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Incompatibility reactions and genotypic identity status of five commercial chicory (Cichorium intybus L.) hybrids
SCIENTIA HORTICULTURR ELSEVIER
Scientia Horticulturae
72
(1997)l-9
Incompatibility reactions and genotypic identity status of five commercial chicory ( Cichorium intybus L.) hybrids C.I. Castafio *, M.A.C. Demeulemeester, Faculty
M.P. De Proft
ofAgricultural and Applied Biological Sciences, Katholieke Uniuersiteit L.euven, Willem de Croylaan 42, B-3001 Heuerlee, Belgium Accepted 24 July 1997
Abstract Incompatibility reactions of chicory plants cv. Flash, cv. Carolus, cv. Pax, cv. Focus and cv. Sigma were determined. Different levels of incompatibility were found. The cv. Carolus was highly self-incompatible, cv. Flash and cv. Pax could be self-compatible or self-incompatible and the cv. Sigma and cv. Focus are self-compatible. All the tested cultivars were intra-compatible (pollinations within the same cultivar) and cross-compatible among them. There was difference in percentage of viable seeds when cultivars were self-pollinated and intra-pollinated. This difference in percentages of viable seeds can be used as a numerical criterion to determine genotypic identity of a cultivar. The higher this difference value, the less uniform the behaviour of the cultivars in the field and during hydroponic forcing for production of the chicory head was observed. The results led to the conclusion that some of the commercial cultivars are not genotypic identical which has unfavorable implications in Belgian endive production and commercialization. There was equal reaction to incompatibility in any sense of crossing (plants used as females compared with the result when plants were used as males in cross-pollinations). 0 1997 Elsevier Science B.V. Keywords: Chicory;
Pollination;
Seed set; Self-incompatibility
1. Introduction Chicory is an important economic plant for its use as a vegetable (Belgian endive) and as an industrial raw material to obtain inulin from roots (Root chicory). Chicory is
* Corresponding author. [email protected]
Tel.:
+ 32
16
322390;
fax:
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)0011 l-8
+ 32
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322966;
e-mail:
2
C.I. Castaiio et al. /Scientia
Horticulturae
72 (1997) l-9
an allogamous species due to the morphology of the flowers and the presence of a sporophytic self-incompatibility system (Coppens d’Eeckenbrugge et al., 1987a). PCcaut suggested two methods to measure the incompatibility level of commercial cultivars of chicory (PCcaut, 1962): (1) Count of viable seeds per flower head after controlled pollination of five flower heads per plant. (2) Examination of the pollen germination on stigma and penetration of pollen tube after controlled pollination. According to Pecaut it is impossible to classify plants as either self-compatible or self-incompatible because the measure of the degree of self-incompatibility within one plant shows continuous variation. It is necessary to sort the plants into classes depending on the different self-compatible degrees. A distribution of plants in groups according to the number of viable seeds/flower head is suggested. Based on experiments about compatibility and incompatibility in chicory, Eenink (198 1) made a distribution of clones over three classes in a rather arbitrary way-formation of fewer than 3 seeds means incompatibility (- ), formation of between 3 and 6 seeds indicates either incompatibility or compatibility ( + ) and formation of more than 6 seeds indicates compatibility. In his work, the mean seed production of 2-4 flower heads over 15 clones from plants of different populations was examined. PCcaut (19621, Eenink (1981) and Varotto et al. (1995) suggested the existence of a sporophytic self-incompatibility system in chicory. The presence in chicory of this system offers possibilities for the production of F, hybrid cultivars. However, if several self-incompatible plants are used to produce inbred lines methods should be available to induce self-fertilization (Eenink, 198 I). On the other hand, full use of self-incompatibility for F, production creates problems for the breeders. The degree of self-incompatibility is often not fully reliable, especially under field conditions (Olesen et al., 1992). Breeding programs require good knowledge of the self-incompatibility status of the material used. In this report, the determination of the self-incompatible degree of five commercial cultivars by self-pollination (the flowers were pollinated with pollen derived from flowers of the same plant), intra-pollination (the flowers were pollinated with pollen derived from flowers of a different plant but of the same cultivar) and cross-pollination between plants of the different cultivars is presented. Comparison of the results obtained produced some interesting information about the genotypic identity of the F, commercial hybrids which are actually used by growers for the production of the vegetable ‘Belgian endive’.
2. Materials and methods 2.1. Plant material Plants of C. intybus L. var. foliosum raised from seeds of the following cultivars were used: cv. Flash (INRA, France), cv. Carolus (Bucomat, Belgium) cv. Sigma (Vilmorin, France), cv. Pax (Rijk Zwaan, Netherlands) and cv. Focus (Nunhems, Netherlands). The seeds were placed in petri dishes in the dark, cold treated at 4°C and relative humidity + 95% for 3 weeks, and transplanted to a hydroponic system (May 1995)
Cl. Castaiio et al. /Scientia Horticulturae 72 (1997) 1-9
under an open plastic greenhouse. 11 cm diameter) filled with fine tubes were placed in a gully (23 in a row being 25 cm. (De Rijck to the seeds, the plants produced
3
The system consisted in PVC tubes (50 cm high and rockwool flocks grodan type 012/519. Two rows of cm wide and 13 m long), with distance between tubes et al., 1993). Due to the vernalization treatment given flowers during the first growing season.
2.2. Controlled pollinations Controlled pollinations were carried out in a manner that was similar to that of PCcaut (Pecaut, 1962). The branches of the plants were enclosed before anthesis in a cellophane bag. Hand pollinations were made by placing the pollen of an open flower, isolated in a bag, to the stigma of an open flower, also isolated in a bag. The enclosure of the branches prevented the intervention of insects in the pollination process. Wind movement in the bags was restricted. 2.3. Experimental
design
Fifteen plants of each cultivar were used. The following treatments were given for each cultivar: 1. Self-pollination: a flower was pollinated with a flower from the same plant 2. Intra-pollination between plants of the same cultivar 3. Cross-pollination among the 5 cultivars. Five flower heads were pollinated in each plant for each treatment. The five flower heads that corresponded to one treatment were all located on one isolated branch. As a result, each plant had six isolated branches: one for self-pollination, one for intra-pollination, and four for the crosses with the other cultivars. The pollinations were carried out during five weeks, from the first days of August until the beginning of September. The weather conditions during the experiment are presented in Table 1. Temperature and RH under open plastic greenhouse can slightly differ. The pollinations were made one hour after anthesis until noon. Eight weeks after the last pollinations (end of October 1995) the branches were collected and the seeds of the marked pollinated flower heads were taken to make the germination test.
Table 1 Weather characteristics per month (during experiment) for the year 1995. Station Herent. (Source: Koninklijk Meteorologisch Instituut van BelgiE, KM1 Klimatologische Dienst) Month
T max (mean “C>
T min (mean “C>
T average (mean “0
RH average (mean %)
Sunshine max (h min)
May June July August September October
19.5 19.9 26.8 26.6 18.9 18.2
8.0 10.8 15.1 13.7 10.7 9.6
13.8 15.4 21.0 20.1 14.8 13.9
72 80 75 69 81 84
1318 1518 1430 1406 1006 0924
4
2.4. Germination
C.I. Castaiio et al./Scientia
Horticulturae 72 (1997) l-9
test of the seeds
In chicory, the fruits are achenes (one-seeded, dry fruits). In this work, the term of seeds refers to achenes. Seeds can be classified as full (embryo is present) or empty. Because it is difficult to distinguish an empty from a full seed, a germination test was done. Seeds that germinated were considered to be viable. The seeds of each plant were placed per treatment in a petri dish on wet filter paper for 3 or 4 days in a culture room at 22 + 2°C 60 pmol mm2 SK’ irradiance (fluorescent lamps Philips 36 W, TLD/54) and 16-h daylength. For cross-pollinations and intra-pollinations, there were approximately 100 total seeds/petri dish per treatment per plant, which corresponded to the five flower heads pollinated in the corresponding treatment. For selfings, the seeds of each flower head were collected separately in petri dishes. As a result, for selfings, there were 5 petri dishes with approximately 20 seeds per plant. 2.5. Statistical analysis The percentage of viable seeds in the total number of seeds (from five flower heads) was calculated per plant in each treatment per cultivar. The means of the percentages were calculated per treatment and per cultivar. For selfings, the percentage of viable seeds per flower head was calculated. The means of the percentages were calculated then per plant, treatment and cultivar. Mean number of total seeds/flower head was 20. To have a better normal distribution of the means of the percentages of the viable seeds, the values were transformed using the inverse parabolic sine. With the transformed percentages, the means of viability were checked for relations between self-, intra- and cross-pollination in each cultivar. The data were statistically analyzed using ANOVA and Duncan test to check the differences of the means. Means with the same letter are not significantly different.
3. Results
3.1. Self-compatibility, cial cultivars
intra-compatibility
and cross-compatibility
of the five commer-
Table 2 shows the mean percentage of viable seeds in the total seeds per plant after self-pollination, intra-pollination and cross-pollination of the five commercial cultivars studied. If the criterion of Eenink (1981) is adopted and an average of 20 seeds per flower head is taken in consideration, a viability percentage of 15% means incompatibility ( - ), 30% indicates compatibility or incompatibility ( f ), and more than 30% indicates compatibility. According to this classification, cv. Carolus is highly self-incompatible (6.2%). The cv. Flash (27.5%) and cv. Pax (24.6%) could be self-compatible or self-incompatible and the cv. Sigma (54.4%) and cv. Focus (50.0%) are self-compatible. All the tested cultivars were intra-compatible with percentages of viable seeds in total seeds per plant ranging from 32.4% for cv. Pax to 77.5% for cv. Sigma. All cultivars were cross-com-
C.I. Castafio et al./Scientia Table 2 The self-incompatibility,
intra-incompatibility
Horticulturae
and cross-incompatibility
72 (1997) l-9
5
of chicory cultivars
Cultivar (female)
Cultivar (male)
Mean (real) %
Duncan
Carolus
Flash Focus Sigma Pax Intra Self
65.9 63.8 52.6 49.9 56.1 6.2
A A A A A B
Flash
Carolus Sigma Focus Pax Intra Self
79.2 52.5 48.2 43.2 50.8 21.5
A B B B B C
Carolus Focus Sigma Flash Intra Self
72.6 42.9 40.3 38.8 32.4 24.6
A B BC BC BC C
Sigma
Carolus Flash Pax Focus Intra Self
75.9 65.9 65.8 57.5 77.5 54.4
A BA BA BA A B
Focus
Flash Carolus Sigma Pax Intra Self
68.3 64.3 55.0 54.4 69.0 50.0
A BA BA BA A B
Mean percentages of viable seeds in total seeds per plant. Means are based on the pollination of 5 flower heads per plant of 15 plants.
patible among themselves. In this respect, the highest percentage of viable seeds in total seeds per plant was observed when cv. Flash was crossed as female with cv. Carolus (79.2%) and the lowest when cv. Pax was crossed as female with cv. Flash (38.8%).
3.2. Differences in the seed set when the plants were used as females result when the plants were used as males in cross-pollinations
compared with the
Mean percentages of viable seeds in total seeds per plant of cv. Carolus, cv. Flash, cv. Pax, cv. Sigma and cv. Focus obtained in the both sense of crossing are shown in Table 3. ANOVA and Duncan analyses of the means when the plants were used as
6
C.I. Castaiio et al./Scientia
Table 3 The cross-incompatibility as males)
of chicory cultivars
in the two directions
Carolus
Flash (mean o/o)
Pax (mean %)
Female Male
65.9 79.2
49.9 72.6
Flash
Carolus (mean %I
Pax (mean o/o)
Female Male
79.2 65.9
43.2 38.8
Pa.%
Flash (mean %)
Female Male
38.8 43.2
Sigma
Flash (mean %)
Female Male
65.9 52.5
Focus
Flash (mean %o)
Pax (mean %I
Female Male
68.3 48.2
54.4 43.0
A A
A A
A A
A A
A B
Horticulturae 72 (1997) 1-9
B A
A A
of pollination
(plants used as females and
Sigma (mean %)
Focus (mean %)
52.6 75.9
63.8 64.3
B A
A A
Sigma (mean %)
Focus (mean %)
52.5 65.9A
48.2 68.3
A A
B A
Carolus (mean %)
Sigma (mean %I
Focus (mean %)
72.6 49.9
40.3 65.8
42.9 54.4
A B
B A
A A
Pax (mean %I
Carolus (mean %o)
Focus (mean %)
65.8 40.3
75.9 52.6
57.5 55.0
A B
A A
A B
A A
Sigma (mean %)
Carolus (mean %)
55.0 57.5
64.3 63.8
A A
A A
Mean percentages of viable seeds in total seeds per plant. Means are based on the pollination of five flower heads per plant of 15 plants.
females and males were carried out, using exclusively the values involved and not the whole data set. Means with the same letter are not significantly different. The five commercial cultivars were cross-compatible among themselves with percentages of viable seeds always higher than 30%. Nevertheless, there were, in some cultivars, significant differences in the degree of compatibility when they were used as either female or male plants.
4. Discussion
4.1. Information incompatibility
about the self- incompatibility, of commercial cultivars
intra-incompatibility
and
cross-
Different levels of self-incompatibility were found. The cv Carolus was highly self-incompatible, the cv. Flash and cv. Pax could be either self-compatible or selfincompatible and the cv. Sigma and cv. Focus were self-compatible. All the tested cultivars were intra-compatible (pollinations within the same cultivar) and crosscompatible among them. As stated by Coppens d’Eeckenbrugge et al. (1987b3) this self-compatibility is ascribed to the presence of heterozygote genotypes composed of S-alleles where the relations between dominance in pollen and the pistil are not clearly defined or homozygote genotypes for the S-allele are very weak in the scale of dominance.
C.I. Castaiio et al. /Scientia Horticulturae 72 (1997) I-9
4.2. Genotypic
I
identity of cultivars
If the commercial cultivars that have been studied are hybrids derived from inbred homozygous plants, genotypic identity of the hybrid plants that belong to the same cultivar would be expected. In this situation, the self-incompatible reaction should be the same whether the pollen used to pollinate a flower head is taken from another flower head of the same plant or from a flower head from a plant within the same cultivar. The statistical analysis shows that there are significant differences between the means of percentages of viable seeds when self-pollinations and intra-pollinations were made in all the cultivars, except in cv. Pax (Table 2). This difference in viability of the seeds could be interpreted as a criterion to define how genotypic identical a cultivar is (genetically homogeneous). The cv. Carolus shows 56.1% of germination when intrapollinated and 6.2% when self-pollinated. The difference between these two values is 50.0%. The differences corresponding to the other cultivars tested are: 23.3% for cv. Flash, 23.1% for cv. Sigma, 19.0% for cv. Focus and 7.8% for cv. Pax. According to these values, the distribution of cultivars regarding their genotypic identity was as follows: cv. Carolus was the less genotypic identical cultivar, followed by cv. Flash, cv. Sigma and cv. Focus. The cultivar cv. Pax proved to be the most genotypic identical (Fig. 1). These results were compared with the uniform behaviour of the cultivars, observed in the field and during forcing at the Research Station of Herent (Claessens G, personal communication). The behaviour of the cultivars, reported by this research station, coincides with the results obtained in the pollination experiments, especially concerning the extreme cases: cv. Pax and cv. Carolus. The cv. Carolus is no longer used for commercial forcing and chicory head production (due to the non-uniform behaviour) and cv. Pax was one of the most uniform cultivars for chicory head production in the period 1995-1996. This comparison leads to the conclusion that some of the commercial cultivars have non-genotypical identical characteristics which have unfavorable implications for Belgian endive production and commercialization. This different value between percentages of viable seeds when cultivars are self-pollinated and intra-pollinated can be used as a numerical criterion. The criterion has not been used or reported in literature by any other author for this purpose. The high level of intra-compatibility between the F, hybrid plants of each of the five cultivars tested (except cv. Pax) suggests that (1) the parents are not homozygous at the S-allele; (2) in the parents, many different S-alleles are present which results in different S-genotypes of the F, plants. The aim of this experimental work was not to specify the number of alleles present. Eenink (1981) made an analysis of the incompatibility system in chicory. For this
Fig. 1. Genotypic
identity of chicory cultivars.
8
C.I. Castaiio et al./Scientia
Horticulturae
72 (1997) 1-9
purpose, Eenink produced two groups of F, plants from known inbred lines and in two experiments crossed each group of F, plants between them and also ,selfed them. When analyzing the result of experiment 1, Eenink found that after self-pollination and intra-pollination the reaction was incompatible in both cases. The experiment 2 of Eenink gave a result of self-incompatibility but intra-compatibility was found. The genotype attributed to the F, plants of the experiment 1 was S,S,. The genotypes attributed to the F, plants of the experiment 2 were S,S,, S,S,, S,S, and S,S,. As can be observed, the intra-compatibility in the experiment 2 can be explained by the higher number of S-alleles involved than in experiment 1. Similar results to those that could be discerned from the work of Eenink (1981) were obtained from the experiments presented in this report. This work produced basic and preliminary results about the genetic identity of cultivars. Modem molecular biology techniques based on RAPD markers and rDNA RFLP enable also to show the degree of genetic purity in a F, population. Since the cultivars analyzed in this work were F, commercial hybrids used by growers for Belgian endive production, information about the genetics of the inbred parents used to derive the F, population was not available. Therefore, a DNA approach in which DNA polymorphism for parents and hybrids should be obtained, was not considered. Nevertheless, our results justify the implementation of direct DNA approach using RAPD or RFLP technique for further studies on establishing the genetic identity of F, hybrids. Genetic purity of F, hybrid chicory samples has been assessed by RFLP analyses with a nuclear rDNA probe (Bellamy et al., 1995). Average percentage of hybrids in the F, hybrid ‘Flash’ was 71.25%. RAPD markers have also been used by Bellamy et al. (1996) to control genetic purity of F, hybrid seed sample. Owing to Mendelian inheritance of RAPD markers, F, hybrid patterns should show the specific pattern of each parent. Analysis from ‘Flash’ sample revealed that only 66.7% of the seedlings were F, hybrids. RAPD appeared to be easier and less time-consuming than RFLP analysis. The results obtained in this work, agree with those of Bellamy et al. (1995, 1996) respecting the no 100% genetic purity of F, hybrid ‘Flash’. 4.3. Seed-set when the plants were used as females plants were used as males in cross-pollinations
compared
with the result when the
The five commercial cultivars were cross-compatible among themselves. Nevertheless, there were, in some cultivars, significant differences in the degree of compatibility when they were used as either female or male plants. The cv. Carolus, cv. Pax and cv. Sigma had significantly different reactions of compatibility when crossed between them as females or as male plants and equal reactions when crossed as female or males with cv. Flash and cv. Focus. The cv. Flash and cv. Focus presented equal behaviour when taken as female or as male plants except when they were crossed with each other. Since the general reaction in all the crossings was cross-compatibility, these significant differences may be attributed to different genotypes presented among each of the commercial F, chicory cultivars. The similar reaction to incompatibility in any sense of crossing agrees with the results of Pecaut (1962) and Eenink (1981).
C.I. Castaiio et al. /Scientia
Horticulturae
72 (1997) I-9
9
Acknowledgements This research was supported by A.B.O.S. through a collaboration program between Katholieke Universiteit Leuven and Jorge Tadeo Lozano University of Santafe de Bogota, Colombia. Authors would like to thank the personnel of the Laboratory of Plant Culture, KUL, for their help with the pollinations in this work.
References Bellamy, A., Mathieu, C., Vedel, F., Bannerot, H., 1995. Cytoplasmic DNAs and nuclear restriction fragment length polymorphisms in commercial witloof chicories. Theor. Appl. Genet. 91, 505-509. Bellamy, A., Vedel, F., Bannerot, H., 1996. Varietal identification in Cichorium intybus L. and determination of genetic purity of Fl hybrid seed samples, based on RAPD markers. Plant Breeding 115, 1288132. Coppens d’Eeckenbrugge, G., Gobbe, J., Evrard, B., 1987a. Fertilite. In Mecanismes de la reproduction chez la chicoree de Bruxelles: fondements et applications a la selection. I.R.S.I.A. 21-26. Coppens d’Eeckenbrugge, Cl., Louant, B.P., Swenne, A., 1987b. Systeme d’incompatibilitt. In: Mecanismes de la Reproduction chez la chicor&. de Bruxelles: fondaments et applications B la selection. 1.R.S.I.A 27-40. De Rijck, G., Schrevens, E., De Proft, M., 1993. Cultivation of chicory plants in hydroponics. Acta Horticulturae 361, 555-564. Eenink, A.H., 1981. Compatibility and Incompatibility in witloof-chicory (Cichorium intybus L.): 2. The incompatibility system. Euphytica 30, 77-85. Olesen, P., Bruun, L., Steen, P., 1992. The use of incompatibility in breeding programs. In: Dattee, Dumas, Gallais (Eds.), Reproductive Biology and Plant Breeding. Springer, Berlin, pp. 177-183. Pecaut, P., 1962. Etude sur le systeme de reproduction de l’endive (Cichorium intybus L.1. Ann. Amelior. Plantes 12 (41, 265-296. Varotto, S., Pizzoli, L., Lucchin, M., Parrini, P., 1995. The incompatibility system in Italian Red Chicory (Cichorium intybus L.1. Plant Breeding 114, 535-538.
Scientia Horticulturae
72
(1997)l-9
Incompatibility reactions and genotypic identity status of five commercial chicory ( Cichorium intybus L.) hybrids C.I. Castafio *, M.A.C. Demeulemeester, Faculty
M.P. De Proft
ofAgricultural and Applied Biological Sciences, Katholieke Uniuersiteit L.euven, Willem de Croylaan 42, B-3001 Heuerlee, Belgium Accepted 24 July 1997
Abstract Incompatibility reactions of chicory plants cv. Flash, cv. Carolus, cv. Pax, cv. Focus and cv. Sigma were determined. Different levels of incompatibility were found. The cv. Carolus was highly self-incompatible, cv. Flash and cv. Pax could be self-compatible or self-incompatible and the cv. Sigma and cv. Focus are self-compatible. All the tested cultivars were intra-compatible (pollinations within the same cultivar) and cross-compatible among them. There was difference in percentage of viable seeds when cultivars were self-pollinated and intra-pollinated. This difference in percentages of viable seeds can be used as a numerical criterion to determine genotypic identity of a cultivar. The higher this difference value, the less uniform the behaviour of the cultivars in the field and during hydroponic forcing for production of the chicory head was observed. The results led to the conclusion that some of the commercial cultivars are not genotypic identical which has unfavorable implications in Belgian endive production and commercialization. There was equal reaction to incompatibility in any sense of crossing (plants used as females compared with the result when plants were used as males in cross-pollinations). 0 1997 Elsevier Science B.V. Keywords: Chicory;
Pollination;
Seed set; Self-incompatibility
1. Introduction Chicory is an important economic plant for its use as a vegetable (Belgian endive) and as an industrial raw material to obtain inulin from roots (Root chicory). Chicory is
* Corresponding author. [email protected]
Tel.:
+ 32
16
322390;
fax:
0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)0011 l-8
+ 32
16
322966;
e-mail:
2
C.I. Castaiio et al. /Scientia
Horticulturae
72 (1997) l-9
an allogamous species due to the morphology of the flowers and the presence of a sporophytic self-incompatibility system (Coppens d’Eeckenbrugge et al., 1987a). PCcaut suggested two methods to measure the incompatibility level of commercial cultivars of chicory (PCcaut, 1962): (1) Count of viable seeds per flower head after controlled pollination of five flower heads per plant. (2) Examination of the pollen germination on stigma and penetration of pollen tube after controlled pollination. According to Pecaut it is impossible to classify plants as either self-compatible or self-incompatible because the measure of the degree of self-incompatibility within one plant shows continuous variation. It is necessary to sort the plants into classes depending on the different self-compatible degrees. A distribution of plants in groups according to the number of viable seeds/flower head is suggested. Based on experiments about compatibility and incompatibility in chicory, Eenink (198 1) made a distribution of clones over three classes in a rather arbitrary way-formation of fewer than 3 seeds means incompatibility (- ), formation of between 3 and 6 seeds indicates either incompatibility or compatibility ( + ) and formation of more than 6 seeds indicates compatibility. In his work, the mean seed production of 2-4 flower heads over 15 clones from plants of different populations was examined. PCcaut (19621, Eenink (1981) and Varotto et al. (1995) suggested the existence of a sporophytic self-incompatibility system in chicory. The presence in chicory of this system offers possibilities for the production of F, hybrid cultivars. However, if several self-incompatible plants are used to produce inbred lines methods should be available to induce self-fertilization (Eenink, 198 I). On the other hand, full use of self-incompatibility for F, production creates problems for the breeders. The degree of self-incompatibility is often not fully reliable, especially under field conditions (Olesen et al., 1992). Breeding programs require good knowledge of the self-incompatibility status of the material used. In this report, the determination of the self-incompatible degree of five commercial cultivars by self-pollination (the flowers were pollinated with pollen derived from flowers of the same plant), intra-pollination (the flowers were pollinated with pollen derived from flowers of a different plant but of the same cultivar) and cross-pollination between plants of the different cultivars is presented. Comparison of the results obtained produced some interesting information about the genotypic identity of the F, commercial hybrids which are actually used by growers for the production of the vegetable ‘Belgian endive’.
2. Materials and methods 2.1. Plant material Plants of C. intybus L. var. foliosum raised from seeds of the following cultivars were used: cv. Flash (INRA, France), cv. Carolus (Bucomat, Belgium) cv. Sigma (Vilmorin, France), cv. Pax (Rijk Zwaan, Netherlands) and cv. Focus (Nunhems, Netherlands). The seeds were placed in petri dishes in the dark, cold treated at 4°C and relative humidity + 95% for 3 weeks, and transplanted to a hydroponic system (May 1995)
Cl. Castaiio et al. /Scientia Horticulturae 72 (1997) 1-9
under an open plastic greenhouse. 11 cm diameter) filled with fine tubes were placed in a gully (23 in a row being 25 cm. (De Rijck to the seeds, the plants produced
3
The system consisted in PVC tubes (50 cm high and rockwool flocks grodan type 012/519. Two rows of cm wide and 13 m long), with distance between tubes et al., 1993). Due to the vernalization treatment given flowers during the first growing season.
2.2. Controlled pollinations Controlled pollinations were carried out in a manner that was similar to that of PCcaut (Pecaut, 1962). The branches of the plants were enclosed before anthesis in a cellophane bag. Hand pollinations were made by placing the pollen of an open flower, isolated in a bag, to the stigma of an open flower, also isolated in a bag. The enclosure of the branches prevented the intervention of insects in the pollination process. Wind movement in the bags was restricted. 2.3. Experimental
design
Fifteen plants of each cultivar were used. The following treatments were given for each cultivar: 1. Self-pollination: a flower was pollinated with a flower from the same plant 2. Intra-pollination between plants of the same cultivar 3. Cross-pollination among the 5 cultivars. Five flower heads were pollinated in each plant for each treatment. The five flower heads that corresponded to one treatment were all located on one isolated branch. As a result, each plant had six isolated branches: one for self-pollination, one for intra-pollination, and four for the crosses with the other cultivars. The pollinations were carried out during five weeks, from the first days of August until the beginning of September. The weather conditions during the experiment are presented in Table 1. Temperature and RH under open plastic greenhouse can slightly differ. The pollinations were made one hour after anthesis until noon. Eight weeks after the last pollinations (end of October 1995) the branches were collected and the seeds of the marked pollinated flower heads were taken to make the germination test.
Table 1 Weather characteristics per month (during experiment) for the year 1995. Station Herent. (Source: Koninklijk Meteorologisch Instituut van BelgiE, KM1 Klimatologische Dienst) Month
T max (mean “C>
T min (mean “C>
T average (mean “0
RH average (mean %)
Sunshine max (h min)
May June July August September October
19.5 19.9 26.8 26.6 18.9 18.2
8.0 10.8 15.1 13.7 10.7 9.6
13.8 15.4 21.0 20.1 14.8 13.9
72 80 75 69 81 84
1318 1518 1430 1406 1006 0924
4
2.4. Germination
C.I. Castaiio et al./Scientia
Horticulturae 72 (1997) l-9
test of the seeds
In chicory, the fruits are achenes (one-seeded, dry fruits). In this work, the term of seeds refers to achenes. Seeds can be classified as full (embryo is present) or empty. Because it is difficult to distinguish an empty from a full seed, a germination test was done. Seeds that germinated were considered to be viable. The seeds of each plant were placed per treatment in a petri dish on wet filter paper for 3 or 4 days in a culture room at 22 + 2°C 60 pmol mm2 SK’ irradiance (fluorescent lamps Philips 36 W, TLD/54) and 16-h daylength. For cross-pollinations and intra-pollinations, there were approximately 100 total seeds/petri dish per treatment per plant, which corresponded to the five flower heads pollinated in the corresponding treatment. For selfings, the seeds of each flower head were collected separately in petri dishes. As a result, for selfings, there were 5 petri dishes with approximately 20 seeds per plant. 2.5. Statistical analysis The percentage of viable seeds in the total number of seeds (from five flower heads) was calculated per plant in each treatment per cultivar. The means of the percentages were calculated per treatment and per cultivar. For selfings, the percentage of viable seeds per flower head was calculated. The means of the percentages were calculated then per plant, treatment and cultivar. Mean number of total seeds/flower head was 20. To have a better normal distribution of the means of the percentages of the viable seeds, the values were transformed using the inverse parabolic sine. With the transformed percentages, the means of viability were checked for relations between self-, intra- and cross-pollination in each cultivar. The data were statistically analyzed using ANOVA and Duncan test to check the differences of the means. Means with the same letter are not significantly different.
3. Results
3.1. Self-compatibility, cial cultivars
intra-compatibility
and cross-compatibility
of the five commer-
Table 2 shows the mean percentage of viable seeds in the total seeds per plant after self-pollination, intra-pollination and cross-pollination of the five commercial cultivars studied. If the criterion of Eenink (1981) is adopted and an average of 20 seeds per flower head is taken in consideration, a viability percentage of 15% means incompatibility ( - ), 30% indicates compatibility or incompatibility ( f ), and more than 30% indicates compatibility. According to this classification, cv. Carolus is highly self-incompatible (6.2%). The cv. Flash (27.5%) and cv. Pax (24.6%) could be self-compatible or self-incompatible and the cv. Sigma (54.4%) and cv. Focus (50.0%) are self-compatible. All the tested cultivars were intra-compatible with percentages of viable seeds in total seeds per plant ranging from 32.4% for cv. Pax to 77.5% for cv. Sigma. All cultivars were cross-com-
C.I. Castafio et al./Scientia Table 2 The self-incompatibility,
intra-incompatibility
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5
of chicory cultivars
Cultivar (female)
Cultivar (male)
Mean (real) %
Duncan
Carolus
Flash Focus Sigma Pax Intra Self
65.9 63.8 52.6 49.9 56.1 6.2
A A A A A B
Flash
Carolus Sigma Focus Pax Intra Self
79.2 52.5 48.2 43.2 50.8 21.5
A B B B B C
Carolus Focus Sigma Flash Intra Self
72.6 42.9 40.3 38.8 32.4 24.6
A B BC BC BC C
Sigma
Carolus Flash Pax Focus Intra Self
75.9 65.9 65.8 57.5 77.5 54.4
A BA BA BA A B
Focus
Flash Carolus Sigma Pax Intra Self
68.3 64.3 55.0 54.4 69.0 50.0
A BA BA BA A B
Mean percentages of viable seeds in total seeds per plant. Means are based on the pollination of 5 flower heads per plant of 15 plants.
patible among themselves. In this respect, the highest percentage of viable seeds in total seeds per plant was observed when cv. Flash was crossed as female with cv. Carolus (79.2%) and the lowest when cv. Pax was crossed as female with cv. Flash (38.8%).
3.2. Differences in the seed set when the plants were used as females result when the plants were used as males in cross-pollinations
compared with the
Mean percentages of viable seeds in total seeds per plant of cv. Carolus, cv. Flash, cv. Pax, cv. Sigma and cv. Focus obtained in the both sense of crossing are shown in Table 3. ANOVA and Duncan analyses of the means when the plants were used as
6
C.I. Castaiio et al./Scientia
Table 3 The cross-incompatibility as males)
of chicory cultivars
in the two directions
Carolus
Flash (mean o/o)
Pax (mean %)
Female Male
65.9 79.2
49.9 72.6
Flash
Carolus (mean %I
Pax (mean o/o)
Female Male
79.2 65.9
43.2 38.8
Pa.%
Flash (mean %)
Female Male
38.8 43.2
Sigma
Flash (mean %)
Female Male
65.9 52.5
Focus
Flash (mean %o)
Pax (mean %I
Female Male
68.3 48.2
54.4 43.0
A A
A A
A A
A A
A B
Horticulturae 72 (1997) 1-9
B A
A A
of pollination
(plants used as females and
Sigma (mean %)
Focus (mean %)
52.6 75.9
63.8 64.3
B A
A A
Sigma (mean %)
Focus (mean %)
52.5 65.9A
48.2 68.3
A A
B A
Carolus (mean %)
Sigma (mean %I
Focus (mean %)
72.6 49.9
40.3 65.8
42.9 54.4
A B
B A
A A
Pax (mean %I
Carolus (mean %o)
Focus (mean %)
65.8 40.3
75.9 52.6
57.5 55.0
A B
A A
A B
A A
Sigma (mean %)
Carolus (mean %)
55.0 57.5
64.3 63.8
A A
A A
Mean percentages of viable seeds in total seeds per plant. Means are based on the pollination of five flower heads per plant of 15 plants.
females and males were carried out, using exclusively the values involved and not the whole data set. Means with the same letter are not significantly different. The five commercial cultivars were cross-compatible among themselves with percentages of viable seeds always higher than 30%. Nevertheless, there were, in some cultivars, significant differences in the degree of compatibility when they were used as either female or male plants.
4. Discussion
4.1. Information incompatibility
about the self- incompatibility, of commercial cultivars
intra-incompatibility
and
cross-
Different levels of self-incompatibility were found. The cv Carolus was highly self-incompatible, the cv. Flash and cv. Pax could be either self-compatible or selfincompatible and the cv. Sigma and cv. Focus were self-compatible. All the tested cultivars were intra-compatible (pollinations within the same cultivar) and crosscompatible among them. As stated by Coppens d’Eeckenbrugge et al. (1987b3) this self-compatibility is ascribed to the presence of heterozygote genotypes composed of S-alleles where the relations between dominance in pollen and the pistil are not clearly defined or homozygote genotypes for the S-allele are very weak in the scale of dominance.
C.I. Castaiio et al. /Scientia Horticulturae 72 (1997) I-9
4.2. Genotypic
I
identity of cultivars
If the commercial cultivars that have been studied are hybrids derived from inbred homozygous plants, genotypic identity of the hybrid plants that belong to the same cultivar would be expected. In this situation, the self-incompatible reaction should be the same whether the pollen used to pollinate a flower head is taken from another flower head of the same plant or from a flower head from a plant within the same cultivar. The statistical analysis shows that there are significant differences between the means of percentages of viable seeds when self-pollinations and intra-pollinations were made in all the cultivars, except in cv. Pax (Table 2). This difference in viability of the seeds could be interpreted as a criterion to define how genotypic identical a cultivar is (genetically homogeneous). The cv. Carolus shows 56.1% of germination when intrapollinated and 6.2% when self-pollinated. The difference between these two values is 50.0%. The differences corresponding to the other cultivars tested are: 23.3% for cv. Flash, 23.1% for cv. Sigma, 19.0% for cv. Focus and 7.8% for cv. Pax. According to these values, the distribution of cultivars regarding their genotypic identity was as follows: cv. Carolus was the less genotypic identical cultivar, followed by cv. Flash, cv. Sigma and cv. Focus. The cultivar cv. Pax proved to be the most genotypic identical (Fig. 1). These results were compared with the uniform behaviour of the cultivars, observed in the field and during forcing at the Research Station of Herent (Claessens G, personal communication). The behaviour of the cultivars, reported by this research station, coincides with the results obtained in the pollination experiments, especially concerning the extreme cases: cv. Pax and cv. Carolus. The cv. Carolus is no longer used for commercial forcing and chicory head production (due to the non-uniform behaviour) and cv. Pax was one of the most uniform cultivars for chicory head production in the period 1995-1996. This comparison leads to the conclusion that some of the commercial cultivars have non-genotypical identical characteristics which have unfavorable implications for Belgian endive production and commercialization. This different value between percentages of viable seeds when cultivars are self-pollinated and intra-pollinated can be used as a numerical criterion. The criterion has not been used or reported in literature by any other author for this purpose. The high level of intra-compatibility between the F, hybrid plants of each of the five cultivars tested (except cv. Pax) suggests that (1) the parents are not homozygous at the S-allele; (2) in the parents, many different S-alleles are present which results in different S-genotypes of the F, plants. The aim of this experimental work was not to specify the number of alleles present. Eenink (1981) made an analysis of the incompatibility system in chicory. For this
Fig. 1. Genotypic
identity of chicory cultivars.
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purpose, Eenink produced two groups of F, plants from known inbred lines and in two experiments crossed each group of F, plants between them and also ,selfed them. When analyzing the result of experiment 1, Eenink found that after self-pollination and intra-pollination the reaction was incompatible in both cases. The experiment 2 of Eenink gave a result of self-incompatibility but intra-compatibility was found. The genotype attributed to the F, plants of the experiment 1 was S,S,. The genotypes attributed to the F, plants of the experiment 2 were S,S,, S,S,, S,S, and S,S,. As can be observed, the intra-compatibility in the experiment 2 can be explained by the higher number of S-alleles involved than in experiment 1. Similar results to those that could be discerned from the work of Eenink (1981) were obtained from the experiments presented in this report. This work produced basic and preliminary results about the genetic identity of cultivars. Modem molecular biology techniques based on RAPD markers and rDNA RFLP enable also to show the degree of genetic purity in a F, population. Since the cultivars analyzed in this work were F, commercial hybrids used by growers for Belgian endive production, information about the genetics of the inbred parents used to derive the F, population was not available. Therefore, a DNA approach in which DNA polymorphism for parents and hybrids should be obtained, was not considered. Nevertheless, our results justify the implementation of direct DNA approach using RAPD or RFLP technique for further studies on establishing the genetic identity of F, hybrids. Genetic purity of F, hybrid chicory samples has been assessed by RFLP analyses with a nuclear rDNA probe (Bellamy et al., 1995). Average percentage of hybrids in the F, hybrid ‘Flash’ was 71.25%. RAPD markers have also been used by Bellamy et al. (1996) to control genetic purity of F, hybrid seed sample. Owing to Mendelian inheritance of RAPD markers, F, hybrid patterns should show the specific pattern of each parent. Analysis from ‘Flash’ sample revealed that only 66.7% of the seedlings were F, hybrids. RAPD appeared to be easier and less time-consuming than RFLP analysis. The results obtained in this work, agree with those of Bellamy et al. (1995, 1996) respecting the no 100% genetic purity of F, hybrid ‘Flash’. 4.3. Seed-set when the plants were used as females plants were used as males in cross-pollinations
compared
with the result when the
The five commercial cultivars were cross-compatible among themselves. Nevertheless, there were, in some cultivars, significant differences in the degree of compatibility when they were used as either female or male plants. The cv. Carolus, cv. Pax and cv. Sigma had significantly different reactions of compatibility when crossed between them as females or as male plants and equal reactions when crossed as female or males with cv. Flash and cv. Focus. The cv. Flash and cv. Focus presented equal behaviour when taken as female or as male plants except when they were crossed with each other. Since the general reaction in all the crossings was cross-compatibility, these significant differences may be attributed to different genotypes presented among each of the commercial F, chicory cultivars. The similar reaction to incompatibility in any sense of crossing agrees with the results of Pecaut (1962) and Eenink (1981).
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Acknowledgements This research was supported by A.B.O.S. through a collaboration program between Katholieke Universiteit Leuven and Jorge Tadeo Lozano University of Santafe de Bogota, Colombia. Authors would like to thank the personnel of the Laboratory of Plant Culture, KUL, for their help with the pollinations in this work.
References Bellamy, A., Mathieu, C., Vedel, F., Bannerot, H., 1995. Cytoplasmic DNAs and nuclear restriction fragment length polymorphisms in commercial witloof chicories. Theor. Appl. Genet. 91, 505-509. Bellamy, A., Vedel, F., Bannerot, H., 1996. Varietal identification in Cichorium intybus L. and determination of genetic purity of Fl hybrid seed samples, based on RAPD markers. Plant Breeding 115, 1288132. Coppens d’Eeckenbrugge, G., Gobbe, J., Evrard, B., 1987a. Fertilite. In Mecanismes de la reproduction chez la chicoree de Bruxelles: fondements et applications a la selection. I.R.S.I.A. 21-26. Coppens d’Eeckenbrugge, Cl., Louant, B.P., Swenne, A., 1987b. Systeme d’incompatibilitt. In: Mecanismes de la Reproduction chez la chicor&. de Bruxelles: fondaments et applications B la selection. 1.R.S.I.A 27-40. De Rijck, G., Schrevens, E., De Proft, M., 1993. Cultivation of chicory plants in hydroponics. Acta Horticulturae 361, 555-564. Eenink, A.H., 1981. Compatibility and Incompatibility in witloof-chicory (Cichorium intybus L.): 2. The incompatibility system. Euphytica 30, 77-85. Olesen, P., Bruun, L., Steen, P., 1992. The use of incompatibility in breeding programs. In: Dattee, Dumas, Gallais (Eds.), Reproductive Biology and Plant Breeding. Springer, Berlin, pp. 177-183. Pecaut, P., 1962. Etude sur le systeme de reproduction de l’endive (Cichorium intybus L.1. Ann. Amelior. Plantes 12 (41, 265-296. Varotto, S., Pizzoli, L., Lucchin, M., Parrini, P., 1995. The incompatibility system in Italian Red Chicory (Cichorium intybus L.1. Plant Breeding 114, 535-538.