J. Japan. Soc. Hort. Sci. 49(1) :1-7. 1980. A Study of Micro-morphology of Grape Berry Surface during Their Development with Special Reference to Stoma Sholchl NAKAGAWA,Harukl KOMATSUand Ei ji YUDA Department of Horticulture, University of Osaka Prefecture, Sakai, Osaka 591 Summary Using seven grape cultivars, `Concord', `Campbell Early', `Delaware', `Takao', `Kyoho' , `Muscat of Alexandria' and `Koshu', changes in the stomatal morphology of berry epidermis accompanied by the growth were observed under scanning electron microscope (SEM). Average number of stomata on the berry surface of `Muscat of Alexandria' at anthesis was 16, that of `Campbell Early', `Delaware', `Kyoho' and `Koshu' was around 12, and those of `Concord' and `Takao' were 7.0 and 2. 4, respectively. The size of stomata in grape berry epidermis at anthesis was not differed among the cultivars, and was 2025 p in width and around 25 p in length. Twenty days before anthesis the guard cells were not yet differentiated, but distinguishable just prior to anthesis. Furrowed feature was seen on the inner wall of opening stomata in this period. After fertilization cracks occurred in the epidermis circulating around some stomata with berry growth, and cork layers developed beneath such stomata, which sometimes protruded from the epidermal layer forming lenticel-like structure. From these observations what were visibly seen as brown specks on grape berry surface at near maturity were confirmed as stomata and the suberized cells adjacent to them. in the grape berry epidermis. On the con- Introduction trary, Kozma P. (4) reported a small number Fruit epidermis is a protecting tissue, of stomata was present in young berries and consisting usually of one layer of cells ac- they would close down in 2-3 weeks after companied sometimes by trichomes or sto- bloom and developed into lenticels. 'There- mata. Most of the epidermal tissues having after, Bessis (1) recognized the presen ce of stomata, their distribution density there stomata in a few grape cultivars and followed differs by kinds and species of fruit, or en- the process of their development through vironmental conditions. For example, the scanning electron microscope (SEM). Swift total number of stomata per fruit varies et al. (9) also observed the stomata in the in a range 40 to 350 in apple cultivars (10), berries of `Sultana' and `Cabernet Sauvi gnon' while in mature sweet cherry, that per cm2 under light microscope. is in a range from 380 to 550, and the This study was therefore conducted to stomata were distributed near the central reconfirm the presence of stomata in the region of fruit (8). Chujo and Ashizawa epidermis of young grape berries just after (2) reported that stomata were not observed bloom, and to observe differences in their in the epidermis of persimmon fruit, though size, external appearance and structual changes the scars of trichomes and cracks were pre- during berry development among common sent. grape cultivars in Japan. In grapes, Nakagawa and Nan jo (96, 7) and Kriedmann (5) did not observe any stomata Materials and Methods Received for publication September 17, 1979. Grape cultivars used for this experiment. 1 2 JOURNAL OF THE JAPANESE SOCIET Y FOR HORTICULTURALSCIENCE were `Campbell Early', `Delaware', `Takao' those at bloom, fruits were used without and `Kyoho', which were grown in an exper- any preparation. For those at maturity, iment vineyard of University of Osaka small sections containing stomata were pre- Prefecture. Fruitlets or fruits of these cul- pared. tivars were collected at weekly intervals, For SEM viewing, each material was fixed in a solution of ethanol: acetic acid= dehydrated in graded ethanol and acetone 3 :1 (v/v) for 24 hr and then stored in 70% series. The materials were critical point ethanol until further used. dried using liquid CO2 in a HCP-1-Critical As for the materials before bloom, only point drier. The dried materials mounted pistils were used removing the flower caps on SEM stubs with ion sputtering (IB-3-Ion (corolla petals) under microscope, and for coater), sputter coated with gold, and viewed ~~••'S>A'(tfNIR Fig. 1. Scanning electron micrographs of ovaries of 4 grape cultivars at anthessis. `Muscat of Alexandria' A : `Campbell Early', B : `Kyoho', C , D : `Koshu'. Table 1. Flowering time, size of ovary, and size of stomata in some cultivars of Vitis. NAKAGAWA ET AL.: A STUDY OF MICRO-MORPHOLOGY OF GRAPE BERRY SURFACE 3 with MSM-4 C-202-SEM using 10 kV acceler- ating potential. The number and side of stomata on the ovary surface at bloom were observed such cultivars as `Concord', `Muscat of Alexan- dria' and `Koshu' besides the four cultivars as mentioned above. In each observation 10 samples were used. Results As shown in Fig. 1 and Table 1, the shape and size of grape pistiles differed by cultivars. `Concord' _ ' oFl4.3~ , `Muscat of Alexandria', and `Ko- Fig. 2. Scanning electron micrograph of `Kyoho ovary shu' were small, and `Delaware' and `Camp- at anthesis. Some stomata are noticeable. bell Early' were medium, while `Kyoho' and `Takao' were largest. Rugged feature was observed on the ovary surface of `Mus- cat of Alexandria', which was not observed in any other cultivars. A few number of stomata was present on the ovary surface in all the cultivars used. Some stomata were observed even on the style surface, but mostly distributed on the ovary surface. Average number of stomata on the ovary surface of `Muscat of Alexandria' was 16.0; whereas that of `Campbell Early', `Delaware, `Kyoho' and `Koshu' was around 12.0; `Concord' was 7. 0, and `Takao' was 2.4 (Table 1). The number of stomata at ma- turity was similar to that at anthesis, while that before bloom was somewhat smaller. Average size of the stomata did not differ so much by cultivars, and the width was 20-25 L and the length was around 25 ~e. The size of `Takao' and `Concord' was slight- ly larger, and was 29.7 x 32.5 , t, 27.0 x 29.3 Fig. 3. Scanning electron micrograph of a stoma 21 days before anthesis (`Campbell Early'). Guard p, respectively. In contrast, that of `Koshu' cells are not differentiated yet. was greater in the ratio of the length/ width. entiated guard cells were clearly obse rved, Changes in stomatal structure of a few though some stomata had not been opened. cultivars were observed, and since marked In this period the epidermal cells were cover- differences were not found among these cul- ed with wavy rod shaped substances (chlo- tivars, only those of `Campbell Early' was roform insoluble), which were not present shown in this paper. in the stomata (Fig. 4). The stomata 20 days before bloom (15 ) An furrowed feature was seen on the inner were slightly larger than the corresponding wall of opening stomata in the same period epidermal cells. The guard cells were not (Fig. 5). Some of the stomata were protrud- differentiated then, though the linear hollow ing from the epidermal layer (Fig.6), but to divide into two guard cells was already others were located on the same level as the seen (Fig. 3). Approaching anthesis, differ- epidermal layer. In `Koshu', many a f the 4 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE Fig. 4. Scanning electron micrograph of a stoma 14 Fig. 6. Scanning electron micrograph of a stoma at days before anthesis (`Campbell Early'). Guard anthesis (`Campbell Early'). The stoma is cells are differentiated, and distinguishable from protruding from the epidermal layer. surrounding epidermal cells. Fig. 5. Scanning electron micrograph of the inner Fig. 7. Scanning electron micrograph of a berry wall of an opening stoma at anthesis (`Campbell surface (`Campbell Early'). A small crack is Early') noticeable near the stoma. NAKAGAWA ET AL.: A STUDY OF MICRO-MORPHOLOGY OF GRAPE BERRY SURFACE 5 Fig. 10. Scanning electron micrograph of cutic ular development around a stoma 49 days a f ter anthesis (`Campbell Early'). Fig. 8. Scanning electron micrograph of a stoma 14 days after anthesis (`Campbell Early'). Cracks are developing. ,~ Mrw+.VVIII... - YYYYI-- -: ...n-~ Fig. 11. Scanning electron micrograph of a vertical section of stoma 49 days after anth Qsis (`Campbell Early'). f..•...rY~ .. x Fig. 9. Scanning electron micrograph of a stoma 23 days after anthesis (`Campbell Early'). Crateriform features develop circumferencially at the stoma. stomata were protruding from the layer. After fertilization the growth of the epi- dermis of immature fruitlet will be taken place through cell division and cell enlarge- Fig. 12. Scanning electron micrograph of a st~ima ment. However, there seems to be clear at maturity (`Campbell Early'). Cracki ngs difference in growth speed between the epi- around the stoma are further progressed and dermal cells and stomata. This difference hypha-like substances were observed. would cause cracking in the epidermal layer (Fig. 7), and suberizat ion might be taken portion of such a crack was enlarged with place in a few layers beneath the crack. The berry growth (Fig. 8). 6 JOURNAL OF THE JAPANESE SOCIET Y FOR HORTICULTURAL SCIENCE Craterif orm features developed circumf er- cultivars of V. vinifera were observed. encially at some stomata (Fig. 9), whereas What we noticed about grape berry stomata some other stomata were collapsed because during their growth were that they should of wound tissue development. be differentiated before bloom and developed As normally occurred on the epidermal up to flowering time. Thereafter, growth cell layers, many of stomata were covered speed of the stomata became slower than that with cuticle, so that the openings were em- of other epidermal cells, resulting in occur- beded with such a substance (Fig. 10). rence of cracks beneath the stomata.