Androgenesis in recalcitrant solanaceae of agronomic interest

At present there are protocols available for androgenic doubled haploid production in more than 250 species of agricultural interest, from grasses such as wheat, barley, rice, rapeseed, tobacco or maize (reviewed in Maluszynski et al., 2003) to woody species as mandarin, bitter orange or oak, among others (reviewed in Srivastava and Chaturvedi, 2008). However, except in model species such as rapeseed, tobacco or barley, the efficiency in obtaining androgenic doubled haploids is still very low (Palmer and Keller, 2005; Seguí-Simarro and Nuez, 2008a; Touraev et al., 2001). This is even more critical in vegetables of high agronomic interest, as those of the Solanaceae family. Of the five major solanaceous crops (pepper, tobacco, potato, eggplant and tomato), only in tobacco sufficient progress have been made to consider this species as a model. Indeed, methods to efficiently produce tobacco double haploids by both anther and isolated microspore cultures are already available, working with an acceptable efficiency (Chupeau et al., 1998; Maluszynski et al., 2003; Touraev et al., 2001). In potato, protocols to induce microspore cultures are also available (Říhová and Tupy, 1999). However, the rest of Solanaceae are considered recalcitrant, as although in some cases doubled haploids have been obtained, we are still far from the efficiency observed in tobacco or rapeseed. In eggplant and pepper only anther culture seems to work, at least for some cultivars, and in tomato none of the two methods is available up to now. Despite the genetic proximity of these five solanaceous species, they seem to respond very differently to androgenic induction. In particular, three of them, tomato (Solanum lycopersicum), eggplant (Solanum melongena) and pepper (Capsicum annuum) are especially recalcitrant. Thus, it is necessary to deepen the study of these systems for a better understand the process, to find new ways of induction and to improve the efficiency of the method in practical application. These are the main objectives of this research line.

In view of our results in recent years, we can conclude that obtaining androgenic doubled haploid microspores culture from recalcitrant Solanaceae such as tomato, eggplant and pepper, is possible but not easy. This will be our main challenge in this research line.


Androgenesis in tomato

Tomato is the first vegetable crop in terms of production and cultivated area, both globally and in the European Union (FAOSTAT, 2009), where only Italy beats Spain in production and acreage (EUROSTAT, 2009). In Spain, tomato is the main vegetable, with 3,615 tons and 55,600 ha in 2007 (FAOSTAT, 2009). However, and despite of its importance, little progress in the tomato DH field as been reported, with no reliable and standardized methods available so far. Over the past 30 years, most of the published research reports on induction of calli (Jaramillo and Summers 1990, 1991) or multicellular structures (Bal and Abak, 2005), or regeneration of roots (Gresshoff and Doy, 1972) or apical shoots (Ma et al., 1999). However, only two laboratories, including ours, have published the complete regeneration of full plants with a haploid or DH origin (Shtereva et al., 1998; Zagorska et al., 1998; Seguí-Simarro and Nuez, 2005, 2006, 2007; Corral-Martínez et al., 2011). In contrast to the high morphological variability of regenerants published previously, our regenerants have high morphological homogeneity, but mixoploidy in a percentage of them and a low general efficiency is still observed (Seguí-Simarro and Nuez, 2007). It is obvious that tomato is extremely recalcitrant, and a much more in-depth study is still essential to obtain successful results.

In recent years, our group has contributed significantly to the understanding of the problem of androgenesis induction in this crop (Seguí-Simarro and Nuez, 2005, 2006, 2007, Corral-Martínez et al. 2011). Until a few years ago, the pollen developmental stage in which this process can be induced in tomato was not clear. We have recently demonstrated that it can be induced in two different stages, although with greatly different results and implications. On the one hand, it is possible to induce meiocytes, before the end of tetrad walling, as long as they are in vitro cultured within the anthers (Seguí-Simarro and Nuez, 2005; Corral-Martínez et al., 2011). This way, haploid, DH, and mixoploid calli can be induced from immature meiocytes (Seguí-Simarro and Nuez, 2007). Moreover, the need for being cultured together with their anther makes possible the occasional appearance of somatic regenerants, coming from the anther wall tissues, not 100% homozygous and therefore not desirable for our purposes. These phenomena, inherent to this technology, represent a major problem when it comes to translating this experimental methodology to production technology, because it requires the genetic evaluation of each single regenerant to determine their origin and ploidy.


Figure 1. Tomato anther culture and doubled haploide plant production. Adapted from Seguí-Simarro, J.M. and Nuez, F. (2005). Acta Physiologiae Plantarum. 27 (4B): 675-685.


Moreover, and as a result of the same investigations, we have also shown that when microspores at the vacuolate stage are isolated and grown in liquid medium, it is possible to induce divisions embryogenic in these microspores (Seguí-Simarro and Nuez, 2007). This alternative would avoid the problems mentioned above, since it eliminates the influence of somatic tissue from the anther and embryos are obtained directly. Microspore-derived embryos are much more genetically stable and less prone to chromosomal instability that microspore-derived calluses (Seguí-Simarro and Nuez 2008b). However, up to now very few genotypes that have been evaluated, and in the few positive results obtained, it has not been possible to go beyond the first divisions in the early development of microspore-derived embryos. For reasons we currently ignore, these embryos arrest, and never progress beyond the early globular stage (Seguí-Simarro and Nuez, 2007). Our work in tomato so far has been a pioneer in Spain in this field and has shed light over some of the most important questions that existed regarding androgenesis in tomato. On the one hand, the meiocyte is confirmed as a inducible stage, but its limited practical application is demonstrated as well. On the other hand, this work opens a hopeful way to the long-awaited androgenic DH through the induction of isolated microspores, which we demonstrated as possible, and the subsequent development of embryos. However, this topic is still largely at its infancy, and much more efforts must be devoted.



Androgenesis in eggplant

Eggplant is another of the most important vegetables worldwide. In Europe, Spain is the leading exporter and second largest producer (FAOSTAT 2009). This species appears to respond better to the induction of embryogenesis, and pure DH lines of some varieties and hybrids have been developed (Rotino, 1996; However, at present only anther cultures seem to work. The results of both our and many other groups have shown that eggplant embryos can be induced from microspores cultured within the anther. Indeed, our group is producing doubled haploids through anther cultures in different eggplant commercial hybrids (Figure 2).


 Figure 2. Anther culture and doubled haploid production in eggplant. Adapted from Seguí-Simarro et al. Plant Cell Reports (2011) 30:765-778

However, anther culture does not exclude the occasional appearance of somatic embryos from anther tissues, as also mentioned for tomato. In addition, we must take into account the uncontrollable secretory effect of the tapetal layer that surrounds the pollen sac, and that prevents us from having a strict control of the culture conditions. In addition, it is well known that anther cultures have a very low efficiency, producing only a few embryos per anther cultivated. In those species in which isolated microspore cultures are well set up, it is possible to get hundreds or even thousands of embryos from the microspores contained in a single anther. As in tomato, the development of a method for androgenesis induction from isolated eggplant microspores would be highly desirable, and would avoid the problems mentioned above of tapetum effect, appearance of somatic regenerants, and low efficiency. In our group we are developing a protocol by which we are getting isolated microspore induction and doubled haploid regeneration (Figure 3) with higher efficiency, well above that previously published.


Figure 3. Isolated microspore cultures and doubled haploid production in eggplant. Adapted from Seguí-Simarro et al. Plant Cell Reports (2011) 30:765-778


Androgenesis in pepper

Pepper is the third solanaceous crop that could be defined as recalcitrant to the induction of androgenesis. Spain ranked first in 2007 of the European Union both in production (1,065 metric tons) and cultivated area of pepper (21,700 ha; FAOSTAT 2009), which gives an idea of the importance that an efficient method to obtain androgenic pure lines would have in economic terms. In pepper, the situation is similar to eggplant. For years, anther cultures have been used as a tool for basic studies (Seguí-Simarro et al., 2006) and genetic improvement programs pepper. In fact, our group is currently involved in a contract for the improvement of pepper varieties, creating androgenic pure lines through anther cultures (Figure 4).


Figure 4. Anther culture and doubled haploide production in pepper.



Worldwide, several hybrid seed companies use this technique to obtain pure lines. Obviously, this technique also involves the problems described in the case of eggplant (tapetum effect, the possibility of somatic regenerants and low efficiency), so it is also desirable to achieve a protocol for obtaining DH through isolated microspore culture. In this respect, in the literature there are several approaches to microspore culture, such as the direct isolation (Supena et al., 2006b, Kim et al., 2008) or the shed-microspore approach, which consists of culturing the anthers in a biphasic (solid-liquid) medium, so as to promote anther dehiscence and microspore shedding (Supena et al. 2006a; Supena et al., 2006b). This way, microspores would be isolated from the anther and trapped in the solid-liquid interface. Some of these approaches have shown a limited efficiency (Supena et al., 2006b). Others have proven efficient for embryo production, but not for the quality of these embryos (Kim et al., 2008). The experience of our group is that it is possible to induce the proliferation of pepper microspores to embryos using a method similar to that of eggplant (Figure 5), although more effort is necessary for this methodology to become applicable on a routine basis.


Figure 5. Isolated microspore cultures and doubled haploid production in pepper.Adapted from Seguí-Simarro et al. Plant Cell Reports (2011) 30:765-778

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