshoff and Doy, 1972; Zamir et al., 1980). In the last 20 years, several reports have described the meiocyte as a time point where normal anther development can be interfered with in order to induce callogenesis (Summers et al., 1992; Shtereva et al., 1998; Brasileiro et al., 1999; Seguí-Simarro and Nuez, 2005) but, to the best of our knowledge, nobody has been able to induce microspore embryogenesis.
In this work, different in vitro-induced developmental alternatives to microsporogenesis have been explored in anthers of eight different tomato cultivars in order to shed light on the poorly understood phenomenon of androgenesis in tomato. The results demonstrate that both meiocytes and vacuolate microspores can be deviated in vitro towards callogenesis and embryogenesis, respectively, in a genotype-dependent manner. Meiocyte-derived callogenesis and subsequent organogenesis, although possible, poses several problems which makes it an inefficient method for obtaining doubled-haploid tomato plants. However, tomato microspore embryogenesis arises as a promising alternative to investigate in order to avoid undesired, non-androgenic products.
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Materials and methods |
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Plant material
As anther and microspore donors, plants of the following cultivars were used: ‘Ailsa Craig’, ‘Van's Early’, ‘Castlemart’, ‘San Marzano’, and a doubled-haploid-derived line of ‘San Marzano’ (‘San Marzano DH’), kindly provided by the Tomato Genetics Resource Center, University of California at Davis, USA; ‘Mercedes’, kindly provided by Zeraim Ibérica, S.A. (Valencia, Spain); and ‘Resaplus’ and a male-sterile line of this cultivar carrying the ms1035 gene, from the COMAV germplasm collection. Plants were grown in the COMAV greenhouses, at the Universidad Politécnica de Valencia, at 18 °C under natural light during the months of September to February.
In vitro anther cultures and plant regeneration
Two different culture procedures, named A and B, were assessed for anther cultures. For procedure A, anthers of all lengths ranging from 
1 mm to 9 mm at 1 mm intervals were considered. Flower buds were excised from donor plants, transported under melting ice, and surface-sterilized. From each dissected bud, one anther was squashed on a glass slide with a drop of 1 mg l–1 4',6-diamidino-2-phenylindole (DAPI)+Tween-20 and observed under the microscope under phase contrast and epifluorescence to characterize the developmental stage. Before plating the anthers, the lengths of the flower bud and their anthers were systematically measured with a caliper. Procedure A consisted of the incubation of anthers on induction medium in a Sanyo MLR 350H growth cabinet at 25 °C in darkness for 1 month, and then under a 16/8 h photoperiod with 100 µM photons m–2 s–1, according to Seguí-Simarro and Nuez (2005). Induction medium consisted of Murashige and Skoog (MS) medium+vitamins, pH 5.7, according to Murashige and Skoog (1962), supplemented with 2.5 g l–1 phytagel (Sigma), 20 g l–1 sucrose, 1 mg l–1 isopentenyladenine, and 2 mg l–1 indole acetic acid. Collection of flower buds for procedure B was identical to that for procedure A, but buds were first pretreated with pretreatment medium at 7 °C for 6 d under darkness, before anther dissection and plating. Pretreatment medium consisted of 2 mg l–1 thiamine-HCl, 5 mg l–1 nicotinic acid, 0.5 mg l–1 D (+)-biotin, 10 mM CaCl2, 100 mg l–1 colchicine, 5 mg l–1 AgNO3, and 0.3 M mannitol, pH 5.7. After pretreatment, anthers were dissected, measured, and checked as in procedure A, and then plated on culture medium and kept at 25 °C under darkness. Culture medium consisted of NLN medium+vitamins according to Lichter (1982), pH 5.7, supplemented with 2.5 g l–1 phytagel, 130 g l–1 sucrose, 0.5 mg l–1 benzylaminopurine and 0.5 mg l–1 naphthylene-1-acetic acid. For each procedure and genotype, 10 buds (50–60 anthers) per length interval were used. Each experiment was repeated three times.
Developing calli from procedure A were transferred to fresh medium on a monthly basis, discarding those anthers which were either necrotic or not responding to the induction treatment. Totally or partially green calli were transferred to regeneration medium (MS medium+vitamins pH 5.7, 2.5 g l–1 Phytagel, 20 g l–1 sucrose, and 0.25 mg l–1 zeatin riboside). Shoots developed from green callus regions were individualized and transferred to glass tubes or magenta pots containing regeneration medium. Non-rooting, developed shoots were transferred to rooting medium (half-strength MS+vitamins and 10 g l–1 sucrose, pH 5.7). Complete, rooted plantlets were transferred to pots with soil and acclimated for 1 week under greenhouse conditions.
Microspore isolation and culture
Procedures A and B, as described above, were adapted to microsporocyte/microspore cultures. Adaptation mostly consisted of microspore isolation in liquid medium prior to culture. Flower buds of different lengths were collected and surface-sterilized as described for anther cultures, and pretreated as described, only for procedure B. Then, anthers were crushed in a glass beaker with induction medium (for procedure A) or culture medium (for procedure B). Both media were identical to those described for anther cultures, except for the gelling agent phytagel, which was excluded in order to make them liquid. Suspensions of microsporocytes/microspores were filtered through a 30 µm nylon mesh and centrifuged at 1000 rpm and 10 °C on a refrigerated centrifuge for 4 min. After three rounds of washing with medium and centrifugation, microsporocyte/microspore concentration was adjusted to 40 000 units ml–1 and distributed in 6 cm (3 ml each) or 9 cm (10 ml each) plastic culture dishes. Dishes were kept under darkness in a Sanyo growth cabinet at 25 °C. For each procedure and genotype, 10 buds (50–60 anthers) per length interval were used. Each experiment was repeated three times in parallel with anther cultures. Microspore cultures were routinely checked and documented under phase contrast and differential interference contrast (DIC) with a Zeiss Axiovert 40CFL inverted microscope.
Viability of microspores was followed at three time points during culture: after bud excision (before bud pretreatment), at the moment of microspore plating, and 1 week after plating. Viability was assessed by incubating isolated microspores for 2 h in medium with calcein AM vital staining (Molecular Probes, Eugene, OR, USA) at a final concentration of 1 µM. After two rounds of centrifugation and washing with fresh medium, pelleted microspores were collected and observed under the fluorescence microscope. Viability was defined as the percentage of viable (calcein-stained) cells of the total counted microspores.
Flow cytometry
Small pieces of cultured young callus and leaves from regenerated plants were chopped at 4 °C with a razor blade in 400 µl of nuclear extraction buffer (NEB) from the CyStain UV precise P kit from Partec GmbH (Münster, Germany). After 1 min incubation in NEB, 1.6 ml of DAPI-based staining buffer from the same kit was added and incubated for 2 min. The extracted nuclear preparation was filtered through 30 µm CellTricks filters (Partec GmbH) and immediately analysed in a Partec PA-I Ploidy Analyzer.
Light and electron microscopy
Samples of anthers were processed at different stages during in vitro culture. For light microscopy, samples were processed as previously described (Seguí-Simarro and Nuez, 2005). Briefly, anthers were fixed in Karnovsky fixative (4% formaldehyde+5% glutaraldehyde in 0.025 M cacodylate buffer, pH 7), dehydrated in an ethanol series and embedded in Technovit 7100 according to the manufacturer's specifications. Thin (2 µm) sections were obtained with a Leica UC6 ultratome and observed under bright field and phase contrast in a Nikon Eclipse E1000 microscope. Sections of anther meiocytes were stained with 0.05% aniline blue, specific for callose, pH 8.5, and observed under fluorescent UV light.
For electron microscopy, samples were fixed in Karnovsky fixative and post-fixed in 2% OsO4 in 0.025 M cacodylate buffer for 4 h at room temperature. After three washes in cacodylate buffer, samples were dehydrated in acetone series and progressively embedded during 3 d in increasing concentrations of Epon resin. Epon resin was polymerized in an oven at 60 °C for 2 d. Ultrathin (80 nm) sections were obtained with a Leica UC6 ultratome, mounted onto formvar- and carbon-coated copper grids, counterstained with uranyl acetate (2% in 70% ethanol) for 7 min and lead citrate for 2 min, and observed in a Philips CM10 transmission electron microscope operating at 100 kV.
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In vitro response of cultured anthers and isolated microspores
The ability of eight different tomato genotypes to generate morphogenic responses from cultured anthers and isolated microspores/microsporocytes was assessed. For the eight cultivars (Fig. 1), anthers of different lengths were cultured, covering the different stages of microsporogenesis. Two different procedures were used in parallel, namely A and B (see Materials and methods for further details), adapted to both anther and microspore/microsporocyte cultures. A summary of procedures, genotypes, and results for both anther and microspore cultures is provided in Table 1. In the case of the ms1035 male-sterile line, no microspore cultures could be performed, since this mutant exhibits delayed meiosis and a late meiotic blockage that prevents microspore release from the tetrad (Zamir et al., 1980).
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In cultured anthers, protocol A (Fig. 1A) induced callus production in six out of the eight tested genotypes. After a few weeks of culture, white, friable callus masses could be observed to emerge from the locule of swollen anthers (Fig. 2A). Cultivars Ailsa Craig and Mercedes showed no response at all. In the responding genotypes, callus production was restricted to a range of anther lengths, approximately between 2 mm and 4 mm. Routine squashings of control anthers of 2–4 mm length confirmed the expected presence of dividing meiocytes in the anther locules as described (Seguí-Simarro and Nuez, 2005). In the case of the ms1035 line, callus production was the highest, and the range of callus response was wider, reaching up to 6 mm anther length. The wider response was due to delayed meiosis, as confirmed by anther squashings which revealed the exclusive presence of meiocytes in longer anthers as well (data not shown). Under the conditions of protocol B, no response at all was observed in the anthers of any genotype at any of the cultured anther lengths (data not shown). After 2 months in culture without any noticeable macroscopic change, anthers progressively became brown and died. Routine squashings of the cultured anthers showed no microscopic signs of callus or embryo production in the microsporocytes/microspores within the locule.
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Microsporocytes/microspores isolated from anthers of different lengths and cultured under procedure A showed no response in terms of young callus or embryo production in any genotype (data not shown). However, procedure B induced the appearance of multicellular embryo-like structures (Fig. 6) in two out of the eight tested genotypes (Fig. 1B). Interestingly, these genotypes were Ailsa Craig and Mercedes, the two cultivars not responding to medium A. The embryogenic response was very low (up to 8.8 embryos per donor anther in cv. Mercedes) and mostly restricted to microspores from anthers ranging from
4 mm to 4.9 mm, which when observed under the microscope mostly corresponded to late, vacuolate microspores (trilobulate, with an off-centre nucleus).
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Once checked, anther and microspore cultures were maintained according to their corresponding protocols, and subsequent development was studied. An analysis of the observed calli, regenerants, and microspore-derived embryos is described in the following sections.
Regeneration of plants from tomato anther cultures through a callus phase
As long as anther cultures were maintained in darkness on inductive medium, most of the white, friable calli (Fig. 2A) quickly proliferated and considerably increased in size, whereas others, which were non-viable, did not grow, became pale brown, and soon died. Upon exposure to light, green regions appeared on the surface of calli from three genotypes: ‘Castlemart’, ‘Resaplus’, and ms1035. Calli from ‘Van's Early’, ‘San Marzano’, and ‘San Marzano DH’ kept growing, with no visible green regions. When viable calli were transferred to regeneration medium, shoots were observed to regenerate and grow within a few weeks from the green regions of the ‘Resaplus’ and ms1035 calli (Fig. 2B). Calli from ‘Castlemart’ rarely developed shoots and they were abnormal. After 6 weeks in culture, they arrested growth and died. Calli from ‘Van's Early’, ‘San Marzano’, and ‘San Marzano DH’ continued a proliferative, undifferentiated growth under regeneration medium, and finally also arrested and died.
Some of the shoots from the ‘Resaplus’ and ms1035 calli spontaneously developed a root system (Fig. 1C) and, after excision and individualization, they soon became fully regenerated plantlets. Some others had to be transferred to rooting medium in order to induce rooting efficiently. After 1 week of acclimatization, plantlets transferred to soil were moved to greenhouses (Fig. 1D), where they grew into full-sized tomato plants.
Flow cytometric analysis of calli and regenerated plants
Next, the ploidy level of calli and regenerants was characterized (Fig. 3). In order to be able to establish relationships between calli and their corresponding callus-derived regenerated plants, only ‘Resaplus’ and ms1035 calli and regenerants were considered. Small pieces of young calli and leaves of the regenerants as well as young leaf samples from the donor plants, to be used as standards, were analysed by flow cytometry. Leaves from the donor plants presented a G1 DNA peak at channel 50 (Fig. 3A) which was set as the standard 2C for diploid cells, together with a small peak at channel 100, characteristic of diploid G2 cells. A total of 77 different calli and 54 regenerated plants were analysed by flow cytometry. Most of the young calli (66%) were mixoploid, presenting two different DNA contents and, in a small percentage (3.89%), even three (Fig. 3E). Among the mixoploid calli, those with 1C+2C (Fig. 3B) and 2C+4C DNA contents (data not shown) were the most frequently found (28.6% and 32.5%, respectively; Fig. 3E). Only 13% of the young calli were clearly haploid (1C DNA content), whereas 14.3% presented only a 2C DNA content (Fig. 3E). In contrast to the frequent mixoploidy observed in calli, only 20.4% of the regenerated plants were mixoploid. Plants with a measured 2C DNA content (Fig. 3C) were the most abundant (46.3%), although 1C (Fig. 3D) and 4C plants were also observed in lower percentages (9.3% and 24.1%, respectively; Fig. 3E).
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These results, in addition to confirming the haploid origin of
10% of the regenerant population coming from the different haploid calli, revealed that mixoploidy is frequent in young callus. However, ploidy frequencies of the regenerants did not match those of young calli, suggesting that although plant regeneration can potentially occur in different regions of the mixoploid callus, it is favoured in those regions with 2C or 4C cells.
Light and electron microscopic analysis of the first stages of anther culture
At the light and electron microscope level, the process of meiosis disruption, under inductive conditions, and the emergence of young calli within the anther locule was followed up. Anthers at the stage where the greatest response was observed carried almost 100% of meiocytes, at a stage between metaphase I (after meiotic recombination) and telophase II (prior to tetrad walling). After 1 d in culture, the tapetal layer and some meiocytes of the anther locule showed clear signs of degeneration and death, whereas some others appeared morphologically normal (Fig. 4A). In a normal (physiological) situation, tomato meiocytes complete meiosis I and II prior to the onset of cytokinesis (Pacini and Juniper, 1984; Ivanova et al., 2000). Since in Solanum there is only one nucleolar organizing region (NOR), located at chromosome 2 (Ivanova et al., 2000), normal tomato haploid products exhibit just one nucleolus after nuclear reconstitution (data not shown). In parallel, post-meiotic cytokinesis proceeds through the assembly of multiple mini-phragmoplasts at the boundaries of each nucleo-cytoplasmic domain, to assist in the assembly of the post-meiotic cell plates that physically isolate each haploid nucleus from the rest (Otegui and Staehelin, 2004; Seguí-Simarro et al., 2006). In contrast, in the present cultures, a number of meiocytes exhibited cells with striking deviations from the described pattern of normal development, including binucleated nucleo-cytoplasmic domains with both nuclei closely apposed (Fig. 4A, B), or peanut/dumbbell-shaped nuclei extending along the whole meiocyte length (Fig. 4C). Of special relevance is the presence of more than one nucleolus in these cells, implying ploidies higher than haploidy. Together, these profiles are highly indicative of nuclear fusion events. In electron micrographs of meiocytes with abnormal nuclear profiles, absent or incomplete cell plates aborted at different stages of cytokinesis were observed (Fig. 4B, C). Incomplete tubular or sheet-like fragments of cell plate intermediates at the boundaries of each nucleo-cytoplasmic domain (Fig. 4B), and ingrowths from the plasma membrane (Fig. 4C, arrowheads) were characteristic of these meiocytes. Additional features included the unexpected presence of residual mini-phragmoplast microtubules and incomplete tubular cell plates, characteristics of early post-meiotic cytokinesis (Otegui and Staehelin, 2004), in the vicinity of the fused macronucleus (Fig. 4D). Such abnormalities were never observed in microsporocytes of anthers under physiological conditions, which undergo normal cytokinesis and complete tetrad walling (JM Seguí-Simarro, F Nuez, unpublished data).
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Within the first 4 d of culture, in a low percentage of microsporocytes, ectopic cell walls were observed to divide meiotic products (Fig. 4E). Interestingly, these ectopic walls did not stain with the callose-specific aniline blue dye as the other callose-rich meiocyte walls did (compare Fig. 4E and E'). Since massive callose deposition is an unequivocal marker of post-meiotic late cell plates and cell walls (Otegui and Staehelin, 2004), callose-free complete walls are suggestive of a somatic-type cytokinesis mechanism (Seguí-Simarro et al., 2004), typical of proliferative divisions (Seguí-Simarro and Staehelin, 2006).
After 7 d in culture, some anthers were observed to swell. Serial sectioning and microscopic analysis through the whole anther depth revealed the presence of proliferating callus masses (Fig. 4F) arising from the anther locule with no physical link to the anther wall, and displacing the residual meiocytes and remnants of the degenerated tapetum to the locular periphery. In parallel, in a significant percentage of the anthers sectioned, the callus was clearly seen to arise from the connective tissues (data not shown), suggesting that in the present culture conditions, callogenesis is also promoted from somatic tissues. Histological analysis of locular calli (not derived from the somatic tissue of the anther) revealed the presence of binucleated cells (Fig. 4G) and nuclear profiles indicative of an immediately occurring or recently occurred process of nuclear fusion, such as pairs of nuclei with closely apposed nuclear envelopes or separated by fragmented cell plates (Fig. 4H), or larger peanut-shaped nuclei with two conspicuous nucleoli (data not shown). These results suggest that young calli, not yet emerged from the anther locule, may also undergo nuclear fusion after a defective/incomplete somatic cytokinesis.
Morphogenesis from anther-derived callus
Young calli just emerged from the anther locule presented a white, amorphous, and friable texture (Fig. 2A). A few days after callus emergence and following an initial period of undifferentiated growth, embryo-like structures could be observed over the surface of some of the friable callus (Fig. 5A, inset). Figure 5A shows a section of an embryo-like structure at a late globular stage, where a well-differentiated protoderm and the developing procambium can be clearly distinguished. However, under these conditions, further development of these structures into mature embryos or germinating seedlings was never observed.
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When older calli, reaching
1 cm in diameter, were transferred to regeneration medium and exposed to the light, growth decreased and several differentiation events were seen in different regions of the callus mass. The callus surface rapidly turned green in certain regions, and small leaf-like primordia arose around dark green areas (Fig. 5B, inset). Histological analysis of the dark green areas revealed the presence of dome-shaped, meristem-like domains with three layers of typically meristematic cells enclosing a central corpus region (Fig. 5B). Meristematic cells were small and polygonal, with a centred nucleus and abundant, dense cytoplasm. The layered disposition of this domain strongly resembled that described for tomato shoot apical meristems (Sekhar and Sawhney, 1985). The inner parts of the callus masses presented parenchymatic, highly vacuolated cells as well as bundles of differentiated vascular tissues where dispersed tracheary elements could be clearly distinguished (Fig. 5C). Besides meristem- and vascular-like structures, which were the most frequently observed morphogenic events, adventitious leaf-like structures (Fig. 5D) and roots (Fig. 5E), as well as pseudofruits (data not shown), were observed to grow from the surface of the differentiating callus, although in a significantly lower proportion. Thus, after a initial phase of callus proliferation where embryogenesis can occasionally occur, a set of different morphogenic responses can be triggered upon transfer to regeneration conditions, shoot organogenesis being the most frequent response.
Direct embryogenesis from isolated microspore cultures
As seen in Fig. 1A and B, two of the assessed cultivars did not respond to the culture of either isolated microsporocytes or microsporocyte-carrying anthers, but they did when microspores at the vacuolate stage (Fig. 6A, A') were isolated from the anther, triggering an embryogenic response (Fig. 6B–G). At the moment of anther excision from the donor plant, microspore viability was 90%, as assessed by calcein vital staining. After 6 d of anther pre-treatment (Fig. 6B, B''), the viability of isolated and plated microspores dropped down to 50%. Among the viable microspores, a number of bicellular microspores were also found to be present in the culture. These enlarged microspores showed a symmetrical pattern of division (Fig. 6B) and two nuclei with a similar level of chromatin condensation, as revealed by the DNA-specific DAPI staining (Fig. 6B'). Within the first week from microspore isolation and plating, 4-celled microspores were observed to emerge from the exine coat (Fig. 6C, C'), whereas microspore viability decreased to 20%. During the second week of culture (10–14 d after plating), the first multicellular structures were identified in both ‘Mercedes’ (Fig. 6D, E) and ‘Ailsa Craig’ (Fig. 6F, G) microspore cultures. Most of these structures strongly resembled young globular embryos (Fig. 6D, F), characterized by a prominent suspensor emerging from the exine coat and a mass of undifferentiated cells at the opposite end. In addition to embryos, suspensorless multicellular structures (Fig. 6E) or abnormal suspensor-only structures (Fig. 6G) were also identified, although at an extremely low frequency. Beyond the second week after plating, cultures were checked weekly to follow embryo progression, and no changes were observed beyond this early globular stage. In some cultures, extra volumes of freshly made culture medium were added, but arrested embryos no longer progressed in growth.
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Discussion |
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It is shown that in cultured tomato anthers two main developmental routes can be induced. On one hand, direct embryogenesis can be promoted when vacuolate microspores are isolated and cultured in vitro. On the other hand, callogenesis can be induced from microsporocyte-carrying anthers. It is postulated that anther-derived callus may have different origins, one of them being incomplete meiocytes entering proliferation. It has been reported previously that the optimal stage for callus induction ranges from metaphase I to telophase II (Seguí-Simarro and Nuez, 2005). Apart from the formation of ectopic somatic walls, no other direct indication of the meiotic origin of the callus could be observed in this study. This was probably due to the rapidly proliferating nature of the callus masses, which can fill the locule and emerge from the anther within a few days. Attempts to culture isolated meiocytes under callus-inducing conditions were unsuccessful, which meant that the process of callus initiation had to be reconstructed from fixed and processed anthers, where the identification of the very early stages of callogenesis is extremely difficult when it occurs at a very low rate, as in this case. Nevertheless, several indirect pieces of evidence point to a microsporophytic origin of at least a percentage of the observed calli: (i) flow cytometry analysis unambiguously revealed that a number of calli and regenerants are haploid or mixoploid, including haploid cells, thus excluding a sporophytic origin for them; (ii) only in meiocyte-containing anthers is such a phenomenon observed; (iii) a percentage of calli clearly grow from the anther locule, with no visible link to the anther connective tissue, as observed in serial sections; (iv) soon after culturing, the (innermost) tapetal layer degenerates and only some meiocytes seem to stay alive; and (v) this fact has been previously reported for these and other tomato cultivars (Shtereva et al., 1998; Zagorska et al., 1998, 2004; Shtereva and Atanassova, 2001; Seguí-Simarro and Nuez, 2005).
It was also shown that the contribution of anther somatic tissue to callus formation cannot be ruled out at all. Indeed, both possibilities can actually co-exist. In such a context, a molecular marker-assisted genetic analysis of regenerants would unequivocally help to clarify their origin. A protocol is currently being set up to analyse tomato plants genetically using microsatellite sequences, but the high homozygosity of old tomato commercial cultivars (nearly pure lines, with no introgressions from related wild species) makes it extremely difficult to find polymorphisms. However, in those cultivars where morphological markers are available, a percentage (14%) of the observed diploid regenerants are homozygous for the studied markers, being heterozygous in the donor plants (JM Seguí-Simarro, F Nuez, unpublished data).
In tomato, the genotype determines the in vitro response towards either callogenesis or embryogenesis
Apart from microspore-derived embryos, in young microsporocyte-derived calli maintained under proliferative conditions, embryo-like structures can differentiate from their surface, whereas tracheogenesis and formation of adventitious shoots, roots, leaves, or pseudofruits can be induced under organogenic conditions. Despite the fact that all of these structures originate from the same type of callus, they appear either at different regions or under different experimental conditions. Thus, it seems that parameters such as the presence and concentration of growth factors, the developmental stage, the cellular environment, or even the different accessibility of a cell to the culture medium can trigger different morphogenic responses, as occurs in other species, including Solanaceae (Dubois et al., 1988; Alizadeh and Mantell, 1991; Decout et al., 1994; Gahan et al., 1994; Mishiba et al., 2001; Kumar and Mathur, 2004).
However, we could not successfully induce all the morphogenic responses in all of the studied genotypes. It was previously demonstrated that the donor genotype is critical for the responsiveness of the cultured anther to meiocyte callogenesis and organogenesis induction (Zagorska et al., 1998). Besides confirming these observations, in this study the relevance of the genotype has been extended not only in terms of the extent of callogenic/organogenic response, but also with respect to the inducible stage and the type of direct response, either callogenesis or embryogenesis. According to this, tomato genotypes could be divided into two categories: those inducible to form callus from meiocytes and those inducible to form embryos from microspores. Both responses seem to be mutually exclusive, since both routes occurring in the same genotype was never observed.
Microsporocyte-derived callogenesis is a route that involves defective cytokinesis and nuclear fusion
It was demonstrated that defective or even absent cell plates, binucleated cells, and nuclear fusion profiles are characteristic of cultured meiocytes and young calli within the anther. These observations suggest a blockage of post-meiotic cytokinesis mechanisms as a consequence of the induction treatment, which may in turn allow for more than one nucleus in the same cytoplasmic domain. Although endoreduplication cannot be absolutely ruled out, it is reasonable to think that the close coalescence of two nuclei in the same cytoplasm without the physical separation of a complete cell wall may end in their fusion and the formation of a larger nucleus with a doubled number of chromosomes. This fact has also been reported for a variety of androgenic systems with an elevated frequency of spontaneous genome doubling, where nuclear fusion is preceded and mediated by defective cytokinesis. Examples of these systems include Datura innoxia (Sunderland et al., 1974), maize (Testillano et al., 2004), barley (Kasha et al., 2001; González-Melendi et al., 2005), and wheat (Hu and Kasha, 1999). It has been pointed out previously that cell plate abnormalities alone may not be sufficient to promote nuclear fusion (González-Melendi et al., 2005). It is thus likely that additional, as yet unknown, processes may also be induced by the culture treatments and assist in the promotion of nuclear fusion in tomato as well as in other species.
Since abnormal cell plates are only observed in meiocytes and the initials of callus proliferation, it is possible that either the effects of the cytokinesis-affecting agents do not persist for a long time in culture or that cells become habituated to the culture conditions. These conditions may not affect all cells equally, since some cells develop intact cell walls whereas others present defects. This fact could be related to the different accessibility to substances, to the distance of the callus cell from the culture medium (Mishiba et al., 2006), or even to a different effect of growth regulators on cells with different ploidies (Jha and Sen, 1990).
Microspore embryogenesis is an emerging and promising alternative for tomato doubled-haploid production
In most plant species, induction of embryogenesis from microspores at the vacuolate stage is the only or the most efficient way to induce androgenesis, either from cultured anthers or from isolated microspores (Touraev et al., 2001). However, an extensive literature search depicts tomato as one of the most recalcitrant species for microspore embryogenesis to be induced, with only two studies reporting, >20 years ago, the occasional appearance of embryo-like structures from cultured microspores (Dao and Shamina, 1978; Varghese and Gulshan, 1986). As an example, in most of the species where embryogenesis has been achieved from isolated microspores, it is also possible to induce it from microspore-containing cultured anthers. However, the environment within tomato anthers does not seem to favour microspore embryogenesis, since it could not be reproduced in anthers cultured under the same conditions.
A protocol capable of inducing microspore embryogenesis in two different tomato cultivars has been set up. Given the extreme recalcitrance of tomato microspores to develop androgenic embryos, in protocol B several stressing agents shown to generate an embryogenic response in other species were successfully combined: a cold pretreatment (Picard and de Buyser, 1973; Reinert et al., 1975; Xu et al., 1981; Cho and Zapata, 1988), mannitol (Roberts-Oehlschlager and Dunwell, 1990; Hu et al., 1995), silver nitrate (Evans and Batty, 1994; Stipic and Campion, 1997), and colchicine (Zaki and Dickinson, 1991; Alemanno and Guiderdoni, 1994; Zhao et al., 1996; Zhou et al., 2002a, b). However, the protocol must still be greatly improved. First, the efficiency of embryo production should be improved. It was demonstrated that microspore viability dramatically drops down to about half after the first week (pretreatment) and subsequently, which must surely explain the observed low efficiency. Such a drop may be accounted for by the use of colchicine in the pretreatment medium. Colchicine is a cytokinesis-disrupting drug widely used in doubled-haploid technology to stimulate the first embryogenic divisions and to induce genome doubling in haploid embryos (reviewed in Shariatpanahi et al., 2006), but its concentration-dependent effects on the cell cycle may greatly affect viability and cell cycle progression (Caperta et al., 2006). Secondly, globular embryos never proceeded beyond the globular stage. This is consistent with the observations on callus-derived embryogenesis and with those of the above-mentioned reports, and constitutes the principal drawback to induction of embryogenesis in tomato anther/microspore cultures. It appears that nutritional requirements of in vitro developed haploid embryos are highly demanding and different from those of the microspores. Thus, efforts should be focused on this matter. Different media designs are currently being assessed to promote further embryo growth.
The different organogenic and embryogenic pathways from tomato anther and microspore culture
Four alternative pathways with the potential of producing embryogenic and/or organogenic responses, including whole plant regeneration, are described here. Considering androgenesis as the competence to generate haploid or doubled-haploid individuals from a male-derived haploid (reduced) nucleus, some of the routes described here should be considered as androgenic routes. These pathways are summarized in Fig. 7, in the context of all of the possible routes including those with a non-androgenic origin.
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It was demonstrated that direct haploid embryogenesis can be induced by culturing isolated vacuolate microspores (‘A’ arrow in Fig. 7). This route has the enormous advantage of excluding the possibility of somatic diploids, which makes it the choice in those systems where an efficient protocol is already developed. Future research should emphasize the improvement of induction efficiency and the maintenance of the proper conditions for embryo development. In parallel, the last meiotic stages can be experimentally disrupted by culturing whole anthers. It is hypothesized that in vitro culture conditions promote defects in tetrad cellularization, leading to tetrads with well-walled meiotic products together with others presenting defective, incomplete cell plates. If a callus arises from the well-walled, haploid cells (‘B’ arrow in Fig. 7), it will be haploid. In turn, haploid callus cells may also undergo cytokinesis impairment, thus promoting nuclear fusion and formation of doubled-haploid cells in the same callus, which becomes mixoploid. Plants regenerated from this callus will be either haploid or doubled-haploid, depending on the region where organogenesis takes place. On the other hand, segregating meiotic products separated by incomplete cell plates may eventually end up with fusion of their nuclei (‘C’ arrow in Fig. 7), forming a diploid nucleus where heterozygous allele combinations may occur. Callus may proliferate from haploid cells, as in the ‘B’ pathway, but it may also arise from the fused, diploid cells. In this case, the mixoploid callus would be formed by cells with different origins. Plants coming from the haploid regions of the callus will have a haploid origin, but those from the diploid region would not. There is also the possibility that haploid callus cells of the ‘C’ pathway undergo nuclear fusion and become doubled-haploids in a callus already containing haploid and diploid (heterozygous) cells. Since few 4x callus cells were observed, it cannot be ruled out that diploid or doubled-haploid cells may undergo a second round of nuclear fusion, becoming tetraploid or ‘quadrupled-haploid’ cells, respectively. Finally, the undesirable appearance of somatic diploid callus from the tissue of the anther must also be considered (‘D’ arrow in Fig. 7).
As seen, tomato anther culture is shown to be a good system to produce efficient organogenesis, but not yet a convenient tool to produce haploids efficiently. From the several possible ways to induce organogenesis and embryogenesis from cultured tomato anthers and microspores, only two will lead to haploidy: proliferation of haploid meiotic products and embryogenesis from microspores. The first one is not common to most of the known androgenic systems, where the late unicellular microspore is the stage most sensitive to induction (Touraev et al., 2001). In this work, 10% of haploid plants were reported, although this percentage may be further increased with the molecular marker-assisted characterization of the initially diploid plants. However, it is also shown that although possible, there are many collateral, undesirable processes that make it difficult to produce doubled-haploids from microsporocytes. It was demonstrated that microspore embryogenesis is possible in tomato, but the system, although promising, must be greatly optimized in order to be exploited as a parallel developmental pathway for the production of doubled-haploids in tomato.
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Acknowledgements |
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We wish to acknowledge the staff of the COMAV greenhouses for their valuable help. Thanks are also due to the Tomato Genetics Resource Center (University of California at Davis, USA) and Zeraim Ibérica, S.A. (Valencia, Spain) for kindly providing seeds of different cultivars. This work was supported by grants GV05-023 from Generalitat Valenciana and AGL2006-06678 from the Spanish Ministry of Education and Science (MEC) to JMSS.
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Abbreviations |
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DAPI, 4',6-diamidino-2-phenylindole; MS, Murashige and Skoog; NEB, nuclear extraction buffer.
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References |
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