Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2010
Volume 13
Issue 4
Topic:
Horticulture
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Zalewska M. , Miler N. , Tymoszuk A. , Drzewiecka B. , Winiecki J. 2010. RESULTS OF MUTATION BREEDING ACTIVITY ON Chrysanthemum × grandiflorum (Ramat.) Kitam. IN POLAND, EJPAU 13(4), #27.
Available Online: http://www.ejpau.media.pl/volume13/issue4/art-27.html

RESULTS OF MUTATION BREEDING ACTIVITY ON CHRYSANTHEMUM × GRANDIFLORUM (RAMAT.) KITAM. IN POLAND

Małgorzata Zalewska1, Natalia Miler2, Alicja Tymoszuk1, Barbara Drzewiecka3, Janusz Winiecki3
1 Department of Ornamental Plants and Vegetable Crops, University of Technology and Life Sciences in Bydgoszcz, Poland
2 Department of Ornamental Plants and Vegetable Crops, Faculty of Agriculture and Biotechnology, University of Science and Technology, Bydgoszcz, Poland
3 Medical Physics Department, Oncology Centre, Bydgoszcz, Poland

 

ABSTRACT

Our researches concerning somatic mutagenesis in Chrysanthemum × grandiflorum (Ramat.) Kitam. induced with ionizing radiation have been carried out since 1977. First significant results were obtained in vivo when irradiated leaves detached from flowering chrysanthemums were applied. Induction of mutations in subsequent experiments was conducted in vitro with the use of the adventitious buds technique for regeneration. Explants were leaves, callus, internodes and single nodes. Except for callus, all explants were taken from irradiated microcuttings growing in vitro during the mutagenic treatment. The dose of X- and gamma- rays involved in our experiments ranged from 15 to 25 Gy. For X-rays therapeutic apparatus THX-250 Medicor was used. Gamma radiation was obtained from Co60 cobalt source generated by Theraton 780 C. Regeneration was conducted on MS medium supplemented with 0.6 mg·dm-3 BAP and 2.0 mg·dm-3 IAA. The most spectacular effects were observed when gamma-rays and in vitro regeneration were used. All of mutants were solid, non-mericlinal and non-sectorial chimeras. Our experiments show that over a short period from a single mother cultivar one can obtain numerous attractive mutants, thus creating new cultivar groups.

Key words: Chrysanthemum × grandiflorum (Ramat.) Kitam., mutation breeding, ionizing radiation, in vitro.

INTRODUCTION

In chrysanthemum induced mutants account for over 50% of all the commercial cultivars. Some characters of this species make the application of mutation breeding techniques fully justifiable. A high level of ploidy and a high level of heterozygocity as well as the phenomenon of self-incompatibility make it difficult to use the traditional breeding method which involves crossing and selection. It is worth noting that in the production of chrysanthemum the propagation is vegetative only and seed setting is in their case – much more difficult.

For the first time an effective use of leaves for irradiation and induction of mutation in chrysanthemum was considered by Broertjes et al. [1]. According to the theory by Broetjes and Keen [2], the adventitious meristem of plants regenerated in vivo and in vitro from leaves finally comes from a single initial cell. Therefore the use of adventitious buds techniques in irradiation breeding makes the regenerated plants, including the mutated ones, consist of genetically homogenous tissues. In that way the creation of chimeras is avoided, which can shorten the breeding process considerably.

The first Polish chrysanthemum mutants, 'Helena' and 'Władysław' cultivars, were created in a spontaneous way at the Department of Ornamental Plants, University of Life Sciences in Poznań in 1976. In 1977 the Department of Ornamental Plants and Vegetable Crops, University of Technology and Life Sciences in Bydgoszcz has launched research into mutagenesis in chrysanthemum,  induced in vivo and in vitro with the ionization irradiation with propagation with the use of adventitious buds.

MATERIAL AND METHODS

In vivo experiments. Our researches concerning somatic mutagenesis in Chrysanthemum × grandiflorum (Ramat.) Kitam. have been carried out since 1977. Mature leaves of 'Bravo' were detached from the flowering plants, exposed to X irradiation at the dose of 15 Gy. The irradiation area included the leaf petioles. The leaves were rooted and the shoots obtained as a result of adventitious regeneration were cut off and then rooted and brought to flowering [12].

A similar regeneration procedure was used for 'Red Nero'. Leaves were irradiated with 5–25 Gy doses of X and gamma rays [13].

In vitro experiments. In the experiments carried out by Jerzy and Zalewska in 1996 [3] microcuttings in vitro were exposed to irradiation and then the leaves were detached. The leaf explants were placed on the MS medium [9], with 0.6 mg·dm-3 BAP and 2.0 mg·dm-3 IAA added. The regenerated adventitious buds were transferred onto the rooting medium containing NAA at the concentration of 0.02 mg·dm-3. The plants were brought to flowering in vivo.

A similar irradiation and regeneration procedure was used for 'Lilac Wonder' the inflorescence of which was violet [4].

In the research reported by Zalewska et al. [16] 3 pot cultivars of chrysanthemum, 'Albugo', 'Alchimist' and 'Satinbleu', were used. The mutagenesis induced in vitro with the gamma irradiation at the dose of 15 Gy involved the use of microcuttings. Single-node fragments, leaves were taken as explants. Also callus obtained on the leaf explant was exposed to irradiation.

The cultivars of Nero, Wonder [8] and Lady [6] Group were exposed to the genome analysis with the RAPD-PCR method and the qualitative and quantitative composition of pigments in ligulate florets in original cultivars and in selected was defined.

Fig. 1. Procedure of breeding with the usage of gamma radiation and adventitious buds method of regeneration in in vitro cultures

To irradiate the plants with the X rays, in all the experiments therapeutic apparatus THX-250 Medicor was used, while gamma irradiation was generated by the source of Co60 apparatus Theraton 780 C. The dose of X and gamma irradiation was, respectively, 0.92–0.93 Gy·min-1 and 1.92–2.14 Gy·min-1. The plant irradiation was carried out in Bydgoszcz-based hospitals. The procedure of breeding with the usage of in vitro cultures and gamma radiation is shown on Figure 1.

RESULTS AND DISCUSSION

In vivo experiments. Our first breeding success occurred in 1979 when 'Paloma', 'Poranek' and 'Promyk' mutants were created [12]. As a result there were obtained mutants with the lilac, pink, bronze and red inflorescences, definitely different from purple, which is found in the mother cultivar 'Bravo'. Silver purple colour was accompanied by a change in shape of florets from ligulate to tubular. Besides also new inflorescence forms were obtained, such as pompon, peony and semi-full, different from the decorative type of the inflorescence of the mother cultivar (Table 1, Fig. 2).

Table 1. The most attractive results of mutagenesis in chrysanthemum induced by ionizing radiation
 

Mother cultivar

Irradiation

The most attractive mutants

phenotype

name

dose
 (Gy)

plant
material

phenotype

name

references

in vivo

purple,
decorative

Bravo

15
X-rays

leaf

lilac, pompon
pink
silver purple, tubular

Paloma
Poranek
Promyk

Stepczyńska et al. [12]

dark red,
strongly growing

Red Nero

25
X-rays

leaf

orange red,
dwarf

Mini Nero

Zalewska and Jerzy [13]

in vitro

purple pink,
decorative

Richmond

15
X-rays
or
γ-rays

leaf

golden beet
golden brown
heather pink, tubular
pink
purple gold
reddish brown
orange
salmon
yellow
white

Lady Apricot
Lady Amber
LadyVitroflora
Lady Pink
Lady Rosy
Lady Bronze
Lady Orange
Lady Salmon
Lady Yellow
Lady White

Jerzy and Zalewska [3]

violet

Lilac Wonder

15
γ-rays

leaf

brown

red

Bronze Wonder
Red Wonder

Jerzy and Zalewska [4]

heather pink,
tubular

Lady Vitroflora

15
γ-rays

leaf

leaf shape

Zalewska and Lema-Rumińska [15]

white,
semiball

Albugo

15
γ-rays

node/ internode

yellow, decorative

Albugo Sunny

Zalewska et al. [16]

dark violet,
semiball

Alchimist

15
γ-rays

leaf

silver violet, tubular

golden beet, decorative

Alchimist Tubular
Alchimist Golden Beet

dark pink,
semiball

Satinbleu

15
γ-rays

node
internode

salmon
pale pink, decorative

Satinbleu Honey
Satinbleu Minty

Also under in vivo conditions in 1988, as a result of the X irradiation treatment at the dose of 25 Gy on the leaf explants, from a strongly growing, cultivar 'Red Nero' a precious mutant was obtained, 'Mini Nero', with an inhibited growth and smaller inflorescences and leaves. As a result a dwarf cultivar was produced, applicable to pot growing (Table 1, Fig. 2). Using gamma rays for inducing mutations was a more effective way of enlarging genetic variation spectrum in plants than using X radiation [13].

Fig. 2. The most attractive results of mutagenesis in chrysanthemum induced by ionizing radiation

Due to the fact that under in vivo conditions the regeneration of adventitious buds is a time-consuming process and as not all the chrysanthemum cultivars are capable of adventitious organogenesis, in vitro cultures are better solution to mutation breeding [17].

In vitro experiments. In the experiments carried out by Jerzy and Zalewska in 1996 [3] there was shown a possibility of obtaining a wide spectrum of variation in 'Richmond' chrysanthemum especially, due to the exposure to mutagenic factors: X and gamma irradiation, at the dose of 15 Gy. As a result there were obtained as many as 21 inflorescence colours which differed significantly from the typical purple pink colour of the initial cultivar. The most interesting ones were the following mutants: golden beet, golden brown, pink, purple gold, reddish brown, salmon, yellow and white in colour as well as the mutant the inflorescence of which was heather pink with ligulate florets grown into tubes (Table 1, Fig. 2).

The range of mutation changes was in the case of cultivar 'Lilac Wonder' much smaller. The research gave rise to two mutants, 'Bronze Wonder' with a bronze and 'Red Wonder' with a red inflorescence (Table 1, Fig. 2).

It is worth mentioning that the cultivars of Nero, Wonder [8] and Lady [6] Group were also exposed to the genome analysis with the RAPD-PCR method, demonstrating the variation which was genetic in character. These radiomutants were also researched as far as the content of pigments was concerned in ligulate florets with the spectrometric method, stating that they differed in the quality and quantity of flavonoids and carotenoids, as compared with the cultivars they have originated from [5, 7]. The Lady group was also used for research which demonstrated that regeneration in vitro with the adventitious buds technique can be not only a way to a potential indication (separation) of periclinal chimeras, based on the change in the phenotype, but also the source of a new variation [14].

The white cultivar, 'Albugo', gave rise to yellow mutant. Dark violet cultivar 'Alchimist' produced two mutants with the following inflorescences: golden beet and silver violet with ligulate florets grown in tubes. Mutants with salmon and pale pink inflorescences were produced by irradiating the dark pink cultivar, 'Satinbleu'. These mutants can quite soon increase the assortment of the existing chrysanthemum cultivars, grown in pots, thus creating cultivar groups [16] (Table 1, Fig. 2).

Another interesting aspect of chrysanthemum mutation breeding can be also the second irradiation of the mutants, which can be seen from our experiments with 'Lady Vitroflora' [15]. The irradiation of leaf explants of this radiomutant produced a phenotype with a significantly changed leaf morphology, maintained in successive vegetative generations (Fig. 2).

Our experiments show, foremost, that over a short period one can obtain attractive changes in the colour of inflorescences. However to obtain a wide spectrum of these changes, it is necessary to select an adequate initial genotype exposed to irradiation. The greatest mutation frequency is recorded after the exposure of pink-flowering chrysanthemum cultivars to the mutagenic factor, while the least effective initial genotypes for induced mutagenesis are plants with yellow inflorescences [11]. This thesis coincides with the unpublished results by Jerzy and Zalewska recorded in 1996 where the mother cultivar for induced mutagenesis was the yellow-flowering 'Mrs. R.C. Pulling'.

In the present mutation breeding one should also note that chrysanthemums sometimes show significant differences across cultivars concerning the ability to adventitious buds regeneration and, additionally, the number of adventitious buds after irradiation can be much lower [10]. The adventitious shoot formation in vitro in chrysanthemum can be also affected by other factors as, e.g. cultivar, age and kind of explant, the way the explant is placed on the medium, medium composition, culture conditions, topophysical location of the explant on the donor plant and regeneration dates [18].

In our experiments into the induced mutagenesis using ionization irradiation in chrysanthemum the frequency of mutants ranged from 2.4 to 11.6%, and the frequency of mutation from 0.3 to 5.0%. The frequency of mutants and mutations were determined against the number of flowering plants [1].

CONCLUSIONS

  1. Our results show that the application of induced mutagenesis in connection with the adventitious buds technique in vitro is an especially useful solution in chrysanthemum breeding.

  2. Gamma irradiation at the dose of 15 Gy has been considered a more effective mutagenic factor than X irradiation.

  3. The mutants produced as a result of the procedure presented are solid. They are neither sectorial nor mericlinal chimeras in character; they can be a new and attractive original chrysanthemum cultivars.


REFERENCES

  1. Broertjes S., Roest S., Bokelmann G.S., 1976. Mutation breeding of Chrysanthemum morifolium Ram. using in vivo and in vitro adventitious bud techniques. Euphytica 25, 11–19.

  2. Broertjes C., Keen A., 1980. Adventitious shoots: do they develop from one cell? Euphytica 29, 73–87.

  3. Jerzy M., Zalewska M., 1996. Polish cultivars of Dendranthema grandiflora  Tzvelev and Gerbera jamesonii Bolus bred in vitro by induced mutations. Mutation Breeding Newsletter 42, 19.

  4. Jerzy M., Zalewska M., 1997. Flower colour recurrence in chrysanthemum and  gerbera mutants propagated in vitro from meristms and leaf explants. Acta Horticulturae 447, 611–614.

  5. Lema-Rumińska J., Zalewska M., 2004. Studies on flower pigments of chrysanthemum mutants: Nero and Wonder Groups. Acta Sci. Pol., Hortorum Cultus 3(1), 125–135.

  6. Lema-Rumińska J., Zalewska M., Sadoch Z., 2004. Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity. Plant Breeding 123, 290–293.

  7. Lema-Rumińska J., Zalewska M., 2005. Changes in flower colour among Lady Group of Chrysanthemum × grandiflorum /Ramat./Kitam. as a result of mutation breeding. Folia Horticulturae 17, 61–72.

  8. Lema-Rumińska J., Zalewska M., Sadoch Z., Jerzy M., 2005. Identification of chrysanthemum (Dendranthema grandiflora Tzvelev) mutants of Nero and Wonder groups using RAPD markers. EJPAU, Horticulture 8(2), www.ejpau.media.pl

  9. Murashige T., Skoog F., 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plant. 15, 473–497.

  10. Rademaker W., de Jong, J., 1990. Genetic variation in adventitious shoot formation in Dendranthema grandiflora (Chrysanthemum morifolium) explants. Proceed. Eucarpia Symp., Wageningen, 34–38.

  11. Shum A., Preil W., 1998. Induced Mutations in Ornamental Plants. In: Somaclonal Variation and Induced Mutations in Crop Improvement. Kluwer Academic Publishers, Dordrecht, Boston, London. Jain, S. M, et all., (eds) 333–336.

  12. Stepczyńska K., Jerzy M., Widacka M., 1980. Mutagenesis in Chrysanthemum cv. Bravo propagated from the X-ray influenced leaf cuttings. Prace Inst. Sadown. Kwiac., Seria B 5, 17–30.

  13. Zalewska, M., Jerzy, M., 1997. Mutation spectrum in Dendranthema grandiflora Tzvelev after in vivo and in vitro regeneration of plants from irradiated leaves. Acta Horticulture 447, 615–618.

  14. Zalewska., M., Lema-Rumińska J., Miler N., 2007. In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum. Sci. Hort. 113, 70–73.

  15. Zalewska M., Lema-Rumińska J., 2007. A mutant of a 'Lady Vitroflora' mutant as a  result of radiomutation breeding of chrysanthemums. In: Spontaneous and induced variation for the genetic improvement of horticultural crops. Nowaczyk, P., (eds.), Univ. Press of Univ. of Technology and Life Sciences in Bydgoszcz. 389–393.

  16. Zalewska M., Tymoszuk A., Miler N. New chrysanthemum cultivars as a result of in vitro mutagenesis with application of different explants types (in preparation).

  17. Chrysanthemum × grandiflorum (Ramat.) Kitam. from leaf cuttings. Propagation of Chrysanthemum × grandiflorum (Ramat.) Kitam Symposium, Poznań, 5th October: 1–8.

  18. Zalewska M., 2010. In vitro adventitious bud techniques as a tool in creation of New chrysanthemum cultivars. W: Datta, S.K., Chakrabarty, D. (eds.). Floriculture. Role of tissue culture and molecular techniques. Pointer Publishers, Jaipur, Indie: 189–218.

 

Accepted for print: 30.11.2010


Małgorzata Zalewska
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Life Sciences in Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 36
email: zalewska@utp.edu.pl

Natalia Miler
Department of Ornamental Plants and Vegetable Crops, Faculty of Agriculture and Biotechnology, University of Science and Technology, Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 22
email: nmiler@utp.edu.pl

Alicja Tymoszuk
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Life Sciences in Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 22
email: alicjaskowronek@wp.pl

Barbara Drzewiecka
Medical Physics Department, Oncology Centre,
Bydgoszcz, Poland
Dr I. Romanowskiej 2, 85-796 Bydgoszcz, Poland
phone: (+48) 52 374 34 99
email: basiad@co.bydgoszcz.pl

Janusz Winiecki
Medical Physics Department, Oncology Centre,
Bydgoszcz, Poland
Dr I. Romanowskiej 2, 85-796 Bydgoszcz, Poland
phone: (+48) 52 374 34 99
email: janusz@co.bydgoszcz.pl

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