It is possible to mimic flowering and fruit set in the woody grapevine (Vitis vinifera) planted under controlled conditions. Both the male and female flowers were similar in the cuttings and field vines.
Phenology
Previous studies on cuttings of grapevine (Mullins, 1966
; Mullins and Rajasekaran, 1981) and apple (Höfer and Lespinasse, 1996) were not focused on the ontogenesis of reproductive organs, although it is a key issue in models of flowering. The grapes could be distinguished at stage 15 (Fig. 1B), whereas each flower bud was separated at stage 17 (Fig. 1C). At least 10 % and 90 % of the flowers were opened, respectively, at stages 21 and 25 (Fig. 1D and E), and thereafter fruit set occurred (Fig. 1F). Development was similar in the two cultivars, and both in vineyards and cuttings (Fig. 2). However, development after stage 17 was more rapid in the field than in the glasshouse.
The PN cuttings had more flowers than the vines, whereas there were no differences in the two types of GW plants (Table 1). There were also no differences between the two cultivars within a plant type. In contrast, the GW plants had more berries than the PN plants, and a higher rate of fruit set. The number of seeds per berry was not affected by cultivar or plant type, with general means of 1·35 ± 0·49 and 1·79 ± 0·97, respectively (Table 1). Final yield was similar in the cuttings and vineyard, with respect to number of flowers, number of fruits and seed set.
Development of reproductive organs
Inflorescences in the cuttings developed more rapidly than those from the vineyard plants, with a difference of 12 and 13 d in GW and PN, respectively (Fig. 2). The rate of flower development is dependent on temperature, with extreme temperatures damaging these tissues (Ebadi et al., 1995; Young et al., 2004). The vineyards are exposed to daily fluctuations of temperature, with relatively low temperatures in the night and early morning, whereas the cuttings were exposed to constant temperature ranging from 20–30 °C.
The chronology of pollen development was identical in the vines and cuttings (Table 2). In GW, stage 12 sporogenous cells (Fig. 3A) multiplied and gave rise to microspore mother cells at stage 15. Male meiosis occurred between stages 15 and 15 + 2 d (Fig. 3B) with microspores released from the tetrads at stage 15 + 8 d (Fig. 3C). Microspores vacuolated thereafter and pollen mitosis took place between stages 17 and 21 (Fig. 3D). At stage 21 (anthesis) mature pollen grains were released from the anthers. Pollen development occurred earlier in PN than in GW (Table 2). Meiosis was registered between stages 12 and 15, whereas pollen mitosis took place between stages 15 + 8 d and 17. These results indicate that cuttings grown under controlled conditions can be used to study pollen development in grape.
There was a slight delay in female meiosis in the cuttings compared with the vines (Table 2). In GW cuttings, within the sporogenous tissue (Fig. 4A) an archespore developed at stage 15 + 1 d (Fig. 4B), which remained until stage 15+3 d. Stage 17 was characterized by the presence of the macrospore mother cell (Fig. 4C) and meiosis occurred thereafter. The embryo sac was formed at stage 21 (Fig. 4D). In the GW vines, the sporogenous tissue multiplied at early stages of development. The macrospore mother cell was seen from stage 15 + 2 d to stage 15 + 8 d. Between stages 15 + 8 d and 17 meiosis occurred, and the three micropylar macrospores degenerated, leading to a single macrospore generating the whole embryo sac at stage 17. At anthesis, the embryo sac contained eight cells and was ready for fertilization.
Female development was faster in PN than in GW (Table 2). In vines, the archespore appeared at stage 15 and meiosis occurred between stages 15 + 2 d and 15 + 8 d. Afterwards, the embryo sac developed between stages 15 + 8 d and 21. In cuttings, meiosis was slightly delayed between stages 15 + 3 d and 17, but the other steps of development were similar to those in vines.
Location of starch in the anther and the ovule
Changes of starch reserves were similar in the two plant types. Starch was found in all the sporophytic tissues of the anther (epidermis, endothecium and tapetum) in the vines (Table 3). In the gametophytic tissue, starch could be seen in PN microspores at all stages, but not at stages 12 and 15+8 d in GW (Table 3). Later on, amylogenesis/amylolysis occurred in the young pollen grains. In the cuttings, starch grains were not detected in sporophytic tissues, except in the endothecium at stages 15 + 3 d and 17 in PN, and only in stage 17 in GW (Table 3). No starch was detected in the gametophytic tissue (Table 3). The anther sporophytic tissues influence the concentrations of sugars in the anther by storing/mobilizing starch and regulate the volume of sugar reaching the pollen grain during development (Clément and Audran, 1995). The difference reported here between vines and cuttings may correspond, in terms of starch in the anther sporophytic tissues, to such buffering action of the sporophytic tissues and obviously does interfere with pollen development.
In the ovules of the vines, concentrations of starch were low except at stage 15 + 8 d in the teguments and nucellus of the PN ovules (not in GW) (
Table 3). Nevertheless, amylaceous reserves were detected in ovaries of both cultivars from stage 12 and 15 + 2 d in PN and GW, respectively, to anthesis. In contrast, starch was not detected in the nucellus and teguments of the cuttings during flower development, except at stage 15+3 d for PN (
Table 3), same as in the vines. In the cuttings, starch accumulation in the ovaries was delayed until stage 17 in GW and stage 15 + 3 d in PN. In grapevine (Lebon
et al., 2004
), as in other fruit trees (Rodrigo and Herrero, 1998
; Rodrigo
et al., 2000
), the presence or absence of starch in the ovule at meiosis has a key influence on fertilization and fruit set. In the cuttings and vines, the variations in concentrations of starch in the inflorescences and ovules were similar.
There were differences in reproduction between GW and PN grown in the vineyard; GW exhibits faster inflorescence development (Fig. 2), more flowers per inflorescence (Table 1), but a lower flower abscission rate, percentage of fruit set and fewer seeds per fruit (Table 1). Moreover, starch has a greater incidence in PN ovules (Lebon et al., 2004). These differences were also detected in the cuttings grown under the conditions described here, further showing that the protocol used is reliable.
In grapevine, uncommitted primordium in the latent bud developed into inflorescence primordium and a potential fully developed inflorescence next spring, depending on environmental conditions (Boss et al., 2002). Rapid shoot growth can produce tendrils rather than inflorescences. Further development of the flowers is influenced by variety and environment (Boss et al., 2003). Flower abscission is poorly described in grapevine but can dramatically diminish the yield (Huglin and Schneider, 1998). The supply of carbohydrates to the flower is a key factor in abscission (Gu et al., 1996). A model of flower development in woody species should assist efforts to understand physiological traits that favour inflorescence initiation and flower development.
Cuttings can be used to develop doubled haploid grapevines. Until recently, attempts to obtain in vitro microspore embryogenesis from field plants generally resulted in diploid callus from the sporophytic tissues of the anther (Mauro et al., 1986). In apple, the success of microspore embryogenesis is dependent on the donor material. Anthers collected from the field did not produce haploid embryos (Höfer and Lespinasse, 1996), whereas those from cuttings gave doubled haploids (Höfer et al., 1999; Höfer, 2004).
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