**3. Application of the use of the microvine**

#### **3.1 Vegetative development**

Several experiments have been conducted outdoor and in controlled environments to characterize the vegetative development of the proleptic axis of the microvine [8]. Different day/night temperature treatments were applied (22/12, 25/15, 30/15, 30/20, 30/25°C), while VPD was maintained constant (about 1 kPa). These experiments showed that the vegetative organogenesis rhythm of the microvine is similar to that of non-dwarf vines. Indeed, its phyllochron (leaf emission rate) is around 24°C, similarly to other varieties of *V. vinifera* such as Grenache [10], and it fluctuates only slightly with temperature and radiation variations between experiments (photosynthetically active radiation (PAR) has been experimented from 19 to 25 mol.m<sup>−</sup><sup>2</sup> d<sup>−</sup><sup>1</sup> ).

The duration of leaf and internode growth of the microvine is also similar to that of non-dwarf vines, lasting ca. 220°C (i.e., 20 days at 25/15°C) for leaves and ca. 150°C (i.e., 14 days under the same conditions) for internodes [9, 10]. The most significant phenotypic difference, induced by *Vvgai1*, is the size limitation of vegetative organs. The leaf area is reduced by half in the microvine compared to non-dwarf vines, and internodes are five times shorter. The dwarf phenotype is thus very valuable to conduct experiments under very well-controlled conditions in small growth chambers. Such property permits to study the impacts of single or combined abiotic factors (radiation, temperature, VPD, CO2) on plant growth and development while minimizing uncontrolled biases arising from environmental fluctuation in field studies on perennial vines.

However, the shortening of the internodes increases leaf shading and promotes the development of fungal diseases as compared to non-dwarf vine. The control of powdery mildew (*Erysiphe necator*) on leaves and green berries or gray mold (*Botrytis*) on ripening fruits requires a strict phytosanitary management.

**9**

simultaneously.

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

To improve the microclimate of the clusters, it is recommended to systematically remove the lateral branches to reduce the plant to a single proleptic axis and to systematically eliminate one leaf out of three, e.g., removing the leaves of all P0 phytomers which do not bear any inflorescence. Also, for the most fertile lines, it is necessary to control the number of ripening berries to avoid source/sink unbalance that could be prejudicial to the growth and the formation of new inflorescences as well as the accumulation of metabolites in the fruits. Because the microvine displays several levels of cluster at ripening stages, a good balance is achieved by limiting the

The reproductive development of the microvine is divided into two distinct and simultaneously occurring patterns: (i) the fructification of proleptic shoots from preformed inflorescence primordia within winter buds and (ii) the continuous fruiting of proleptic and sylleptic axes resulting from the conversion of tendrils into

In the grapevine, as for many other perennial fruit crops, fruit formation occurs during 2 consecutive years. The first step starts with the initiation and differentiation of inflorescence primordia in the winter buds prior to endo-dormancy until approximately the end of summer or beginning of autumn. During the subsequent cycle after the break of dormancy, approximately 2 weeks before budburst, the inflorescences resume their development and complete flower organogenesis and subsequently flowering in spring [6]. The level of differentiation of microvine winter buds (i.e., the number of preformed phytomers and inflorescence primordia) was analyzed during 80 days of growth under controlled environmental conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h). Two imaging methods were compared, the classic microscopy dissection and the noninvasive X-ray micro-tomography [11], with a resolution of 9 m. These observations showed that winter buds of the microvine harbor a complex formed of primary, secondary, and tertiary buds of decreasing fertility, as non-dwarf vines [12]. The maximum fertility of the primary buds is two inflorescences in the microvine, whereas it can reach three or even four in some non-dwarf varieties. These inflorescences are inserted into phytomers n°4 to n°6 with an acropetal development as for macrovines [12, 13]. The lignification of the stem which develops from the vegetative axis base is concomitant with the slowdown of bud development and probably

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

number of ripening berries to 8–15 per cluster.

*3.2.1 Fruiting from winter buds (two successive seasons)*

its entry into endo-dormancy, similarly as for non-dwarf vines [14].

The microvine has the particularity to develop inflorescences from tendrils along proleptic and sylleptic axes (**Figure 4**), which result in a continuous flowering and fruiting processes. A gradient of reproductive development stages is thus present simultaneously along the proleptic axis from the differentiation of inflorescences until maturity. This characteristic offers the opportunity to evaluate abiotic or biotic stress impacts on all reproductive stages of development along the proleptic axis

Top right, section of a winter bud analyzed by tomography. Bottom right, a longitudinal section of a winter bud exhibiting a lateral inflorescence primordium (IP) on the primary bud axis and a secondary preformed vegetative axis on the left side.

*3.2.2 Continuous flowering and fruiting (one single growing season)*

**3.2 Reproductive development**

inflorescences.

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

To improve the microclimate of the clusters, it is recommended to systematically remove the lateral branches to reduce the plant to a single proleptic axis and to systematically eliminate one leaf out of three, e.g., removing the leaves of all P0 phytomers which do not bear any inflorescence. Also, for the most fertile lines, it is necessary to control the number of ripening berries to avoid source/sink unbalance that could be prejudicial to the growth and the formation of new inflorescences as well as the accumulation of metabolites in the fruits. Because the microvine displays several levels of cluster at ripening stages, a good balance is achieved by limiting the number of ripening berries to 8–15 per cluster.

#### **3.2 Reproductive development**

*Advances in Grape and Wine Biotechnology*

when triggered by gibberellins [5].

program which is similar to non-dwarf varieties.

**3. Application of the use of the microvine**

d<sup>−</sup><sup>1</sup> ).

**3.1 Vegetative development**

mented from 19 to 25 mol.m<sup>−</sup><sup>2</sup>

fluctuation in field studies on perennial vines.

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

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

Several experiments have been conducted outdoor and in controlled environments to characterize the vegetative development of the proleptic axis of the microvine [8]. Different day/night temperature treatments were applied (22/12, 25/15, 30/15, 30/20, 30/25°C), while VPD was maintained constant (about 1 kPa). These experiments showed that the vegetative organogenesis rhythm of the microvine is similar to that of non-dwarf vines. Indeed, its phyllochron (leaf emission rate) is around 24°C, similarly to other varieties of *V. vinifera* such as Grenache [10], and it fluctuates only slightly with temperature and radiation variations between experiments (photosynthetically active radiation (PAR) has been experi-

The duration of leaf and internode growth of the microvine is also similar to that of non-dwarf vines, lasting ca. 220°C (i.e., 20 days at 25/15°C) for leaves and ca. 150°C (i.e., 14 days under the same conditions) for internodes [9, 10]. The most significant phenotypic difference, induced by *Vvgai1*, is the size limitation of vegetative organs. The leaf area is reduced by half in the microvine compared to non-dwarf vines, and internodes are five times shorter. The dwarf phenotype is thus very valuable to conduct experiments under very well-controlled conditions in small growth chambers. Such property permits to study the impacts of single or combined abiotic factors (radiation, temperature, VPD, CO2) on plant growth and development while minimizing uncontrolled biases arising from environmental

However, the shortening of the internodes increases leaf shading and promotes the development of fungal diseases as compared to non-dwarf vine. The control of powdery mildew (*Erysiphe necator*) on leaves and green berries or gray mold (*Botrytis*) on ripening fruits requires a strict phytosanitary management.

**8**

The reproductive development of the microvine is divided into two distinct and simultaneously occurring patterns: (i) the fructification of proleptic shoots from preformed inflorescence primordia within winter buds and (ii) the continuous fruiting of proleptic and sylleptic axes resulting from the conversion of tendrils into inflorescences.

#### *3.2.1 Fruiting from winter buds (two successive seasons)*

In the grapevine, as for many other perennial fruit crops, fruit formation occurs during 2 consecutive years. The first step starts with the initiation and differentiation of inflorescence primordia in the winter buds prior to endo-dormancy until approximately the end of summer or beginning of autumn. During the subsequent cycle after the break of dormancy, approximately 2 weeks before budburst, the inflorescences resume their development and complete flower organogenesis and subsequently flowering in spring [6]. The level of differentiation of microvine winter buds (i.e., the number of preformed phytomers and inflorescence primordia) was analyzed during 80 days of growth under controlled environmental conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h). Two imaging methods were compared, the classic microscopy dissection and the noninvasive X-ray micro-tomography [11], with a resolution of 9 m. These observations showed that winter buds of the microvine harbor a complex formed of primary, secondary, and tertiary buds of decreasing fertility, as non-dwarf vines [12]. The maximum fertility of the primary buds is two inflorescences in the microvine, whereas it can reach three or even four in some non-dwarf varieties. These inflorescences are inserted into phytomers n°4 to n°6 with an acropetal development as for macrovines [12, 13]. The lignification of the stem which develops from the vegetative axis base is concomitant with the slowdown of bud development and probably its entry into endo-dormancy, similarly as for non-dwarf vines [14].

#### *3.2.2 Continuous flowering and fruiting (one single growing season)*

The microvine has the particularity to develop inflorescences from tendrils along proleptic and sylleptic axes (**Figure 4**), which result in a continuous flowering and fruiting processes. A gradient of reproductive development stages is thus present simultaneously along the proleptic axis from the differentiation of inflorescences until maturity. This characteristic offers the opportunity to evaluate abiotic or biotic stress impacts on all reproductive stages of development along the proleptic axis simultaneously.

Top right, section of a winter bud analyzed by tomography. Bottom right, a longitudinal section of a winter bud exhibiting a lateral inflorescence primordium (IP) on the primary bud axis and a secondary preformed vegetative axis on the left side.

#### **Figure 4.**

*Vegetative and reproductive development of the ML1 somaclone n°7, a microvine line regenerated from pinot Meunier cl. ENTAV 8 according to the method described by Torregrosa [15]. Top left, longitudinal section of an apex showing the preformation of 7–9 phytomers before emergence of caulinar organs. Upper middle, emergence of young inflorescences just below the apex. We note the very hairy appearance of the apex of the microvine ML1. On the middle, an 8-month-old ML1 microvine displaying all the sequences of the reproductive development from flowering to fruit ripening. Bottom left, a focus on the phytomers carrying bunches shifting from green to ripening stages and the concomitant lignification of the shoot (leaves have been removed for the clearness of the photograph). Top right, section of a winter bud analyzed by tomography. Bottom right, a longitudinal section of a winter bud exhibiting a lateral inflorescence primordium (IP) on the primary bud axis and a secondary preformed vegetative axis on the left side.*

The synchronism between vegetative development and fruiting of the microvine also simplifies the study of their interactions compared to macrovines. The impact of contrasted source/sink balance on fruiting can be easily studied by manipulating shoot or fruit load (number of growing axes and/or number of leaves/inflorescence per axis). The continuous fruiting was found to be stable under standard environmental conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h) and when the leaf area to fruit fresh weight was less than 1 m2 .kg<sup>−</sup><sup>1</sup> . On the contrary, the capacity of flowering is strongly altered in the presence of abiotic or biotic stresses. High temperature (> 33°C), low radiation levels (PAR < 15 mol.m<sup>−</sup><sup>2</sup> .j<sup>−</sup><sup>1</sup> ), or high VPD (>3 kPa) can induce inflorescences abortion and disrupt the continuity of the reproductive gradient along stem axes. The sensitivity of inflorescence development was found higher when the C reserves (starch) were reduced, in particular, in young plants. Thus, although it is possible to obtain fruiting organs from 5-monthold microvine cuttings, it is advisable to use 1-year-old or older plants that are much less susceptible to inflorescence abortion [16]. In experiments conducted in our lab, we obtained successive cycles of fruiting for at least 5 years without repotting.

The size of inflorescences of microvines is smaller (10–50 berries per cluster in average) than that of macrovines [17–19]. However, flowers and young fruits of the microvine do not display a very high abscission rate as observed in non-dwarf varieties. The development of flowers and berries is identical to non-dwarf vines. Flowering (50% of open flowers) occurs 320°C GDD (growing degree days) after the phytomer emission (i.e., 30 days at 25/15°C), which is comparable to the duration between budburst and flowering in the non-dwarf vines [18]. Ripening (onset of sugar loading) starts at ca. 500°C GDD (i.e., 47 days at 25/15°C) after flowering, and the physiological ripening (when metabolite loading stops) is reached at ca. 900°C GDD (i.e., 80 days at 25/15°C) after flowering or 30 days after the start of sugar loading. This behavior is similar in macrovines [18, 20]. Thus, berries of the

**11**

**Figure 5.**

*per fruit unit.*

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

ML1 microvine reach a final individual size of 1.2 g, comparable to that of cv. Pinot meunier, from which this line derives. At physiological ripening, berries contain about 0.8 mmol berry-1 of soluble sugars in non-limiting water supply conditions

The microvine provides different advantages over non-dwarf vines to speed up or facilitate genetics. Since the mutation is transmissible by hybridization and has a codominant effect, it is possible to cross microvines (*VvGAI1/Vvgai1*) or picovines (*Vvgai1/Vvgai1*) with non-dwarf genotypes, i.e., without the mutation (*VvGAI1/ VvGAI1*), to create microvine segregating populations. In the first case, 50% of individuals will display the microvine phenotype, while using picovines as parent,

The *VvGAI1* gene is located on chromosome n°1, while the QTL determining grapevine flower sex is located on chromosome n°2. That means both loci segregate independently, and it is therefore possible to use female microvines or picovines, which facilitates crosses by avoiding the time-consuming emasculation and reducing the risk of selfing [19]. On the other hand, when a female microvine (f/f) is crossed with a hermaphrodite genotype (H/f, the most common genotype in *V. vinifera* varieties), the population will be composed of 50% of female plants and 50% of hermaphroditic plants. For instance, by crossing between the

*Spatiotemporal distribution of the reproductive developmental stages from flowering to ripening. On the abscissa, the calendar time in DAF (days after flowering) was recalculated for each phytomer converting the corresponding plastochron index in thermal time and inferred in calendar time with the phyllochron. Kinetics of fresh fruit weight and the contents of major primary metabolites and potassium are presented in quantity* 

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

**3.3 Genetics and genomics**

*3.3.1 Genetic mapping and pre-breeding*

100% of the progeny exhibit a dwarf behavior.

which is similar to other varieties of *V. vinifera* (**Figure 5**).

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

ML1 microvine reach a final individual size of 1.2 g, comparable to that of cv. Pinot meunier, from which this line derives. At physiological ripening, berries contain about 0.8 mmol berry-1 of soluble sugars in non-limiting water supply conditions which is similar to other varieties of *V. vinifera* (**Figure 5**).

#### **3.3 Genetics and genomics**

*Advances in Grape and Wine Biotechnology*

The synchronism between vegetative development and fruiting of the microvine also simplifies the study of their interactions compared to macrovines. The impact of contrasted source/sink balance on fruiting can be easily studied by manipulating shoot or fruit load (number of growing axes and/or number of leaves/inflorescence per axis). The continuous fruiting was found to be stable under standard environmental conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h)

*Vegetative and reproductive development of the ML1 somaclone n°7, a microvine line regenerated from pinot Meunier cl. ENTAV 8 according to the method described by Torregrosa [15]. Top left, longitudinal section of an apex showing the preformation of 7–9 phytomers before emergence of caulinar organs. Upper middle, emergence of young inflorescences just below the apex. We note the very hairy appearance of the apex of the microvine ML1. On the middle, an 8-month-old ML1 microvine displaying all the sequences of the reproductive development from flowering to fruit ripening. Bottom left, a focus on the phytomers carrying bunches shifting from green to ripening stages and the concomitant lignification of the shoot (leaves have been removed for the clearness of the photograph). Top right, section of a winter bud analyzed by tomography. Bottom right, a longitudinal section of a winter bud exhibiting a lateral inflorescence primordium (IP) on the primary bud* 

the capacity of flowering is strongly altered in the presence of abiotic or biotic stresses. High temperature (> 33°C), low radiation levels (PAR < 15 mol.m<sup>−</sup><sup>2</sup>

high VPD (>3 kPa) can induce inflorescences abortion and disrupt the continuity of the reproductive gradient along stem axes. The sensitivity of inflorescence development was found higher when the C reserves (starch) were reduced, in particular, in young plants. Thus, although it is possible to obtain fruiting organs from 5-monthold microvine cuttings, it is advisable to use 1-year-old or older plants that are much less susceptible to inflorescence abortion [16]. In experiments conducted in our lab, we obtained successive cycles of fruiting for at least 5 years without repotting.

The size of inflorescences of microvines is smaller (10–50 berries per cluster in average) than that of macrovines [17–19]. However, flowers and young fruits of the microvine do not display a very high abscission rate as observed in non-dwarf varieties. The development of flowers and berries is identical to non-dwarf vines. Flowering (50% of open flowers) occurs 320°C GDD (growing degree days) after the phytomer emission (i.e., 30 days at 25/15°C), which is comparable to the duration between budburst and flowering in the non-dwarf vines [18]. Ripening (onset of sugar loading) starts at ca. 500°C GDD (i.e., 47 days at 25/15°C) after flowering, and the physiological ripening (when metabolite loading stops) is reached at ca. 900°C GDD (i.e., 80 days at 25/15°C) after flowering or 30 days after the start of sugar loading. This behavior is similar in macrovines [18, 20]. Thus, berries of the

.kg<sup>−</sup><sup>1</sup>

. On the contrary,

.j<sup>−</sup><sup>1</sup> ), or

and when the leaf area to fruit fresh weight was less than 1 m2

*axis and a secondary preformed vegetative axis on the left side.*

**10**

**Figure 4.**

#### *3.3.1 Genetic mapping and pre-breeding*

The microvine provides different advantages over non-dwarf vines to speed up or facilitate genetics. Since the mutation is transmissible by hybridization and has a codominant effect, it is possible to cross microvines (*VvGAI1/Vvgai1*) or picovines (*Vvgai1/Vvgai1*) with non-dwarf genotypes, i.e., without the mutation (*VvGAI1/ VvGAI1*), to create microvine segregating populations. In the first case, 50% of individuals will display the microvine phenotype, while using picovines as parent, 100% of the progeny exhibit a dwarf behavior.

The *VvGAI1* gene is located on chromosome n°1, while the QTL determining grapevine flower sex is located on chromosome n°2. That means both loci segregate independently, and it is therefore possible to use female microvines or picovines, which facilitates crosses by avoiding the time-consuming emasculation and reducing the risk of selfing [19]. On the other hand, when a female microvine (f/f) is crossed with a hermaphrodite genotype (H/f, the most common genotype in *V. vinifera* varieties), the population will be composed of 50% of female plants and 50% of hermaphroditic plants. For instance, by crossing between the

#### **Figure 5.**

*Spatiotemporal distribution of the reproductive developmental stages from flowering to ripening. On the abscissa, the calendar time in DAF (days after flowering) was recalculated for each phytomer converting the corresponding plastochron index in thermal time and inferred in calendar time with the phyllochron. Kinetics of fresh fruit weight and the contents of major primary metabolites and potassium are presented in quantity per fruit unit.*

PV00C001V0008 [19] and the fleshless berry mutant of the ugni blanc [21], a range of genotypes and phenotypes can be obtained [5].

This progeny is composed of 100% microvines (since the female parent has a *Vvgai1/Vvgai1* genotype) and a very small proportion of individuals with both hermaphrodite flowers and pigmented berries. Indeed, these two characters are present at the homozygous recessive state in one parent (f/f and n/n) and in the heterozygous dominant state in the other (H/f and N/n). It should be noted that since ugni blanc is heterozygous at the sex locus (H/f), while the picovine is f/f, selecting hermaphrodite individuals leads to a segregation distortion in the progeny of the genetic traits determined on the chromosome n°2.

As the microvine produces inflorescences as long as vegetative growth is maintained, it becomes possible to cross all year around without being hampered by seasonality. Under standard thermal and photoperiodic conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h), the microvine produces two to three new inflorescences per week, which enables to make hybridizations during long periods in repeating the crosses on the same plants. This also reduces the number of plants required for crosses and therefore experimental space while spreading the hybridization effort over selected and potentially long periods.

One to two months after a cross, it is possible to start harvesting seeds [22] to rescue zygotic embryos, which makes possible to establish a population maintained and amplifiable by micropropagation or microcuttings [23]. After a few micropropagation cycles, in vitro plants can be acclimatized to greenhouse conditions, and the first grapes are obtained within 12 months after the crosses. Thus, in less than a year, it is possible to start the study of the characteristics of the fruits and to proceed to new crossings to recover F2 populations. These speed up genetic mapping studies because it becomes possible to link a genotype and a phenotype in either F1 or F2 progenies in a few months instead of several years when using macrovines [23, 24].

Moreover, if a trait can be inherited through such crosses, it is possible to recover non-dwarf phenotypes (*GAI1/GAI1*) that can be directly proposed as breeding material. Indeed, 50% of the individuals from a cross between a microvine (*VvGAI1/Vvgai1*) and a macrovine (*VvGAI1/VvGAI1*) exhibit the same biological properties as conventional non-dwarf varieties. Thus, the microvine can be used both for the identification of QTLs of interest and also to combine or pyramid characters of interest in a pre-breeding perspective.

#### *3.3.2 Functional genomics*

The biological properties of the microvine are also of great interest for functional genomics [26]. Indeed, grapevine, as other perennial plants, is a difficult plant model to study the genes regulating the development of reproductive organs. The difficulty comes from its long juvenile period, its discontinuous fructification from winter buds, and the handling of large plants. The genetic transformation of classical varieties [28] requires several years to obtain adult plants and study the phenotypes linked to the ectopic expression of candidate genes.

With microvine, starting from embryogenic tissues compatible to *Agrobacterium tumefaciens*-mediated transformation (**Figure 6**), it is possible to recover transgenic fruiting plants in less than 1 year [19]. As for classical genetics, it is then easy to derive F2 lines to establish transgenic loci at homozygous state for further studies. In addition, the microvines have a very good aptitude for transformation by *Agrobacterium rhizogenes*, allowing to obtain transgenic organs stabilizable in axenic culture in a few weeks [25, 29, 30].

**13**

**axis**

**Figure 6.**

development.

**4.1 Temporal conversion of spatial profiles**

*configurations or combine them with various other transgenic traits or not.*

after the emission of the corresponding phytomer.

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

**4. Temporal inference of spatial observations obtained on the proleptic** 

*From competent embryogenic tissues (top left), it is possible to regenerate transgenic plants in a few months and obtain reproductive organs in less than a year. This allows the study of the regulation of flower and fruit development within shorter delays than with the non-dwarf vines. On the right, a microvine line V9 overexpressing the gene VvHB was identified as a major regulator of the development of the flesh in grapevine fruit [27]. Using genetically modified microvines, it is possible to segregate the transgenes in different genotypic* 

We have tested the possibility of converting spatial observations (along the proleptic axis) into temporal dynamics at a given stage of vegetative or reproductive

Under controlled and stable environment (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h), the development of the proleptic axis of the microvine is stable. The phyllochron is constant reaching ca. 24°C. The growing dynamics of leaves (surface) and berries (volume) from continuous fructification was found to be constant at a given level of phytomer, regardless of the date of bud break [20]. The growth durations of leaves and berries (herbaceous phase) are ca. 220°C after the emission of the phytomer and 500°C after flowering, respectively, as mentioned in Section 2.2. The development of these organs is also spatially stable: the dynamics of leaf area and berry volumes (herbaceous phase) for all levels of phytomer are superimposed when they are represented as a function of cumulative thermal time

Based on these outcomes, the conversion of spatial dynamics of leaf and berry development along the stems into time profiles was tested (**Figure 7**). For this purpose, the positions of the phytomers along the axis were converted into cumulated thermal time after their emission by multiplying their plastochron index (or rank position from the apex) by the phyllochron. The temporal profiles of leaf area and berry volume (green growth phase) resulting from this spatiotemporal conversion are similar to the real temporal profiles obtained at a given level of phytomer [8, 20, 31]. This property makes it possible to reconstruct temporal dynamics of

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

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

#### **Figure 6.**

*Advances in Grape and Wine Biotechnology*

tially long periods.

macrovines [23, 24].

*3.3.2 Functional genomics*

culture in a few weeks [25, 29, 30].

of genotypes and phenotypes can be obtained [5].

of the genetic traits determined on the chromosome n°2.

PV00C001V0008 [19] and the fleshless berry mutant of the ugni blanc [21], a range

This progeny is composed of 100% microvines (since the female parent has a *Vvgai1/Vvgai1* genotype) and a very small proportion of individuals with both hermaphrodite flowers and pigmented berries. Indeed, these two characters are present at the homozygous recessive state in one parent (f/f and n/n) and in the heterozygous dominant state in the other (H/f and N/n). It should be noted that since ugni blanc is heterozygous at the sex locus (H/f), while the picovine is f/f, selecting hermaphrodite individuals leads to a segregation distortion in the progeny

As the microvine produces inflorescences as long as vegetative growth is maintained, it becomes possible to cross all year around without being hampered by seasonality. Under standard thermal and photoperiodic conditions (25/15°C day/night temperature, VPD 1 kPa, photoperiod 12 h), the microvine produces two to three new inflorescences per week, which enables to make hybridizations during long periods in repeating the crosses on the same plants. This also reduces the number of plants required for crosses and therefore experimental space while spreading the hybridization effort over selected and poten-

One to two months after a cross, it is possible to start harvesting seeds [22] to rescue zygotic embryos, which makes possible to establish a population maintained and amplifiable by micropropagation or microcuttings [23]. After a few micropropagation cycles, in vitro plants can be acclimatized to greenhouse conditions, and the first grapes are obtained within 12 months after the crosses. Thus, in less than a year, it is possible to start the study of the characteristics of the fruits and to proceed to new crossings to recover F2 populations. These speed up genetic mapping studies because it becomes possible to link a genotype and a phenotype in either F1 or F2 progenies in a few months instead of several years when using

Moreover, if a trait can be inherited through such crosses, it is possible to recover

The biological properties of the microvine are also of great interest for functional genomics [26]. Indeed, grapevine, as other perennial plants, is a difficult plant model to study the genes regulating the development of reproductive organs. The difficulty comes from its long juvenile period, its discontinuous fructification from winter buds, and the handling of large plants. The genetic transformation of classical varieties [28] requires several years to obtain adult plants and study the

With microvine, starting from embryogenic tissues compatible to *Agrobacterium tumefaciens*-mediated transformation (**Figure 6**), it is possible to recover transgenic fruiting plants in less than 1 year [19]. As for classical genetics, it is then easy to derive F2 lines to establish transgenic loci at homozygous state for further studies. In addition, the microvines have a very good aptitude for transformation by *Agrobacterium rhizogenes*, allowing to obtain transgenic organs stabilizable in axenic

non-dwarf phenotypes (*GAI1/GAI1*) that can be directly proposed as breeding material. Indeed, 50% of the individuals from a cross between a microvine (*VvGAI1/Vvgai1*) and a macrovine (*VvGAI1/VvGAI1*) exhibit the same biological properties as conventional non-dwarf varieties. Thus, the microvine can be used both for the identification of QTLs of interest and also to combine or pyramid

characters of interest in a pre-breeding perspective.

phenotypes linked to the ectopic expression of candidate genes.

**12**

*From competent embryogenic tissues (top left), it is possible to regenerate transgenic plants in a few months and obtain reproductive organs in less than a year. This allows the study of the regulation of flower and fruit development within shorter delays than with the non-dwarf vines. On the right, a microvine line V9 overexpressing the gene VvHB was identified as a major regulator of the development of the flesh in grapevine fruit [27]. Using genetically modified microvines, it is possible to segregate the transgenes in different genotypic configurations or combine them with various other transgenic traits or not.*
