Impaginato


106

Adv. Hort. Sci., 2011  25(2): 106-111

(1) Corresponding author: skuhf07@yahoo.co.in
Received for publication 4 April 2011.
Accepted for publication 10 May 2011.

In vitro mutagenesis and detection of variability among
radiomutants of chrysanthemum 

using RAPD

A. Kaul, S. Kumar*(1), M. Ghani
Department of Biotechnology, University of Horticulture and Forestry, Solan 173 230 (H.P.), India.

Key words: Dendranthema grandiflora, in vitro mutagenesis, radiomutants, somaclones.

Abstract: The present study was undertaken to induce mutations in Dendranthema grandiflora cv. Snow Ball
through in vitro mutagenesis by exposing the in vitro shoots to 5, 10, 20 and 30 Gy gamma radiation. RAPD analy-
sis was used to detect genetic polymorphism among the variants and the control. Morphological variations were
not observed with the 5 Gy gamma dose during the first season. Shoot regeneration, rooting and survival of
shoots were affected by gamma ray doses. 10 Gy of gamma irradiation was the most effective in inducing muta-
tions in flower colour through direct in vitro mutagenesis. The shoots irradiated with 20 and 30 Gy gamma radi-
ation did not root and died. Twenty RAPD primers were used to amplify DNA segments from the genomic DNA
of the control and its 10 variants, and the genetic similarity among them ranged from 0.06 to 0.79 revealing high
genetic diversity.

1. Introduction

Chrysanthemum is a major horticultural crop and it
is the second largest in terms of cut flowers after rose,
among the ornamental plants traded in the global
flower market (Kumar et al., 2006). The common gar-
den chrysanthemum is hexaploid with 54 chromo-
somes (Wolff, 1996). It is propagated vegetatively and
has a strong self incompatibility system (Richard,
1986), hence new cultivars are difficult to obtain by
crossing. Traditionally, new cultivars have been
obtained from spontaneous mutations in vegetative
reproduction, sports, being some variations more stable
than others (Miñano et al., 2009). In the last few years,
induced mutations and somaclonal variations derived
from the tissue culture process have been employed as
a new source of variability (Schum, 2003; Datta et al.,
2005; Jain and Spencer, 2006; Zalewska et al., 2007;
Jain, 2010; Barakat et al., 2010).

Although extensive work has been carried out to
develop novelties in chrysanthemum through induced
mutations using physical and chemical mutagens
(Broetjes and Van Harten, 1978), there is always a need
to explore the possibility of new variety for floriculture
trade. Mutation breeding by radiation has been widely
used to upgrade well-adapted plant varieties and also to

develop new variations within improved agricultural
characteristics. Since most cultivated chrysanthemum
cultivars are polyploids with high genetic heterogene-
ity, mutants with allied flower colour, shape, floral size
and shape are often recovered.  Allied flower colours
with chimeric tissue can be easily induced by radiation
and can be isolated using in vitro tools (Kumar et al.,
2006). Identification and characterization of cultivars
is extremely important in horticultural crops in order to
protect the plant breeder’s rights. Traditionally, identi-
fication has been based on morphological characters;
however the development of new technologies has
made it possible to base this analysis on DNA informa-
tion. One approach is a PCR-based technique - RAPD
(William et al., 1990) - that has been widely used for
plant germplasm characterization (Wolff et al., 1994;
Huang et al., 2000; Martin et al., 2002; Martin and
Gonzalez-Benito, 2005). The aim of the present study
was to induce mutation in chrysanthemum cv. Snow
Ball through in vitro mutagenesis by treating the in
vitro shoots with gamma radiation and to apply RAPD
analysis for the detection of genetic polymorphism
among the mutants and control.

2. Materials and Methods

Plant material and in vitro mutagenesis
Nodal segments (2-3 cm) of Chrysanthemum gran-

diflora (Tzelev) cv. Snow Ball, collected from one-



107

year-old mother plants maintained in the glasshouse of
the Department of Biotechnology, University of Horti-
culture and Forestry Solan (Himachal Pradesh), India,
were used as explants. The explants were surface ster-
ilized with 5% sodium hypochlorite (NaOCl2) for 10min, washed thoroughly three to four times with steril-
ized water and cultured on MS (Murashige and Skoog,
1962) medium supplemented with 2 mg/l benzylade-
nine (BA), 0.5 mg/l  α-naphthalene acetic acid (NAA),
30 g/l sucrose (w/v) and 8 g/l agar (w/v; Sigma-
Aldrich, Bangalore). The culture without growth regu-
lators served as control. The pH of the medium was
adjusted to 5.8 before autoclaving at 121oC at a pres-
sure of 1.1 kg cm-2 for 15 min. The cultures were main-
tained under 16-hr photoperiod with a light intensity of
50-60 µmol cm-2 s-1 provided by cool, white fluores-
cent lamps at 24±2oC. After four weeks of culture, the
shoots were subcultured on shoot multiplication medi-
um consisting of MS salts supplemented with 0.5 mg/l
BA, 0.1 mg/l indole acetic acid (IAA) and 1 mg/l gib-
berellic acid (GA3). Shoots about 2.5-3.0 cm long wereirradiated in a gamma cell (60 cobalt source) with 5,
10, 20 and 30 Gy doses. After four weeks, the shoots
were multiplied and 3-4 cm long shoots were trans-
ferred to 1/2 strength MS medium supplemented with
0.3 mg/l indolebutyric acid (IBA) and 0.2% activated
charcoal for rooting. The shoots with well developed
roots were removed from the culture vessel, washed
with running tap water to remove the adhering agar and
transferred to plastic pots (25 cm diameter) filled with
soil:sand:FYM in 1:1:1 ratio and kept in the glasshouse
with 80-90% relative humidity. After acclimatization
for four weeks, the plants were observed for any varia-
tions in morphological characters from the control and
PCR analysis was carried out using the genomic DNA.

Statistical analysis
The data recorded for different parameters were

subjected to completely randomized design (Gomez
and Gomez, 1984). The statistical analysis based on
mean value per treatment was made using analysis of
variance (ANOVA). The LSD multiple range test (p ≤
0.05) was used to determine differences between treat-
ments.

Polymerase chain reaction
Genomic DNA was isolated from fresh, young

green leaves of the control and gamma ray-irradiated
plants following the method of Doyle and Doyle
(1987) with some modifications. For amplification,
reaction mixtures (21.8 µl) contained 1 µl oligonu-
cleotide, 2.5 µl 10X Taq polymerase buffer, 1.5 nM
MgCl2, 2 µl each of dNTPs, 3 µl genomic DNA and 3
µl Taq polymerase (3 units/µl). Random oligonu-
cleotide primers were used for RAPD amplification
(Bangalore Genei, Bangalore, India). Amplification
was performed in a  thermocycler (MJ Research, USA)
programmed for a first denaturation step of 3 min at

94oC followed by 36 cycles of 94oC for 30 s for denat-
uration, 50oC for 30 s for annealing, and 72oC for 2 min
(extension). Twenty Operon random primers (Operon
Technologies, Inc., USA), ten of the OPA series (1-10)
and ten of the OPB series (1-10), were employed for
amplification using the cycling condition mentioned
above.

All samples were given 10 min at 73oC for post-
amplification. PCR products were separated on a 1.4%
agarose gel using 1 x TAE buffer and were stained with
ethidium bromide. In all amplification reactions, a
reaction mixture with water instead of genomic DNA
was used as negative control.  1 kb DNA ladder (Fer-
mantas, Lithuania) was used as the size marker. The
amplified products were visualized using a UV transil-
luminator and photographed using gel documentation
system (Alpha Imager, USA). Amplified DNA was
scored as either presence (1) or absence (0) of band.
Pairwise comparison between the control and variants
was performed to calculate similarity (J) between the
samples (Jaccard, 1908) using SIMQUAL programme
of NTSYS-PC (version 2). A dendrogram was pro-
duced from the resulting similarity using UPGMA
method.

3. Results 

Effect of gamma radiation on in vitro cultures 
The data presented in Table 1 reveal that the sur-

vival of shoots was affected by gamma ray doses. A
decrease in survival was observed with an increase of
irradiation dose; a significantly higher survival per-
centage was observed in the control shoots. The varia-
tion in survival percentage between 5 and 10 Gy was
statistically insignificant. A decrease was also observed
on shoots forming roots. A lethal effect of higher doses
(20 and 30 Gy) was noticed on rooting of shoots. The
shoots did not root, hence they turned yellowish brown
and died. No variation was observed in shoots forming
roots with 5 and 10 Gy irradiation. A significant differ-
ence in percent root initiation was observed between
the control and irradiated shoots. The rooted plants
were acclimatized as explained above and allowed to
grow in the glasshouse till flowering.

Table 1 - Effect of radiation doses on survival of in vitro shoots in D.
grandiflora

Dose strength Survival(%)
Number of shoots

producing roots (%)
0
5
10
20
30
LSD0.05

90.47 (79.93)
66.60 (59.25)
52.38 (46.39)
42.85 (40.83)
19.05 (20.15)
25.11 (21.89)

100.00 (90.90)
82.60 (65.48)
82.35 (65.40)

0(0)
0(0)

6.25 (4.88)
Figures within parentheses are arc sine transformed values.



108

Effect of gamma radiation on morphological charac-
ters

Morphological variations were not observed in the
acclimatized plants exposed to 5 Gy gamma radiation.
All the plants flowered true to the mother floret
colour/shape. Therefore, selections were made among
the plants exposed to 10 Gy gamma radiation for agro-
nomic and molecular characterization of mutants.

At flowering stage, plants in the glasshouse were
observed for any variations in morphological charac-
ters. Results revealed that there were 10 variants with
different morphological characters compared to the
control. The data presented in Table 2 reveal that mor-
phological characters of D. grandiflorum cv. Snow Ball
and its mutant were statistically, highly significantly
affected by gamma ray doses.

Plant height 
Plant height was significantly reduced in the vari-

ants compared to control (Table 2). A significant varia-
tion in plant height was also observed among the vari-
ants (Table 3). Variant V10 gave the significantly high-
est value for plant height (69.00 cm), compared with
the control (62.10 cm). Variant V8 showed the lowest
value for plant height, followed by variant V2. The
plant height was significantly lower in most of the vari-
ants as compared to the control.

Leaf number and leaf area (cm2)
The number of leaves was significantly reduced in

the variants compared to control (Table 2). There was a
significant increase in the number of leaves in variant

V4. In general, the number of leaves in the variants was
lower compared to the control. Variant V2 had the
fewest number of leaves, followed by variants V7 and
V8. Statistically leaf area did not differ between the
variants and control (Table 2), however leaf area dif-
fered among the variants (Table 3). Variant V7 had
significantly higher mean values of leaf area, fol-
lowed by variants V10, V2 and V9 in comparison to
the control.

Number of flower buds and flowers
Table 2 reports that the number of flower buds was

significantly different between the variants, whereas
the difference in the number of flowers was not signif-
icant. The number of flower buds and flowers varied
among the variants (Table 3) with variants V3 and V8
having a significantly lower number of flower buds and
flowers, followed by variant V7. It was observed that in
most of the variants about 82-83% of buds opened into
flowers while in variant V3 only 50% of the buds
opened into flowers. 

Flower diameter and flower colour
The variation in flower diameter between the control

and the variants was statistically not significant (Tables
2 and 3). The original flower colour of the cv. Snow
Ball was white with incurve ray florets (Fig. 1A),
whereas one branch of variant V9 produced yellow-
coloured flowers with incurve ray florets (Fig. 1B). The
results indicate that the irradiation dose of 10 Gy was an
effective dose in inducing mutations in flower colour,
but no changes were observed in flower shape and size.

Table 2 - Analysis of variance of the morphological characters of control and variants of chrysanthemum
Treatment Plant

height
(cm)

Number of
leaves

Leaf area 
(cm2)

Number of
buds

Number of
flower

Flowers
diameter

(cm2)
Control
Variants
LSD0.05

62.10
54.80

2.97

38.70
31.60

4.18

8.60
10.93

2.35

15.10
11.70

4.05

11.40
8.10
3.44

6.60
6.50
1.58

Table 3 - Effect of gamma radiation (10 Gy) on morphological characters in D. grandiflora
Treatment Plant height

(cm)
Number of

leaves
Leaf area 

(cm2)
Number of

buds
Number of

flower
Diameter
flowers 
(cm2)

Control
V1
V2
V3
V4
V5
V6
V7
V8
V9
V10

62.10
56.00
40.00
60.00
58.00
60.00
55.00
58.00
37.00
55.00
69.00

38.70
23.00
16.00
40.00
49.00
38.00
35.00
19.00
19.00
40.00
37.00

8.61
7.39

13.00
8.48
7.87

10.85
10.48
15.16

9.32
12.56
14.15

15.10
15.00

5.00
14.00
16.00
14.00
10.00

7.00
5.00

13.00
18.00

11.40
10.00

4.00
7.00
9.00

11.00
7.00
5.00
3.00

10.00
15.00

6.60
5.50
6.00
8.00
5.50
6.60
7.30
6.00
7.50
6.80
6.60



109

Characterization
Twenty decamer primers (Table 4) were used for the

amplification of genomic DNA of control and mutants of
Dendranthema grandiflora Snow Ball. The number of
DNA fragments amplified ranged from one to five
depending upon the primer and the DNA sample with a
mean value of 2.90 bands per primer (Table 4). The
amplification product ranged from 250 to 3000 bp. A
total number of 58 markers were produced by the 20
primers. A total of 100% of the 58 scored bands were
polymorphic in 11 genotypes (one control and its ten

Fig. 1 - Induced somatic mutations in chrysanthemum cv. Snow Ball
after 10 Gy gamma radiation treatment.  (A) control,  white-
coloured  flowers;  (B) mutated, yellow-coloured flower.

Table 4 - Nucleotide sequences and RAPD amplification results of the primer used in the PCR amplification
Primer Sequence (5’-3’) Scored

bands
Polymorphic

bands
Polymorphism

(%)
OPA-1
OPA-2
OPA-3
OPA-4
OPA-5
OPA-6
OPA-7
OPA-8
OPA-9
OPA-10
OPB-1
OPB-2
OPB-3
OPB-4
OPB-5
OPB-6
OPB-7
OPB-8
OPB-9
OPB-10
Total

GTTTCGCTCC
TGATCCCTGG
CATCCCCCTG
GGACTGGAGT
TGCGCCCTTC
TGCTCTGCCC
GGTGACGCAG
GTCCACACGG
TGGGGGACTC
CTGCTGGGAC
CAGGCCCTTC
TGCCGAGCTG
AGTCAGCCAC
AATCGGGCTG
AGGGGTCTTG
GGTCCCTGAC
GAAACGGGTG
GTGACGTAGG
GGGTAACGCC
GTGATCGCAG

3
4
3
1
4
5
4
4
0
2
4
1
1
3
2
2
4
3
3
5

58

3
4
3
1
4
5
4
4
0
2
4
1
1
3
2
2
4
3
3
5

100
100
100
100
100
100
100
100

0
100
100
100
100
100
100
100
100
100
100
100

58

Fig. 2 - RAPD profile with primer OPB-4 showing polymorphism in
control and its variants in chrysanthemum cv. Snow Ball.
Lanes 1- 10: variants (V1 to V10); Lane 11: control; Lane 12:
DNA ladder (1kb).

variants). Figure 2 shows the amplification profiles, gen-
erated by primer OPB-4 across the chrysanthemum geno-
types. The RAPD markers produced by the 20 primers
were used to construct a similarity matrix (Table 5). Five
clusters can be observed. The first cluster includes only

Table 5 - Jaccard’s similarity matrix in control (C) and mutated (V) plants based on RAPD analysis

C
V1
V2
V3
V4
V5
V6
V7
V8
V9
V10

C
1.00
0.32
0.31
0.31
0.28
0.37
0.20
0.29
0.20
0.06
0.28

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10

1.00
0.79
0.67
0.76
0.37
0.30
0.65
0.50
0.27
0.62

1.00
0.54
0.65
0.37
0.26
0.68
0.54
0.26
0.61

1.00
0.67
0.37
0.32
0.53
0.37
0.28
0.44

1.00
0.42
0.32
0.72
0.50
0.29
0.51

1.00
0.37
0.40
0.26
0.24
0.31

1.00
0.45
0.37
0.26
0.32

1.00
0.62
0.33
0.62

1.00
0.25
0.59

1.00
0.35 1.00



110

yellow-coloured flowers (Group 5C, Fan 1) (British
Colour Council, 1938). In order to check the stability
of flower colour, the mutated branch was propagated
vegetatively. The plants were grown in a glasshouse,
where they expressed the same colour/shape.

Over recent years, RAPD analysis has been used in
ornamental breeding to characterize genotypes and to
identify genes controlling important traits (Huang et
al., 2000; Rumińska et al., 2004; Kumar et al., 2006;
Miñano et al., 2009). In the present study, a high level
of polymorphism was observed in 10 radiomutants of
chrysanthemum. Wolff and Van Rijn (1993) also
noticed a high degree of polymorphism in chrysanthe-
mum cultivars using RAPD markers. This high level of
polymorphism may be due to the strict out-crossing,
resulting in a higher level of heterozygosity in chrysan-
themum (Wolff et al., 1994). RAPD markers were used
to construct a similarity matrix and the results of char-
acterization analysis revealed a high diversity between
the control and its somaclones. The greatest genetic
distance between control and variant 9 (V9) may be
due to the fact that V9 is a somaclone where one branch
was mutated and produced yellow-coloured flower
heads. Although the plants differed in flower colour,
bands specific for flower colour could not be distin-
guished due to the resolution capacity of the tested
primers. Wolff (1996) reported that the choice of the
primers may be an important factor in obtaining a rapid
discrimination between samples. Barakat et al. (2010)
reported that mutants with different flower colour
could be identified at the molecular level using RAPD
technique, holding promise to identify unique genes as
SCAR markers. Bhattacharya and Teixeira da Silva
(2006) attempted to understand the molecular system-
atic and genetic difference between 10 original
chrysanthemum cultivars and 11 mutants and reported
that similarity ranged from 0.17 to 0.90 using rapid
analysis. Kumar et al. (2006) reported genetic dis-
tances from 0.43 to 0.96 between 13 chrysanthemum
cultivars.  A great genetic distance among the different
cultivars showed the existence of introgressing new
and novel genes from the chrysanthemum gene pool. It
may be suggested, with regard to the present study, that
by using RAPD markers, it is possible to differentiate
newly evolved chrysanthemum cultivars from their
parents which can be a useful tool to supplement the
distinctness, uniformity and stability analysis for plant
variety protection in future. Our results are in accor-
dance with those of other workers who reported RAPD
as a powerful tool in the assessment of genetic vari-
ability as well as for genetic characterization, allowing
differentiation of chrysanthemum mutants/cultivars
(Huang et al., 2000; Rumińska et al., 2004; Kumar et
al., 2006; Chatterjee et al., 2006; Miñano et. al., 2009).

Therefore, it may be concluded that 10 Gy gamma
irradiation was most effective in inducing variations in
flower colour and other morphological characters.
Morphological variations were not observed with the 5

Fig. 3 - Dendrogram of control and mutants of chrysanthemum cv.
Snow Ball

variant 9 (V9); the second includes the control (C); the
third includes variants 5 and 6 (V5 and V6); the fourth
includes variants 8 and 10 (V8 and V10), and the last
cluster includes variants 3, 4, 7, 2 and 1 (V3, V4, V7,
V2, V1). It can be seen from figure 3 that the shortest
genetic distance was found between variant 1 (V1) and
variant 2 (V2), whereas the greatest distance was
observed between the control and variant 9 (V9).

4. Discussion and Conclusions

Analysis of explant sensitivity is one of the basic
requirements for an effective use of mutation induction
in plant breeding programmes (Walther and Sauer,
1986). In the present study, the radio sensitivity of
shoots was assessed by survival and rooting of shoots
after irradiation in order to select a suitable dose of
gamma irradiation. Predieri (2001) also reported the
necessity to identify an appropriate dose to apply in
mutagenic treatments. The effect of gamma radiation
on in vitro cultures in crop breeding has been studied
by many workers (Shen et al., 1990; Charbaj and
Nabul, 1999; Predieri and Gatti, 2000; Datta et al.,
2005; Barakat et al., 2010).

Reductions in survival, plant height, number of
leaves and flowers were recorded after gamma irradia-
tion. Mutation in flower colour was detected as
chimera in one branch of the plant, which produced



111

Gy gamma dose and a lethal effect of 20 and 30 Gy
doses was observed on rooting and survival of in vitro
shoots. One hundred percent polymorphism was
observed among the radiomutants using PCR tech-
nique. The present results also indicate the applicabili-
ty of gamma radiation in crop breeding, and the assess-
ment of genetic variability and characterization of
radiomutants at genomic level by RAPD can be a use-
ful tool in breeding programs aimed to improve orna-
mental characters of chrysanthemum cultivars.

References
BRITISH COLOUR COUNCIL, 1938 - Horticultural Colour

Chart I and II. - The British Colour Council, London, pp. 1-200.
BARAKAT M.N., ABDEL-FATTAH R.S., BADR M., EL-

TORKY G., 2010 - “In vitro” mutagenesis and identification of
new variants via RAPD markers for improving Chrysanthe-
mum morifolium. - African J. Agric. Res., 5: 748-757.

BHATTACHARYA A., TEIXEIRA DA SILVA J.A., 2006 - Molec-
ular systematics in Chrysanthemum x grandiflorum (Ramat)
Kitamura. - Sci. Hort., 109: 379-385.

BROETJES C., VAN HARTEN A.M., 1978 - Application of muta-
tion breeding methods in the improvement of vegetatively prop-
agated crops. An Interpretative Literature Review. - Elsevier,
Amsterdam, The Netherlands, pp. 324.

CHARBAJ I., NABUL S., 1999 - Mutational analysis of racial dif-
ferentiation in Oryza sativa. - Curr. Sci., 32: 451-453.

CHATTERJEE J., MANDAL A.K.A., RANADE S.A., TEIXEIRA
DA SILVA J.A., DATTA S.K., 2006 - Molecular systematics in
Chrysanthemum x grandiflorum (Ramat) Kitamura. - Sci.
Hort., 119: 373-378.

DATTA S.K., MISHRA P., MANDAL A.K.A., 2005 - “In vitro”
mutagenesis - a quick method for establishment of solid mutant
in chrysanthemum. - Curr. Sci., 88: 155-158.

DOYLE J.J., DOYLE J.L., 1987 - A rapid DNA isolation proce-
dure for small amounts of fresh leaf tissue. - Phytochem. Bull.,
19: 11-15.

GOMEZ K.A., GOMEZ A.A., 1984 - Statistical procedure for
agricultural research. - John Wiley and Sons, New York, USA.

HUANG S.C., TSAI C.C., SHEU C.S., 2000 - Genetic analysis of
Chrysanthemum hybrids based on RAPD molecular markers. -
Bot. Bull. Acad. Sin., 41: 257-262.

JACCARD P., 1908 - Nouvelles recherches sur la distribution flo-
rale. - Bull. Soc. Vaud. Sci. Natl., 44: 223-270.

JAIN S.M., 2010 - Mutagenesis in crop improvement under the cli-
mate change. - Romanian Biotechnol. Lett., 15: 88-106.

JAIN S.M., SPENCER M.M., 2006 - Biotechnology and mutagen-
esis in improving ornamental plants, pp. 589-600. - In: TEIX-
EIRA DA SILVA J.A. (ed.) Floriculture, ornamental and plant

biotechnology: advances and topical issues, vol. I. Global Sci-
ence Books, Isleworth, UK, pp. 2481.

KUMAR S., PRASAD K.V., CHOUDHARY M.L., 2006 - Detec-
tion of genetic variability among chrysanthemum radiomutants
using RAPD markers. - Curr. Sci., 90: 1108-1113.

MARTIN C., GONZÁLEZ-BENITO M.E., 2005 - Survival and
genetic stability of Dendranthema grandiflora Tzvelev shoot
apices after cryopreservation by vitrification and encapsula-
tion-dehydration. - Cryobiology, 51: 281-289.

MARTIN C., UBERHUAGA S., PEREZ C., 2002 - Application of
rapid markers in the characterization of chrysanthemum vari-
eties and assessment of somaclonal variation. - Euphytica,
127: 247-253.

MIÑANO H.S., GONZÁLEZ-BENITO M.E., MARTIN C., 2009
- Molecular characterization and analysis of somaclonal vari-
ation in chrysanthemum cultivars using RAPD markers. - Sci.
Hort., 122: 238-243.

MURASHIGE T., SKOOG F., 1962 - Revised medium for the
rapid growth and bioassay with tobacco tissue culture. - Phys-
iol. Plant., 15: 473-479.

PREDIERI S., 2001 - “In vitro” propagation and breeding of orna-
mental plants: Advantages and disadvantages of variability. -
Euphytica, 115: 12-14.

PREDIERI S., GATTI J., 2000 - Pear mutagenesis: “in vitro”
treatment with gamma-rays and field selection for vegetative
form trials. - Euphytica, 114: 79-90.

RICHARD A.J., 1986 - Plant breeding systems. - George Allen and
Unwin, London, UK.

RUMIŃSKA J.L., ZALEWSKA M., SADOCH Z., 2004 -
Radiomutants of chrysanthemum (Dendranthema grandiflora
Tzvelev) of the lady group RAPD analysis of the genetic diver-
sity. - Plant Breeding, 123: 290-293.

SCHUM A.R., 2003 - Mutation breeding in ornamentals: an effi-
cient breeding method? - Acta Horticulturae, 612: 47-60.

SHEN T., LI W., DAI C., CHEN  J.Y., 1990 - The induction of
sports in chrysanthemum by gamma radiation. - Theor. Appl.
Genet., 88: 129-136.

WALTHER F., SAUER A., 1986 - “In vitro” mutagenesis in roses. -
Acta Horticulturae, 189: 37-46.

WILLIAM G.K., KUBELIK A.R., LIVAK K.L., RAFALSKI J.A.,
TINGEY S.V., 1990 - DNA polymorphisms amplified by arbi-
trary primers are useful as genetic markers. - Nucleic Acid
Res., 18: 159-164.

WOLFF K., 1996 - RAPD analysis of sporting and chimerism in
chrysanthemum. - Euphytica, 88: 159-164.

WOLFF K., VAN PETER R.J., HOFSTRA H., 1994 - RFLP analy-
sis in Chrysanthemum L. Probe and primer development. -
Theor. Appl. Genet., 88: 472-478.

WOLFF K., VAN RIJN P.J., 1993 - Rapid detection of genetic
variability in chrysanthemum (Dendranthema grandiflora
Tzvelev) using random primers. - Heredity, 71: 336-341.

ZALEWSKA M., RUMIŃSKA L.J., MILLER N., 2007 - “In vitro”
propagation using adventitious bud techniques as a source of
new variability in Chrysanthemum. - Sci. Hort., 113: 70-73.