**2. Biological origin of the microvine**

#### **2.1 Tissue chimerism and phenotypic consequences**

The meristem of higher plants is organized in several cell layers. The outermost, which corresponds to epidermal cells, results from anticlinal divisions (i.e., following a plane of division perpendicular to the surface). This tissue which covers all the organs of the shoot system develops as a single cell layer [1]. Underneath, a multicellular zone, called L2 cell layer, is at the origin of all subepidermal tissues, following multidirectional divisions (i.e., primary structures but also lateral meristems, vascular cambium, phellogen, and their derivative tissues). No further, deeper cell layer (L3 cell layer), which forms in some species the core of shoot organs (pith), has been clearly identified in the grapevine yet [2].

In general, these cell lines that derive from initial cells located at the tip of the apical dome do not mix, unless there is an accident during cells multiplication. The organization in L1 and L2 cell layers is found in the various organs that derive from the shoot apical meristem (SAM) and in particular in the axillary meristems at the origin of caulinar organs. Because a somatic mutation is initially a single cellular event, it leads to the setting of chimeric tissues or organs, i.e., composed of cells of different genotypes and potentially displaying some phenotypic diversity [2]. When a somatic mutation appears laterally to a meristem, changes can only be distributed in the sector of the mutated organ. If the mutation occurs in an initial cell of a meristem, it can spread to all the tissues derived from the mutated cell. The resulting structure is a chimeric and periclinal genotype, i.e., including cell layers that are not all genetically identical. Periclinal chimeras can be stabilized by vegetative propagation, i.e., by cuttings or by grafting.

A somatic mutation can invade all the cell layers and spread uniformly to all derivative tissues, provided that the three following conditions are fulfilled: (i) the mutation is not lethal for the plant, (ii) the mutation appears in an initial cell within a meristem, and (iii) the mutation is established, by cell substitution in both L1 and L2 cell layers [2]. The probability of simultaneous occurrence of these three

**5**

**Figure 1.**

*The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics*

conditions being very low, most of the mutations therefore develop sectorially or

Plants regenerated from L1 or L2 cells exhibited very different phenotypes. The plants obtained from the deepest cell layer (L2) no longer had a mutation at *VvGAI1* locus and presented phenotypic traits very close to Pinot Noir. Conversely, the plants derived from L1 cells that retained a mutated version of *Vvgai1* associated with a wild-type allele *VvGAI1* were dwarf and hairy and displayed a full conversion of all tendrils into inflorescences (**Figure 2**). This phenotype has been called

Thus, the microvine has the *Vvgai1* mutation present in both cell layers that confers a very different phenotype from the Pinot Meunier from which it derives and which only bears the mutation in the L1 cell layer. Another interesting feature is related to the genetic status of the mutation in the microvine. Although it is present in both cell layers, the *VvGAI* locus is heterozygous, i.e., each cell is carrying a mutated allele *Vvgai1* is associated with a wild-type allele *VvGAI1*. Because *Vvgai1* is

*Genetic structures of pinot noir and pinot Meunier and their respective apex phenotypes. Pinot Meunier is a somatic variant of pinot noir, which carries the mutation (Vvgai1) at heterozygous status. Localized in the epidermal cells (L1 cell layer), the mutation exacerbates the hairiness of vegetative organs of this variety* 

*(http://plantgrape.plantnet-project.org/en), without any other significant phenotypic change.*

In the 1990s, thanks to the use of codominant genetic markers (microsatellites, RFLP), the existence of genetic chimerism has been demonstrated in several vine varieties. As such, Franks et al. [3] showed that Pinot Meunier can display up to three alleles for some loci, whereas a vine, having a diploid genome, can theoretically only show one allelic form per homozygous locus and two allelic forms for a heterozygous locus. Boss and Thomas [4] were able to de-chimerise Pinot Meunier by somatic embryogenesis. They characterized the resulting L1 and L2 genotypes and studied the associated phenotypes. This work showed that Pinot Meunier carries a mutation in *VvGAI1* gene in the L1 layer which confers the hairy phenotype to

*DOI: http://dx.doi.org/10.5772/intechopen.86166*

microvine, due to the small size of the mutant.

the variety (**Figure 1**).

periclinally and give rise to chimeric tissues and organs.

#### *The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics DOI: http://dx.doi.org/10.5772/intechopen.86166*

conditions being very low, most of the mutations therefore develop sectorially or periclinally and give rise to chimeric tissues and organs.

In the 1990s, thanks to the use of codominant genetic markers (microsatellites, RFLP), the existence of genetic chimerism has been demonstrated in several vine varieties. As such, Franks et al. [3] showed that Pinot Meunier can display up to three alleles for some loci, whereas a vine, having a diploid genome, can theoretically only show one allelic form per homozygous locus and two allelic forms for a heterozygous locus. Boss and Thomas [4] were able to de-chimerise Pinot Meunier by somatic embryogenesis. They characterized the resulting L1 and L2 genotypes and studied the associated phenotypes. This work showed that Pinot Meunier carries a mutation in *VvGAI1* gene in the L1 layer which confers the hairy phenotype to the variety (**Figure 1**).

Plants regenerated from L1 or L2 cells exhibited very different phenotypes. The plants obtained from the deepest cell layer (L2) no longer had a mutation at *VvGAI1* locus and presented phenotypic traits very close to Pinot Noir. Conversely, the plants derived from L1 cells that retained a mutated version of *Vvgai1* associated with a wild-type allele *VvGAI1* were dwarf and hairy and displayed a full conversion of all tendrils into inflorescences (**Figure 2**). This phenotype has been called microvine, due to the small size of the mutant.

Thus, the microvine has the *Vvgai1* mutation present in both cell layers that confers a very different phenotype from the Pinot Meunier from which it derives and which only bears the mutation in the L1 cell layer. Another interesting feature is related to the genetic status of the mutation in the microvine. Although it is present in both cell layers, the *VvGAI* locus is heterozygous, i.e., each cell is carrying a mutated allele *Vvgai1* is associated with a wild-type allele *VvGAI1*. Because *Vvgai1* is

#### **Figure 1.**

*Genetic structures of pinot noir and pinot Meunier and their respective apex phenotypes. Pinot Meunier is a somatic variant of pinot noir, which carries the mutation (Vvgai1) at heterozygous status. Localized in the epidermal cells (L1 cell layer), the mutation exacerbates the hairiness of vegetative organs of this variety (http://plantgrape.plantnet-project.org/en), without any other significant phenotypic change.*

*Advances in Grape and Wine Biotechnology*

quantitative trait loci (QTLs) of agronomic traits.

**2.1 Tissue chimerism and phenotypic consequences**

has been clearly identified in the grapevine yet [2].

propagation, i.e., by cuttings or by grafting.

**2. Biological origin of the microvine**

proleptic axis.

supplies) is possible, in contrast with experiments under vineyard conditions. Indeed, it is possible to grow the vines up to densities of 15–30 plants/m<sup>2</sup>

limit their height to 1.2 m. Under such conditions, the most advanced fruits are mature 5–6 months after plantation of cuttings or seedlings, and the vegetative axis displays all developmental stages from young inflorescences (distal phytomers) to flowering, berry growth, and ripening (proximal phytomers). Under stable controlled conditions, the spatial gradients of vegetative and reproductive development of the microvine mimic well the temporal development of each phytomer, which allows to infer kinetic data from one-off spatial information along the

In controlled conditions, microvine allows to experiment on berry development all year long, which greatly accelerates studies on physiology and molecular biology. Furthermore, by reducing the time lag between two generations and by increasing the precision of phenotyping, genetic approaches are much more efficient than the ones generally performed with macrovines. In the first section of the paper, we describe the genetic and molecular mechanisms underlying the phenotypes of the microvine and derived lines. Then, we review typical experimental designs that can be designed with the microvine. In the last section, we review recent project using this model to study grapevine development and fruit physiology and to identify

The meristem of higher plants is organized in several cell layers. The outermost, which corresponds to epidermal cells, results from anticlinal divisions (i.e., following a plane of division perpendicular to the surface). This tissue which covers all the organs of the shoot system develops as a single cell layer [1]. Underneath, a multicellular zone, called L2 cell layer, is at the origin of all subepidermal tissues, following multidirectional divisions (i.e., primary structures but also lateral meristems, vascular cambium, phellogen, and their derivative tissues). No further, deeper cell layer (L3 cell layer), which forms in some species the core of shoot organs (pith),

In general, these cell lines that derive from initial cells located at the tip of the apical dome do not mix, unless there is an accident during cells multiplication. The organization in L1 and L2 cell layers is found in the various organs that derive from the shoot apical meristem (SAM) and in particular in the axillary meristems at the origin of caulinar organs. Because a somatic mutation is initially a single cellular event, it leads to the setting of chimeric tissues or organs, i.e., composed of cells of different genotypes and potentially displaying some phenotypic diversity [2]. When a somatic mutation appears laterally to a meristem, changes can only be distributed in the sector of the mutated organ. If the mutation occurs in an initial cell of a meristem, it can spread to all the tissues derived from the mutated cell. The resulting structure is a chimeric and periclinal genotype, i.e., including cell layers that are not all genetically identical. Periclinal chimeras can be stabilized by vegetative

A somatic mutation can invade all the cell layers and spread uniformly to all derivative tissues, provided that the three following conditions are fulfilled:

(i) the mutation is not lethal for the plant, (ii) the mutation appears in an initial cell within a meristem, and (iii) the mutation is established, by cell substitution in both L1 and L2 cell layers [2]. The probability of simultaneous occurrence of these three

and to

**4**

#### **Figure 2.**

*By somatic embryogenesis from anthers of pinot Meunier, it is possible to obtain two types of plants. One, which no longer carries the mutation of VvGAI in the L1 and L2 cell layers, has a phenotype similar to pinot noir (large size, juvenility period, main production of clusters from proleptic axes, i.e., winter buds). The other, which carries the mutation of VvGAI in all the tissues, displays a miniaturized phenotype and extreme hairiness and produces inflorescences both in the winter buds and from the conversion of tendrils in inflorescences. In the figure, the numbers associated with VvGAI allele correspond to the nucleotide base length (bp) of the VVS2 microsatellite marker [4].*

not a lethal mutation nor for the sporophyte or the gametophyte, this status can be rearranged by selfing in three genotypes:


**7**

signaling.

**Figure 3.**

*The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics*

*VvGAI1*), it is possible to recover 50% of individuals with a microvine phenotype and 50% of individuals with the characteristics of a non-dwarf grapevine.

*The three genotypes/phenotypes that can be obtained by selfing from the microvine (VvGAI1/Vvgai1): left, extremely miniaturized vines that carries the homozygous locus Vvgai1/Vvgai1, called picovines; middle, individuals with the same phenotype as the microvine, heterozygous for the mutation (VvGAI1/Vvgai1); and right, normal-sized plants that no longer carry mutated alleles, homozygous for the non-mutated form of the gene (VvGAI1/VvGAI1).*

The comparison of the allelic VvGAI forms present in Pinot Meunier and the microvine [4, 5] showed that the mutation corresponds to a modification of a single nucleotide in the DELLA motif of the protein, which is important for gibberellin

**2.2 Molecular mechanisms associated with the mutation** *Vvgai1*

*DOI: http://dx.doi.org/10.5772/intechopen.86166*

Another interesting feature, linked to the heterozygous status *VvGAI/Vvgai1*, is the possibility to return to non-dwarf phenotype. Indeed, by crossing a microvine (*VvGAI1/Vvgai1*) with a classic grapevine variety, i.e., a macrovine (*VvGAI1/*

*The Microvine: A Versatile Plant Model to Boost Grapevine Studies in Physiology and Genetics DOI: http://dx.doi.org/10.5772/intechopen.86166*

#### **Figure 3.**

*Advances in Grape and Wine Biotechnology*

not a lethal mutation nor for the sporophyte or the gametophyte, this status can be

*By somatic embryogenesis from anthers of pinot Meunier, it is possible to obtain two types of plants. One, which no longer carries the mutation of VvGAI in the L1 and L2 cell layers, has a phenotype similar to pinot noir (large size, juvenility period, main production of clusters from proleptic axes, i.e., winter buds). The other, which carries the mutation of VvGAI in all the tissues, displays a miniaturized phenotype and extreme hairiness and produces inflorescences both in the winter buds and from the conversion of tendrils in inflorescences. In the figure, the numbers associated with VvGAI allele correspond to the nucleotide base length* 

i.Homozygous *VvGAI1/VvGAI1*, which corresponds to a vine without any mutation at the locus. The phenotype associated with this genetic status is

ii.Heterozygote *VvGAI1/Vvgai*, which corresponds to the same genotype and

iii.Homozygotous *Vvgai1/Vvgai1*, which corresponds to plants carrying both alleles in a mutated version. The phenotype associated with this status, called picovine, corresponds to an extreme dwarfism, with plants displaying very

Another interesting feature, linked to the heterozygous status *VvGAI/Vvgai1*, is the possibility to return to non-dwarf phenotype. Indeed, by crossing a microvine (*VvGAI1/Vvgai1*) with a classic grapevine variety, i.e., a macrovine (*VvGAI1/*

non-dwarf, similar to classical macrovine varieties.

(dwarf) phenotype than the original microvine ML1.

miniaturized shoot organs [4] (**Figure 3**).

rearranged by selfing in three genotypes:

*(bp) of the VVS2 microsatellite marker [4].*

**6**

**Figure 2.**

*The three genotypes/phenotypes that can be obtained by selfing from the microvine (VvGAI1/Vvgai1): left, extremely miniaturized vines that carries the homozygous locus Vvgai1/Vvgai1, called picovines; middle, individuals with the same phenotype as the microvine, heterozygous for the mutation (VvGAI1/Vvgai1); and right, normal-sized plants that no longer carry mutated alleles, homozygous for the non-mutated form of the gene (VvGAI1/VvGAI1).*

*VvGAI1*), it is possible to recover 50% of individuals with a microvine phenotype and 50% of individuals with the characteristics of a non-dwarf grapevine.

#### **2.2 Molecular mechanisms associated with the mutation** *Vvgai1*

The comparison of the allelic VvGAI forms present in Pinot Meunier and the microvine [4, 5] showed that the mutation corresponds to a modification of a single nucleotide in the DELLA motif of the protein, which is important for gibberellin signaling.

After transient transformation of epidermal onion cells, green fluorescent protein (GFP) fusions to *VvGAI1* and *Vvgai1* sequences responded differently to gibberellin applications. The GFP signal of the GAI1::GFP fusion disappears rapidly from the nucleus under the effect of gibberellins, which indicates its degradation following the hormonal stimulus. On the contrary, the gai1::GFP translational protein fusion remains insensitive to hormonal signaling, which indicates that the mutation in the DELLA motif abolishes the property of the protein to be degraded when triggered by gibberellins [5].

The GAI gene is known to be an important regulator of vegetative growth and reproductive development [6]. In grapevine, gibberellins, produced under shade, stimulate growth and inhibit the formation of inflorescences [7]. This effect is mediated by the nuclear protein GAI1, which, in its mutated form gai1, no longer transmits the hormonal signaling [5]. Thus, vegetative growth and the inhibition of the conversion of tendrils into inflorescences are no longer maintained which explains the dwarf phenotype and the continuous fructification along the stems. The characterization of the expression profiles of different isogenes of *VvGAI* revealed that *Vvgai1* is mainly expressed in vegetative organs such as buds and young leaves, while other forms are expressed in reproductive organs (unpublished data). For instance, *Vvgai2*, which does not have any mutation in the DELLA protein motif, is expressed in reproductive organs from flowering to ripening [5]. This explains why *Vvgai1* mutation does not interfere directly with berry developmental program which is similar to non-dwarf varieties.
