Cellular and molecular basis of androgenesis.

It is known that crop productivity can be increased through the use of hybrid seeds, produced by crossings between homozygous (pure) lines with defined characters. These lines are traditionally generated by selfing and selection techniques based on classical breeding, which involves much time and resources. However, in the last years alternative biotechnological approaches are used in some species. These techniques, mainly based on exploiting a phenomenon known as androgenesis (Seguí-Simarro, 2010), present a number of advantages over traditional methods.

Androgenesis is defined as the process of generation of an individual whose genetic background is derived exclusively from a nucleus of male origin. That is, androgenesis is the generation of a plant exclusively from a male, haploid gamete precursor (gametophyte). This experimental route, alternative to the normal development of pollen, was discovered in 1964 by Guha and Maheshwari. Practical exploitation of this experimental phenomenon allows for the production of double haploid, pure lines, through the induction of haploid embryogenesis or callus formation, generally from the immature male gametophyte (the microspore). Once haploid embryos or calli are produced, doubled haploid plants can be regenerated by common in vitro culture techniques. Microspores can be deviated from their original gametophytic route and induced to form a haploid embryo or callus in two ways (Seguí-Simarro and Nuez, 2008a): either isolating them from the anther and culturing in liquid medium (isolated microspore culture), or directly culturing the microspore-containing anther in solid medium (anther culture).

Androgenesis can be induced in several species of angiosperms, both monocots and dicots. Following induction, whole plants can be regenerated either directly through haploid embryogenesis or indirectly through an intermediate callus phase. Plants will be either haploid (in which chromosomal duplication should be induced; Seguí-Simarro and Nuez, 2008b) or doubled haploid (by not induced doubling of the haploid genome). In both cases the resulting androgenic plants have a genetic background derived exclusively from the donor individual, being 100% homozygous, for of their characters. They are pure lines, with the same genetic variability that donors.

Figure 1. Induction of androgenesis in microspores/pollen. The vacuolate microspores and/or young bicellular pollen may be deviated from its natural pathway, the gametophytic pathway (blue background), to embryogenesis (yellow background). As an alternative to embryogenesis, microspores may proliferate as calli (green background), capable of regenerating haploid or doubled haploid plants by organogenesis. In addition, other microspores in culture may adopt a pollen-like gametophytic development, prior to arrest and death (white-pink background). Many other microspores are arrested and die directly, without being induced (pink background). Image adapted from Seguí-Simarro J.M. and Nuez, F. (2008). Physiologia Plantarum. 134: 1-12.

 

From the point of view of plant breeding, this alternative reduces the typical 7-8 selfing generations needed to stabilize a hybrid genotype, to only one in vitro. It is therefore much faster and cheaper. In plant breeding, these lines are also essential for genetic mapping of complex traits such as production or quality. These traits are the most agronomically important, and at present cannot be addressed by other techniques. They are also a powerful tool in transgenesis, to avoid hemizygous and save time and resources in the production of plants transformed with the transgene in both alleles. Moreover, from a basic scientific point of view, these lines are also very useful for basic studies of linkage and estimates of recombination frequencies. Although these studies can also be approached by conventional genetic techniques (backcross or F2), double haploid lines have the advantage of being self-perpetuating, ie can be perpetuated simply from selfed seed. They are also a useful tool for genetic selection and screening of recessive mutants, because the phenotype of the resulting plants is not affected by the effects of dominance, and the characters determined by recessive genes can be readily identified. Another advantage is the ability to serve as a model system for studying in vitro embryogenic development without the interference of maternal tissue, as zygotic embryogenesis and microspore-derived haploid embryogenesis present a large number of similarities.

For the study and advance in the understanding of androgenesis, rapeseed (canola) has played a key role. Rapeseed (Brassica napus) is a cruciferous crop extremely important worldwide for seed-derived oil production. Besides, its high response to androgenesis induction treatments make B. napus a model system to study the induction and development of androgenic embryos (Chupeau et al., 1998; Friedt and Zarhloul, 2005, Seguí-Simarro and Nuez, 2008a). This system has been demonstrated over the years as the most efficient from a practical point of view and easily inducible from an experimental point of view. In fact, the majority of studies on microspore embryogenesis induction and haploid embryo development have been conducted on B. napus, generating a wealth of results useful to improve the process in other species, less sensitie to induction. Since this system is where most cellular and molecular data are available, it is easier and useful to deepen their study. This approach is being used successfully by our group since its inception. In fact, we have extensive experience in using this model system to study this phenomenon. As an example, we recently managed to refine our technique, inducing the differentiation of the embryo suspensor (Seguí-Simarro and Nuez, 2008a), thus saving one of the major differences that still exist today between both types of embryogenic development.

 

 

Figure 2. Comparison between the different stages of haploid embryogenesis (androgenic, Figures A-F) and zygotic embryogenesis (Figures G-K) in rapeseed (Brassica napus). Clear similarities can be observed at the anatomical and morphological level between the two types of embryogenic developmental pathways. Image adapted from Seguí-Simarro, J.M. and Nuez, F. (2008). Physiologia Plantarum. 134: 1-12.

 

In short, one of the research lines of our group is the study of the factors involved in the process of androgenesis induction in B. napus. Using a parallel experimental approach based on a combination of techniques of molecular and cellular biology, we try to identify useful markers to identify those microspores effectively induced, and new factors involved in the induction, potentially applicable to improve the induction in other species, more recalcitrant to androgenesis induction.

 

Bibliography

  • Chupeau Y, Caboche M, Henry Y (1998) Springer-Verlag, Berlin, Heidelberg. Androgenesis and haploid plants.
  • Friedt W, Zarhloul MK (2005) Haploids in the improvement of crucifers, in ,(Palmer CE, Keller WA, Kasha KJ eds), vol 56, pp 191-213. Springer-Verlag, Berlin.Haploids in crop improvement II
  • Guha S, Maheshwari SC (1964) In vitro production of embryos from anthers of Datura. Nature 204:497.
  • Seguí-Simarro JM (2010) Androgenesis Revisited. The Botanical Review 76(3):377-404.
  • Seguí-Simarro JM, Nuez F (2008a) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiologia Plantarum 134:1-12.
  • Seguí-Simarro JM, Nuez F (2008b) Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenetic and Genome Research 120(3-4):358-369.

 

 

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