Kiwifruit size and CPPU response are influenced by the time of anthesis

Scientia Horticulturae 95 (2002) 23–30

Kiwifruit size and CPPU response are influenced by the time of anthesis J.G. Cruz-Castilloa,*, D.J. Woolleyb, G.S. Lawesb a

Centro Regional Universitario Oriente, Universidad Auto´noma Chapingo, Apartado 49, Huatusco, Veracruz 94100, Mexico b Institute of Natural Resources, College of Sciences, Massey University, Palmerston North, New Zealand Accepted 18 December 2001

Abstract The cytokinin-active compound, N1-(2-chloro-4-pyridyl)-N3-phenylurea (CPPU), applied at different flowering dates, affected final ‘Hayward’ kiwifruit size. Ovaries from early opening flowers had significantly greater ðP  0:05Þ length, diameter, and fresh and dry weight than late ovaries. Cell number and cell size in the inner and outer pericarp of the ovary at anthesis were similar for early and late opening flowers but core cell number was significantly higher ðP  0:05Þ in the early flowers. Fruit shape and ovary dry matter percentage at harvest was unaffected by the time of anthesis. When fruitlets from both types of flowers were treated with CPPU at 15 ml l1 there was a significant interaction, with the early flowers achieving a much larger commercial fruit size (153 g) than fruit from later flowers (126 g). CPPU-treated fruit from the two bloom dates achieved higher cell number in the outer pericarp at harvest. In contrast, the cell size in the inner pericarp of early and late untreated fruits was higher than CPPU-treated fruit. Pre-anthesis factors and early fruit growth were important in determining final fruit size. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Actinidia deliciosa; Cytokinin; Fruit growth; Forchlorfenuron; Flower quality

1. Introduction Fruits of apple (Marguery and Sangwan, 1993), citrus (Praloran et al., 1981), peach (Scorza et al., 1991), grape (Coombe, 1973), persimmon (Hasegawa and Nakajima, 1990), and strawberry (Cheng and Breen, 1992) from early opening flowers are larger at maturity than those from later blooms. Therefore, the potential final size in some fruits may be determined before anthesis. Lai et al. (1990), Cruz-Castillo et al. (1991), Smith et al. (1992), and Patterson et al. (1999) found that kiwifruit from early flowers are larger than * Corresponding author. Tel.: þ52-273-40764; fax: þ52-273-40764. E-mail address: [email protected] (J.G. Cruz-Castillo).

0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 3 8 4 - 3

24

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

fruit from late flowers at harvest. However, Gould et al. (1992), did not find any relationship between timing of kiwifruit flower opening and fruit weight at harvest. Cell numbers of mature fleshy fruits depend on the number of cell divisions that occur before (and often after) anthesis (Coombe, 1976). In tomato (Ho et al., 1984) and apricot (Jackson and Combe, 1966) the final fruit size is closely related to the number of cells in the flower ovary. They considered the differences between fruits from early and late flowers to have arisen from the different number of cell divisions within the ovaries before anthesis. In the present work, the number and size of cells in ovaries and fruit with different times of anthesis were investigated. The synthetic bioregulator N1-(2-chloro-4-pyridyl)-N3-phenylurea (CPPU) is an urea-derivative cytokinin that promotes fruit growth in apple (Tartarini et al., 1993), grape (Retamales et al., 1993), and muskmelon (Hayata et al., 2000). In kiwifruit, Lawes et al. (1991), Costa et al. (1997), and Famiani et al. (1997) showed that the application of CPPU dramatically increased the final size of the fruit. In this study the interaction between date of anthesis and CPPU was studied in relation to cell number and size, and final weight of the fruit.

2. Materials and methods Experiments were carried out on T-bar trained ‘Hayward’ kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson var. deliciosa) vines at Massey University, Palmerston North, New Zealand. In the fruit growth season of 1989–1990, early (day 2 of the vine’s flowering period) and late flowers (days 11 and 12) on shoots of approximately 30 cm length with three fruits and a leaf:fruit ratio of 3:1 were tagged on 4-year-old vines. Ovary length (L), diameter (maximum (W1), and minimum (W2)), fresh and dry weight (including stigma tissue) were recorded on 50 early and 50 late flowers at anthesis on three contiguous vines in the orchard showing both type of flowers. At harvest, fruit weight, length, diameter (maximum, and minimum) were recorded on 120 fruits each from early, and late flowers spread over the orchard. A sample of 50 fruits from each of the two treatments were cut into quarters transversely, and the seed number counted on the six surfaces. The data were analyzed using the procedure TTEST of SAS for independent samples (SAS Institute, 1989). In the 1990–1991 season, early (day 2), and late (day 11) flowers in an orchard block were tagged on each of 20 vines aged 6 years having similar floral anthesis dates, and pollinated manually by brushing fresh male flowers. Ovary fresh and dry weight, and percentage ovary dry matter (Woolley et al., 1991) were recorded at anthesis on 12 flowers selected randomly for each of the two treatments. A t-test for independent samples using the TTEST procedure of SAS was carried out on each of the variates measured on those flowers. CPPU (Sitofex EC 1.0%; SKW, Trostberg, Germany) at 15 ml l1 a.i. was applied as a 5 s fruit dip 23 days after anthesis to a total of 60 fruit from each early and late flowers. The four treatments were fruit from two periods of anthesis each with or without CPPU. Data were analyzed as a randomized complete factorial design using the procedure ANOVA of SAS (SAS Institute, 1989). Orthogonal contrasts were carried out for means differentiation. Cell number and size were investigated on ovaries at anthesis, and for fruit harvested in May. At anthesis 12 each early and late ovaries were selected randomly, and at

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

25

harvest six fruit from each of the four treatments were used. Transverse sections of ovary, and fruit were fixed in formalin:acetic acid:ethyl alcohol (60%) (1:1:20) (Hopping, 1976). After fixation, the ovary and 3 mm thick fruit sections were processed in labeled embedding cassettes in a Shandon Hypercenter tissue processor. The full cycle of the tissue embedding processing took about 14 h, and was carried out as follows: 70% alcohol (1 h), 95% alcohol (1 h), 100% alcohol (2  1 h), 100% alcohol (2  2 h), xylene (3  45 min) and wax (2  2 h). Prior to wax embedding the tissue was evacuated at 1.72 bar for 4–5 h to remove all air bubbles in the cells. The wax used was Paraplast (medium) melting point 56 8C. The two wax baths were under a vacuum of 1.03 bar. A Heitz 1512 microtome was used to cut ovary and fruit transverse sections of 8 mm thickness which were then dried overnight in an oven at 60 8C. The process of staining was as follows: dewax sections in xylene (2  5 min), hydrate through alcohol 70% to water, stain in Gills haematoxylin (3 min), wash in tap water (30 s), wash in Scotts tapwater (30 s), wash in tap water (60 s), dehydrate through alcohol 95%, clear in xylene, and mount in DPX (BDH). The cell numbers in a transverse section were counted across the outer pericarp, inner pericarp between the locules, and right across the narrowest core diameter using a Reichert microscope equipped with Nomarski differential interference optics. Cell counts represented number of cells in a straight line across the outer pericarp and inner pericarp on one side of the flower ovary and fruit but the total number across the whole core. Cell size was determined by a division between the number of cells and the length of the section evaluated.

3. Results At anthesis, the ovaries from the early flowers were significantly greater (P  0:05) than late flowers in length, diameter, fresh weight, and dry weight (Table 1). There was no significant difference in ovary percentage dry matter for the two types of flowers (Table 1). The shape of the ovaries determined by L/W1, and W2/W1 were similar for both types of flowers. At harvest, fruit from early flowers had significantly greater fresh weight (Table 1). The fruit surface seed number, and fruit elongation (L/W1), and symmetry (W2/W1) were not affected significantly by the time of anthesis (Table 1). In the following season ovary fresh weight with stigmas at anthesis was significantly higher (P  0:05) for the early flowers (Table 2), but there were not significant treatment differences in ovary dry weight and percentage dry matter. There was a significant interaction (P  0:05) between the time of anthesis and the CPPU application on final fruit fresh weight. The highest fruit fresh weight was achieved by fruit from early flowers treated with CPPU. Untreated fruit from late flowers had the lowest fresh weight (Table 2). All treatments had similar fruit surface seed number, and fruit W2/W1 ratios (Table 2). Fruit from early flowers were the most elongate, and CPPU-treated late bloom fruit the most square (Table 2). Cell number, and cell size across both the outer and inner pericarp of the ovary at anthesis were similar for early and late flowers (Table 3). The core cell number was significantly higher for ovaries from early flowers. There were not significant differences in

26

Flower type

Ovary length (mm)

Ovary diameter (mm)

Ovary L/W1b

Ovary W2/W1c

Ovary fresh weight (g)d

Ovary dry weight (g)d

Ovary dry matter (%)

Fruit fresh weight (g)

Fruit surface seed number

Fruit L/W1b

Fruit W2/W1c

Early Late

8.62 a 7.39 b

8.04 a 7.08 b

1.07 a 1.08 a

0.93 a 0.92 a

0.531 a 0.459 b

0.075 a 0.066 b

14.1 a 14.4 a

100.0 a 86.8 b

204 a 202 a

1.15 a 1.13 a

0.92 a 0.90 a

a

Numbers in the same column with different letters significantly differ by t-test at P  0:05. Length/maximum width. c Minimum width/maximum width. d Ovaries with stigmas. b

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

Table 1 Ovary (at anthesis) and fruit size for fruit from early and late flowers of ‘Hayward’ kiwifruit (1989–1990 season)a

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

27

Table 2 The effect of bloom date and CPPU application on ‘Hayward’ kiwifruit characteristics (1990–1991 season) Flower type (without stigmas)

Ovary fresh Ovary dry Ovary dry Fruit fresh Fruit surface weight at weight at matter at weight (g)b seed numberb anthesis (g)a anthesis (g)a anthesis (%)a

Fruit Fruit L/W1b,c W2/W1b,c

Early Early þ CPPU Late Late þ CPPU

0.384 a – 0.362 b –

1.20 1.17 1.16 1.13

0.051 a – 0.048 a –

13 a – 13 a –

101 153 92 126

c a d b

229 221 222 218

a a a a

a ab ab b

0.84 0.80 0.79 0.78

a a a a

a

Numbers in the same column with different letters significantly differ by t-test at P  0:05. Numbers in the same column with different letters significantly differ by orthogonal contrasts at P  0:05. c Length/maximum width or minimum width/maximum width. b

Table 3 Cell number, and cell size (m) of the outer pericarp (OP), inner pericarp (IP), and core (C) of ‘Hayward’ kiwifruit ovaries from early and late flowers at anthesis (1990–1991 season)a Flower type

Cell number OP

Cell size OP

Cell number IP

Cell size IP

Cell number C Cell size C

Early Late

36 a 38 a

24 a 23 a

21 a 24 a

48 a 44 a

90 a 76 b

a

27 a 25 a

Numbers in the same column with different letters significantly differ by t-test at P  0:05.

Table 4 Cell number and size (m) in the outer pericarp (OP), inner pericarp (IP), and core (C) of commercially harvested ‘Hayward’ kiwifruit as influenced by bloom date and CPPUa Flower type

Cell number OP Cell size OP Cell number IP Cell size IP

Cell number C Cell size C

Early Early þ CPPU Late Late þ CPPU

69 78 60 75

115 132 105 122

a

b a c ab

106 96 100 100

a a a a

57 59 52 49

a a a a

204 176 220 175

a b a b

a a a a

72 67 76 73

a a a a

Numbers in the same column with different letters significantly differ by orthogonal contrasts at P  0:05.

core cell size between the two different ovary types (Table 3). At harvest, CPPU treatment increased cell number in the outer pericarp for both early and late flowers (Table 4). However, there were not significant differences among treatments for cell number in the inner pericarp and core. The cell size in the outer pericarp and core were not affected significantly by any treatment. In contrast, the cell size of the inner pericarp was significantly reduced for the chemically treated fruit (Table 4).

4. Discussion At harvest the fresh weight of mature fruit from early flowers at harvest was significantly higher than fruit from late flowers by 13 g (Table 1) and 9 g (Table 2) in each of the two

28

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

seasons, respectively. In both the seasons, the size increase was independent of seed number (Tables 1 and 2). In contrast, Gould et al. (1992), did not find any relationship between timing of kiwifruit flower opening and fruit weight at harvest. They presented no data on fruit seed number, and the lack of an effect may have been due to high variability of fruit seed number, or fruit location on the vine, or variability between vines. The ovaries of early flowers were larger and had higher fresh weight than those of flowers with a late anthesis (Tables 1 and 2). Early flowers had higher ovary core cell number at anthesis than late flowers (Table 3). The development of more cells and larger flower parts suggests a competitive advantage of flowers growing first, and a greater initial rate of metabolic activity. Thus, flowering date and hence the timing of flower bud development, influenced flower size and their potential to produce large size fruit. Harvest fruit from early flowers showed significantly higher cell numbers in the outer pericarp than in fruit from late flowers although mean cell size was the same (Table 4). In the two seasons studied, seed number was similar between early and late flowers but fruit from early flowers was significantly larger. In apple it was found that early opening flowers had a greater subtending expanded leaf area than later opening flowers (Denne, 1963). Spurs with less leaf area seem to require significant import of photosynthesis from other parts of the branch to maintain apple fruit development 3 weeks after bloom (Grappadelli et al., 1994). Therefore, in addition to pre-anthesis factors (e.g. large ovary cell number or more developed vascular tissue) early flowers may have a larger supply of resources with respect to late opening flowers. In this work no data on vine leaf area was recorded. Although it has been suggested that the carbon resources available to a flower bud probably determine the time of anthesis of a flower (Lai et al., 1990), further research is required to elucidate the relative importance of factors that influence flower development such as the timing of cane growth, time and severity of pruning, and cane angle. Lai et al. (1990) demonstrated that when the time of flowering within a vine varied by less than 6 days the difference in final size for fruit from early and late flowers was not significant. The larger size of fruit from early flowers in comparison to late flowers (Tables 1 and 2) suggests a short bloom period within both a vine and an orchard may result in an increased number of larger sized export fruit, or at least a more uniform fruit size range. Spraying vines with hydrogen cyanamide (Patterson et al., 1999), or late spring tipping of canes contracts the blooming period and promotes large fruit size (Snelgar and Manson, 1992). More attention to other vine management practices that may reduce the duration of the period of bloom such as the use of specific rootstocks (Wang et al., 1994) is required. Significantly higher cell number was observed in the outer pericarp of fruit when they were treated with CPPU (Table 4). Cell size in the outer pericarp, and core were not significantly affected by CPPU, but the cell size of the inner pericarp was significantly reduced (Table 4). However, Antognozzi et al. (1997) showed that CPPU increased cell size in the inner pericarp. Kurosaki and Mochisuki (1990) concluded that in CPPU-treated ‘Monty’ kiwifruit there was no change in the cell size, and the fruit size increase was due to a raised cell number. In contrast, Patterson et al. (1993), found that CPPU stimulates only cell expansion in kiwifruit. Woolley et al. (1991), studying the outer pericarp of treated fruit found increases in both cell number, and cell expansion. In the present work, CPPU increased only cell number but cell count measurements based on a straight line across the

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

29

tissue in only one dimension do not account for the variation in size, and shape of the cells. Our results do not clearly resolve if cell number or cell size effects are the basis of the kiwifruit growth response to CPPU. For example, after treatment with CPPU early and late flowers have similar cell number and cell size. Perhaps the accumulation of small, nonsignificant differences in cell number in all the three kiwifruit tissues accounted for the differences in fruit size at commercial harvest (Table 4). The final fruit size response to CPPU was 17 g higher in fruit from early flowers than in fruit from late flowers with respect to untreated fruit for each time of anthesis. This fresh weight increase was independent of their seed number (Table 2). Thus, the response to CPPU was more effective on fruit showing a higher number of cells at early anthesis.

Acknowledgements The authors thank SKW-Trostberg, Germany for providing CPPU.

References Antognozzi, E., Famiani, F., Proietti, P., Ferranti, F., Frenguelli, G., 1997. Effect of CPPU (cytokinin) treatments on fruit anatomical structure and quality in Actinidia deliciosa. Acta Hort. 444 (2), 459–465. Cheng, G.W., Breen, P.J., 1992. Cell count and size in relation to fruit size among strawberry cultivars. J. Am. Soc. Hort. Sci. 117, 946–950. Coombe, B.G., 1973. The regulation of set and development of the grape berry. Acta Hort. 34, 261–273. Coombe, B.G., 1976. The development of fleshy fruits. Annu. Rev. Plant Physiol. 27, 207–228. Costa, G., Succi, F., Quadretti, R., 1997. Effect of CPPU on fruiting performance, fruit quality and storage life of kiwifruit (cv. Hayward). Acta Hort. 444 (2), 467–472. Cruz-Castillo, J.G., Lawes, G.S., Woolley, D.J., 1991. The influence of the time of anthesis, seed factor(s), and the application of a growth regulator mixture on the growth of kiwifruit. Acta Hort. 297, 475–480. Denne, M.P., 1963. Fruit development and some tree factors affecting it. N.Z. J. Bot. 1, 265–294. Famiani, F., Antognozzi, E., Battistelli, A., Moscatello, S., 1997. Effects of altered source–sink relationships on fruit development and quality in Actinidia deliciosa. Acta Hort. 444 (1), 355–360. Gould, K.S., Watson, M., Patterson, K.F., Barker, K.A., 1992. Fruit cell type-juice or flavour. New Zealand Kiwifruit. Special Publication No. 4, New Zealand Kiwifruit Marketing Board, Rotorua, New Zealand, pp. 32–33. Grappadelli, L.C., Lasko, A.N., Flore, J.A., 1994. Early season patterns of carbohydrate partitioning in exposed and shaded apple branches. J. Am. Soc. Hort. Sci. 119, 596–603. Hasegawa, K., Nakajima, Y., 1990. Effects of bloom date, seediness, GA treatment and location of fruits in the foliar canopy on the fruit quality of persimmon (Diospyros kaki Thunb.). J. Jpn. Soc. Hort. Sci. 59, 263– 270. Hayata, Y., Niimi, Y., Inove, K., Kondo, S., 2000. CPPU and BA, with and without pollination, with and without pollination, affect set, growth, and quality of muskmelon fruit. HortScience 35, 868–870. Ho, L.C., Bangerth, F., Ebert, A., Flore, J.A., Glifford, R.M., Hoad, G.V., Sjut, V., 1984. Factors affecting final size of fruits. Annual Report for 1982. Glasshouse Crops Research Institute, pp. 45–50. Hopping, M.E., 1976. Structure and development of fruit and seeds in Chinese gooseberry (Actinidia chinensis Planch). N.Z. J. Bot. 14, 63–68. Jackson, D.I., Combe, B.G., 1966. The growth of apricot fruit. I. Morphological changes during development and the effects of various tree factors. Aust. J. Agric. Res. 17, 465–477. Kurosaki, T., Mochisuki, T., 1990. Effect of KT-30 treatment on fruit growth and some components of Monty kiwifruit. J. Jpn. Soc. Hort. Sci. 59, 43–50.

30

J.G. Cruz-Castillo et al. / Scientia Horticulturae 95 (2002) 23–30

Lai, R., Woolley, D.J., Lawes, G.S., 1990. The effect of inter-fruit competition, type of fruiting lateral and time of anthesis on the fruit growth of kiwifruit (Actinidia deliciosa). J. Hort. Sci. 65, 87–96. Lawes, G.S., Woolley, D.J., Cruz-Castillo, J.G., 1991. Field responses of kiwifruit to CPPU (cytokinin) application. Acta Hort. 297, 351–356. Marguery, P., Sangwan, B.S., 1993. Sources of variation between apple fruits within a season, and between seasons. J. Hort. Sci. 68, 309–315. Patterson, K.J., Mason, K.A., Gould, K.S., 1993. CPPU (N-(2-chloro-4-pyridyl)-N0 -phenylurea) on fruit growth, maturity, and storage quality of kiwifruit. N.Z. J. Crop Hort. Sci. 21, 253–261. Patterson, K.J., Snelgar, W.P., Richardson, A.C., McPherson, H.G., 1999. Flower quality and fruit size of Hayward kiwifruit. Acta Hort. 498, 143–150. Praloran, J.C., Vullin, G., Jaquemond, C., Depierre, D., 1981. Observations sur la croissance des cle´ mentines en Corse. Fruits 36, 755–767. Retamales, J., Cooper, T., Bangerth, B., Callejas, R., 1993. Efecto de aplicaciones de CPPU y GA3 en el crecimiento y calidad de uva de mesa cv. Sultanina. Revista Frutı´cola 14, 89–94. SAS Institute, 1989. SAS User’s Guide: Statistics, Version 6, 4th Edition, Vol. 1. SAS Institute Inc., Cary, NC. Scorza, R., May, L.G., Purnell, B., Upchurch, B., 1991. Differences in number and area of mesocarp cells between small-and-large-fruited peach cultivars. J. Am. Soc. Hort. Sci. 116, 861–864. Smith, G.S., Gravett, I., Curtis, J., 1992. The position of fruit in the canopy influences fruit quality. New Zealand Kiwifruit. Special Publication No. 4, New Zealand Kiwifruit Marketing Board, Rotorua, New Zealand, pp. 38–41. Snelgar, W.P., Manson, P., 1992. Canopy management, vine yield and fruit quality. New Zealand Kiwifruit. Special Publication No. 4, New Zealand Kiwifruit Marketing Board, Rotorua, New Zealand, pp. 7–10. Tartarini, S., Sansavini, S., Ventura, M., 1993. CPPU control of morphogenesis in apple. Sci. Hort. 53, 273–279. Wang, Z.Y., Patterson, K.J., Gould, K.S., Lowe, R.G., 1994. Rootstock effects on budburst and flowering in kiwifruit. Sci. Hort. 57, 187–199. Woolley, D.J., Lawes, G.S., Cruz-Castillo, J.G., 1991. The growth and the competitive ability of Actinidia deliciosa Hayward fruit: carbohydrate availability and response to the cytokinin-active compound CPPU. Acta Hort. 297, 467–473.