Development and implementation of an in vitro culture system for intact detached grape berries

[Abstract] Grape composition depends on the metabolites accumulated and synthesized during grape development. It is of paramount importance for grape growers because of its major role in shaping wine quality. Therefore, understanding the regulation mechanisms that control the accumulation of quality-related metabolites in grape is of both scientific and agronomical interests. The composition of grape berry at harvest is under complex regulation and can be affected by many factors (Conde et al. , 2007). The study of the effects of these factors on berries still attached to intact plants can be highly challenging because of the large size of the plants, interplant, intercluster and interberry variability; and because it is complicated to precisely control the nutrients and hormones imported by the berries, and the environment. Therefore, in vitro cultured grape berries are a good model system, which better represents berry anatomy structure (skin and flesh) than grape cell suspensions and nevertheless largely reduces the system complexity compared to whole plant (Bravdo et al. , 1990; Pérez et al. , 2000; Gambetta et al. , 2010). To this end, an in vitro culture system of intact detached grape berries has been developed by coupling greenhouse fruiting-cuttings production and in vitro organ culture techniques (Dai et al. , 2014). The cultured berries are able to actively absorb and utilize carbon and nitrogen from the culture medium, and exhibit fruit ripening features such as color changing and softening. This in vitro system may serve to investigate the response of berry composition to environmental and nutrient factors.

[Abstract] Grape composition depends on the metabolites accumulated and synthesized during grape development. It is of paramount importance for grape growers because of its major role in shaping wine quality. Therefore, understanding the regulation mechanisms that control the accumulation of quality-related metabolites in grape is of both scientific and agronomical interests. The composition of grape berry at harvest is under complex regulation and can be affected by many factors (Conde et al., 2007). The study of the effects of these factors on berries still attached to intact plants can be highly challenging because of the large size of the plants, interplant, intercluster and interberry variability; and because it is complicated to precisely control the nutrients and hormones imported by the berries, and the environment. Therefore, in vitro cultured grape berries are a good model system, which better represents berry anatomy structure (skin and flesh) than grape cell suspensions and nevertheless largely reduces the system complexity compared to whole plant (Bravdo et al., 1990;Pérez et al., 2000;Gambetta et al., 2010). To this end, an in vitro culture system of intact detached grape berries has been developed by coupling greenhouse fruiting-cuttings production and in vitro organ culture techniques (Dai et al., 2014). The cultured berries are able to actively absorb and utilize carbon and nitrogen from the culture medium, and exhibit fruit ripening features such as color changing and softening. This in vitro system may serve to investigate the response of berry composition to environmental and nutrient factors.

Materials and Reagents
1. Grapevine berries from greenhouse-grown fruiting-cuttings of Vitis vinifera L. cv.
Cabernet Sauvignon at various developmental stages (e.g. pea size, green berry, veraison, or later stages). The fruiting-cuttings (i.e. only one primary shoot axis with a single cluster per plant) were prepared as described in Mullins and Rajasekaran (1981) and grown in a naturally illuminated and semi-regulated greenhouse (mean seasonal temperature amplitude 20-35 °C) with fungicide treatments every two weeks   Figure 3A and B).
2. Prepare polystyrene cuboids corresponding to the size of tip plates ( Figure 3C).
3. Put the polystyrene cuboids on the two sides of the tip plates to form a floater ( Figure   3D-F).
4. Sterilize floaters with 10% NaClO and 90% ethanol during 20 min each following three rinses with sterile water and then install them in the tissue culture box containing liquid medium.  Figure 4A).
2. Grape clusters were excised from the mother plant, and berries were subsequently excised from peduncle with berry pedicel (about 3 mm) and dropped immediately into tap water ( Figure 4B).
3. Keep tap water running for 15 min to clean the berries.

Put berries into 70% ethanol for 2 sec and transfer immediately into NaClO with 2%
available chlorine with a sterilized colander ( Figure 4D).

Note: Dependent on the status of the greenhouse-grown berries, this step can be extended to 3-5 min. Longer sterilization will hurt berry skin.
6. Transfer berries into sterilized de-ionized water with colander ( Figure 4D) and shake at 350-450 rpm for 2 min.
8. Transfer fully rinsed berries into 20 mM EDTA solution, which is contained in a big culture dish ( Figure 4E).
9. Cut berry pedicel again to about 2 mm under EDTA solution ( Figure 4E), in order to exclude cavitations and to prevent plugging of sieve tubes by callose synthase, a strictly calcium-dependent enzyme.
10. Gently open the culture boxes and quickly put the berries on the solid or liquid culture medium with berry pedicel rooting into culture medium (see Recipes, Figure 4F and Figure 5).
11. Gently put the cover of culture box ( Figure 4G) and transport all boxes to culture room with constant temperature of 26 ± 0.5 °C, light period 16 h/8 h day/night, and light ~50 μmol m -2 s -1 ( Figure 4H). 12. Alternative culture methods depending on the desired experiment might be chosen ( Figure 5).
13. The berry culture can be maintained as long as 3 months. Since the medium volume is much greater than the berries, there is no need for change of medium. However, it will be very easy to change the medium composition of liquid medium with syringe injection.
Note: Berries are not very tightly fixed on the culture medium, so any movement and transport should be done gently to avoid berry slanting. Adjust pH to 5.8 with 0.5 M NaOH Complete to 1 L with deionized water Add 9 g agar for solid medium Autoclave (120 °C, 20min) For medium containing agar, pour 4 ml medium to each well of the 6-well plate ( Figure   5B); or 50 ml medium to the tissue culture box with special filters allowing gas http://www.bio-protocol.org/e1510 Vol 5, Iss 12, Jun 20, 2015 7 exchange ( Figure 4G), before medium concretion and inside of the laminar flow cabinet.
For liquid medium, pour 150 ml medium the tissue culture box with special filters ( Figure 5A).