Euphytica (2005) 144: 207–213DOI: 10.1007/s10681-005-5813-8 C© Springer 2005

Differential pollen tube growth in inbred self-compatiblealmond genotypes

Jose Manuel Alonso & Rafel Socias i Company∗Unidad de Fruticultura, Centro de Investigacion y Tecnologıa Agroalimentaria de Aragon,Apartado 727, 50080 Zaragoza, Spain (∗author for correspondence: e-mail: rsocias@aragon.es)

Received 8 November 2004; accepted 20 April 2005

Key words: almond, inbreeding, pollen tube growth, self-compatibility

Summary

Pistil and pollen behavior during self- and cross-pollinations in 10 inbred almond seedlings with self-compatiblegenotypes and self-incompatible phenotypes were studied. Pollen from these inbred seedlings was examined forthe pollen tube growth in self and non-self styles, while pistils from these seedlings were tested for their ability tosupport self and non-self pollen tube growth. Pistils and pollen of inbred genotypes were compatible with unrelatedgenotypes but the pistils were unable to support the growth of related pollen, which showed a slower tube growth rate.This may be a consequence of inbreeding, resulting in a silenced self-compatibility or cryptic self-incompatibility insome genetically self-compatible genotypes. This reaction would be a mechanism favoring crossing with unrelatedgenotypes and reducing inbreeding in future generations.

Introduction

Almond [Prunus amygdalus Batsch syn. P. dulcis(Mill.) D.A. Webb], with very few exceptions, is anobligate outcrosser, due to the presence of gameto-phytic self-incompatibility (Socias i Company et al.,1976). This incompatibility is expressed mostly by thearrest of pollen tube growth in the middle third of thestyle. However, the introduction of the S f allele fromthe Puglia almond population into most breeding pro-grammes is changing the reproductive behavior of thespecies (Socias i Company, 2002) and has raised therisk of inbreeding depression in many breeding pro-genies (Grasselly & Olivier, 1981, 1988). Inbreedingaffords the expression of lethal and deleterious genes,which could also be evident at the pistil level affectingpollen tube behavior.

Pollination efficiency as measured by the pollentube growth is an important selection criterion in al-mond (Socias i Company & Alonso, 2004) becausein commercial self-compatible cultivars self-pollenmust show a similar behavior as foreign pollen. Dis-crepant results have been reported when observing

pollen tube growth after self- and cross-pollinationsin self-incompatible cultivars (Certal et al., 2002; Egeaet al., 2001; Eti et al.,1994; Pimienta & Polito 1983;Socias i Company, 1982, 2001; Vezvaei, 1997), in self-compatible cultivars (Cousin & El Maataoui, 1998;Dicenta et al., 2001; Godini, 1981; Oukabli et al., 2000;Socias i Company, 2001; Socias i Company & Felipe,1992; Vasilakakis & Porlingis, 1984) and in breedinglines (Ben Njima & Socias i Company, 1995; Sociasi Company et al., 1976; Socias i Company & Felipe,1987), thus raising the question of the real pollinationefficiency of different genotypes.

Previous observations in offspring from reciprocalcrosses ‘Ferralise’ (S1S3) × ‘Tuono’ (S1S f ), where theinbreeding coefficient of 0.125 is expected in all plants,have shown different levels of inbreeding depression,including a scale of vigor from dwarf to very vigor-ous plants. Inbreeding has been shown to affect boththe morphology and the physiology, including vigorreduction, a decrease in the number of flowers, and aconsiderable sterility of flowers (Grasselly & Olivier,1988). Retarded growth of the self-pollen tube un-der laboratory conditions observed in some genetically

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self-compatible plants of these populations may be an-other symptom of inbreeding depression.

Retarded growth of the self-pollen tube must bedue to another mechanism masking the genetic ef-fect of the S f allele on the pollen tube growth. Thus,our objective was to evaluate the pollination efficiencyof these genotypes by comparing the ability of theirpollen to grow in self- and non-self-pistils, as wellas the ability of their pistils to support the growth ofself- and non-self-pollen, in order to elucidate if thepollen–pistil interaction in these inbred genotypes isdependent on more factors than the presence of the S f

allele.

Material and methods

Plant material

Ten plants from reciprocal crosses ‘Ferralise’ בTuono’ and ‘Tuono’ × ‘Ferralise’ were selectedbecause of their self-compatible genotype and self-incompatible phenotype. These plants are referred toas seedlings because they were raised from seed, as op-posed to the usual vegetative propagation in fruit trees,and the term does not define their stages of maturity.The genotypes of these seedlings were determined bythe analyses of their stylar S-RNases (Boskovic et al.,1998) and by partial amplification of the S-genes byPCR (Channuntapitat et al., 2001, 2003). Their pheno-type was assessed by microscopic observation of pollentube growth after self-pollination (data not shown). Theorigin and S genotype of the seedlings are shown inTable 1.

‘Ferralise’ (S1S3) is an inbred almond cultivar, withthe inbreeding coefficient of 0.25 (Lansari et al., 1994),having originated from a cross of two full sibs, ‘Fer-raduel’ (S1S4) and ‘Ferragnes’ (S1S3) (Crossa-Raynaud& Grasselly, 1985). ‘Tuono’ (S1S f ) is a self-compatiblecultivar (Crossa-Raynaud & Grasselly, 1985) with un-known pedigree. ‘Ferralise’ and ‘Tuono’ share the S1

allele, which could be identical by descent becausethe S1 allele of ‘Ferralise’ has been inherited from

Table 1. Origin and genotype of the seedlings studied

Family S-genotype Tested seedlings Self-compatible full sibs

‘Ferralise’ S1S3 × ‘Tuono’ S1S f S1S f ‘L-2-77’, ‘L-3-47’, ‘L-2-79’, ‘L-2-88’, ‘L-3-18’ ‘L-3-37’

S3S f ‘L-3-32’, ‘L-3-40’ ‘L-3-63’

‘Tuono’ S1S f × ‘Ferralise’ S1S3 S3S f ‘M-2-57’, ‘M-2-60’, ‘M-2-70’ ‘M-2-75’

‘Cristomorto’ (S1S2), a cultivar which originated inthe same Italian region of Puglia as ‘Tuono’. As aconsequence, inbreeding is expected in the families ob-tained by crossing these two cultivars.

The behavior of these 10 seedlings was com-pared with that of three additional seedlings from thesame populations with identical S genotypes (Table 1).These three seedlings showed no inbreeding symp-toms and were chosen as controls because of their self-compatible genotypes and phenotypes, as assessed byconsistent pollen tube growth and the presence of alarge number of pollen tubes at the style base afterself-pollination.

Additionally, two commercial self-incompatiblecultivars unrelated to the parents of the seedlingswere included: the early blooming Spanish ‘Marcona’(S11S12 genotype; Boskovic et al., 1998), which couldonly be used as pollen donor; and the late bloom-ing Californian ‘Titan’ (S8S14 genotype, Ortega et al.,2005).

Pollination treatments

Six different pollination treatments were made underlaboratory conditions. One was self-pollination, to con-firm the previous results (Alonso & Socias i Company,2005). Each seedling was pollinated with pollen fromthree different sources: ‘Marcona’; ‘Titan’; and fromanother full sib with the same S-genotype and self-compatible phenotype. This was done to test the abil-ity of the pistils to support the growth of foreign pollentubes of unrelated and related pollinators. The two othertreatments also involved cross-pollinations by pollen ofeach seedling on flowers of ‘Titan’ and of another fullsib with the same S genotype. This tested the ability ofthe pollen of each seedling to grow in foreign pistils,both on unrelated and related genotypes.

Methods

For pollen collection, unopened flowers at stage D(Felipe, 1977) were taken from all seedlings and pollendonors. The anthers were removed from flowers and

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left to dry on paper trays for 48 h. Pollen samples werestored in aliquot parts in a freezer (at 2–4 ◦C) untiluse. For laboratory pollinations, flowers were emascu-lated and placed in a water-filled-tray supported by asemi-rigid plastic mesh allowing contact of the flowerpeduncles with water in the tray. Whenever possible,pistils were prepared in blocks of 20 for each pollina-tion treatment and each block was pollinated 48 h afteremasculation. Trays were kept in controlled temper-ature chambers at 12 ◦C. Pistils were collected 5 daysafter pollination, a time sufficient for compatible pollentubes to reach the ovaries at 12 ◦C (Socias i Companyet al., 1976). Pistils kept under these conditions main-tain viability as good as that in the field, and supportpollen tube growth with the same compatibility be-havior as under natural conditions (Socias i Company,2001). Pistils were autoclaved for 20 min at a pressureof 1.2 kg cm−2 in small glass bottles containing 5 ml ofa 5% solution of Na2SO3 and maintained at 4 ◦C untilobservation.

For microscopic observation, pistils were preparedaccording to Socias i Company (1979): the outer partsof the pistils and the ovary were removed, leavingonly the transmitting tissue through which pollen tubesgrow. The pollen tube growth was assessed on squashedpreparations observed under a UV microscope by thefluorescence of the callose deposits of pollen tubesstained with a solution of 0.1% aniline blue in 0.1 Npotassium phosphate (Linskens & Esser, 1957). Thenumber of pollen tubes in each style base was recordedusing a Leitz Ortholux II microscope (Leitz, Wetzlar,Germany) with an Osram HBO 200 W/4 mercury lamp(Osram GmbH, Munich, Germany).

A minimum of 12 pistils were observed for eachtreatment. Single pistils without pollen on the stigmaor with very poor pollen germination were discarded.The numbers of pollen tubes were estimated whenthey reached the style base. An analysis of variancewas applied to the data using SAS V8, with angulartransformation for the analysis of percentages (SAS,1989).

Results

Inbreeding symptoms

As expected, some seedlings of the populations understudy showed symptoms of inbreeding such as dwarf-ing, reduction of the number of flowers, and a highpercentage of sterile flowers, thus making pollination

studies on them difficult. Flower sterility was dueto different levels of pistil sterility (Socias i Com-pany, 1983). However, these flowers produced normalamounts of viable pollen (data not shown). Pistil steril-ity could be an expression of inbreeding (Charlesworth& Charlesworth, 1987; Lande & Schemske, 1985) inthis highly heterozygous species (Kester et al., 1991),where gametophytic self-incompatibility and tradi-tional seed propagation have favored a high level ofheterozygosity (McCubbin & Kao, 2000) and main-tained, most often not expressed, a large number oflethal and deleterious genes.

Self-pollinations

Self-pollinations confirmed the erratic pollen tubegrowth of these inbred seedlings (Tables 2 and 3). Onlytwo seedlings (‘M-2-57’ and ‘M-2-60’) had pollentubes at the base of all pistils observed, whereas inthe remaining seedlings the percentages of pistils withpollen tubes at their base ranged from 17% (‘L-3-32’)to 67% (‘L-3-18’, ‘L-3-47’, and ‘M-2-70’). These per-centages are higher than those in previous observationsand may be due to a delay in sampling, 5 days afterpollination instead of 4 days. However, the number ofpollen tubes reaching the style base was very low, with amaximum of 1.2 pollen tubes in ‘M-2-60’. This is muchlower than the mean observed in the full-sibs with self-compatible phenotype, which was about three pollentubes at the style base (Alonso & Socias i Company,2005).

Cross-pollinations on pistils of the seedlings

A sharp difference was observed between pollinationsby unrelated pollen and by pollen with the same S geno-type (Tables 2 and 3). Pollinations by ‘Marcona’ and‘Titan’ resulted in very high percentages of styles withpollen tubes at their bases (93 and 96% on the aver-age) and very high numbers of pollen tubes at stylebase (on average, 5.6 and 7.8), confirming full compat-ibility of these pollinations. However, pollinations bypollen from seedlings with the same S genotype gavethe same level of erratic results as self-pollinations,with lower percentages of pistils with pollen tubes attheir bases, 23 as compared to 59% (Tables 2 and 4),and a similar number of pollen tubes, 0.7 (Tables 3 and5). The differences in the behavior of different pollenin these pistils (Table 4) were highly significant.

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Table 2. Percentage of pistils with pollen tubes at their base for the different pollination treatments

Pollination by Pollination on

Origin S-genotype Plant Self-pollination ‘Marcona’ ‘Titan’ ‘L-3-37’ S1S f ‘Titan’ ‘L-3-37’ S1S f

Ferralise × Tuono S1S f ‘L-2-77’ 47 94 100 0 53 100

‘L-3-47’ 67 100 100 37 79 100

‘Marcona’ ‘Titan’ ‘L-3-63’ S3S f ‘Titan’ ‘L-3-63’ S3S f

S3S f ‘L-2-79’ 50 92 100 21 73 100

‘L-2-88’ 25 83 92 0 80 92

‘L-3-18’ 67 90 100 100 80 100

‘L-3-32’ 17 100 100 0 82 100

‘L-3-40’ 53 100 71 36 81 100

‘Marcona’ ‘Titan’ ‘M-2-75’ S3S f ‘Titan’ ‘M-2-75’ S3S f

Tuono × Ferralise S3S f ‘M-2-57’ 100 100 100 26 47 92

‘M-2-60’ 100 78 92 20 41 100

‘M-2-70’ 67 89 92 0 50 100

Table 3. Mean number of pollen tubes at the style base for the different pollination treatments

Pollination by Pollination on

Origin S-genotype Plant Self-pollination ‘Marcona’ ‘Titan’ ‘L-3-37’ S1S f ‘Titan’ ‘L-3-37’ S1S f

Ferralise × Tuono S1S f ‘L-2-77’ 0.6 4.1 8.8 0 1 6.6

‘L-3-47’ 0.8 9 8.2 0.7 2.5 7

‘Marcona’ ‘Titan’ ‘L-3-63’ S3S f ‘Titan’ ‘L-3-63’ S3S f

S3S f ‘L-2-79’ 0.5 3.3 >10 1.4 1.6 >10

‘L-2-88’ 0.3 2.5 7.2 0 1.8 8.5

‘L-3-18’ 0.8 3.5 9.8 3.7 1.6 4.4

‘L-3-32’ 0.2 9.3 7.8 0 3 9.2

‘L-3-40’ 0.5 7.7 2.2 0.6 5 >10

‘Marcona’ ‘Titan’ ‘M-2-75’ S3S f ‘Titan’ ‘M-2-75’ S3S f

Tuono × Ferralise S3S f ‘M-2-57’ 1 5.6 9 0.5 0.7 8

‘M-2-60’ 1.2 4 7.7 0.6 1.9 8.6

‘M-2-70’ 0.7 6.5 7.7 0 1.3 7.6

Table 4. Pistil behavior of the inbred seedlings as shown by the mean percentage of pistils with pollen tubes at their base and mean number ofpollen tubes at the pistil base for the different pollination treatments

Number of tested Mean of percentage of pistils with Mean number of pollenPollen treatment seedlings pollen tubes at their basea tubes at the pistil basea

‘Titan’ 10 95a 7.8a

‘Marcona’ 10 93a 5.6b

Selfed 10 59b 0.7c

Same S-genotype 10 23c 0.7c

aValues followed by different letters are significantly different at P > 0.05 by Duncan’s test.

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Table 5. Pollen behavior from the inbred seedlings as shown by the mean percentage of the differentpollinated pistils by pollen of the tested seedlings with pollen tubes at their base and the mean number ofpollen tubes at the pistil base

Number of tested Mean of percentage of pistils with Mean number of pollenPollinated pistils seedlings pollen tubes at their basea tubes at the pistil basea

Same S-genotype 10 98a 8.0a

‘Titan’ 10 66b 1.9b

Selfed 10 59b 0.7c

aValues followed by different letters are significantly different at P > 0.05 by Duncan’s test.

Cross-pollinations by pollen of the seedlings

Pollen behavior on unrelated pistils or on pistils withthe same S genotype was vigorous (Tables 2 and 3). Onthese latter, and unlike the reciprocal cross, pollen tubegrowth was very vigorous with 98% of pistils showingpollen tubes at their bases and high average numbers ofpollen tubes. However, pollen tube growth on ‘Titan’pistils was poorer than expected in a theoretically cross-compatible pollination, with one-third of the pistils notshowing pollen tubes at their bases (Tables 2 and 4),and with the average number of 1.9 tubes when theydid show (Table 3 and 5).

Discussion

Pistil behavior

The low percentage of pistils with pollen tubes at theirbases and the low number of pollen tubes after self-pollination in the inbred seedlings studied here are notdue to the inability of pistils to sustain the pollen tubegrowth. Unrelated pollen tubes were able to reach thestyle bases in high numbers in most pistils. Thus, thephysiological self-incompatibility observed could notbe due to a deficient reserve accumulation in the pis-til transmitting tissue (de Graaf et al., 2001), becausepistils were able to release all the nourishment forattraction, guidance, and signalling of foreign pollentubes (Cheung, 1996a,b; Cheung et al., 1995). Con-sequently, inbreeding was not affecting pistil behav-ior. Pistils supported an excellent growth of ‘Marcona’and ‘Titan’ pollen in terms of pollination efficiency,but this was only expressed against pollen of the samegenotype, thus showing a behavior similar to crypticself-incompatibility (Bateman, 1956).

Compared to our previous observations in labora-tory self-pollinations (Alonso & Socias i Company,2005), a higher percentage of pistils showed pollentubes at their bases, and with higher numbers of tubes.

This increase may be due to a delay in pistil sampling,5 days instead of four. A longer period of pollen tubegrowth may account for some increase in the averagenumbers of tubes reaching the bottoms of pistils. How-ever, in unrelated pollinations, the percentages of pis-tils with pollen tubes and the numbers of tubes werealready very high. This difference may be explainedby a slower growth rate of the self and related pollentubes, as compared to the unrelated ones. Self and re-lated pollen tubes were thinner and showed less thanthe unrelated pollen tubes, thus appearing less vigor-ous. This is similar to what has been seen in partialself-compatibility (Socias i Company & Felipe, 1988).

Pollen behavior

Pollen from the inbred seedlings showed a slowergrowth rate only when grown in its own pistils. When itwas applied to stigmas from plants without inbreedingsymptoms, both of the related and unrelated genotypes,the pollen tube growth was normal. In fact, the besttreatment (Table 5) was cross-pollination onto the pis-tils with the same S genotype, when theoretically thispollination is only semi-compatible because only thepollen grains carrying the S f allele should be able togrow (Socias i Company & Alonso, 2004). Surpris-ingly, cross-pollination on unrelated pistils (‘Titan’)gave only average results in terms of percentages ofpistils with pollen tubes and of pollen tube numbers.In spite of these results, pollen from the tested inbredgenotypes showed a good ability to develop normallyand accomplish an efficient pollination.

Inbreeding effects

Both the pistil and the pollen of the inbred geno-types studied were able to produce efficient pol-linations when matched with unrelated genotypes.Consequently, the pollination failures observed inthe experiments could be due to the pistil–pollen

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interaction in inbred pistils, where a negative effect forrelated pollen could be activated. The negative reac-tion was expressed against self-pollen and also againstrelated pollen with the same genotype, whereas it wasnot observed in the control seedlings showing highlyself-compatible phenotypes (Table 1) that were able toefficiently support the pollen tube growth of the testedinbred genotypes.

The observed differences may also affect ovule de-velopment and viability, and induce a late-acting self-incompatibility (Seavey & Bawa, 1986). A general re-duction in fruit set was observed after self-pollinationin one inbred selection (Socias i Company et al., 2004)as well as a higher embryo set after cross-pollinationthan after self-pollination in ‘Tuono’, probably due toa delay in the development of the female gametophyte(Oukabli et al., 2000).

These results confirm that even the genotypes pos-sessing the S f allele show a self-incompatible phe-notype, indicated by a very low number of pollentubes at the bases of a low number of pistils, as wellas by a slower pollen tube growth rate as comparedto compatible pollinations, whether in self- or cross-pollinations. The slower tube growth rate of self andrelated pollen could be the expression of a silenced self-compatibility or cryptic self-incompatibility in geneticinbred self-compatible genotypes, which would favorcrossing with unrelated genotypes through pollen se-lection due to a differential pollen tube growth. Withthis mechanism, further inbreeding would be avoidedand heterosis restored in the offspring of these pseudo-self-compatible genotypes. Furthermore, these resultsdemonstrate the need to avoid inbreeding in almondbreeding programmes, an increasing risk for a tradi-tionally obligate outcrosser such as almond, a speciesshowing a high risk of inbreeding depression due tothe utilization of a reduced number of parents in theseprogrammes, mainly as donors for self-compatibility.

Acknowledgments

This work was supported by the grant AGL2001-1054-C03-02 of the Spanish CICYT. Technical assistance byJ. Bubal, A. Escota, J.M. Anson, and M.T. Espiau ishighly appreciated.

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