Self-fertilization in homozygous and heterozygous self-compatible almonds

Scientia Horticulturae 109 (2006) 288–292 www.elsevier.com/locate/scihorti

Self-fertilization in homozygous and heterozygous self-compatible almonds E. Ortega *, F. Dicenta Dpto. Mejora Vegetal, CEBAS-CSIC, Apdo. correos 164, E-30100 Espinardo, Murcia, Spain Received 7 November 2005; received in revised form 5 April 2006; accepted 18 April 2006

Abstract In homozygous self-compatible genotypes 100% of the pollen grains are potentially able to grow through their own pistil, and thus the rate of self-fertilization could be higher than in heterozygous self-compatible genotypes. To evaluate the advantages of growing homozygous selfcompatible almonds, pollen tube growth along the pistil at different times following self-pollination, and fruit set were studied in four homozygous and four heterozygous self-compatible seedlings. The results showed important differences between homozygous and heterozygous individuals for the percentages of pollen tubes in the third section of the style at 24 and 48 h, the pollen tube growth rate being higher in the homozygous. Twentyfour hours following self-pollination only the homozygous individuals showed pollen tubes in the ovary. However, at 72 and 96 h those values were similar for both genotypes, suggesting that space and availability of nutrients become the main limiting factors, overcoming the genetic interactions between pollen and pistil. In general, fruit set was similar in homozygous and heterozygous individuals. Interestingly, one of the homozygous individuals showed problems of fruit development, which might be explained by its inbred origin. # 2006 Elsevier B.V. All rights reserved. Keywords: Almond; Self-compatibility; Self-fertilization; Prunus dulcis; Pollen tube growth; Fruit set

1. Introduction Almond [Prunus dulcis (Mill.) D.A. Webb] is a selfincompatible species with a gametophytic system controlled by a multi-allelic S locus (Gagnard, 1954). In this type of system, the self-incompatibility alleles (Si) are expressed in the style as ribonucleases (S-RNases), which specifically reject those pollen tubes with the same S genotype (Kao and McCubbin, 1996). However, self-compatible almond cultivars have been described in the Italian region of Apulia (Stazione Agraria Sperimentale di Bari, 1957; Jaouani, 1973; Grasselly and Olivier, 1976; Godini, 1977; Godini et al., 1992; Palasciano and Godini, 2001), Portugal (Almeida, 1945), and India (Kumar and Kumar, 2000). To explain the origin of self-compatibility in almond cultivars from Apulia two different hypotheses have been proposed. Grasselly and Olivier (1976) suggested that some of those cultivars had a mutation, which was transmitted to other cultivars within the Apulian population during the

* Corresponding author. Tel.: +34 968396237; fax: +34 968396213. E-mail address: [email protected] (E. Ortega). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.04.017

selection process conducted by the growers. Other authors pointed out that self-compatibility was probably transmitted by natural hybridization of the cultivated almond species with the self-compatible wild species [Prunus webbii (Spach) Vierh.] (Godini, 1979; Socias i Company, 1984; Reina et al., 1986; Yamashita et al., 1987). More recently, Bosˇkovic´ et al. (1999) demonstrated that self-compatibility in almond is due to the absence of ribonuclease activity in the style, and suggested a mutation either in almond or in P. webbii as its origin. Growing self-compatible almonds in single cultivar orchards would be desirable, as lower costs in crop management and higher yields are expected. For this reason, self-compatibility is one of the main objectives in almond breeding programmes (Socias i Company and Felipe, 1988; Duval and Grasselly, 1994; Godini and Palasciano, 1997; Vargas et al., 1997; Gradziel and Kester, 1998; Dicenta et al., 2002a). Experimentally, this characteristic has been introduced in almond by hybridization with other Prunus species such as peach [P. persica (L.) Batsch] (Anderson, 1972; Zaiger, 1978) and the wild Prunus species P. webbii, P. mira and P. fenzliana (Gradziel and Kester, 1998; Gradziel et al., 2001). However, most breeding programmes have achieved this aim by crossing a self-compatible cultivar and a self-incompatible cultivar of

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good agronomic characteristics (Felipe and Socias i Company, 1987; INRA, 1991; Socias i Company and Felipe, 1999; Duval, 1999; Egea et al., 2000). For this reason, the self-compatible individuals selected in such programmes are heterozygous for self-compatibility (SiSf ). Homozygous self-compatible almond cultivars (Sf Sf ) are of great interest as in these genotypes 100% of the pollen grains are potentially able to grow down their own pistil following self-pollination, unlike the heterozygous in which 50% of selfpollen grains are rejected in the style. In addition, homozygous self-compatible genotypes could be used in breeding to assure self-compatibility in the progeny (Ortega and Dicenta, 2003). In this work we have studied pollen tube growth and fruit set following self-pollination of homozygous and heterozygous self-compatible almond cultivars to evaluate the possible advantages of homozygous self-compatible cultivars regarding self-fertilization, and thus the convenience of growing single cultivar orchards.

germinated pollen grains in vitro, following the procedure indicated by Remy (1953). In all cases the percentage of germination was higher than 65% (data not shown). The pistils were then self-pollinated using a paintbrush and kept again under the same controlled conditions. A sample of 10 pistils per individual was collected from the trays at 24, 48, 72 and 96 h after pollination, then fixed in FAA solution and prepared for fluorescence microscopy observation as indicated in Ortega et al. (2002). For each pistil the number of germinated pollen grains in the stigma, the number of pollen tubes in the first, second and third section of the style and the number of pollen tubes in the ovary were determined using an Olympus BH2 microscope with a UV light-adapted system BH2-RFL-T2, with illumination from an Osram HBO 100 W/2 mercury lamp. Pollen tubes at each section of the style were expressed as a percentage of the number of germinated pollen grains in the stigma for data analysis.

2. Materials and methods

2.3. Fruit set determination

2.1. Plant material

For each individual 60–100 flower buds at ‘D’ stage were emasculated on branches of these trees and then self-pollinated by hand. Self-pollinated branches on each tree were labelled and the percentages of initial and final fruit set were determined 30 and 60 days after pollination, respectively.

Pollen tube growth and fruit set were studied in four homozygous (‘A1473’, ‘A2198’, ‘A2206’, ‘A2416’) and four heterozygous (‘A1177’, ‘A1194’, ‘A1775’, ‘A2321’) selfcompatible almond seedlings following self-pollination by hand. The seedlings were obtained in 1993 by bagging branches of self-compatible almond selections from CEBAS-CSIC breeding programme (Murcia, Spain). The S genotype of these individuals was determined by non-equilibrium pH gradient electrofocusing (NEPHGE) of stylar ribonucleases (Dicenta et al., 2002b). Table 1 summarises information on the parentage and the S genotype of these seedlings. 2.2. Fluorescence microscopy A sample of 40 flowers at stage ‘D’ (Felipe, 1977) was collected for each individual. The same day the flowers were emasculated, the pistils were placed in trays with the calyx inserted on wet floral foam and kept under controlled conditions (22  2 8C, 12 h photoperiod and 75–80% relative humidity), and the anthers were placed on Petri dishes at room temperature for dehiscence. After 24 h, pollen viability was determined for each individual by observing the percentage of

2.4. Statistical analysis Differences between self-compatible genotypes (homozygous and heterozygous) and between individuals within each genotype were analysed following a nested general linear model procedure using SAS software (SAS Institute, 1989). In order to homogenize variances, the percentages of pollen tubes in each section of the style and in the ovary were previously transformed by calculating the angular transformation (arc sin value of the square root), and mean values were analysed by Duncan’s multiple range test. 3. Results and discussion 3.1. Pollen tube growth The analysis of variance (data not shown) indicated the presence or absence of differences for the number of pollen

Table 1 Parentage and S genotype (determined by NEPHGE) of the studied self-compatible almond seedlings F1 parentage

F2 parentage

Seedling (F2)

S genotype (NEPHGE)

‘Ferragne`s’  ‘Genco’ ‘Tuono’  ‘Genco’ ‘Tuono’  ‘Genco’ ‘Genco’  ‘Tuono’ ‘Tuono’  ‘Ferragne`s’ ‘Tuono’  ‘Ferragne`s’ ‘Ferragne`s’  ‘Tuono’ ‘Genco’  ‘Ferragne`s’

C1123  C1123 C1328  C1328 C1328  C1328 C3105  C3105 C1010  C1010 C1011  C1011 C1192  C1192 C2062  C2062

A1473 A2198 A2206 A2416 A1177 A1194 A1775 A2321

SfSf SfSf SfSf SfSf S3Sf S3Sf S1Sf S1Sf

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grains germinated in the stigma between homozygous and heterozygous self-compatible individuals, and between individuals of the same genotype, depending on the time following self-pollination. Within each genotype differences between the four individuals were observed, although they disappeared with time and along the style. Regarding differences between homozygous and heterozygous genotypes, the number of germinated pollen grains in the stigma was in all cases higher for the homozygous than for the heterozygous individuals (Table 2), although only at 24 and 96 h differences were significant. This could be explained by the action of S-RNases in the stigma in the heterozygous individuals, since the number of pollen grains placed on the stigma was theoretically similar in all cases (each stigma was pollinated with a paintbrush completely saturated with pollen), and as previously indicated pollen viability was high in all cases. Significant differences between homozygous and heterozygous individuals were also observed for the percentage of pollen tubes in the third section of the style and in the ovary at 24 and 48 h. At 72 h differences were significant in the third section of the style, but not in the ovary. However, from 72 h differences were not observed, since in almond most pollen tubes reach the ovary at this time. The mean values indicated in Table 2 corroborate these results. Thus, at 24 h homozygous self-compatible individuals showed significantly higher values than the heterozygous for the percentage of pollen tubes in the third section of the style (9/0.3) and the ovary (4/0). These differences were also observed at 48 h (14/8 in the third section of the style and 7/3 in the ovary), but disappeared after 72 h, when they were not significant. These results agree with those observed by Socias i Company et al. (1976), Certal et al. (2002) and Dicenta et al. (2002c) following self-pollination and cross-pollination of self-compatible and self-incompatible almond genotypes, and could be explained by a lower growth rate of the incompatible pollen (which corresponds to 50% in the case of the heterozygous individuals) against the compatible pollen. In addition, in the case of the heterozygous individuals, the reduction of the number of incompatible tubes at each section of the style may consequently result in a slower growth of the compatible tubes (Sf ), since pollen tube growth rate may

depend on the total number of pollen tubes in the transmitting tissue, as observed by Visser et al. (1988) in various rosaceous species. Under particular weather conditions that significantly reduce ovule viability, such as high temperatures (Williams, 1966; Tonutti et al., 1991; Burgos et al., 1991; Tromp and Borsboom, 1994; Gonza´lez et al., 1995), and due to the advanced maturity stage of almond ovules at anthesis (Egea and Burgos, 2000), the higher pollen tube growth rate observed here for the homozygous self-compatible individuals would imply higher possibilities of successful self-fertilization of those genotypes against the heterozygous self-compatible ones. On the other hand, the fact that between the homozygous individuals significant differences for the number of pollen tubes in the ovary were observed, suggests that other genetic interactions (pollen–pollen and pollen–pistil) apart from the self-incompatibility reaction may affect self-fertilization, as proposed by Hormaza and Herrero (1996, 1999) for sweet cherry (P. avium L.). According to that hypothesis, pollen grains will have different aptitude to reach the ovary and likewise some pistils would be more suitable than others for pollen tube growth. The absence of differences between homozygous and heterozygous individuals after 72 h of self-pollination seems to indicate that once pollen grains germinate on the stigma and grow along the style, the space and nutrients available become the main limiting factors, overcoming genetic interactions between pollen and pistil including self-incompatibility (Ortega et al., 2002). For this reason, although self-pollen in the homozygous genotypes seems to grow faster than in the heterozygous, the number of pollen tubes in the ovary was similar for both genotypes at 72 and 96 h. Moreover, it has to be considered that this study was performed under optimal conditions for pollen tube growth. Thus, self-pollination by hand assures enough and similar amounts of pollen grains on the stigma for all individuals, and the controlled temperature (22  1 8C) was within the optimal temperature range (15– 25 8C) for pollen tube growth in almond (Socias i Company et al., 1976). However, under field conditions a lower temperature regime may occur and then a slower pollen tube growth rate would be expected.

Table 2 Mean number of germinated pollen grains in the stigma and percentage of pollen tubes at each section of the style and ovary at different times following selfpollination by hand in homozygous (Hm) and heterozygous (Ht) self-compatible almond genotypes Genotype

Time 24 h

48 h

Hm Stigma Style-1 Style-2 Style-3 Ovary

90 a 40 21 9a 4a

a

72 h

96 h

Ht

Hm

Ht

Hm

Ht

Hm

Ht

54b 36 17 0.3 b 0b

83 42 28 14 a 7a

73 44 28 8b 3b

79 47 33 17 a 8

73 50 32 13 b 8

81 a 51 38 21 9

67 b 55 38 17 11

a For the stigma, each section of the style and the ovary, different letters mean significant differences between genotypes at each time, according to Duncan’s multiple range test (P = 0.05).

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Table 3 Percentage of penetrated ovaries at different times after self-pollination under controlled conditions in the laboratory, and percentage of initial and final fruit set following self-pollination by hand under field conditions in homozygous and heterozygous self-compatible almond genotypes Sf genotype

Homozygous (SfSf)

Heterozygous (SiSf)

Individual

Laboratory

Field

Penetrated ovaries (%)

Number of pollinated flowers

24 h

48 h

72 h

96 h

A1473 A2198 A2206 A2416

0 89 100 44

78 100 100 100

89 100 100 100

80 100 100 100

Mean

58

95

97

A1177 A1194 A1775 A2321

0 0 0 0

100 56 67 0

Mean

0

56

Fruit set (%) Initial

Final

73 81 58 82

25 11 38 16

25 0 34 7

95

74

23

17

89 90 100 80

100 100 100 100

98 75 58 104

26 69 36 13

26 67 31 13

90

100

84

36

34

3.2. Percentage of penetrated ovaries and fruit set Table 3 shows the percentage of penetrated ovaries at 24, 48, 72 and 96 h following self-pollination under laboratory conditions, and the percentage of fruit set at 30 and 60 days after self-pollination by hand, corresponding to the initial and final fruit set, respectively, for the homozygous and heterozygous self-compatible individuals studied. Differences for the percentage of penetrated ovaries were observed between individuals of each genotype, although in general the pollen tubes reached the ovary faster in the homozygous than in the heterozygous individuals. Three of the homozygous individuals showed a high percentage of penetrated ovaries 24 h after self-pollination (100% in the case of ‘A2206’) and all at 48 h. However, in the case of the heterozygous individuals there were no pollen tubes in the ovary at 24 h. Despite this delay, after 48 h differences decrease and finally disappeared at 96 h, with values of 100% in almost all cases. Despite these results, the percentages of fruit set following self-pollination were higher for the heterozygous than the homozygous genotypes. Differences were less important for the initial fruit set (36% against 23%), but higher for the final fruit set (34% and 17%). These differences are mainly due to the low fruit set of the homozygous individuals ‘A2198’ and ‘A2416’, and the high fruit set of the heterozygous individual ‘A1194’. Fruit set observed in the other homozygous individuals did not highly differ from heterozygous individuals. In the particular case of the homozygous individual ‘A2198’, the low initial fruit set and later drop of all the fruits seems to indicate problems for a normal fruit development following self-pollination. However, this individual showed an acceptable fruit set following open pollination (data not shown). These problems could be an expression of the high level of inbreeding in this particular individual, as the parents of the selection from which it was obtained are the self-compatible cultivars ‘Tuono’ and ‘Genco’ (both originated in Apulia).

As observed in other species, fruit drop is sometimes associated with embryo abortion (Crane and Iwakiri, 1980; Furukawa and Bukovac, 1989), which in addition has been attributed to deficiencies in endosperm development (Bradley and Crane, 1975). In the self-compatible almond cultivar ‘Tuono’, Oukabli et al. (2000) observed degeneration of the endosperm and subsequent embryo abortion following selffertilization. These results were explained as a consequence of inbreeding effects at post-zygotic stage. However, in prezygotic stage differences were not observed for pollen tube growth along the pistil following self-pollination and crosspollination. These results greatly disagree with those of Alonso and Socias i Company (2005), who found that inbred selfcompatible almonds had a very low number of pollen tubes at the base of their styles following self-pollination, and also a very slow growth rate. 4. Conclusion We conclude that pollen tube growth rate following selfpollination by hand was higher in the homozygous than in the heterozygous individuals. However, 72 h after self-pollination the percentages of pollen tubes in the ovary were similar for both genotypes. Consequently, the efficiency of fruit set following self-pollination does not seem to be related to homozygous or heterozygous self-compatibility genotypes, since important differences between them where not observed. Some homozygous individuals showed problems related to fruit development, which could be an expression of inbreeding. Acknowledgement This work has been financed by the project ‘‘Mejora gene´tica del almendro’’ (AGF98-0211-C03-02) from the ‘‘Plan Nacional de I+D’’ of the Spanish Ministry of Education and Culture.

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