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EuphyticaInternational Journal of PlantBreeding ISSN 0014-2336Volume 178Number 2 Euphytica (2010) 178:203-214DOI 10.1007/s10681-010-0298-5

Pollen morphology as fertility predictor inhybrid tea roses

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Pollen morphology as fertility predictor in hybrid tea roses

Luca Pipino • Marie-Christine Van Labeke •

Andrea Mansuino • Valentina Scariot •

Annalisa Giovannini • Leen Leus

Received: 23 February 2010 / Accepted: 1 November 2010 / Published online: 15 November 2010

� Springer Science+Business Media B.V. 2010

Abstract Fertility of hybrid tea roses is often

reduced due to their interspecific origin but also to

intensive inbreeding. New genotypes used as pollen

donors represent an economic risk for a breeding

programme, as their influence on seed production is

unknown. In this study 11 cut rose genotypes were

selected from a company database as high fertile or

low fertile male parents, according to the number of

seeds per hybridisation. Pollen morphology and in

vitro germination of the selected genotypes were

characterised. Pollen was either small (mean diameter

\30 lm), shrunken, and irregular (abnormal), or

large (mean diameter[30 lm), elliptical and crossed

by furrows (normal). High correlations were found

between the number of seeds produced per hybrid-

isation and the pollen diameter (r = 0.94) or the

percentage of normal pollen (r = 0.96). In order to

evaluate the predictive power of the models, we

conducted regression analyses and performed a

validation experiment on genotypes not present in

the database and without background information on

fertility. Pollen diameter and percentage of normal

pollen were characterised and fitted in the regression

models for seed set predictions. Validation with an

independent dataset gave a good prediction for 83.3%

of the data. This indicates that using either the mean

pollen diameter or the percentage of normal pollen

resulted in effective fertility prediction. This tool

could enhance the genetic variability in crossings

between hybrid tea roses, thus creating possibilities

for less economically risky exploitation of new

tetraploid genotypes as male parents.

Keywords Breeding � Hybridisation �Pollen diameter � Rosa � Seed production

Introduction

Cut rose breeding programmes in the twentieth

century focused mainly on introduction of new flower

colours, thornless stems, higher production and a

L. Pipino � V. Scariot

Department of Agronomy, Forest and Land Management,

University of Turin, Via Leonardo da Vinci 44,

10095 Grugliasco, TO, Italy

L. Pipino � M.-C. Van Labeke

Department of Plant Production, Ghent University,

Coupure Links 653, 9000 Ghent, Belgium

A. Mansuino

NIRP International, Az. Agricola di Ghione L. & Figli,

via San Rocco 1, Fraz. Bevera, 18039 Ventimiglia,

IM, Italy

A. Giovannini

CRA-FSO, Research Unit for Floriculture and Ornamental

Species, Corso Inglesi 508, 18038 Sanremo, IM, Italy

L. Pipino � L. Leus (&)

Plant Sciences Unit, ILVO, Institute for Agricultural and

Fisheries Research, Caritasstraat 21, 9090 Melle, Belgium

e-mail: leen.leus@ilvo.vlaanderen.be

123

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DOI 10.1007/s10681-010-0298-5

Author's personal copy

good post-harvest performance. Yet, the fertility of cut

roses, typically tetraploid (2n = 2x = 28) hybrid tea

roses, is frequently reduced probably due to their

interspecific origin but also to intensive inbreeding (de

Vries and Dubois 1996; Debener et al. 2000). To avoid

the risk of very low seed production, breeders hybri-

dise using high pollen loads of a limited number of

male parents with the desired characteristics (Zlesak

2006), chosen for their known fertility (i.e., number of

seeds produced per hybridisation). New pollen donors

represent an economic risk for a breeding programme,

as their influence on seed production is unknown.

However, generating wider populations in terms of

number of seedlings is only one of the aspects that

contribute to the overall efficiency of a breeding

programme. The successful introduction of a new

parent into a breeding programme depends on its

potential to transmit the desired traits to the offspring.

It may be more efficient in some cases to choose a

parent with moderate fertility if it produces offspring

with a higher rate of advanced selections and,

ultimately, cultivars. In any case, recognising param-

eters related to fertility could increase the overall

efficiency of a breeding programme.

Since the end of the 1970s, several studies

concerning pollen quality in vitro (germination and

pollen tube elongation) or in vivo (considering the

hip set or the mean number of seeds per hip) were

carried out with the aim of improving sexual

reproduction of roses (Visser et al. 1977a, b; de

Vries and Dubois 1987; Gudin and Arene 1991, 1992;

Gudin et al. 1991a, b; Zlesak et al. 2007). These

studies mainly focused on the environmental effects

which influenced pollen quality, in order to define the

best conditions to pollinate or to store the pollen. The

integration of this knowledge on pollen quality

increased the number of seedlings obtained per

hybridisation by more than 200% (Gudin 1995).

Visser et al. (1977b) observed that pollen viability of

hybrid tea roses is influenced by the season and is

positively correlated to seedling production. Gudin

et al. (1991a) observed seasonal influence on pollen

fertility and also on pollen size. Temperatures

between 23 and 30�C and a relative humidity of

about 60–65% are the best conditions for in vitro

pollen germination and pollen tube elongation (Gudin

et al. 1991b). De Vries and Dubois (1987) observed

how in vivo temperatures between 22 and 26�C

improve pollination results.

In the past, pollen size has been widely studied by

means of pollen diameter. This morphological char-

acteristic has been correlated to several other param-

eters but not to seed set. Darwin (1884) measured the

pollen diameter of 44 species, observing how pollen

size was positively correlated to the distance between

the surface of the stigma and the transmission tissue of

the style. More recently, Cruden (2009) confirmed this

correlation, suggesting that after germination pollen

grows autotrophically through the stigma to reach the

resources in the style. Erlanson (1931a) studied the

relation between percentage of empty pollen grains and

the ploidy level in Rosa species; the same author

proposed a positive relation between microspore

diameter and ploidy (1931b). In roses, 2n pollen has

a diameter 1.2–1.3 times larger than n pollen

(Darlington 1937). Crespel et al. (2006) used this

information to confirm the presence of 2n pollen in a

population of diploid interspecific hybrids. Most

studies on fertility have been performed on cut roses,

as they are the most important commercial group.

Nevertheless, it can be expected that this data on

fertility can be useful for other types of roses. For

instance, Zlesak (2009) created a model of ploidy

prediction measuring the pollen diameter of a broad

range of different rose cultivars, species and breeding

lines.

No studies carried out on roses have focused on the

screening of breeding databases to aid the individu-

ation of fertility related markers, and therefore no

reliable model of fertility prediction has yet been

published. The aim of our study was to investigate the

relationship between fertility and pollen characteris-

tics. We selected genotypes of hybrid tea roses from a

cut rose breeding database based on the number of

seeds produced per hybridisation. We then investi-

gated pollen morphology and germination of the

selected genotypes and correlated this data with

fertility parameters, i.e. seeds per hybridisation, mean

seed germination and mean seed germination per hip.

Materials and methods

Genotype selection, plant material and pollen

sampling

Hybrid tea roses were selected among 213 genotypes

from a database, consisting of 14,109 hybridisations

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performed from 1994 to 2007 at the company NIRP

International (Cuers, France; Ventimiglia, Italy). The

ploidy level of a subset of 30 genotypes was assessed

by flow cytometry according to De Schepper et al.

(2001), recognising all genotypes as tetraploid.

Considering the origin of hybrid tea roses, the general

knowledge on their ploidy level and the fact that most

breeders choose to work with tetraploid genotypes

(Tackholm 1923; Erlanson 1938; Krussmann 1981;

de Vries and Dubois 1996; Crespel et al. 2002; Zlesak

2006), we assumed that the cut roses used at NIRP

International were tetraploid. We introduced three

criteria to reduce the bias created by cross combina-

tions using different seed parents on the character-

isation of the fertility of a genotype. Namely, we

excluded male parents from the database screening if

they were used with less than 20 different seed

parents, in less than 100 crossings and during less

than six crossing years. These restrictions provided a

subset of 107 tetraploid genotypes, used as male

parent in the breeding programmes. For these rose

genotypes, the number of seeds produced per hybrid-

isation was calculated. Four of the most fertile

genotypes (mean number of seeds produced per

hybridisation [15) and seven of the least fertile

(mean number of seeds produced per hybridisation

\7) were chosen. These two groups were called

‘‘high fertile’’ and ‘‘low fertile’’, respectively

(Table 1). For each of the 11 selected genotypes

two other parameters were calculated: mean seed

germination (%) meaning the germination per total

number of obtained seeds (values originating from

less than 10 hips per female parent were not

considered) and the mean seed germination (%) per

hip, calculated as the ratio between the seed germi-

nation (%) and the number of hips originated from

the male parent. Flow cytometry analysis confirmed

the tetraploidy of the 11 selected genotypes.

Twenty plants of each genotype were cultivated in

single pots with perlite at NIRP International (Cuers,

France) and fertilised according to the company’s

practises. The glasshouses were unheated but the

monthly mean outside day/night temperatures were

never less than 5.9�C. During June 2008, the monthly

mean outside temperatures were 24 ± 7.1�C (data of

the meteorological station of Cuers, Chambre d’Agri-

culture du Var, France). Pollen of the selected geno-

types was obtained as a bulk of at least 20 flowers (each

one collected from a different plant of the same

genotype) by gathering the anthers during June 2008.

Following the pollination method used at NIRP, the

anthers were placed at room temperature in open Petri

Table 1 Fertility parameters of the 11 male parents selected

from the database (number of seeds per hybridisation, mean

seed germination, mean seed germination per hip) and pollen

characteristics (in vitro germination, diameter, diameter and

percentage of normal pollen)

Fertility Pollen

Total Normal

Group Genotype

code

Seeds/hyb. Seed germ. (%) Seed germ.

(%)/hip

Germ. (%) Diam. (lm) Diam. (lm) (%)

High fertile 1848 21.3 ± 10.7A aB 29.9 ± 16.9 a 5.9 ± 9.8 a 31.8 ± 1.6 b 40.8 ± 8.6 a 43.1 ± 6.9 a 73.1

2518 20.0 ± 12.6 a 24.3 ± 13.7 a 2.7 ± 1.7 a 46.5 ± 2.6 a 40.2 ± 9.6 a 43.6 ± 7.3 a 68.8

2756 15.5 ± 14.8 a 37.1 ± 12.4 a 3.7 ± 2.3 a 22.9 ± 1.5 b 39.4 ± 10.2 ab 43.3 ± 8.1 a 59.7

2491 16.0 ± 17.9 a 36.3 ± 14.5 a 4.2 ± 5.5 a 22.8 ± 3.7 b 35.1 ± 9.3 b 40.1 ± 6.2 b 63.5

Low fertile 2376 4.4 ± 5.2 b 24.7 ± 18.6 a 1.2 ± 1.3 a 14.6 ± 2.3 c 29.3 ± 10.1 c 39.6 ± 6.8 b 27.5

1331 6.3 ± 8.4 b 19.3 ± 15.1 a 1.8 ± 3.0 a 18.9 ± 0.9 c 28.3 ± 9.0 c 38.0 ± 5.4 c 24.4

1999 1.1 ± 2.3 b 22.8 ± 16.8 a 1.0 ± 1.8 b 18.4 ± 1.5 c 28.0 ± 10.0 c 38.9 ± 6.5 c 25.5

2695 2.7 ± 3.2 b 36.0 ± 19.5 a 2.6 ± 2.1 a 0 d 27.4 ± 8.6 cd 37.1 ± 5.6 c 19.3

1985 5.1 ± 6.3 b 23.0 ± 13.3 a 2.0 ± 2.7 a 13.6 ± 0.2 c 26.6 ± 8.6 d 37.2 ± 5.0 c 26.7

2145 3.8 ± 6.1 b 18.5 ± 12.8 a 2.4 ± 5.6 a 5.8 ± 1.6 d 24.8 ± 7.0 e 35.4 ± 4.4 c 10.2

1627 1.9 ± 3.9 b 15.4 ± 16.7 b 1.3 ± 5.5 a 4.6 ± 0.5 d 21.3 ± 7.3 f 36.2 ± 4.8 c 9.5

A Mean ± standard deviationB Different letter indicates significant differences at the 0.05 level

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dishes in order to favour release and drying of the

pollen (Crespel and Mouchotte 2003). After two days

the Petri dishes were sealed and stored at -20�C.

Pollen was sent to ILVO (Belgium) for analysis.

Pollen germination

In vitro pollen germination of the 11 selected genotypes

was assessed using germination medium (40 mg l-1

H3BO3; 152 mg l-1 CaCl2(H2O); 150 g l-1 sucrose;

7 g l-1 agar; pH 5.6; Leus 2005) using one Petri dish

(diameter 9 cm) for each replicate. Three replicates

were performed for each rose genotype. Pollen was

spread on the medium with a small paint brush.

Germination was evaluated after 24 h incubation at

22 ± 2�C in dark conditions using a Leitz Laborlux D

Binocular Phase Contrast microscope (Leica Micro-

systems GmbH, Wetzlar, Germany) at a magnification

of 1009. Pollen was considered germinated when a

pollen tube reached a length of at least 1.5 times the

pollen diameter (Leus 2005). The mean pollen

germination (%) was calculated as the germination

per total number of observed pollen grains (200) on

the Petri dish. Pollen tube length was also observed

for each genotype.

Pollen diameter

Dry pollen was dusted onto a glass slide without a

cover slip. No hydration or embedding was per-

formed, because imbibed pollen swells, resulting in

significant changes in pollen diameter and a globular

shape (data not shown). Microscopic observations

(1609) were made using a Leica DMIRB microscope

(Leica Microsystems GmbH, Wetzlar, Germany).

Stored digital images of the pollen were used to

analyse the pollen size with LAS software (Leica

Application Suite). To measure the pollen diameter,

the major axis (transverse diameter) of the ellipsoidal

dry pollen grain was considered. In the text below,

the term ‘diameter’ describes ‘the major axis’. The

diameter of at least 400 pollen grains of the collected

bulk of each genotype was measured.

Paternal effect on seed set and offspring

germination

One high fertile and one low fertile female parent

(genotypes 1985 and 1627, respectively) as well as

one high fertile and one low fertile male parent

(genotypes 1848 and 1999, respectively) were selected

from the database. The same pollen samples used for

the in vitro germination experiment and the diameter

measurement were used for the crossings. The high

fertile seed parent 1985 was crossed with the high

fertile pollen parent 1848 (334 pollinations) and the

low fertile pollen parent 1999 (60 pollinations). The

same pollen donors were used on the low fertile seed

parent 1627, leading to 198 and 213 pollinations,

respectively. The crossings were carried out during

the first 10 days of June 2008, at the NIRP Interna-

tional glasshouses (Cuers, France). Seeds were gath-

ered and counted at the beginning of October 2008.

Afterwards seeds were stratified in sand at 18 ± 2�C

for 22 days and then transferred at 5 ± 1�C for

40 days. Per cross combination, four replicates of one

hundred seeds were sown in perlite beds at a

germination density of 209 seeds m-2. The experi-

mental set-up of the germination experiment was a

randomized complete block design. Germinations

were counted after 90 days.

Statistical analysis

Pollen germination and diameter measurements of

the genotypes were analysed using a one-way

ANOVA and Tukey’s HSD test for multiple com-

parisons. Student’s t-test was used to compare the

means of the high and low fertility group. The

frequency distribution of normal and abnormal pollen

was studied using a Chi-square test (Pearson).

Correlations (Pearson) were calculated between

the pollen characteristics (pollen diameter (lm),

normal pollen (%), normal pollen diameter (lm),

pollen germination (%)) and fertility parameters

(seeds/hybridisation, mean seed germination (%)

and mean seed germination (%)/hip). If correlations

were higher than r = 0.90 regression analysis was

conducted on the data. Analyses were performed with

SPSS 15.0 (SPSS Inc., Chicago, IL).

Validation experiment

To evaluate the predictive power of the regression

analysis, 18 genotypes not present in the database and

without background information on fertility were

introduced in the breeding programme, using random

seed parents for the crossings. The pollen of these

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genotypes, gathered during June 2008, was dried and

analysed as described above. In this experiment the

diameter of at least 100 pollen grains per genotype

was measured. Seeds were gathered and counted in

October to evaluate the actual number of seeds

produced per hybridisation by each genotype. The

pollen data of these genotypes were then used as

predictors for the number of seeds produced per

hybridisation using the regression equation. The

predictions were validated by checking which values

of actual number of seeds produced per hybridisation

exceeded the prediction interval (95%).

Results

Genotype selection

For the 107 genotypes used as pollen donors and

matching the minimal hybridisation standards in the

database analysis, the mean number of seeds produced

per hybridisation was 9.13 ± 9.30. The fertility

parameters for the selected subset of 11 genotypes

are given in Table 1. For the two groups established

(‘‘high fertile’’ and ‘‘low fertile’’) the number of seeds

per hybridisation differed significantly (F10,325 =

17.79, P \ 0.001) (t = 1.98, df = 111, P =

9.27E-16). Mean seed germination, as a percentage

of the obtained seeds, was only significantly lower for

genotype 1627 compared to the other genotypes

(F10,193 = 4.65, P \ 0.001). The mean seed germina-

tion per hip, calculated as the ratio between the seed

germination (%) and the number of hips originated

from the same male parent, was only significantly

lower for genotype 1999 (F10,374 = 2.96, P = 0.001)

(Table 1). If data were clustered for fertility, the high

fertile group showed a significantly higher amount of

germinated seeds (mean 32.1 ± 15.2%) than those of

the low fertile group (mean 22.4 ± 17.2%) (t = 1.97,

df = 202, P = 3.8E-05). Also for mean seed germi-

nation per hip, the genotypes of the high fertile group

had a significantly higher percentage of germinated

seeds per hybridisation (mean 4.1 ± 5.8%) compared

to the low fertile group (mean 1.7 ± 4.3%) (t = 1.98,

df = 131, P = 0.0002).

Pollen germination

The mean pollen germination varied significantly

between the 11 genotypes and ranged from 0%

(genotype 2695) to 46.5% (genotype 2518) (F10,22 =

52.96, P \ 0.001) (Table 1). A repeat experiment

(June 2009) confirmed that pollen of genotype 2695

does not germinate (data not shown). The high fertile

group was characterised by a significantly higher mean

pollen germination (31.02 ± 10.73%) compared to the

mean of the low fertile group (10.85 ± 7.22%)

(t = 2.10, df = 17, P = 2.12E-05).

Pollen diameter

The mean pollen diameter varied significantly among

the 11 genotypes studied (F10,10147 = 530.83,

P \ 0.001) (Table 1). The group of high fertile

genotypes was characterised by larger diameters

(mean 38.43 ± 9.79 lm) than low fertile genotypes

(mean 26.15 ± 9.13 lm) (t = 1.96, df = 10156,

P = 0). Microscopic observations revealed two types

of pollen grains: undeveloped with a diameter smaller

than 30 lm and ellipsoidal with a diameter larger

than 30 lm (Fig. 1). Therefore the distribution of the

pollen in two classes, i.e., abnormal pollen (diameter

smaller than 30 lm) and normal pollen (diameter

larger than 30 lm), was calculated for both the high

and low fertile group (Table 2). A significantly

higher percentage of normal pollen was found for

Fig. 1 Pollen of a low

fertile (a) and a high fertile

(b) genotype

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the high fertile group (Pearson Chi-square = 2075.1,

P \ 0.001). Only 20.6% of the pollen of the low

fertile group was normal, while the high fertile group

had 65.6% normal pollen.

The mean diameter of the normal pollen was

calculated for each genotype as well as its percentage

of the whole population (Table 1). The 11 genotypes

were characterised by percentages of normal pollen

varying between 9.5 and 73.1%. When the genotypes

were analysed for their mean normal pollen diameter,

three significantly different groups could be distin-

guished: one composed of three high fertile genotypes

(1848, 2518 and 2756), a second composed of one

high and one low fertile genotype (2491 and 2376)

and a third composed of six low fertile genotypes

(1331, 1999, 2695, 1985, 2145 and 1627) (F10,4873 =

76.04, P \ 0.001) (Table 1). When data were clus-

tered for fertility, the diameter of the normal pollen of

the high fertile group (mean 42.37 ± 7.29 lm) was

also significantly higher than the normal pollen of the

low fertile group (mean 37.79 ± 5.79 lm) (t = 1.96,

df = 4504, P = 5.21E-122).

Paternal effect on seed set and offspring

germination

In order to strengthen the database observation by

evaluating the paternal effect on seed set and

germination, a crossing experiment was performed

with four genotypes selected from the database

(Table 3). According to the database, the mean number

of seeds per hybridisation of the two female parents

1985 and 1627 was 19.6 ± 18.7 and 2.1 ± 3.0,

respectively. The mean number of seeds per hybrid-

isation of the two male parents 1848 and 1999 was

21.3 ± 10.7 and 1.1 ± 2.3, respectively. When pollen

of the high fertile male parent (1848) was used on both

female parents the number of seeds produced per

pollination was very high compared to the low fertile

male parent (1999) and to its value in the database. In

crosses with pollen donor 1999, the number of seeds

produced per pollination was very low for genotype

1985 and almost equal to zero for the low fertile seed

parent 1627 (Table 3). Seeds of the high fertile

genotype 1985 9 1848 reached 45.8% of germination,

while no germination occurred among the progeny

sired from pollen donor 1999. Only 13.6% of the seeds

of 1627 9 1848 germinated, and no seeds of the

crossing with 1999 germinated (Table 3).

Data analysis

The correlations (Pearson) for the variables pollen

diameter (lm), normal pollen (%), normal pollen

diameter (lm), pollen germination (%), seeds/hybrid-

isation, mean seed germination (%) and mean seed

germination (%)/hip are presented in Table 4. Seeds

per hybridisation correlated strongly with the mean

pollen diameter and percentage of normal pollen

(r [ 0.90); a strong correlation was also obtained

with the mean diameter of normal pollen (r = 0.87).

The correlations of mean seed germination per hip

with pollen diameter, percentage of normal pollen

and seeds per hybridisation were still high (respec-

tively r = 0.75, r = 0.78 and r = 0.83). The

Table 2 Frequency distribution of normal pollen among the

genotypes sorted into fertility groups

Group Normal pollen Total (counts)

counts %

High fertile 2,607 65.6 3,974

Low fertile 1,274 20.6 6,184

Table 3 Paternal effect on seed set (number of seeds per pollination) and offspring germination (mean seed germination): a crossing

experiment

Seed parent Pollen donor

High fertile (1848) Low fertile (1999)

Seeds/pollination Seed germ. (%) Seeds/pollination Seed germ. (%)

High fertile (1985) 60.2 45.8 ± 4.6A 1.80 0

Low fertile (1627) 9.0 13.6 ± 4.9 0.05 0

A Mean ± standard deviation

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correlation of mean seed germination per hip with the

mean diameter of normal pollen was not significant.

The correlations of mean pollen diameter, percentage

of normal pollen and the mean diameter of normal

pollen with mean seed germination were moderate.

Percentage of pollen germination correlated signifi-

cantly with mean pollen diameter (r = 0.75), per-

centage of normal pollen (r = 0.78) and mean

diameter of normal pollen (r = 0.80).

Based on the correlation analysis, percentage of

normal pollen (r = 0.96) and mean pollen diameter

(r = 0.94) were selected as the most suitable putative

predictors of seeds per hybridisation and a simple

linear regression was performed (Fig. 2). Moreover,

the mean diameter was entered as an independent

variable (Fig. 2a) and showed a significant linear

regression (R2 = 0.88), even though the model

obtained using the percentage of normal pollen

(Fig. 2b) was the most significant (R2 = 0.93).

Therefore, both pollen parameters were chosen as

an independent variable for the validation of the

linear model.

Validation

The 18 genotypes chosen for the validation of the

linear model (Table 5) were characterised by mean

pollen diameters varying between 17.4 ± 6.4 and

30.9 ± 10.2 lm and percentages of normal pollen

varying between 4.1 and 35% (Table 5). The diam-

eters formed 3 different subsets of data (F18,3465 =

21.83, P \ 0.001) (Table 5). Analysis of the diame-

ters of the normal pollen showed that only genotype

3360 separated significantly from the other genotypes

(F18,1018 = 6.1, P \ 0.001).

Of the predictions, 83.3% were validated using

either the mean diameter or the percentage of normal

pollen as independent variable (Fig. 2a, b). Only two

predictions (genotypes 3820 and 3825) were vali-

dated by one model and excluded by the other, and in

both cases the values were very near to the prediction

interval. Therefore both parameters showed a high

percentage of validation and a rather good accuracy

as predictors of number of seeds produced per

hybridisation.

Discussion

In order to characterise the fertility of pollen donors,

this study used pollen morphology to predict the number

of seeds produced per hybridisation in tetraploid rose

genotypes. Company breeding databases have never

been studied to assess fertility using pollen charac-

teristics in roses. In previous studies to evaluate

fertility, it has been commonplace to work with a

small number of parents (Visser et al. 1977b; de Vries

and Dubois 1987; Ogilvie et al. 1991; Crespel et al.

2006). Our breeding database analysis enabled the

selection of genotypes that are accurately character-

ised by high or low fertility in terms of seeds

produced per hybridisation. Furthermore, robust

characterisation of fertility of a specific genotype

increases with the number of crossings performed on

different genotypes and in different years. However,

cross combinations using different seed parents will

Table 4 Correlations between the pollen characteristics (diam-

eter, percentage of normal pollen, diameter of normal pollen, in

vitro germination) and fertility parameters (number of seeds

per hybridisation, mean seed germination and mean seed

germination per hip) of the 11 genotypes selected from the

database

Diam.

(lm)

Normal

pollen (%)

Normal pollen

diam. (lm)

Pollen

germ. (%)

Seeds/

hyb.

Seed

germ. (%)

Seed germ

(%)/hip

Diam. (lm) 1

Normal pollen (%) 0.97** 1

Normal pollen diam. (lm) 0.96** 0.93** 1

Pollen germ. (%) 0.75** 0.78** 0.8** 1

Seeds/hyb. 0.94** 0.96** 0.87** 0.74** 1

Seed germ. (%) 0.66** 0.63* 0.64* 0.36 0.53 1

Seed germ. (%)/hip 0.75** 0.78** 0.6 0.35 0.83** 0.45 1

** Correlation (Pearson) is significant at the 0.01 level (2-tailed)

* Correlation (Pearson) is significant at the 0.05 level (2-tailed)

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still bias the characterisation of the fertility of a

genotype. To reduce this bias, thresholds of exclusion

were introduced to select the genotypes. In our study

the results of the fertility analysis of the database with

an overall mean number of seeds per hybridisation of

9.13 ± 9.30 are in agreement with the number of

seeds per hybridisation reported in literature. Ogilvie

et al. (1991) hybridised 17 hybrid tea roses as seed

parents with two winter hardy cultivars used as male

parents, reporting for male parents a mean value of

3.83 and 10.83 seeds obtained per hybridisation. De

Vries and Dubois (1987) obtained 8.6 seeds per hip

(with a single pollination) hybridising two hybrid tea

roses. In agreement with the values reported for the

fertility of the low fertile genotypes, Werlemark et al.

(2009) consider 3.0–5.2 seeds per hip a low fertility

value for progenies sired from an interspecific

crossing between the pentaploid R. rubiginosa used

as seed parent and several tetraploid garden roses.

Breeding programmes with other types of roses, such

as garden roses, include a broader range of genotypes

for crossing, e.g., interploidy crosses and (re)intro-

duction of species. Despite their greater genetic

diversity, data on pollen fertility would likely be a

very valuable addition for these breeding programs

and validation experiments to confirm the utility of

pollen diameter and germination on fecundity are

warranted.

In our study, pollen grains with a diameter up to

30 lm almost always appeared shrunken with either

an irregular or spherical shape, with no furrows on

the surface-typical of aborted pollen. Pollen grains

with a diameter greater than 30 lm always looked

ellipsoidal, with furrows on the surface typical of

normal pollen. In contrast with Crespel et al. (2006)

and Zlesak (2009), who used immersed pollen, our

pollen size analysis was performed on dry pollen as

used for pollination in breeding. Immersed pollen

changes shape from ellipsoidal to spherical and

increases in size (Cruden 2009). In preliminary

experiments we observed that pollen grains imbibed

in water showed a significantly lower variance in the

diameter measurements (results not shown). Normal

pollen (diameter more than 30 lm) clearly swells to a

spherical shape, while small abnormal pollen main-

tains more or less the original shrunken and irregular

shape in most cases. Moreover, we found a significant

positive correlation between percentage of normal

pollen and mean percentage of pollen germination

(r = 0.78). Therefore, immersion in water could be a

simple test to evaluate germinability or calculate the

amount of normal pollen. This test is comparable to

what happens after pollination when rose pollen

comes in contact with the exudates of the stigma

surface: pollen grains absorb water and swell to start

the germination process (Jacob and Ferrero 2003).

Normal pollen (%)0 10 20 30 40 50 60 70 80

seed

s / h

ybrid

isat

ion

-10

0

10

20

30

High fertile genotypeLow fertile genotypeLinear regressionConfidence interval (95%)Prediction interval (95%)Validation

(b)

474.2307.0 −= xy

(a) Diameter (µm)

20 30 40

seed

s / h

ybrid

isat

ion

-10

0

10

20

30

High fertile genotypeLow fertile genotypeLinear regressionConfidence interval (95%)Prediction interval (95%)Validation

839.25184.1 −= xy

Fig. 2 Linear regression analyses of the genotypes selected

from the database. Graphs show the models created fitting the

mean diameter (a) and the percentage of normal pollen (b).

Black triangles in the graphs represent the actual fertility of the

genotypes used for the validation of the model

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Several authors report that abnormal pollen does not

stain during viability tests or significantly changes

shape when immersed (Cole 1917; Erlanson 1931a;

Calvino Mameli 1951; Visser et al. 1977a, b; Crespel

et al. 2006). In our study pollen of the low fertile

genotype 2695 never germinated in vitro. However,

this genotype is able to produce offspring. Therefore,

it should be remarked that in some cases in vitro

pollen germination does not completely reflect ger-

mination capacity in vivo.

To more accurately relate the pollen characteristics

to the fertility parameters of the database, we

conformed as much as possible to the conditions of

gathering, storage and use of pollen as listed in the

company’s protocols. This created robust and high

correlations between the number of seeds per hybrid-

isation and the measured pollen diameters, the

percentage of normal pollen and the diameter of

normal pollen. In this study, mean diameter and

percentage of normal pollen are highly correlated

parameters (r = 0.97). Within the normal pollen, the

mean diameter of normal pollen grains also differed

for the high and low fertile genotypes. The more

fertile genotypes were characterised by larger pollen

grains. Low fertile genotypes produce more abnormal

pollen, which is shrunken and in some cases clearly

aborted. The high fertile genotypes showing larger

diameters had in some observations also longer

pollen tubes than the low fertile genotypes (results

not shown). Cole (1917) studied the pollen of 37

species belonging to the genus Rosa and observed,

though not determining the ploidy level of the

species, that a large amount of pollen is abortive or

sterile and has a great variability in size already

before the anthers dehiscence. Cole assumed that the

amount of abnormal pollen is correlated to a certain

degree of hybridism within these species. Calvino

Mameli (1951) studied the pollen of 48 genotypes of

roses (18 were diploid species and 30 were tetraploid

hybrid teas), also highlighting differences in the grain

Table 5 Hybridisation performances (number of crossings,

number of seed parents, and number of seeds per hybridisation)

and pollen characteristics (mean diameter, mean diameter and

percentage of normal pollen) of the genotypes used for the

validation experiment

Hybridisations Pollen

Total Normal

Genotype code Pollinations (n) Seed parents (n) Seeds/hyb. Diam. (lm) Diam. (lm) (%)

3820 15 1 2.6 17.4 ± 6.4A cB 36.9 ± 6.4 a 4.1

3823 29 2 0.7 20.8 ± 7.2 c 34.8 ± 5.6 a 4.3

3360 234 11 1.8 23.8 ± 7.1 b 34 ± 3 b 6.6

2817 289 14 1.5 21.1 ± 7.6 c 39.7 ± 7.2 a 6.8

4083 66 3 0.7 26.2 ± 6.7 b 34.3 ± 3.9 a 8.2

3518 138 8 2.1 24.1 ± 8.3 b 35.5 ± 5.2 a 9.0

4096 20 1 0.5 22.4 ± 7.3 c 34.9 ± 4.9 a 9.1

3817 116 8 0.3 24.4 ± 8.1 b 34.6 ± 3.6 a 9.9

3816 43 6 10.7 24.2 ± 8.9 b 36.7 ± 5.9 a 11.1

3741 33 3 0.1 27.3 ± 10.1 b 36 ± 5.9 a 19.7

3742 132 7 13.1 27.4 ± 10.3 a 39.7 ± 6.9 a 20.6

4092 37 2 0.3 25.3 ± 7.4 b 35.4 ± 3.6 a 21.5

4087 31 2 2.1 27.9 ± 9.3 a 36 ± 5.4 a 21.8

3826 137 9 4.9 26.2 ± 12.5 b 39 ± 7.6 a 24.5

3824 144 11 4.3 28.1 ± 10.2 a 38.7 ± 5.8 a 26.8

3740 261 11 4.1 28.6 ± 10.2 a 36.8 ± 5.3 a 26.9

4100 309 21 9.1 29 ± 9.3 a 38.2 ± 6.8 a 30.6

3825 175 15 14.3 30.9 ± 10.2 a 39.2 ± 6.3 a 35.0

A Mean ± standard deviationB Different letter indicates significant differences at the 0.05 level

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sizes before the anthers dehiscence, and reported that

the length of the major axis of normal pollen grains of

hybrid tea roses, when immersed in bergamot oil

(which does not alter the pollen shape for the first 2 h

after immersion), ranges between 32.2 and 66.4 lm.

More recently, other authors related the co-existence

of sub-populations of pollen grains with higher sizes

to the occurrence of 2n pollen in a dihaploid rose

(El Mokadem et al. 2002), in three tetraploid hybrid

tea roses (Crespel and Gudin 2003) and in 10

individuals of a population of 53 diploid rose hybrids

derived from a dihaploid rose and R. wichurana

(Crespel et al. 2006). The diameter values that are

related to the occurrence of 2n pollen refer to

immersed pollen, and are not comparable with our

values that refer to dry pollen. Therefore, we cannot

compare our diameter values with those reported in

literature. No subpopulations differing in pollen size

were observed. It should be remarked that pollen size

and shape also changes in relation to the genotype

and to environmental conditions (Gudin et al. 1991a;

Crespel et al. 2006; Zlesak 2009). The evidence in the

genus Rosa of a positive correlation between pollen

grain size and ploidy level was proved by Jacob and

Pierret (2000) and by Zlesak (2009).

In our study only moderate correlations of pollen

diameter and percentage of normal pollen with mean

seed germination per hip were found. This could be

related to maternal effects on seed germination. For

example, the progeny’s germination value of male

parent 1848 was higher when sired from a high fertile

female parent (1985) than a low fertile one (1627).

Empty seeds or seeds with desiccated embryos are

found in hybrid tea roses, in which germination

capacity is also related to seed density (Gudin et al.

1992). These results are in agreement with the

general knowledge of predominant maternal influ-

ence on seed development, germination and early

seedling growth in plants (Roach and Wulff 1987;

Schmid and Dolt 1994; Thiede 1998). Our crossings

were not performed as a full diallel scheme, and

therefore the maternal effect cannot be excluded. In

addition, all the genotypes showed high standard

deviation values for mean seed germination, probably

due to the possible maternal effect as well as to the

annual environmental influences on seed germina-

tion. This also explains why the correlation between

mean seed germination and seeds per hybridisation

was not high. Nevertheless, our results clearly

indicate that high fertile male parents have a positive

influence on seed germination. Ogilvie et al. (1991)

also found that the number of seeds produced per

pollination varied according to the male parent. The

authors suggest that the use of most distantly related

cultivars in rose breeding would probably give a

higher seed set, but also suggest that there are

probably other factors that may influence pollination

success. Rose hybridisation is a part of a process of

unnatural selection, in which the application on the

surface of the stigma of very large amounts of pollen

of only one genotype could completely suppress

sexual selection among male parents. Breeders of

hybrid tea roses compensate low male parent perfor-

mances by adding more pollen during the pollination

process. De Vries and Dubois (1987) showed that the

number of seeds obtained per pollinated flower

(pollination index) did not further increase after five

pollinations between hybrid teas. Moreover, if the

ratios between the number of seeds per hip and

the number of pollinations are calculated, these

values already started to decrease as of the second

pollination.

Given the parameters that might influence pollen

diameter besides male genotype, we judged the

validation results to be good. In this study 83.3% of

the predicted seeds per hybridisation were within the

95% prediction intervals of both regression models.

The mean pollen diameter explained 87.9%

(R2 = 0.879) of the variation in seed set while the

percentage of normal pollen explained 93.2%

(R2 = 0.932). This indicates that either the mean

pollen diameter or the percentages of normal pollen

can be used as fertility predictors for hybrid tea roses.

Both parameters showed that all the roses used for the

validation are low fertile genotypes. The effective-

ness of the predictive tool for these low fertile

genotypes has been demonstrated and can success-

fully increase the efficiency of breeding programmes

with new tetraploid genotypes. Creating a prediction

of the number of seeds per hybridisation for new

genotypes by calculating the percentage of normal

pollen grains or measuring the mean pollen diameter

is reliable, but should be carefully standardised. Our

model for pollen fertility prediction is built on the

basis of hybridisations performed for 14 years at the

same location, by the same breeders and standard-

ised protocols of storage and manipulation of the

pollen. A different location, pollen manipulation and

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pollination conditions could affect the effectiveness

of the model. Nevertheless, the efficiency of the

equation was validated for genotypes which were

never used in the company breeding programmes and

were therefore not present in the database. For this

reason breeders could apply the method developed on

the basis of their own database information and create

a prediction model valid for their location and their

protocols of gathering, storage and usage of pollen.

Uncomplicated pollen observations can improve the

efficiency of breeding programmes. Our proposal for

narrowing down the pool of potential male parents in

a breeding programme is to indicate the fecundity

level of a male parent by measuring percentage of

normal pollen and pollen diameter and selecting

those with the greatest likelihood of fertility. The

quality of the offspring raised from the male parent

and the prioritisation of traits will then determine

whether the male will be suitable for continued use in

the breeding programme.

Acknowledgments The authors wish to thank all the

technical staff of NIRP International, Azienda Agricola di

Ghione Luciano & Figli s.s. (Ventimiglia, Italy).

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