Changes of abscisic acid, indole-3-acetic acid and gibberellin-like substances in the flowers and developing fruitlets of citrus cultivar ‘Hyuganatsu’

SCIENTIA HOI?llCUlTuR& ELSEVIER

Scientia Horticuhurac 65 (1996) 263-272

Changes of abscisic acid, indole-3-acetic acid and gibberellin-like substances in the flowers and developing fruitlets of citrus cultivar ‘ Hyuganatsu’ Kiyohide Kojima Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 604, Japan

Accepted 16 January 1996

Abstract Endogenous hormonal changes associated with fruit set and growth were studied in ‘Hyuganatsu’ (Citrus tamuruna [Hart.] ex. Tanaka), a species which has no parthenocarpic ability. Concentrations and contents of abscisic acid (ABA), indole-3-acetic acid acid (IAA) and gibberillin (GA)-like substances were measured before, during and after anthesis, in several floral and fruitlet parts. On the day of anthesis, the ABA concentration in stamens had decreased to about 30% as compared to 5 days before, while the IAA concentration in stamens and petals had increased about twofold. Eight days after anthesis (DAA), the contents of ABA and IAA in the styles and in the fruitlets increased markedly, particularly in the pollinated flowers in which were found the highest values per fresh weight recorded in this study. The contents of GA-like substances rose continuously in the pollinated fruitlets, whereas in unpollinated ones no increase was found from 8 DAA. These changes in hormone levels are discussed in relation to the development of the floral parts and the early growth of the fruitlets. Keywords: Abscisic acid; Citrus; Fruit set; Gibberellins; Indole-3-acetic acid; Pollination

1. Introduction In non-parthenocarpic species, a burst of ovary growth occurs after successful pollination and fruit development begins, usually with a simultaneous wilting and abscission of the petals (Leopold and Kriedemann, 1975). These changes are believed to be regulated by phytohormones (Schwabe and Mills, 1981). During flowering, abscisic

Abbreviations:

ABA, abscisic

0304-4238/%/$15.00 Copyright PII SO304-4238(96)00882-5

acid; DAA, days after anthesis;

GA, gibberellin;

0 1996 Elsevier Science B.V. All rights reserved.

IAA, indole-3-acetic

acid

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K. Kojima / Scientia Horticulturae 65 (I 996) 263-272

acid (ABA) levels increased dramatically in the styles of parthenocarpic oranges ‘Shamouti’ (Goldschmidt, 1980) and ‘Washington’ navel (Harris and Dugger, 1986), but the relationship between this ABA increase and pollination was not determined. The effect of exogenous hormonal applications on reproductive organs in citrus have been widely studied in relation to fruit set and growth. Powell and Krezdom (1977) reported that the application of gibberellin (GA) to pistils of citrus flowers increased mobilization of metabolites to fruitlets. Guardiola and Lazaro (1987) reported that the application of synthetic auxins had a direct stimulatory effect on the growth of mandarin fruitlets. The endogenous phytohormone contents of citrus reproductive organs have also been studied. Takahashi et al. (1975) determined the ABA and indole-3-acetic acid (IAA) levels during fruit set, and suggested that the hormonal balance may affect abscission of the fruits. Results of Garcia-Papi and Garcia-Martinez (1984) with seeded and seedless ‘Clementine’ mandarin fruits suggest that more than one hormone may be involved in fruit set. Hofman (1990) showed that there was little difference in ABA and GA levels in ‘Valencia’ orange fruitlets from leafy and leafless inflorescences and that those levels were fairly constant. Talon et al. (1990) determined the gibberellins (GAS) and free and conjugated ABA and IAA levels of two related species of seedless mandarins differing in their extent of parthenocarpy, and concluded that parthenocarpic ability was mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Talon et al. (1992) suggested that endogenous GA content in developing ovaries was the limiting factor controlling parthenocarpy. Overall, no clear relationship between the hormone levels and fruit set and growth has been established. In this report, changes in the levels of ABA, IAA and GA-like substances in the reproductive organs of both pollinated and unpollinated flowers of ‘Hyuganatsu’ were quantified, with the aim to determine the roles of these phytohormones in fruit set and growth.

2. Materials and methods 2.1. Plant material Nine 20-year-old ‘Hyuganatsu’ trees (Citrus tamurana [Hot-t.] ex. Tanaka) grafted on trifoliate orange (Poncirus trifoliata Raf.) growing at the Kuchinotsu Branch in Nagasaki, Japan, were used in this study. Flowers and fruitlets developing during the spring flush were used. The day the stigma could be seen through a crack in the petals was designated ‘the day of anthesis’. The flower buds were covered with paper bags and sufficient number of them were hand-pollinated with the pollen of cultivar ‘Kawachibankan’ (Citrus kawachiensis [Hot-t.] ex. Tanaka). ‘Hyuganatsu’ is a self-incompatible cultivar, and has no potential for setting parthenocarpic fruits (Yamashita, 1978). Emasculation was therefore unnecessary. Fruit retention and diameter were determined on 20 pollinated and 20 unpollinated flowers. For hormonal analysis, at least 60 organs were sampled at random from nine trees at - 5,0, 8, 15, 23 and 31 days after anthesis

K. Kojima / Scientia Horticulturae 65 (1996) 263-272

265

Expction (80% EtOH with PVP + internal standards) Filtration (paper filter) , Reduce to aqueous phase Discard Adjust to pH 2.5 residue Filtration (membrane filter)

I

Discard residue

Par;tition(petroleum ether) Partition (diethyl ether) 1

: fraction fraction

Diethyl ether extract

Sepralyte DEA HPLCbDS

Ethyl acetate extract

Micro-drop biAssay (GAS) Fig. 1. FIow diagram for the purification, fractionation and determination of phytohormones in citrus. PVP, polyvinylpyrrolidone; SIM, specific ion monitoring; ECD, electron capture detection; other abbreviations as in the text.

(DAA). After sampling, the flowers and fruits were immediately separated into their component parts. The ‘sepals’ fraction included flora1 disks, sepals and receptacles. All separation procedures were performed in a chilled room (10°C). The separated tissues were immediately weighed, frozen in liquid nitrogen, lyophilized and stored at -75°C until analysed. 2.2. Hormone purification and fractionation Fig. 1 shows the hormone fractionation procedure followed (Kojima, 1995; Kojima et al., 1995). Briefly, soluble polyvinylpyrrolidone and the internal standards ([3HlABA and [13C]IAA> were added to the sample, which was homogenized in 80% ethanol. Ethanol in the filtrates was evaporated off; the pH of the aqueous phase was adjusted to 2.5, and it was then filtered through membrane filters (0.22 p.m pore size). The aqueous filtrate was partitioned three times against petroleum ether and another three times against diethyl ether. The combined organic layers were evaporated to dryness. The dried extracts were dissolved in 25% CH,CN, and were fractionated with a high-performance liquid chromatography (HPLC) system equipped with ultraviolet and

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65 (1996) 263-272

fluorescence detectors. The temperature of the HPLC column (Inertsil ODS-2, 1.50 mm x 6.0 mm; GL Sciences, Tokyo) was maintained at 40°C. The sample was eluted with a gradient of CH,CN in 20 mM acetic acid at a flow rate of 1.6 ml min- ‘. Initially it was eluted isocratically with the starting solvent (25% CH,CN) for 12 min, then the CH,CN concentration was increased linearly to 50% until 16 min, then held at 50% until 22 min. The effluent corresponding to the retention times of ABA and IAA was collected and methylated with diazomethane. The remainder of the effluent was collected at 22 min for GA analysis. The aqueous phase obtained after the diethyl ether extraction was partitioned three times against ethyl acetate. The ethyl acetate layer was partitioned twice against 0.5 M K,I-IPO,. The pH of the aqueous phase was then adjusted to 2.5 and was partitioned twice against ethyl acetate. The ethyl acetate layer was dried over anhydrous Na,SO, overnight. The dried ethyl acetate layer and the effluent from the HPLC (except the ABA and IAA fractions) in the ether extract were combined and purified with Sepralyte DEA (Analytichem International, Harbor City, CA, USA). After purification the extract was separated by HPLC as described above. The effluent was collected for 22 min for GA analysis. 2.3. ABA and IAA analysis Determinations of ABA and IAA contents were performed according to Kojima et al. (1994b). Briefly, the methylated ABA fraction was injected into a gas chromatography (GC) system equipped with a 63Ni electron capture detector. A fused silica glass capillary column (Methyl silicone, 0.53 mm ID X 10 m; Quadrex, New Haven, CT) was used. The column temperature was programmed from 150 to 210°C at the rate of 5°C rnin- ’ . The flow rate of nitrogen carrier gas was 40 ml min- ’ . A portion of the methylated sample was injected into the HPLC system for collection of the methylated ABA. The radioactivity of the collected fraction was measured in a scintillation counter. Data were corrected according to the recovery of the internal standard. The methylated IAA fraction was injected into a GC coupled to a mass spectrometer using the splitless technique. The column used was a fused silica capillary column (CBP 1, 25 m X 0.22 mm ID, 0.25 pm film thickness; Shimadzu Inc.). The oven was programmed from 2 min at 100°C to 280°C at 30°C min- ’ and then held at 280°C for 15 min. IAA content was calculated by the methods of Cohen et al. (1986) using [ 13C6DAA as the internal standard. 2.4. Determination

of GA-like activity

The bioassay procedure used was similar to the ‘modified micro-drop bioassay’ (Nishijima and Katsura, 1989). Briefly, seeds of dwarf rice (Oryza sativa L., cultivar ‘Tan-ginbozu’) were sterilized and soaked in water with S-3307 (Sumitomo Chemical Co., Takarazuka, Hyogo, Japan) for 24 h at 30°C. When the coleoptiles were approx. 2 mm in length, seedlings were planted on 0.8% (w/v> agar. GA activity calculated from the standard curve tended to increase until the threshold for measurement of GA activity, possibly because the substances that inhibit GA activity were diluted (Kojima et al.,

K. Kojima/Scientia

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Table 1 Fresh weights (FW) and hormone concentrations of ABA, IAA and GA-like substances in parts of flower buds of ‘Hyuganatsu’ -5 days after antbesis (DAA) and of flowers 0 DAA

organ

FW and (DW) (mg per organ)

ABA’ (pm01 g - ’ FW)

DAA:

-5

0

-5

0

-5

0

-5

0

Styles c Stamen Petal

23 (5) 140 (26) 181 (21)

38 (10) 102 (25) 235 (32)

33OOf 150 13OOf 160 130+24

24OO* 150 390*25 450* 14

5052 120?3 130* 13

67i-10 240!~23 23Okl5

1.4 4.5 4.7

-d 3.5 1.1

IAA a (pm01 g- ’ FW)

GAb (pm01 g- ’ FW)

a Values for ABA and IAA levels are the means f SE of three determinations. b GA-like substances are expressed as GA, equivalents detected by the dwarf rice bioassay. ’ Styles include stigmas. d Below detectable level ( < 0.29 pmol gg ’ FW).

1995). Thus, collected samples after drying were successively diluted three times with 50% acetone. After the application of the standard (GA,) and each of the gradually diluted samples in 50% acetone, seedlings were incubated under continuous irradiation for 48 h at 32°C. Ten seedlings were used for each replication. The highest value of all the values obtained from the gradually diluted samples above the threshold level was used as the value of each GA-like substance. 3. Results 3.1. Weight and phytohormone

levels in non-persistent power parts

Table 1 shows the fresh weights and concentrations of ABA, IAA and GA-like substances in the component organs, excluding ovaries of flower buds and flowers. Styles and petals increased in both fresh and dry weight from - 5 to 0 DAA. Stamens decreased in fresh weight, but had a constant dry weight, indicating drying. The ABA concentration in styles was the highest of all floral parts examined. From - 5 to 0 DAA, the ABA concentration in stamens decreased to about one-third, while that in petals increased about threefold. The IAA concentration in stamens and petals increased about twofold. The GA-like substances in styles and petals decreased considerably. Table 2 shows the fresh weights and the concentrations of ABA, IAA and GA-like substances in styles 8 DAA. ABA and IAA concentrations in pollinated styles were

Table 2 Fresh weights (FW) and concentrations of ABA, IAA and GA-like substances in styles (includes stigmas) of ‘Hyuganatsu’ 8 days after anthesis Treatment

FW (mg per organ)

ABAl (pm01 g - ’ FW)

IAA ’ (pm01 g- ’ FW

GAb (pm01 g- ‘FW)

Unpollinated Pollinated

43 41

39OOf210 11000*1000

46*6 150f4

0.20 0.34

a Values for ABA and IAA levels are the mean f SE of three determinations. b GA-like substances are expressed as GA, equivalents detected by the dwarf rice bioassay.

K. Kojima / Scientia Honiculturae 65 f1996) 263-272

268

25

May 12

Date June2 10

18

20 3 15 5 2 10 n

5I-

q

10 ‘: 8

I

[

10-i

-o-UnpOllinated fruitlet -O-Pollinated fruitlet -a-Unpoliinated ‘sepals’ -+Pollinatexl ‘sepals’

lo-*

I

10 20 Days after anthesis

0

B

.

I

30

Fig. 2. Changes in the retention number (squares) and diameter (circles) of the reproductive organs (A), and fresh weight (FW) of reproductive organs and ‘sepals’ (B) in ‘Hyuganatsu’. ‘Sepals’ include floral disks, sepals and receptacles. ?he downward arrow shows date of pollination. The upper horizontal axis shows sampling date for hormone analysis.

about three times higher than those in unpollinated controls, while the concentration of GA-like substances was 70% higher in the pollinated styles. 3.2. Growth and abscission in fruitlets Fig. 2(A) shows changes of retention number and diameter in the reproductive organs of ‘Hyuganatsu’. The pollinated fruitlets showed no abscission from 10 to 31 DAA, but unpollinated fruitlets dropped completely by 36 DAA, confirming the lack of parthenocarpic ability in ‘Hyuganatsu’. The diameter of the pollinated fruitlets increased dramatically, while that of unpollinated fruitlets increased slightly. Fig. 2(B) shows changes of fresh weights in the reproductive organs and ‘sepals’. The fresh weight of pollinated fruitlets increased linearly on a logarithmic scale from 8 DAA, while that of unpollinated fruitlets increased at a lower rate. There was little difference in the fresh weights of the ‘sepals’ between the pollinated and unpollinated treatments. 3.3. Phytohonnones in fruitlets and ‘sepals’ Fig. 3 shows the changes of ABA, IAA and GA-like substances in the developing reproductive organs and ‘sepals’. Because the fresh weight of ‘sepals’ was nearly

K. Kojima / Scientia Horticulturae 65 11996) 263-272

Content 12 104e .May ’ ’

Date 18 May 12 I

25 June 2 10 ! 1 1

269

Concentration 25 June2

10

18

103

-O-Unpollinated fruitlet -O-Pollinated fruitlet

-a-Unpollinated ‘sepals’ +Pollinated ‘sepals’

I .

I

.

I .

I

GA-like substances

I 0

.

I

10

.

I

20

.

I

30 Days after anthesis

Fig. 3. Changes in the contents and concentrations of ABA (A, D), IAA (B, E) and GA-like substances (C, F) of the developing ovaties/fndtlets and ‘sepals’ of ‘Hyuganatsu’. GA-like substances are expressed as GA, equivalents detected by the dwarf rice bioassay. The downward arrows show date of pollination. For ABA and IAA, means of three determinations and their SE are shown; where vertical bars are not shown the limits are within the dimensions of the symbols.

constant in both pollinated and unpollinated fruitlets (Fig. 2(B)), the changing patterns of phytohormone data expressed per fresh weight are identical with those expressed per organ. In the pollinated fruitlets, ABA and IAA contents 8 DAA were more than 10 times higher than at anthesis (Fig. 3(A) and Fig. 3(B)) and ABA and IAA concentrations had the highest peak throughout the experimental period (Fig. 3(D) and Fig. 3(E)). ABA and IAA contents in unpollinated fruitlets increased to a lesser extent. In ‘sepals’, ABA concentration peaked 8 days after pollination; this concentration was the highest recorded in this experiment (Fig. 3(D)). In contrast, ABA concentration in ‘sepals’ of unpollinated flowers declined to low levels. IAA concentration in ‘sepals’ after pollination peaked at 23 DAA (Fig. 3(E)). The content of GA-like substances of pollinated

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fruitlets continued to increase until 31 DAA, but there was little increase in unpollinated fi-uitlets from 8 DAA (Fig. 3(C)). There was no difference of concentration of GA-like substances in the ‘sepals’ after pollination and unpollination, both peaking at anthesis (Fig. 3(F)).

4. Discussion Gillaspy et al. (1993) divided early development of most fruits into three phases: phase I is the period of ovary development, fertilization and fruit set; phase II is the period of cell division, seed formation and early embryo development; phase III is the period of cell expansion and embryo maturation. In citrus, cell division occurs in phases I and II (Bain, 1958). In this study, phase I corresponds to the period from 0 to 10 DAA; Yamashita (1978) reported that, during cross-pollination, some of the fast growing pollen tubes penetrate into ovaries of ‘Hyuganatsu’ 5 days after pollination and that pollinated fruitlets stop dropping from 10 DAA (Fig. 2(A)). Phase II corresponds to the period from 10 to 31 DAA; Bain (1958) reported that the period of cell division corresponds to about 1 month of early development in orange fruit. It was suggested that the sink strength of an organ is the product of sink activity per unit mass and the mass of the organ (Daie, 1985). Because the former may be related to concentration of phytohormones and the latter to content, the content of a phytohormone is almost equally as important as its concentration for organ growth (Hofman, 1990). Therefore, in this study, phytohormones are presented both per organ and per unit fresh weight. In non-persistent floral parts, the increase in ABA concentration in petals and decrease in stamens on anthesis (Table 1) confirm the results of Goldschmidt (1980). In tomato it has also been reported that ABA concentration in stamens decreased during flowering (Kojima et al., 1993). IAA concentration in stamens of ‘Hyuganatsu’ increased at anthesis, as has been reported in tomato (Kojima et al., 1994a). It is possible that the ABA decrease and IAA increase in stamens at anthesis are related to the drying of the stamens and the maturation of pollen within the anther, although the physiological roles of ABA and IAA in stamens are not clear. A marked increase in ABA concentration in the styles at flower opening has been reported in ‘Shamouti’ (Goldschmidt, 1980) and ‘Washington’ navel oranges (Harris and Dugger, 1986). However, in these studies, the relationship between ABA increase and pollination was not determined. The results in the present study clearly show that pollination induces the ABA and IAA increases in the styles (Table 2). IAA concentrations in both pollinated and unpollinated fruitlets also attained their highest value during phase I (Fig. 3(E)), similar to the increase reported in parthenocarpic mandarin (Takahashi et al., 1975; Talon et al., 1990). The endogenous levels of IAA found in ‘Hyuganatsu’ are in reasonable agreement with those reported. Guardiola and Lazaro (1987) observed that application of synthetic auxins had a direct stimulatory effect on the growth of mandarin fruitlets. Thus, this increase in IAA probably acts on preventing abscission and promoting the growth of fruitlets in phase I.

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In phase II, pollinated fruitlets which contained seeds had higher contents of IAA and GA-like substances than unpollinated fruitlets (Fig. 3(B) and Fig. 3(C)). Nitsch (1950) showed that, in strawberry, seeds provided auxin to stimulate fruit growth, while Leopold and Kriedemann (1975) pointed out that developing seeds were a notable source of GAS. It seems that the increased amounts of these hormones in pollinated fruitlets in ‘Hyuganatsu’ are provided by the developing seeds. The GA-like substances of pollinated fruitlets increased dramatically 15 DAA (Fig. 3(C)). The application of GA, to pistils of citrus flowers increased the mobilization of metabolites to the fruitlets (Powell and Krezdom, 1977), while it has been suggested that, in the parthenocarpic cultivars, the endogenous GA content in the developing ovaries is the limiting factor controlling fruit set (Talon et al., 1992). The increase of endogenous GAS found in phase II may play a major role in the growth of fruitlets of ‘Hyuganatsu’. ABA was originally discovered as an abscission-inducing hormone, but it is an open question whether ABA directly promotes the abscission process in citrus (Goren, 1993). This possibility is not supported by the present results (Fig. 2(A), Fig. 3(A) and Fig. 3(D)). In view of the foregoing, it is likely that sequential hormone action is involved in the prevention of abscission and growth of fruitlets of ‘Hyuganatsu’. IAA probably acts in phase I, while GA action predominates in phase II. Further studies on cytokinins, which play an important role in cell division, are necessary in order to understand physiological mechanism in fruit set and growth.

Acknowledgements We thank Y. Yamada and Dr. M. Yamamoto of Kuchinotsu Branch, Fruit Tree Research Station for valuable suggestions and the supply of samples, trainees of Kuchinotsu Branch for assistance in sampling, Prof. R. Rajagopal of the Royal Veterinary and Agriculture University (Denmark) for correcting the manuscript, Prof. N. Sakurai of Hiroshima University and Dr. Goto of Kuchinotsu Branch for the use of facilities, and C. Kojima for technical assistance.

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Goldschmidt, E.E., 1980. Abscisic acid in citrus flower organs as related to floral development and function. Plant Cell Physiol., 21: 193-195. Goren, R., 1993. Anatomical, physiological, and hormonal aspects of abscission in citrus. Hortic. Rev., 15: 145- 182. Guardiola, J.L. and Lazaro, E., 1987. The effect of synthetic auxins on fruit growth and anatomical development in Satsuma mandarin. Scientia Hortic., 3 1: 119- 130. Harris, M.J. and Dugger, W.M., 1986. Levels of free and conjugated abscisic acid in developing floral organs of the navel orange (Citrus sinensis IL.1 Osbeck cv Washington). Plant Physiol., 82: 1164-1166. Hofman, P.J., 1990. Abscisic acid and gibberellins in the fruitlets and leaves of ‘Valencia’ orange in relation to fruit growth and retention. Scientia Hortic., 42: 257-267. Kojima, K., Km&hi, S., Sakurai, N. and Fusao, K., 1993. Distribution of abscisic acid in different parts of the reproductive organs of tomato. Scientia Hortic., 56: 23-30. Kojima, K., Sakurai, N. and Tsurusaki, K., 1994a. Distribution of IAA within tomato flower and fruit. HortScience, 29: 1200. Kojima, K., Yamada, Y. and Yamamoto, M., 1994b. Distribution of ABA and IAA within a developing Valencia orange fruit and its parts. J. Jpn. Sot. Hortic. Sci., 63: 335-339. Kojima, K., 1995. Simultaneous measurement of ABA, IAA and GAS in citrus-role of ABA in relation to sink ability. JARQ, 29: 179-185. Kojima. K., Goto, A. and Yamada, Y., 1995. Simultaneous measurement for ABA, IAA and GAS in citrus fruits. Bull. Fruit Tree Res. Sm., 27: l-10. Leopold, A.C. and Kriedemann, P.E., 1975. Plant Growth and Development. McGraw-Hill, New York, pp. 305-336. Nishijima, T. and Katsura, N., 1989. A modified micro-drop bioassay using dwarf rice for detection of femtomol quantities of gibberellins. Plant Cell Physiol., 30: 623-627. Nitsch, J.P., 1950. Growth and morphogenesis of the strawberry as related to auxin. Am. 1. Bot., 37: 211-215. Powell, A.A. and Krezdom, A.H., 1977. Influence of fruit-setting treatment on translocation of “C-metabo lites in citrus during flowering and fruiting. J. Am. Sot. Hortic. Sci., 102: 709-714. Schwabe, W.W. and Mills, J.J., 1981. Hormones and partbenocarpic fruit set. Hortic. Abst., 51: 661-698. Takahashi, N., Yamaguchi, I., Kono, T., Igoshi, M., Hirose, K. and Suzuki, K., 1975. Characterization of plant growth substances in Cirrus unshiu and their changes in fruit development. Plant Cell Physiol., 16: 1101-1111. Talon, M., Zacarias, L. and Primo-Millo, E., 1990. Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability. Physiol. Plant, 79: 400-406. Talon, M., Zacarias, L. and Primo-Millo, E., 1992. Gibberellins and partbenocarpic ability in developing ovaries of seedless mandarins. Plant Physiol., 99: 1575- 1581. Yamashita, K., 1978. Studies on self-incompatibility of Hyuganatsu, Citrus tamuranu Hon. ex Tanaka. J. Jpn. Sot. Hortic. Sci.. 47: 188- 194.