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Microwave treatment can induce chrysanthemum phenotypic and genetic changes
Scientia Horticulturae 227 (2018) 223–233
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Research Paper
Microwave treatment can induce chrysanthemum phenotypic and genetic changes ⁎
Natalia Miler , Dariusz Kulus
MARK
⁎
UTP University of Science and Technology, Faculty of Agriculture and Biotechnology, Department of Ornamental Plants and Vegetable Crops, Laboratory of Biotechnology, Bernardyńska 6, 85-029, Bydgoszcz, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Adventitious shoot Breeding Callus Chrysanthemum × grandiflorum/Ramat./Kitam. Magnetron Mutagenesis
The impact of microwaves on the DNA of plant cells is still ambiguous, but the usage of this electromagnetic radiation as a source of variation in mutation breeding could be very advantageous. The aim of the study was to examine the influence of microwave radiation on the in vitro regeneration and acclimatization efficiencies, as well as on the genetic and phenotypical variation of chrysanthemum ‘Alchimist’. Leaf explants with or without callus were subjected to microwave treatment for various periods and in different milieus. Microwaves were generated by a domestic magnetron at 2.45 GHz, 800 W·cm−2. Irradiation negatively affected shoot formation if applied for longer periods. On the other hand, it did not affect the rooting and acclimatization steps which were fully successful. Chrysanthemums produced from MW-treated explants had longer shoots with inflorescences of greater diameter and altered phenotypes. It was also noticed, that MW-treatment affected the generative phase by prolonging the bud colouration period. The evaluation of genetic variation was performed with the RAPD technique by using 10 primers for all regenerated shoot; a total of 116 genotypes were analysed. Approximately 22% of plants regenerated from MW-treated explants showed band profiles different from the reference control. Most plants (86%) with altered band profiles originated from irradiated callus. These plants also showed higher mean genetic distance coefficient. In conclusion, microwaves can be considered as an efficient and easy-to-access tool in mutation breeding of chrysanthemum ‘Alchimist’.
1. Introduction Microwaves (MW) are a part of electromagnetic radiation with the spectrum of frequencies ranging from 300 MHz to 300 GHz and the wavelengths of 1 m–1 mm (Halmagyi et al., 2017). Besides their wide industrial and communication applications, MW are useful in some laboratory work and in life sciences (Chen, 2006), mainly for sterilization purposes (Tisserat et al., 1992), as well as in soil and biological material ultra-rapid drying (Diprose, 2001). In crop production, MW are applied in special devices allowing for non-chemical weed elimination in field conditions (Velazquez-Marti et al., 2008) and for tray sterilization in order to prevent fungal and viral infections in a greenhouse plant production (Soriano-Martin et al., 2006). Oza et al. (2008) applied MW in the estimation of crop area, growth and phonological information, crop condition and productivity assessment. Halmagyi et al. (2017), on the other hand, used microwaves for rewarming of cryopreserved Sequoia explants. Up to date, however, MW were not used for in vitro plant production and breeding purposes. The application of MW for laboratory and horticultural purposes
⁎
utilizes mainly the effect of dielectric heating (Diprose, 2001). When the electromagnetic microwave radiation from oscillating electric fields is absorbed in tissues, it provokes a rotation of water molecules, which leads to heating (Khalafallah and Sallam, 2009). It is known that this increase in temperature can lead to the death of cells (Diprose, 2001). However, the precise microwave effects on living organisms are still ambiguous (Cretescu et al., 2013). Due to their high availability, MW, especially at 2.45 GHz which is a frequency commonly used in popular microwave ovens, could be an efficient and cost-effective alternative for user-harmful chemical mutagens or less available gamma or X-radiation in plant mutation breeding programs. This particular radiation is absorbed by water molecules, present in all living cells (Cretescu et al., 2013). However, knowledge on the subject whether MW can induce significant changes in the DNA of plant cells and what are the best conditions for inducing such variation is still poor. There are some findings on the change of gene expression and mutations induction after the microwave irradiation in Vigna aconitifolia (Jacq.) Marechal (Jangid et al., 2010). It was also found that in rat brains there was a significant increase in DNA
Corresponding authors. E-mail addresses: [email protected] (N. Miler), [email protected] (D. Kulus).
http://dx.doi.org/10.1016/j.scienta.2017.09.047 Received 7 August 2017; Received in revised form 18 September 2017; Accepted 20 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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solidified medium. Longer treatments lead to a considerable medium heating-up (to 40 °C) and necrosis of the tissues.
double-strand breaks after the exposure to 2.45 GHz MW (Kesari et al., 2010). As for wheat and barley, the microwave radiation promoted grains germination, accelerated seedling development and growth, as well as enhanced metabolic biosynthesis by increasing the level of photosynthetic pigments (Khalafallah and Sallam, 2009; Cretescu et al., 2013). MW also altered the anatomical features of maize leaves by increasing midrib vascular bundle length, wing and assimilating layer thickness (Khalafallah and Sallam, 2009). Therefore, it can be assumed that it is possible to at least partially substitute chemical plant growth regulators (PGRs) with physical ones which could reduce the costs of in vitro culture systems or avoid pollution in the field. This, however, still requires much research. Our hypothesis was that chrysanthemum explants exposed to MW will behave differently than those unexposed. The aim of this study was to address for the first time the question whether the microwaves generated in a domestic oven (for different periods and in different milieu) can influence the in vitro regeneration potential, acclimatization efficiency, phenotype and/or the genetic stability in chrysanthemum.
2.3. Experiment I: the influence of explant type, irradiation time and milieu on chrysanthemum in vitro shoot regeneration The experiment involved an irradiation of the two explant types with MW for 0 (control); 2; 4; 6 or 8 s. During the exposure, half of the explants were entirely covered with sterile distilled water (+H2O, for a total of 4 min; Fig. 1C) and half were treated without the cooling water jacket (–H2O; Fig. 1A,B). The changes of the medium temperature during the irradiation are shown in Table 2. After the treatment of explants with MW, the cooling water was removed. As for the nonirradiated control, half of the explants were kept in water for 4 min and half were not. The number of in vitro regenerated adventitious shoots (at least 0.5 cm long; Fig. 1D), as well as the share [%] of regenerating explants were recorded after 10 weeks of culture. The total number of inoculated explants was considered 100%.
2. Material and methods 2.4. Experiment II: the influence of explant type and the number of irradiation cycles on the in vitro regeneration of chrysanthemum shoots, their acclimatization and stability
2.1. Plant material and tissue culture conditions The in vitro-grown chrysanthemum (Chrysanthemum × grandiflorum/ Ramat./Kitam.) ‘Alchimist’ was the source of plant material. The applicability of this purple blooming cultivar was earlier confirmed in the experiment using gamma radiation to induce mutation (Zalewska et al., 2011). The plant material was multiplied in vitro via the single-node method (Zalewska et al., 2012) on the PGRs-free MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and solidified with 0.8% agar (Biocorp), at pH 5.8 (set after adding all of the compounds, prior to autoclaving at 105 kPa and 121 °C for 20 min). Whole-leaf explants (Table 1) were dissected form the produced mother plants and inoculated vertically in 350-mL glass jars containing 40 mL of the solid MS medium supplemented with 2.0 mg·dm−3 of 3indoleacetic acid (11.42 μM IAA, Sigma-Aldrich) and 0.6 mg·dm−3 N6benzyladenine (2.66 μM BA, Sigma-Aldrich). The explants were kept on the medium during the entire in vitro experiment. The cultures were maintained in a growth room at 23 ± 1 °C under 16/8 h (day/night) photoperiod and photosynthetic photon flux density of an average 26.1 μmol·m−2·s−1 provided by the TLD Philips 36W/54 fluorescent lamps.
The two explant types (leaves and leaves with callus) were irradiated with MW for 8 s in repeated cycles, as following: 1 × 8 s, 2 × 8 s (a total of 16 s), 3 × 8 s (a total of 24 s) or 4 × 8 s (a total of 32 s). In order to maintain a constant room temperature between the successive irradiations, the jars with the explants, were cooled in a water bath at the temperature of 10 °C for 1 min. Leaves not treated with MW (0 s) were considered as the control. All of the 116 shoots regenerated in the 2nd experiment (i.e. 20 control and 96 recovered from MW-exposed explants) were rooted, acclimatized and subjected to further genetic and phenotypic stability analysis. 2.4.1. Rooting, acclimatization and glasshouse cultivation For acclimatization, the shoots 5 cm in length were excised and placed on the rooting ½MS medium containing half-strength macronutrients and supplemented with 2 mg·dm−3 (11.42 μM) of IAA (Sigma-Aldrich) for 10 days. The two-week-long acclimatization was conducted in June in natural light conditions in a glasshouse. The plants were grown in plastic trays filled with a disinfected (0.2% v/w Dithane) mixture of peat and perlite (2:1), sprayed and covered with perforated foil and geo-cover. The effectiveness of rooting and acclimatization [%] were assessed. The total number of plantlets transferred to ex vitro conditions was considered 100%. Next, in July, the plants were transferred to plastic pots (19 cm in diameter, 0.8 dm3, 3 clones of each genotype per pot) filled with professional peat substrate for chrysanthemum cultivation. Cultivation was carried under controlled light and temperature conditions according to Jerzy and Borkowska (2004). After reaching a minimum height of 20 cm, in order to stimulate flowering, the plants were shaded (photoperiod: 10-h light and 14-h dark). Chrysanthemums were grown with a standard method, i.e. one stem with a single inflorescence.
2.2. Irradiation – general conditions The SAMSUNG MW-73B-S microwave oven with the power of 800 W·cm−2 and the frequency of 2.45 GHz was used as the source of radiation. The irradiation with microwaves was done: 1) immediately on the following day after explant inoculation (the irradiated objects were typical leaves; Fig. 1A), and 2) three weeks after culture initiation (the irradiated objects were leaves with callus forming a 0.5 cm3 lump at the base of the leaf petiole; Fig. 1B). By those means two types of explants were analysed, named as: 1) leaf and 2) callus. In the preliminary experiments it was found that 8 s is the maximum time of a single microwave treatment of explants placed on the agarTable 1 Morphometric parameters of leaf explants used in the in vitro experiments. Leaf area [cm2]
Perimeter [cm]
Vertical length [cm]
Horizontal width [cm]
Average horizontal width [cm]
Aspect ratio (W/L)
Blade length [cm]
Petiole length [cm]
0.62a
3.60
2.1
1.34
0.78
0.83
1.28
0.82
a
Measurements performed with the EPSON Perfection V800 Photo scanner and the WinFolia software.
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Fig. 1. Explants used in the experiments. A – leaves inoculated on the MS medium for one day prior to irradiation; B – explants with callus lumps at the base of leaf petiole after three-week preculture prior to irradiation; C – leaf explants in a cooling water jacket during irradiation. D – adventitious shoots regenerating from explants three weeks after irradiation; 1 bar = 1 cm.
at 36 °C for annealing, and 2 min at 72 °C for DNA extension. The last cycle was followed by a final extension step of 4 min at 72 °C. The amplified DNA fragments were separated horizontally on 1.5% (w/v) agarose gel, in a TBE buffer (90 mM TRIS, 90 mM boric acid, 2 mM EDTA, pH = 8.0) first at 90 V for 10 min, then at 120 V for 110 min (Biometra P25), and detected by staining the gel with 18 μL ethidium bromide at a concentration 10 mg∙mL−1 for 300 mL of gel. Molecular weights of the fragments were estimated by using a 100–5000 bp DNA Ladder (DNA GeneRuler Express DNA Ladder, Fermentas). The PCR product visualization was performed in the UV light transilluminator. Gel images were recorded and analysed using the GelAnalyzer 2010 software. For every primer used the number of reference (control), polymorphic (present in the electrophoretic profile of more than one individual) and specific/unique (present in the electrophoretic profile of a single individual) loci was estimated.
Table 2 Temperature [°C] of culture medium after various MW irradiation times, with (+H2O) or without (−H2O) a cooling water jacket. Milieu
−H2O +H2O
Irradiation time [s] 0
2
4
6
8
24.5 24.6
24.5 26.6
28.2 28.6
33.1 29.8
35.2 32.6
2.4.2. Phenotypical analysis The inner and outer colour of ray florets of fully developed inflorescences (i.e. of maximal diameter) of control plants and all irradiation-derived chrysanthemums were estimated using the RHSCC (Royal Horticultural Society Colour Chart) in the natural light. Moreover, the morphometric analyses included: the height of plants [cm], the shape and diameter [cm] of inflorescences, the share [%] and level of ray florets integration into a tube, the presence [%] of spurs in ray florets, and the date and length of blooming [days]: since potting of plants until flower bud appearance (named as: stage I), through bud colouration (beginning of flowering; stage II), until full opening of inflorescence (stage III).
2.5. Statistical analysis As for the 1st experiment, a three-factor analysis referred to the effect of two milieu conditions, five times of irradiation and two explant types. As for the 2nd experiment a two-factor analysis referred to five irradiation treatments and two explant types. The experiments were set up in a completely randomized design with five (1st experiment) or six (2nd experiment) replications; a single replication consisted of one jar with five explants dissected from a single microshoot. A total of 800 explants were used (500 in the 1st and 300 in the 2nd experiment) during the in vitro step and 116 plants were analysed genetically and in the glasshouse. The results were statistically verified with the analysis of variance (ANOVA), and the comparisons of means were made with HSD Tukey test (P = 0.05) using Statistica 10.0 and ANALWAR-5.2-FR tools. The tables and graphs with results provide real numerical data, while alphabet letters point to homogeneous groups after the statistical calculations based on transformed data. As for the results of the molecular analysis, the banding patterns were recorded as 0–1 binary matrices, where “1” indicates the presence and “0” – the absence of a given fragment, followed by statistical analysis. Population groups were distinguished based on the analysis of molecular variance (AMOVA) estimates and population principle
2.4.3. Molecular analysis The total genomic DNA was isolated from fresh leaves using a readyto-use Genomic Mini AX Plant column kit (A & A Biotechnology). The concentrated stocks of DNA were suspended in the TE buffer (10 mM TRIS and 1 mM EDTA, pH = 8.0) and stored at −20 °C, while the working solutions, with the concentration of 20 ng DNA·μL−1, were based on 10 mM TRIS at pH = 8.0. DNA concentration and purity was monitored spectrophotometrically (UV–vis 1610 SCHIMADZU). In order to evaluate the genetic variation, the Randomly Amplified Polymorphic DNA (RAPD) marker system was applied based on 10 primers (Table 7). Each 25 μL reaction volume contained: 0.25 mM dNTPs mix, 1 μM single primer, 1.25 U DNA Taq polymerase, 2 mM MgCl2, 20 ng template DNA (0.8 ng·μL−1) and deionised water to volume. Amplification was performed in a NyxTechnic ATC401 thermocycler, in the following conditions: one cycle of 4 min at 94 °C for initial DNA denaturation; 45 cycles of 1 min at 94 °C for denaturation, 1 min 225
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Table 3 Influence of explant type and irradiation time on the mean number of shoot regenerating from one explant after 10 weeks of culture. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
0.7 ± 02 aa 0.7 ± 0.2 a 0.7 ± 0.2 A
0.6 ± 0.3 ab 0.6 ± 0.1 ab 0.6 ± 0.2 AB
0.4 ± 0.2 ab 0.5 ± 0.1 ab 0.5 ± 0.2 AB
0.5 ± 0.1 ab 0.4 ± 0.1 ab 0.4 ± 0.1 AB
0.4 ± 0.1 ab 0.2 ± 0.1 b 0.3 ± 0.1 B
0.5 ± 0.2 A 0.5 ± 0.1 A
a Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 4 Influence of explant type and irradiation time on the height [cm] of ex vitro-grown chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
35.6 ± 0.6 b 35.8 ± 0.8 b 35.7 ± 0.7 B
38.4 ± 1.0 ab 35.7 ± 1.1 b 37.2 ± 1.0 B
36.2 ± 0.8 ab 39.5 ± 1.2 ab 38.1 ± 1.0 AB
39.1 ± 1.1 ab 36.4 ± 1.9 ab 38.4 ± 1.5 AB
42.8 ± 1.1 a 36.0 ± 1.5 ab 41.2 ± 1.3 A
38.6 ± 0.9 A 36.9 ± 1.3 B
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 5 Influence of explant type and irradiation time on the inflorescence diameter [cm] of ex vitro-grown chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
6.5 ± 0.2 d 6.5 ± 0.3 d 6.5 ± 0.2 B
7.4 ± 0.3 ad 8.2 ± 0.4 ab 7.8 ± 0.4 A
6.9 ± 0.5 bd 8.5 ± 0.3 a 7.8 ± 0.4 A
6.8 ± 0.2 cd 7.4 ± 0.3 ad 7.1 ± 0.2 A
8.2 ± 0.2 ab 7.0 ± 0.6 ad 7.9 ± 0.4 A
7.1 ± 0.3 A 7.9 ± 0.4 A
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 6 Influence of explant type and irradiation time on the length [days] of the second developmental stage (from potting till bud colouration) of chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
143.8 ± 0.5 ab 144.0 ± 0.5 ab 143.9 ± 0.5 B
145.2 ± 0.3 ab 144.2 ± 0.3 ab 144.7 ± 0.3 B
145.3 ± 0.5 b 142.8 ± 0.6 c 144.1 ± 0.6 B
144.0 ± 0.4 ab 144.8 ± 0.7 ab 144.4 ± 0.6 B
145.8 ± 0.3 b 148.3 ± 0.8 a 147.1 ± A
144.8 ± 0.4 A 144.8 ± 0.6 A
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 7 Sequence of primers used in the RAPD analysis, the number of bands they generated and the number of plants with polymorphic bands. Symbol
Reference
Sequence
No of reference/polymorphic/unique bands
No of plants with polymorphism detected
A B C
Lema-Rumińska et al. (2004)
5′-GGG AAT TCG G-3′ 5′-GAC CGC TTG T-3′ 5′-GGA CTG GAG T-3′
4/4/4 6/6/6 6/6/11
3 1 4
D E
Shibata et al. (1998)
5′-GCT GCC TCA GG-3′ 5′-TAC CCA GGA GCG-3′
3/3/0 10/10/5
1 19
F G
Wolff (1996)
5′-CAA TCG CCG T-3′ 5′-GGT GAC GCA G-3′
3/3/3 6/4/2
1 1
H I
Martín and González-Benito (2005)
5′-CCC AGT CAC T-3′ 5′-TGG CGT CCT T-3′
7/5/6 7/7/4
1 1
J
Chattarjee et al. (2006)
5′-AGC GTG TCT G-3′
6/3/7
4
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Fig. 2. Influence of the tested main effects on the number of shoots produced from one explant and the share of regenerating explants after 10 weeks of culture. *Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the number of shoots; upper-case letters refer to the share of regenerating explants.
Fig. 3. Share [%] of inflorescence variants with different colours of inner [A] and outer [B] side of ray florets produced from untreated control and MW-irradiated explants.
Fig. 4. Different shapes of chrysanthemum ‘Alchimist’ inflorescences (A–D) and ray floret types (E–I): A – flat semi-full; B – flat full; C – convex semi-full; D – convex full; E – ray floret with yellow discolouration; F – ray floret with a spur (indicated with an arrow); G–I – ray florets overgrown into a tube in ¼ (G); ½ (H) and ¾ (I) of their length; 1 bar = 1 cm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results
cluster analysis (PCoA) performed with the use of GeneAlex 6.5 software (Peakall and Smouse, 2012). The coefficient of genetic distance based on Nei and Li algorithm (1979) was calculated by a comparison of the predominant band pattern of the control plants with the band patterns of the plants regenerated from the explants treated with microwaves. On the base of genetic distance values, a dendrogram was created with the AHC unweighted pair group method (UPGMA) for clustering, using Treecon 1.3 software (Van de Peer and De Wachter, 1994).
3.1. Morphological response of explants In both experiments, callus formation began during the second week of culture on all inoculated explants. Primarily, it was green and compact, however, in the following weeks it turned brown. There were no visual signs of explant tissue damage after MW treatment observed in the first experiment. Some necrotic changes were only occasionally 227
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Fig. 5. Share [%] of various inflorescence shapes produced in the experiment.
Fig. 6. Influence of explant type and irradiation time main effects on the share of ray florets with spurs and discolouration. *Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to spurs; upper-case letters refer to the discolouration.
Fig. 7. Share [%] of ray florets with various degree of overgrowth into a tube.
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Fig. 8. Influence of experimental factors on the length of the first (from potting till flower bud appearance) and the third developmental stage (from potting until full flowering) of chrysanthemum. * Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the I stage; upper-case letters refer to the III stage.
control plants reached 35.7 cm, while the ones produced after four cycles of 8 s irradiation −41.2 cm). Three variants of both inner and outer colour of ray florets were observed (Fig. 3). As for the inner side of the flowers (Fig. 3A), the dark pink inflorescences (RHSCC code: 75C) were the most numerous in both control and MW-derived plants (63 and 40%, respectively). A similar share of light purple flowers (RHSCC: 77D) was observed in both groups (31%). The light pink inflorescences (RHSCC: 69C) were the least frequent, however, in the untreated control they accounted for only 6%, while in the MW-treated plants, their share was nearly fivefold higher and reached 29%. The variation in the outer side colour of ray florets was not as evident (Fig. 3B). Within the untreated control and MWderived chrysanthemums, the share of purple (RHSCC: 70A), light purple (RHSCC: 78B) and dark purple (RHSCC: 71A) inflorescences was as follows: 50/42; 25/20 and 25/38%, respectively. Four shapes of chrysanthemum inflorescences were observed (Figs. 4 4A–D and 5 ). Most of the non-treated control plants produced flat full inflorescences (44%; Fig. 4B), while the least numerous were the ones with convex full phenotype (6%; Fig. 4D). On the other hand, most of the MW-treated plants produced convex full inflorescences (32.4%) and the flat full type (31.6%), while the convex semi-full ones (Fig. 4C) were the most scarce ones (8.8%). All of the plants regenerated from MW-treated explants produced inflorescences of a greater diameter (7.1–7.9 cm) then the untreated control (6.5 cm) (Table 5). Yellow discolouration could be observed on the outer part of ray florets of approximately 41% of both non-treated control (30.5%) and the irradiated (43.7%) chrysanthemum inflorescences (Fig. 4E). There was, however, no influence of the explant type or the irradiation time on this feature noticed (Fig. 6). On the other hand, it was observed that MW treatment lead to a more frequent formation of spurs in ray florets (Figs. 4F and 6) but with no interaction with the second factor. It was noticed that the control chrysanthemums had ray florets overgrown into a tube in ¼ or ½ of their length (44% of both types) (Figs. 4G–I and 7 ). As for the MW-derived inflorescences, the ray florets overgrown in ½ were dominant (65.1%), while the ones overgrown in ¼ were the least frequently observed (15.5%). None of the experimental factors influenced the pace of the first (from potting till flower bud appearance) and the third developmental stage (from potting until full flowering) of chrysanthemums (Fig. 8). On the other hand, the time of irradiation and the interaction of the two tested factors affected the length of the second developmental stage – beginning of flowering (bud colouration) (Table 6). Four cycles of irradiation (4 × 8 s) lead to slower flower bud colouration (over
observed in the second experiment after applying the highest irradiation dosages (3 × 8 and 4 × 8 s). Adventitious shoots were forming mostly between the first and the fifth week of culture, regardless of the experimental conditions. Their number remained constant afterwards (data not shown). All of the shoots regenerated from the MW-irradiated explants were morphologically similar to the control ones during the in vitro stage. 3.2. Experiment I Only the type of explant had an influence on the regeneration of adventitious shoots. Leaves which were covered with callus prior to MW irradiation, regenerated more shoots in comparison to leaves without a previous 3-week preculture (1.0 and 0.7 shoot per explant, respectively; Fig. 2). Neither the milieu conditions nor the time of irradiation had a significant influence on this parameter. There were also no interactions between the tested experimental factors observed. Moreover, no differences in the share of explants regenerating shoots were reported (approximately 47%), regardless of the conditions (Fig. 2). 3.3. Experiment II Approximately 40% of both explant types regenerated adventitious shoots, however, MW treatment decreased the share of regenerating explants (26%) in comparison to the untreated control (60%; data not shown). Similarly it was observed that the longest irradiation decreased more than twice the number of regenerating shoots (0.7 shoot per one non-treated explant and 0.3 after 32 s exposure), especially after callus irradiation (Table 3). 3.3.1. Acclimatization efficiency Both, the rooting and acclimatization efficiency were 100% for the control and the irradiation-derived plants. In the end, 20 control and 96 MW-treated plants were acclimatized. No differences in the morphology or deformations within those two groups were visually observed during that stage. 3.3.2. Ex vitro growth and phenotypical analysis Both of the tested factors affected the length of the produced chrysanthemum shoots (Table 4). The explants which were precultured for three weeks prior to irradiation (callus) produced shorter shoots (36.9 cm) in comparison to the leaves (38.6 cm). It was also observed that longer MW-treatment positively affected the growth of plants (the 229
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Fig. 9. Example band profiles received as a result of electrophoresis of the DNA amplification products obtained with: J; E; C; A; J; E primers (from the top left). Outermost lanes are DNA bp ladders. Arrows point to the profiles which differ from the reference.
plant with a changed band profile. The share of mutated plants was higher among the chrysanthemums regenerated from irradiated callus (37%) than among the control plants (10%) and the ones received from irradiated leaves (16%; Table 8). The highest percentage of plants with changed band profiles was obtained from callus exposed to MW treatment for 24 s (86%). The mean values of the coefficients of genetic distance (compared to predominant monomorphic band pattern in control plants) ranged from 14.7% (for plants regenerated from callus irradiated for 3 × 8 s) to null in plants regenerated from leaves irradiated for 8 and 2 × 8 s (Table 9). Plants regenerated from MW-treated callus showed higher mean coefficient of genetic distance (3.01%) in comparison to the ones regenerated from irradiated leaves (0.41%), what was confirmed by the PCoA analysis (Fig. 10). On the base of genetic distance values a dendrogram was created for
147 days since potting) in comparison to all other treatments (144–145 days). Chrysanthemums produced from explants which were precultured for three weeks prior to MW-treatment for 32 s, required most time (148.3 days since potting) to start flowering. 3.3.3. Evaluation of the genetic variation The amplification products reached a size from 289 bp to 2954 bp. The highest number of amplicons was generated by the E primer (10 bands per reference), while the lowest number – by primers D and F (3 bands) (Table 7). A total of 106 different loci was detected by ten primers. A total of 25 plants with an altered band pattern (2 control and 23 MW-treated) were observed (Fig. 9). The E primer was most efficient in detecting variation (polymorphism observed within 19 genotypes; Table 7). On the other hand, primers: B, D, F, G, H and I denoted only 1 230
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variation detected with RAPD markers is of interindividual origin, while 11% results from variation between groups.
Table 8 Number and share [%] of plants with an altered band profile in comparison with a predominant band profile of control plants. Explant type
Irradiation time [s]
Number of plants analysed
number
%
Control Leaf
0 8 2×8 3×8 4×8 total
20 17 12 19 10 58
2 0 0 4 5 9
10 0 0 21 50 16
Callus
8 2×8 3×8 4×8 total
14 14 7 3 38
4 3 6 1 14
29 21 86 33 37
116
25
22
Total
4. Discussion
Plants with an altered RAPD band profile
The horticultural market constantly demands flower novelties; hence mutation breeding is commonly used for rapid improvement of commercially important ornamental plants. There are many findings on chrysanthemum mutation breeding with gamma and X-radiation, as well as with chemical agents (Teixeira da Silva and Kulus, 2014; Oladosu et al., 2016). On the other hand, there are no information about the use of microwaves in breeding of ornamentals. Our studies suggest that MW treatment can be a useful tool in modern horticulture. Due to a relatively high ease in inducing variation, chrysanthemum is a suitable object for studying the effects of mutation breeding. However, various cultivars may differ in terms of susceptibility to mutation occurrence. Pink- or purple-flowering chrysanthemums undergo mutations most often (due to a high share of dominant alleles responsible for flower colour), while yellow ones – mutate the least frequently (Kulus, 2017). For this reason, the purple-flowering ‘Alchimist’ was selected as the donor of the biological material. In the first experiment a more efficient shoot regeneration was obtained from irradiated callus, which may be a result of the additional 3week preculture of explants prior to MW-treatment. The influence of explant type on the chrysanthemum and other ornamental plant species in vitro morphogenetic response was also reported by Zalewska et al. (2010, 2011), Teixeira da Silva and Kulus (2014) and Lema-Rumińska and Kulus (2014). On the other hand, there was no influence of the milieu conditions on the explant survival, callus formation and further regeneration of shoots observed. As for Vigna aconitifolia, immersion of explants in water during radiation affected their survival and callus development (Jangid et al., 2010). Similarly, an increase of temperature during preculture influenced the regeneration of callus and shoots in Brassica napus L. (Burbulis et al., 2004), which was not observed with chrysanthemum. A positive influence of magnetic field on the explant regeneration rate was observed with Paulownia tomentosa (Thunb.) Steud. (Alikamanoğlu et al., 2007) and Glycine max L. Merrill (Atak et al., 2003). Microwave treatment stimulated the in vivo germination of Phaseolus vulgaris L. seeds (Jakubowski, 2015). Zalewska (2010), on the other hand, described that treatment with ionizing radiation leads to a decrease in chrysanthemum shoot regeneration efficiency. In our experiment, microwave treatment for over 8 s also reduced the share of
Table 9 The highest, lowest and mean values [%] of the coefficient of genetic distance between the control and MW-treated plants. Explant
Time of irradiation [s]
Highest
Lowest
Mean
Control Leaf
0 8 2×8 3×8 4×8 Mean
2.65 0 0 2.65 0.87
0 0 0 0 0
0.27 0 0 0.41 0.35 0.41
Callus
8 2×8 3×8 4×8 Mean
10.53 1.75 83.02 3.45
0 0 0 0
1.01 0.25 14.70 1.15 3.01
all analysed genotypes (Fig. 11). The most distant from monomorphic control plants were two individual plants regenerated from callus MWtreated for 8 and 24 s. Other polymorphic plants were gathered into seven minor clusters not surpassing 10% of genetic distance among each other. The AMOVA analysis revealed that 89% of the total genetic
Fig. 10. Graph of principal coordinates analysis of control and MW-treated plant populations.
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Fig. 11. Dendrogram based on the estimation of genetic distance coefficients and UPGMA clustering presenting the relationships between non-treated control and MW-radiated genotypes, revealed by the RAPD analysis. All genotypes representing the same band pattern as the predominant control plants are collected within a single group named ‘Monomorphic’. The scale above shows a real genetic distance value.
the formation of three new phenotypes: silver-purple (RHSCC code: 77C), light-purple (77B) and claret-gold (60C). The application of MW has broaden the possible variation with new phenotypes (69C; 71A; 70A and 78B). It is worth mentioning that some of the obtained plants with yellow discolouration might have been mericlinal or sectorial chimeras, valuable in further breeding programmes (Kulus, 2017). Those findings may contribute to a wider application of microwaves as a cheap and commonly available source of variation, acceptable by the society. In order to detect genetic variation we have applied the RAPD marker system. The technique is simple, fast and cost-efficient. Moreover, it does not require knowledge of the genome sequence (Grover and Sharma, 2016). In the past, it was thought that RAPDs cannot be used with closely related cultivars and that they have a low repeatability. However, research conducted by Wolff (1996), Shibata et al. (1998) and Miler and Zalewska (2014) showed that this marker system can be successfully applied not only to distinguish closely related individuals, but also genetically different tissues of the same chimerical plant even at early developmental stage with only 1–2 primers. The technique was also successfully utilized in the detection of variation in cryopreservation-recovered chrysanthemums (Martín et al., 2011). Genetic similarity coefficients based on RAPD analysis were calculated previously for the somaclones of chrysanthemum ‘Alchimist’ by Miler and Zalewska (2014). This justifies the suitability of its use in mutagenesis research. The RAPD marker system used in our study revealed small changes (genetic distance at the level of 2.7%) in the band patterns of 10% untreated control plants. As for the MW-treatment, variation was detected in even 86% of chrysanthemums regenerated from callus irradiated for 24s. In comparison, Zalewska et al. (2011) obtained only 5.4% plants with altered genetic profiles by applying X and gamma radiation to ‘Alchimist’ cultivar, while the treatment of ‘Ingrid’ chrysanthemum with ethylsulphonate resulted in a 10.4% of changed plants (Latado et al., 2004). This clearly indicates that MW can
regenerating explants, as well as the shoot number if dozed for 32 s, especially to callus, which seems to be more sensitive in comparison to leaves. Similarly, the negative impact of microwaves (800 W) on the in vitro regeneration efficiency was reported with Vigna aconitifolia. As for this species, however, a MW-treatment of leaf explants for only 1–7 s resulted in a twofold decrease in the number of regenerating shoots in comparison to the untreated control (Jangid et al., 2010). Moreover, a significant influence of the increasing irradiation periods on the explants mortality was observed. As for chrysanthemum, no visual signs of tissue damage were observed after explant irradiation for up to 16 s, whereas longer treatments lead to minor tissue necrosis. There was also no influence of MW-treatment on chrysanthemum callus formation, which is in contrast with the reports of Jangid et al. (2010). Therefore, it can be assumed that chrysanthemum explants are tolerant to lower MW doses. It is known, that mutagens can influence on both the vegetative and the generative growth phases of plants (Qui et al., 2011). Khan et al. (2014) reported that combined treatment of Brasica rapa L. seeds with gamma radiation and sodium azide increased the length of the produced shoots by as much as 39%. In the present study, a similar phenomenon was observed (increase by 15%). Moreover, MW radiation lead to the formation of inflorescences of greater diameter (by 21.5%) and of altered shape in comparison to the control. The production of bigger more attractive inflorescences may be considered a positive change from the horticultural point of view. Moreover, the longest MW treatment prolonged the time of flower bud colouration by four days. Similar results were observed with ‘Lalima’ chrysanthemum subjected to 0.5 Gy gamma radiation (Misra et al., 2003). A protraction of time required from bud appearance till the beginning of flowering is positive since it may improve the post-harvest quality of plants. Altering the colour of the flowers is one of the most important goals of breeding. In the research conducted by Zalewska et al. (2011), the treatment of chrysanthemum ‘Alchimist’ leaf explants with gamma rays resulted in 232
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Tisserat, B., Galletta, P.D., Jones, D., 1992. Microwave sterilization of plant tissue culture media. HortScience 27 (4), 358–361. Van de Peer, Y., De Wachter, Y., 1994. TREECON for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment. Comput. Appl. Biosci. 10, 569–570. Velazquez-Marti, B., Gracia-Lopez, C., de la Puerta, R., 2008. Work conditions for microwave applicators designed to eliminate undesired vegetation in a field. Biosys. Eng. 100, 31–37. http://dx.doi.org/10.1016/j.biosystemseng.2008.01.010. Wolff, K., 1996. RAPD analysis of sporting and chimerism in chrysanthemum. Euphytica 89, 159–164. Zalewska, M., Miler, N., Wenda-Piesik, A., 2010. Effect of in vitro topophysis on the growth, development, and rooting of chrysanthemum explants (Chrysanthemum × grandiflorum/Ramat./Kitam). J. Hortic. Sci. Biotechnol. 85 (4), 362–366. http://dx.doi.org/10.1080/14620316.2010.11512681. Zalewska, M., Tymoszuk, A., Miler, N., 2011. New chrysanthemum cultivars as a result of in vitro mutagenesis with the application of different explant types. Acta Sci. Polonorum Hortic. Cult. 10 (2), 109–123. Zalewska, M., Antkowiak, M., Tymoszuk, A., 2012. Micropropagation of Ajania pacifica (Nakai) Bremer et Humpries with single-node method. Sci. Nat. Technol. 6, 1–7. Zalewska, M., 2010. In vitro adventitious bud techniques as a tool in creation of new chrysanthemum cultivars. In: Datta, S.K., Chakrabarty, D. (Eds.), Floriculture; Role of Tissue Culture and Molecular Techniques. Pointer Publishers, Jaipur, pp. 189–218.
be a useful tool in modern breeding, although after proper optimization. In the present study, the share of plants with band patterns different from the reference control plants was higher in the plants regenerated from microwaved callus in comparison to leaves, which is a common phenomenon (Betekhtin et al., 2017). Most of the changed band profiles showed several additional or missing amplicons, which is in contrast with the findings of Jangid et al. (2010) and suggests that DNA changes after microwave treatment are quite expanded. Therefore, it can be assumed that mutations occurring in meristematic centres are not lost but can spread out as the meristemoid continues to grow into an adventitious shoot. The genetic distance at the level of 2.7% present in control plants and in some plants regenerated from irradiated explants, on the other hand, may be a result of somaclonal variation, which is often observed in the in vitro cultured plants, especially if the nonmeristematic explant is used, and in the presence of plant growth regulators (Miler and Zalewska, 2014). 5. Conclusions The present research confirms that MW-treatment of leaf explants with preliminary regenerated callus can be used for the induction of genetic and phenotypic variation in chrysanthemum ‘Alchimist’. In order to obtain a high share of mutated plants, a prolonged irradiation (three or four cycles, 8 s, each) is recommended. The obtained variation (alternation in flower shape and colour, increase in inflorescence diameter and prolongation of bud colouration) can be considered positive from a horticultural point of view. In the future the use of other explant types for MW-treatment is recommended, e.g. petals which have been indicated recently as the most suitable for variation induction in chrysanthemum (Teixeira da Silva et al., 2015). Conflict of interest The authors declare no conflict of interest. The authors wish to acknowledge Mr. Marek Mayka and Mrs. Katarzyna Milczek for their technical support in performing the experiments. References Alikamanoğlu, S., Yaycılı, O., Atak, Ç., Rzakoulieva, A., 2007. Effect of magnetic field and gamma radiation on Paulownia tomentosa tissue culture. Biotechnol. Biotechnol. Equip. 21 (1), 49–53. http://dx.doi.org/10.1080/13102818.2007.10817412. Atak, Ç., Emiroglu, Ö., Alikamanoglu, S., Rzakoulieva, A., 2003. Stimulation of regeneration by magnetic field in soybean (Glycine max L. Merrill) tissue cultures. J. Cell Mol. Biol. 54 (4), 703–706. Betekhtin, A., Rojek, M., Jaskowiak, J., Milewska-Hendel, A., Kwasniewska, J., Kostyukova, Y., Kurczynska, E., Rumyantseva, N., Hasterok, R., 2017. Nuclear genome stability in long-term cultivated callus lines of Fagopyrum tataricum (L.) Gaertn. PLoS One 12 (3), e0173537. http://dx.doi.org/10.1371/journal.pone. 0173537. Burbulis, N., Kupriene, R., Zilenaite, L., 2004. Embryogenesis, callogenesis and plant regeneration from anther cultures of spring rape (Brassica napus L.). Acta Univ. Latv. Biol. 676, 153–158. Chattarjee, J., Mandal, A.K.A., Ranade, S.A., Teixeira da Silva, J.A., Datta, S.K., 2006. Molecular systematic in Chrysanthemum × grandiflorum (Ramat.) Kitamura. Sci. Hortic. 110, 373–378. http://dx.doi.org/10.1016/j.scienta.2006.06.004. Chen, Y.P., 2006. Microwave treatment of eight seconds protects cells of Isatis indigotica from enhanced UV-B radiation lesions. Photochem. Photobiol. 82 (2), 503–507. http://dx.doi.org/10.1562/2005-06-29-RA-595. Cretescu, I., Rodica, C., Velicevici, G., Ropciuc, S., Buzamat, G., 2013. Response of barley seedlings to microwaves at 2.45 GHz. Anim. Sci. Biotechnol. 46 (1), 185–191. Diprose, M.F., 2001. Some considerations when using a microwave oven as a laboratory research tool. Plant Soil 229, 271–280. Grover, A., Sharma, P.C., 2016. Development and use of molecular markers: past and present. Critic. Rev. Biotechnol. 36 (2), 290–302. http://dx.doi.org/10.3109/ 07388551.2014.959891. Halmagyi, A., Surducan, E., Surducan, V., 2017. The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage. J. Biol. Phys. 43 (3), 367–379. http://dx.doi.org/10.1007/s10867-0179457-4. Jakubowski, T., 2015. Evaluation of the impact of pre-sowing microwave stimulation of bean seeds on the germination process. Agric. Eng. 19 (2), 45–56. http://dx.doi.org/
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Research Paper
Microwave treatment can induce chrysanthemum phenotypic and genetic changes ⁎
Natalia Miler , Dariusz Kulus
MARK
⁎
UTP University of Science and Technology, Faculty of Agriculture and Biotechnology, Department of Ornamental Plants and Vegetable Crops, Laboratory of Biotechnology, Bernardyńska 6, 85-029, Bydgoszcz, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Adventitious shoot Breeding Callus Chrysanthemum × grandiflorum/Ramat./Kitam. Magnetron Mutagenesis
The impact of microwaves on the DNA of plant cells is still ambiguous, but the usage of this electromagnetic radiation as a source of variation in mutation breeding could be very advantageous. The aim of the study was to examine the influence of microwave radiation on the in vitro regeneration and acclimatization efficiencies, as well as on the genetic and phenotypical variation of chrysanthemum ‘Alchimist’. Leaf explants with or without callus were subjected to microwave treatment for various periods and in different milieus. Microwaves were generated by a domestic magnetron at 2.45 GHz, 800 W·cm−2. Irradiation negatively affected shoot formation if applied for longer periods. On the other hand, it did not affect the rooting and acclimatization steps which were fully successful. Chrysanthemums produced from MW-treated explants had longer shoots with inflorescences of greater diameter and altered phenotypes. It was also noticed, that MW-treatment affected the generative phase by prolonging the bud colouration period. The evaluation of genetic variation was performed with the RAPD technique by using 10 primers for all regenerated shoot; a total of 116 genotypes were analysed. Approximately 22% of plants regenerated from MW-treated explants showed band profiles different from the reference control. Most plants (86%) with altered band profiles originated from irradiated callus. These plants also showed higher mean genetic distance coefficient. In conclusion, microwaves can be considered as an efficient and easy-to-access tool in mutation breeding of chrysanthemum ‘Alchimist’.
1. Introduction Microwaves (MW) are a part of electromagnetic radiation with the spectrum of frequencies ranging from 300 MHz to 300 GHz and the wavelengths of 1 m–1 mm (Halmagyi et al., 2017). Besides their wide industrial and communication applications, MW are useful in some laboratory work and in life sciences (Chen, 2006), mainly for sterilization purposes (Tisserat et al., 1992), as well as in soil and biological material ultra-rapid drying (Diprose, 2001). In crop production, MW are applied in special devices allowing for non-chemical weed elimination in field conditions (Velazquez-Marti et al., 2008) and for tray sterilization in order to prevent fungal and viral infections in a greenhouse plant production (Soriano-Martin et al., 2006). Oza et al. (2008) applied MW in the estimation of crop area, growth and phonological information, crop condition and productivity assessment. Halmagyi et al. (2017), on the other hand, used microwaves for rewarming of cryopreserved Sequoia explants. Up to date, however, MW were not used for in vitro plant production and breeding purposes. The application of MW for laboratory and horticultural purposes
⁎
utilizes mainly the effect of dielectric heating (Diprose, 2001). When the electromagnetic microwave radiation from oscillating electric fields is absorbed in tissues, it provokes a rotation of water molecules, which leads to heating (Khalafallah and Sallam, 2009). It is known that this increase in temperature can lead to the death of cells (Diprose, 2001). However, the precise microwave effects on living organisms are still ambiguous (Cretescu et al., 2013). Due to their high availability, MW, especially at 2.45 GHz which is a frequency commonly used in popular microwave ovens, could be an efficient and cost-effective alternative for user-harmful chemical mutagens or less available gamma or X-radiation in plant mutation breeding programs. This particular radiation is absorbed by water molecules, present in all living cells (Cretescu et al., 2013). However, knowledge on the subject whether MW can induce significant changes in the DNA of plant cells and what are the best conditions for inducing such variation is still poor. There are some findings on the change of gene expression and mutations induction after the microwave irradiation in Vigna aconitifolia (Jacq.) Marechal (Jangid et al., 2010). It was also found that in rat brains there was a significant increase in DNA
Corresponding authors. E-mail addresses: [email protected] (N. Miler), [email protected] (D. Kulus).
http://dx.doi.org/10.1016/j.scienta.2017.09.047 Received 7 August 2017; Received in revised form 18 September 2017; Accepted 20 September 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
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solidified medium. Longer treatments lead to a considerable medium heating-up (to 40 °C) and necrosis of the tissues.
double-strand breaks after the exposure to 2.45 GHz MW (Kesari et al., 2010). As for wheat and barley, the microwave radiation promoted grains germination, accelerated seedling development and growth, as well as enhanced metabolic biosynthesis by increasing the level of photosynthetic pigments (Khalafallah and Sallam, 2009; Cretescu et al., 2013). MW also altered the anatomical features of maize leaves by increasing midrib vascular bundle length, wing and assimilating layer thickness (Khalafallah and Sallam, 2009). Therefore, it can be assumed that it is possible to at least partially substitute chemical plant growth regulators (PGRs) with physical ones which could reduce the costs of in vitro culture systems or avoid pollution in the field. This, however, still requires much research. Our hypothesis was that chrysanthemum explants exposed to MW will behave differently than those unexposed. The aim of this study was to address for the first time the question whether the microwaves generated in a domestic oven (for different periods and in different milieu) can influence the in vitro regeneration potential, acclimatization efficiency, phenotype and/or the genetic stability in chrysanthemum.
2.3. Experiment I: the influence of explant type, irradiation time and milieu on chrysanthemum in vitro shoot regeneration The experiment involved an irradiation of the two explant types with MW for 0 (control); 2; 4; 6 or 8 s. During the exposure, half of the explants were entirely covered with sterile distilled water (+H2O, for a total of 4 min; Fig. 1C) and half were treated without the cooling water jacket (–H2O; Fig. 1A,B). The changes of the medium temperature during the irradiation are shown in Table 2. After the treatment of explants with MW, the cooling water was removed. As for the nonirradiated control, half of the explants were kept in water for 4 min and half were not. The number of in vitro regenerated adventitious shoots (at least 0.5 cm long; Fig. 1D), as well as the share [%] of regenerating explants were recorded after 10 weeks of culture. The total number of inoculated explants was considered 100%.
2. Material and methods 2.4. Experiment II: the influence of explant type and the number of irradiation cycles on the in vitro regeneration of chrysanthemum shoots, their acclimatization and stability
2.1. Plant material and tissue culture conditions The in vitro-grown chrysanthemum (Chrysanthemum × grandiflorum/ Ramat./Kitam.) ‘Alchimist’ was the source of plant material. The applicability of this purple blooming cultivar was earlier confirmed in the experiment using gamma radiation to induce mutation (Zalewska et al., 2011). The plant material was multiplied in vitro via the single-node method (Zalewska et al., 2012) on the PGRs-free MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and solidified with 0.8% agar (Biocorp), at pH 5.8 (set after adding all of the compounds, prior to autoclaving at 105 kPa and 121 °C for 20 min). Whole-leaf explants (Table 1) were dissected form the produced mother plants and inoculated vertically in 350-mL glass jars containing 40 mL of the solid MS medium supplemented with 2.0 mg·dm−3 of 3indoleacetic acid (11.42 μM IAA, Sigma-Aldrich) and 0.6 mg·dm−3 N6benzyladenine (2.66 μM BA, Sigma-Aldrich). The explants were kept on the medium during the entire in vitro experiment. The cultures were maintained in a growth room at 23 ± 1 °C under 16/8 h (day/night) photoperiod and photosynthetic photon flux density of an average 26.1 μmol·m−2·s−1 provided by the TLD Philips 36W/54 fluorescent lamps.
The two explant types (leaves and leaves with callus) were irradiated with MW for 8 s in repeated cycles, as following: 1 × 8 s, 2 × 8 s (a total of 16 s), 3 × 8 s (a total of 24 s) or 4 × 8 s (a total of 32 s). In order to maintain a constant room temperature between the successive irradiations, the jars with the explants, were cooled in a water bath at the temperature of 10 °C for 1 min. Leaves not treated with MW (0 s) were considered as the control. All of the 116 shoots regenerated in the 2nd experiment (i.e. 20 control and 96 recovered from MW-exposed explants) were rooted, acclimatized and subjected to further genetic and phenotypic stability analysis. 2.4.1. Rooting, acclimatization and glasshouse cultivation For acclimatization, the shoots 5 cm in length were excised and placed on the rooting ½MS medium containing half-strength macronutrients and supplemented with 2 mg·dm−3 (11.42 μM) of IAA (Sigma-Aldrich) for 10 days. The two-week-long acclimatization was conducted in June in natural light conditions in a glasshouse. The plants were grown in plastic trays filled with a disinfected (0.2% v/w Dithane) mixture of peat and perlite (2:1), sprayed and covered with perforated foil and geo-cover. The effectiveness of rooting and acclimatization [%] were assessed. The total number of plantlets transferred to ex vitro conditions was considered 100%. Next, in July, the plants were transferred to plastic pots (19 cm in diameter, 0.8 dm3, 3 clones of each genotype per pot) filled with professional peat substrate for chrysanthemum cultivation. Cultivation was carried under controlled light and temperature conditions according to Jerzy and Borkowska (2004). After reaching a minimum height of 20 cm, in order to stimulate flowering, the plants were shaded (photoperiod: 10-h light and 14-h dark). Chrysanthemums were grown with a standard method, i.e. one stem with a single inflorescence.
2.2. Irradiation – general conditions The SAMSUNG MW-73B-S microwave oven with the power of 800 W·cm−2 and the frequency of 2.45 GHz was used as the source of radiation. The irradiation with microwaves was done: 1) immediately on the following day after explant inoculation (the irradiated objects were typical leaves; Fig. 1A), and 2) three weeks after culture initiation (the irradiated objects were leaves with callus forming a 0.5 cm3 lump at the base of the leaf petiole; Fig. 1B). By those means two types of explants were analysed, named as: 1) leaf and 2) callus. In the preliminary experiments it was found that 8 s is the maximum time of a single microwave treatment of explants placed on the agarTable 1 Morphometric parameters of leaf explants used in the in vitro experiments. Leaf area [cm2]
Perimeter [cm]
Vertical length [cm]
Horizontal width [cm]
Average horizontal width [cm]
Aspect ratio (W/L)
Blade length [cm]
Petiole length [cm]
0.62a
3.60
2.1
1.34
0.78
0.83
1.28
0.82
a
Measurements performed with the EPSON Perfection V800 Photo scanner and the WinFolia software.
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Fig. 1. Explants used in the experiments. A – leaves inoculated on the MS medium for one day prior to irradiation; B – explants with callus lumps at the base of leaf petiole after three-week preculture prior to irradiation; C – leaf explants in a cooling water jacket during irradiation. D – adventitious shoots regenerating from explants three weeks after irradiation; 1 bar = 1 cm.
at 36 °C for annealing, and 2 min at 72 °C for DNA extension. The last cycle was followed by a final extension step of 4 min at 72 °C. The amplified DNA fragments were separated horizontally on 1.5% (w/v) agarose gel, in a TBE buffer (90 mM TRIS, 90 mM boric acid, 2 mM EDTA, pH = 8.0) first at 90 V for 10 min, then at 120 V for 110 min (Biometra P25), and detected by staining the gel with 18 μL ethidium bromide at a concentration 10 mg∙mL−1 for 300 mL of gel. Molecular weights of the fragments were estimated by using a 100–5000 bp DNA Ladder (DNA GeneRuler Express DNA Ladder, Fermentas). The PCR product visualization was performed in the UV light transilluminator. Gel images were recorded and analysed using the GelAnalyzer 2010 software. For every primer used the number of reference (control), polymorphic (present in the electrophoretic profile of more than one individual) and specific/unique (present in the electrophoretic profile of a single individual) loci was estimated.
Table 2 Temperature [°C] of culture medium after various MW irradiation times, with (+H2O) or without (−H2O) a cooling water jacket. Milieu
−H2O +H2O
Irradiation time [s] 0
2
4
6
8
24.5 24.6
24.5 26.6
28.2 28.6
33.1 29.8
35.2 32.6
2.4.2. Phenotypical analysis The inner and outer colour of ray florets of fully developed inflorescences (i.e. of maximal diameter) of control plants and all irradiation-derived chrysanthemums were estimated using the RHSCC (Royal Horticultural Society Colour Chart) in the natural light. Moreover, the morphometric analyses included: the height of plants [cm], the shape and diameter [cm] of inflorescences, the share [%] and level of ray florets integration into a tube, the presence [%] of spurs in ray florets, and the date and length of blooming [days]: since potting of plants until flower bud appearance (named as: stage I), through bud colouration (beginning of flowering; stage II), until full opening of inflorescence (stage III).
2.5. Statistical analysis As for the 1st experiment, a three-factor analysis referred to the effect of two milieu conditions, five times of irradiation and two explant types. As for the 2nd experiment a two-factor analysis referred to five irradiation treatments and two explant types. The experiments were set up in a completely randomized design with five (1st experiment) or six (2nd experiment) replications; a single replication consisted of one jar with five explants dissected from a single microshoot. A total of 800 explants were used (500 in the 1st and 300 in the 2nd experiment) during the in vitro step and 116 plants were analysed genetically and in the glasshouse. The results were statistically verified with the analysis of variance (ANOVA), and the comparisons of means were made with HSD Tukey test (P = 0.05) using Statistica 10.0 and ANALWAR-5.2-FR tools. The tables and graphs with results provide real numerical data, while alphabet letters point to homogeneous groups after the statistical calculations based on transformed data. As for the results of the molecular analysis, the banding patterns were recorded as 0–1 binary matrices, where “1” indicates the presence and “0” – the absence of a given fragment, followed by statistical analysis. Population groups were distinguished based on the analysis of molecular variance (AMOVA) estimates and population principle
2.4.3. Molecular analysis The total genomic DNA was isolated from fresh leaves using a readyto-use Genomic Mini AX Plant column kit (A & A Biotechnology). The concentrated stocks of DNA were suspended in the TE buffer (10 mM TRIS and 1 mM EDTA, pH = 8.0) and stored at −20 °C, while the working solutions, with the concentration of 20 ng DNA·μL−1, were based on 10 mM TRIS at pH = 8.0. DNA concentration and purity was monitored spectrophotometrically (UV–vis 1610 SCHIMADZU). In order to evaluate the genetic variation, the Randomly Amplified Polymorphic DNA (RAPD) marker system was applied based on 10 primers (Table 7). Each 25 μL reaction volume contained: 0.25 mM dNTPs mix, 1 μM single primer, 1.25 U DNA Taq polymerase, 2 mM MgCl2, 20 ng template DNA (0.8 ng·μL−1) and deionised water to volume. Amplification was performed in a NyxTechnic ATC401 thermocycler, in the following conditions: one cycle of 4 min at 94 °C for initial DNA denaturation; 45 cycles of 1 min at 94 °C for denaturation, 1 min 225
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Table 3 Influence of explant type and irradiation time on the mean number of shoot regenerating from one explant after 10 weeks of culture. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
0.7 ± 02 aa 0.7 ± 0.2 a 0.7 ± 0.2 A
0.6 ± 0.3 ab 0.6 ± 0.1 ab 0.6 ± 0.2 AB
0.4 ± 0.2 ab 0.5 ± 0.1 ab 0.5 ± 0.2 AB
0.5 ± 0.1 ab 0.4 ± 0.1 ab 0.4 ± 0.1 AB
0.4 ± 0.1 ab 0.2 ± 0.1 b 0.3 ± 0.1 B
0.5 ± 0.2 A 0.5 ± 0.1 A
a Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 4 Influence of explant type and irradiation time on the height [cm] of ex vitro-grown chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
35.6 ± 0.6 b 35.8 ± 0.8 b 35.7 ± 0.7 B
38.4 ± 1.0 ab 35.7 ± 1.1 b 37.2 ± 1.0 B
36.2 ± 0.8 ab 39.5 ± 1.2 ab 38.1 ± 1.0 AB
39.1 ± 1.1 ab 36.4 ± 1.9 ab 38.4 ± 1.5 AB
42.8 ± 1.1 a 36.0 ± 1.5 ab 41.2 ± 1.3 A
38.6 ± 0.9 A 36.9 ± 1.3 B
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 5 Influence of explant type and irradiation time on the inflorescence diameter [cm] of ex vitro-grown chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
6.5 ± 0.2 d 6.5 ± 0.3 d 6.5 ± 0.2 B
7.4 ± 0.3 ad 8.2 ± 0.4 ab 7.8 ± 0.4 A
6.9 ± 0.5 bd 8.5 ± 0.3 a 7.8 ± 0.4 A
6.8 ± 0.2 cd 7.4 ± 0.3 ad 7.1 ± 0.2 A
8.2 ± 0.2 ab 7.0 ± 0.6 ad 7.9 ± 0.4 A
7.1 ± 0.3 A 7.9 ± 0.4 A
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 6 Influence of explant type and irradiation time on the length [days] of the second developmental stage (from potting till bud colouration) of chrysanthemums. Explant type
Irradiation time [s]
Leaf Callus Mean
Mean
0
8
2×8
3×8
4×8
143.8 ± 0.5 ab 144.0 ± 0.5 ab 143.9 ± 0.5 B
145.2 ± 0.3 ab 144.2 ± 0.3 ab 144.7 ± 0.3 B
145.3 ± 0.5 b 142.8 ± 0.6 c 144.1 ± 0.6 B
144.0 ± 0.4 ab 144.8 ± 0.7 ab 144.4 ± 0.6 B
145.8 ± 0.3 b 148.3 ± 0.8 a 147.1 ± A
144.8 ± 0.4 A 144.8 ± 0.6 A
* Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the interaction of the two factors; upper-case letters refer to the independent main effects.
Table 7 Sequence of primers used in the RAPD analysis, the number of bands they generated and the number of plants with polymorphic bands. Symbol
Reference
Sequence
No of reference/polymorphic/unique bands
No of plants with polymorphism detected
A B C
Lema-Rumińska et al. (2004)
5′-GGG AAT TCG G-3′ 5′-GAC CGC TTG T-3′ 5′-GGA CTG GAG T-3′
4/4/4 6/6/6 6/6/11
3 1 4
D E
Shibata et al. (1998)
5′-GCT GCC TCA GG-3′ 5′-TAC CCA GGA GCG-3′
3/3/0 10/10/5
1 19
F G
Wolff (1996)
5′-CAA TCG CCG T-3′ 5′-GGT GAC GCA G-3′
3/3/3 6/4/2
1 1
H I
Martín and González-Benito (2005)
5′-CCC AGT CAC T-3′ 5′-TGG CGT CCT T-3′
7/5/6 7/7/4
1 1
J
Chattarjee et al. (2006)
5′-AGC GTG TCT G-3′
6/3/7
4
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Fig. 2. Influence of the tested main effects on the number of shoots produced from one explant and the share of regenerating explants after 10 weeks of culture. *Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the number of shoots; upper-case letters refer to the share of regenerating explants.
Fig. 3. Share [%] of inflorescence variants with different colours of inner [A] and outer [B] side of ray florets produced from untreated control and MW-irradiated explants.
Fig. 4. Different shapes of chrysanthemum ‘Alchimist’ inflorescences (A–D) and ray floret types (E–I): A – flat semi-full; B – flat full; C – convex semi-full; D – convex full; E – ray floret with yellow discolouration; F – ray floret with a spur (indicated with an arrow); G–I – ray florets overgrown into a tube in ¼ (G); ½ (H) and ¾ (I) of their length; 1 bar = 1 cm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results
cluster analysis (PCoA) performed with the use of GeneAlex 6.5 software (Peakall and Smouse, 2012). The coefficient of genetic distance based on Nei and Li algorithm (1979) was calculated by a comparison of the predominant band pattern of the control plants with the band patterns of the plants regenerated from the explants treated with microwaves. On the base of genetic distance values, a dendrogram was created with the AHC unweighted pair group method (UPGMA) for clustering, using Treecon 1.3 software (Van de Peer and De Wachter, 1994).
3.1. Morphological response of explants In both experiments, callus formation began during the second week of culture on all inoculated explants. Primarily, it was green and compact, however, in the following weeks it turned brown. There were no visual signs of explant tissue damage after MW treatment observed in the first experiment. Some necrotic changes were only occasionally 227
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Fig. 5. Share [%] of various inflorescence shapes produced in the experiment.
Fig. 6. Influence of explant type and irradiation time main effects on the share of ray florets with spurs and discolouration. *Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to spurs; upper-case letters refer to the discolouration.
Fig. 7. Share [%] of ray florets with various degree of overgrowth into a tube.
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Fig. 8. Influence of experimental factors on the length of the first (from potting till flower bud appearance) and the third developmental stage (from potting until full flowering) of chrysanthemum. * Means ± standard errors indicated with the same letters do not vary at P = 0.05; lower-case letters refer to the I stage; upper-case letters refer to the III stage.
control plants reached 35.7 cm, while the ones produced after four cycles of 8 s irradiation −41.2 cm). Three variants of both inner and outer colour of ray florets were observed (Fig. 3). As for the inner side of the flowers (Fig. 3A), the dark pink inflorescences (RHSCC code: 75C) were the most numerous in both control and MW-derived plants (63 and 40%, respectively). A similar share of light purple flowers (RHSCC: 77D) was observed in both groups (31%). The light pink inflorescences (RHSCC: 69C) were the least frequent, however, in the untreated control they accounted for only 6%, while in the MW-treated plants, their share was nearly fivefold higher and reached 29%. The variation in the outer side colour of ray florets was not as evident (Fig. 3B). Within the untreated control and MWderived chrysanthemums, the share of purple (RHSCC: 70A), light purple (RHSCC: 78B) and dark purple (RHSCC: 71A) inflorescences was as follows: 50/42; 25/20 and 25/38%, respectively. Four shapes of chrysanthemum inflorescences were observed (Figs. 4 4A–D and 5 ). Most of the non-treated control plants produced flat full inflorescences (44%; Fig. 4B), while the least numerous were the ones with convex full phenotype (6%; Fig. 4D). On the other hand, most of the MW-treated plants produced convex full inflorescences (32.4%) and the flat full type (31.6%), while the convex semi-full ones (Fig. 4C) were the most scarce ones (8.8%). All of the plants regenerated from MW-treated explants produced inflorescences of a greater diameter (7.1–7.9 cm) then the untreated control (6.5 cm) (Table 5). Yellow discolouration could be observed on the outer part of ray florets of approximately 41% of both non-treated control (30.5%) and the irradiated (43.7%) chrysanthemum inflorescences (Fig. 4E). There was, however, no influence of the explant type or the irradiation time on this feature noticed (Fig. 6). On the other hand, it was observed that MW treatment lead to a more frequent formation of spurs in ray florets (Figs. 4F and 6) but with no interaction with the second factor. It was noticed that the control chrysanthemums had ray florets overgrown into a tube in ¼ or ½ of their length (44% of both types) (Figs. 4G–I and 7 ). As for the MW-derived inflorescences, the ray florets overgrown in ½ were dominant (65.1%), while the ones overgrown in ¼ were the least frequently observed (15.5%). None of the experimental factors influenced the pace of the first (from potting till flower bud appearance) and the third developmental stage (from potting until full flowering) of chrysanthemums (Fig. 8). On the other hand, the time of irradiation and the interaction of the two tested factors affected the length of the second developmental stage – beginning of flowering (bud colouration) (Table 6). Four cycles of irradiation (4 × 8 s) lead to slower flower bud colouration (over
observed in the second experiment after applying the highest irradiation dosages (3 × 8 and 4 × 8 s). Adventitious shoots were forming mostly between the first and the fifth week of culture, regardless of the experimental conditions. Their number remained constant afterwards (data not shown). All of the shoots regenerated from the MW-irradiated explants were morphologically similar to the control ones during the in vitro stage. 3.2. Experiment I Only the type of explant had an influence on the regeneration of adventitious shoots. Leaves which were covered with callus prior to MW irradiation, regenerated more shoots in comparison to leaves without a previous 3-week preculture (1.0 and 0.7 shoot per explant, respectively; Fig. 2). Neither the milieu conditions nor the time of irradiation had a significant influence on this parameter. There were also no interactions between the tested experimental factors observed. Moreover, no differences in the share of explants regenerating shoots were reported (approximately 47%), regardless of the conditions (Fig. 2). 3.3. Experiment II Approximately 40% of both explant types regenerated adventitious shoots, however, MW treatment decreased the share of regenerating explants (26%) in comparison to the untreated control (60%; data not shown). Similarly it was observed that the longest irradiation decreased more than twice the number of regenerating shoots (0.7 shoot per one non-treated explant and 0.3 after 32 s exposure), especially after callus irradiation (Table 3). 3.3.1. Acclimatization efficiency Both, the rooting and acclimatization efficiency were 100% for the control and the irradiation-derived plants. In the end, 20 control and 96 MW-treated plants were acclimatized. No differences in the morphology or deformations within those two groups were visually observed during that stage. 3.3.2. Ex vitro growth and phenotypical analysis Both of the tested factors affected the length of the produced chrysanthemum shoots (Table 4). The explants which were precultured for three weeks prior to irradiation (callus) produced shorter shoots (36.9 cm) in comparison to the leaves (38.6 cm). It was also observed that longer MW-treatment positively affected the growth of plants (the 229
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Fig. 9. Example band profiles received as a result of electrophoresis of the DNA amplification products obtained with: J; E; C; A; J; E primers (from the top left). Outermost lanes are DNA bp ladders. Arrows point to the profiles which differ from the reference.
plant with a changed band profile. The share of mutated plants was higher among the chrysanthemums regenerated from irradiated callus (37%) than among the control plants (10%) and the ones received from irradiated leaves (16%; Table 8). The highest percentage of plants with changed band profiles was obtained from callus exposed to MW treatment for 24 s (86%). The mean values of the coefficients of genetic distance (compared to predominant monomorphic band pattern in control plants) ranged from 14.7% (for plants regenerated from callus irradiated for 3 × 8 s) to null in plants regenerated from leaves irradiated for 8 and 2 × 8 s (Table 9). Plants regenerated from MW-treated callus showed higher mean coefficient of genetic distance (3.01%) in comparison to the ones regenerated from irradiated leaves (0.41%), what was confirmed by the PCoA analysis (Fig. 10). On the base of genetic distance values a dendrogram was created for
147 days since potting) in comparison to all other treatments (144–145 days). Chrysanthemums produced from explants which were precultured for three weeks prior to MW-treatment for 32 s, required most time (148.3 days since potting) to start flowering. 3.3.3. Evaluation of the genetic variation The amplification products reached a size from 289 bp to 2954 bp. The highest number of amplicons was generated by the E primer (10 bands per reference), while the lowest number – by primers D and F (3 bands) (Table 7). A total of 106 different loci was detected by ten primers. A total of 25 plants with an altered band pattern (2 control and 23 MW-treated) were observed (Fig. 9). The E primer was most efficient in detecting variation (polymorphism observed within 19 genotypes; Table 7). On the other hand, primers: B, D, F, G, H and I denoted only 1 230
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variation detected with RAPD markers is of interindividual origin, while 11% results from variation between groups.
Table 8 Number and share [%] of plants with an altered band profile in comparison with a predominant band profile of control plants. Explant type
Irradiation time [s]
Number of plants analysed
number
%
Control Leaf
0 8 2×8 3×8 4×8 total
20 17 12 19 10 58
2 0 0 4 5 9
10 0 0 21 50 16
Callus
8 2×8 3×8 4×8 total
14 14 7 3 38
4 3 6 1 14
29 21 86 33 37
116
25
22
Total
4. Discussion
Plants with an altered RAPD band profile
The horticultural market constantly demands flower novelties; hence mutation breeding is commonly used for rapid improvement of commercially important ornamental plants. There are many findings on chrysanthemum mutation breeding with gamma and X-radiation, as well as with chemical agents (Teixeira da Silva and Kulus, 2014; Oladosu et al., 2016). On the other hand, there are no information about the use of microwaves in breeding of ornamentals. Our studies suggest that MW treatment can be a useful tool in modern horticulture. Due to a relatively high ease in inducing variation, chrysanthemum is a suitable object for studying the effects of mutation breeding. However, various cultivars may differ in terms of susceptibility to mutation occurrence. Pink- or purple-flowering chrysanthemums undergo mutations most often (due to a high share of dominant alleles responsible for flower colour), while yellow ones – mutate the least frequently (Kulus, 2017). For this reason, the purple-flowering ‘Alchimist’ was selected as the donor of the biological material. In the first experiment a more efficient shoot regeneration was obtained from irradiated callus, which may be a result of the additional 3week preculture of explants prior to MW-treatment. The influence of explant type on the chrysanthemum and other ornamental plant species in vitro morphogenetic response was also reported by Zalewska et al. (2010, 2011), Teixeira da Silva and Kulus (2014) and Lema-Rumińska and Kulus (2014). On the other hand, there was no influence of the milieu conditions on the explant survival, callus formation and further regeneration of shoots observed. As for Vigna aconitifolia, immersion of explants in water during radiation affected their survival and callus development (Jangid et al., 2010). Similarly, an increase of temperature during preculture influenced the regeneration of callus and shoots in Brassica napus L. (Burbulis et al., 2004), which was not observed with chrysanthemum. A positive influence of magnetic field on the explant regeneration rate was observed with Paulownia tomentosa (Thunb.) Steud. (Alikamanoğlu et al., 2007) and Glycine max L. Merrill (Atak et al., 2003). Microwave treatment stimulated the in vivo germination of Phaseolus vulgaris L. seeds (Jakubowski, 2015). Zalewska (2010), on the other hand, described that treatment with ionizing radiation leads to a decrease in chrysanthemum shoot regeneration efficiency. In our experiment, microwave treatment for over 8 s also reduced the share of
Table 9 The highest, lowest and mean values [%] of the coefficient of genetic distance between the control and MW-treated plants. Explant
Time of irradiation [s]
Highest
Lowest
Mean
Control Leaf
0 8 2×8 3×8 4×8 Mean
2.65 0 0 2.65 0.87
0 0 0 0 0
0.27 0 0 0.41 0.35 0.41
Callus
8 2×8 3×8 4×8 Mean
10.53 1.75 83.02 3.45
0 0 0 0
1.01 0.25 14.70 1.15 3.01
all analysed genotypes (Fig. 11). The most distant from monomorphic control plants were two individual plants regenerated from callus MWtreated for 8 and 24 s. Other polymorphic plants were gathered into seven minor clusters not surpassing 10% of genetic distance among each other. The AMOVA analysis revealed that 89% of the total genetic
Fig. 10. Graph of principal coordinates analysis of control and MW-treated plant populations.
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Fig. 11. Dendrogram based on the estimation of genetic distance coefficients and UPGMA clustering presenting the relationships between non-treated control and MW-radiated genotypes, revealed by the RAPD analysis. All genotypes representing the same band pattern as the predominant control plants are collected within a single group named ‘Monomorphic’. The scale above shows a real genetic distance value.
the formation of three new phenotypes: silver-purple (RHSCC code: 77C), light-purple (77B) and claret-gold (60C). The application of MW has broaden the possible variation with new phenotypes (69C; 71A; 70A and 78B). It is worth mentioning that some of the obtained plants with yellow discolouration might have been mericlinal or sectorial chimeras, valuable in further breeding programmes (Kulus, 2017). Those findings may contribute to a wider application of microwaves as a cheap and commonly available source of variation, acceptable by the society. In order to detect genetic variation we have applied the RAPD marker system. The technique is simple, fast and cost-efficient. Moreover, it does not require knowledge of the genome sequence (Grover and Sharma, 2016). In the past, it was thought that RAPDs cannot be used with closely related cultivars and that they have a low repeatability. However, research conducted by Wolff (1996), Shibata et al. (1998) and Miler and Zalewska (2014) showed that this marker system can be successfully applied not only to distinguish closely related individuals, but also genetically different tissues of the same chimerical plant even at early developmental stage with only 1–2 primers. The technique was also successfully utilized in the detection of variation in cryopreservation-recovered chrysanthemums (Martín et al., 2011). Genetic similarity coefficients based on RAPD analysis were calculated previously for the somaclones of chrysanthemum ‘Alchimist’ by Miler and Zalewska (2014). This justifies the suitability of its use in mutagenesis research. The RAPD marker system used in our study revealed small changes (genetic distance at the level of 2.7%) in the band patterns of 10% untreated control plants. As for the MW-treatment, variation was detected in even 86% of chrysanthemums regenerated from callus irradiated for 24s. In comparison, Zalewska et al. (2011) obtained only 5.4% plants with altered genetic profiles by applying X and gamma radiation to ‘Alchimist’ cultivar, while the treatment of ‘Ingrid’ chrysanthemum with ethylsulphonate resulted in a 10.4% of changed plants (Latado et al., 2004). This clearly indicates that MW can
regenerating explants, as well as the shoot number if dozed for 32 s, especially to callus, which seems to be more sensitive in comparison to leaves. Similarly, the negative impact of microwaves (800 W) on the in vitro regeneration efficiency was reported with Vigna aconitifolia. As for this species, however, a MW-treatment of leaf explants for only 1–7 s resulted in a twofold decrease in the number of regenerating shoots in comparison to the untreated control (Jangid et al., 2010). Moreover, a significant influence of the increasing irradiation periods on the explants mortality was observed. As for chrysanthemum, no visual signs of tissue damage were observed after explant irradiation for up to 16 s, whereas longer treatments lead to minor tissue necrosis. There was also no influence of MW-treatment on chrysanthemum callus formation, which is in contrast with the reports of Jangid et al. (2010). Therefore, it can be assumed that chrysanthemum explants are tolerant to lower MW doses. It is known, that mutagens can influence on both the vegetative and the generative growth phases of plants (Qui et al., 2011). Khan et al. (2014) reported that combined treatment of Brasica rapa L. seeds with gamma radiation and sodium azide increased the length of the produced shoots by as much as 39%. In the present study, a similar phenomenon was observed (increase by 15%). Moreover, MW radiation lead to the formation of inflorescences of greater diameter (by 21.5%) and of altered shape in comparison to the control. The production of bigger more attractive inflorescences may be considered a positive change from the horticultural point of view. Moreover, the longest MW treatment prolonged the time of flower bud colouration by four days. Similar results were observed with ‘Lalima’ chrysanthemum subjected to 0.5 Gy gamma radiation (Misra et al., 2003). A protraction of time required from bud appearance till the beginning of flowering is positive since it may improve the post-harvest quality of plants. Altering the colour of the flowers is one of the most important goals of breeding. In the research conducted by Zalewska et al. (2011), the treatment of chrysanthemum ‘Alchimist’ leaf explants with gamma rays resulted in 232
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be a useful tool in modern breeding, although after proper optimization. In the present study, the share of plants with band patterns different from the reference control plants was higher in the plants regenerated from microwaved callus in comparison to leaves, which is a common phenomenon (Betekhtin et al., 2017). Most of the changed band profiles showed several additional or missing amplicons, which is in contrast with the findings of Jangid et al. (2010) and suggests that DNA changes after microwave treatment are quite expanded. Therefore, it can be assumed that mutations occurring in meristematic centres are not lost but can spread out as the meristemoid continues to grow into an adventitious shoot. The genetic distance at the level of 2.7% present in control plants and in some plants regenerated from irradiated explants, on the other hand, may be a result of somaclonal variation, which is often observed in the in vitro cultured plants, especially if the nonmeristematic explant is used, and in the presence of plant growth regulators (Miler and Zalewska, 2014). 5. Conclusions The present research confirms that MW-treatment of leaf explants with preliminary regenerated callus can be used for the induction of genetic and phenotypic variation in chrysanthemum ‘Alchimist’. In order to obtain a high share of mutated plants, a prolonged irradiation (three or four cycles, 8 s, each) is recommended. The obtained variation (alternation in flower shape and colour, increase in inflorescence diameter and prolongation of bud colouration) can be considered positive from a horticultural point of view. In the future the use of other explant types for MW-treatment is recommended, e.g. petals which have been indicated recently as the most suitable for variation induction in chrysanthemum (Teixeira da Silva et al., 2015). Conflict of interest The authors declare no conflict of interest. The authors wish to acknowledge Mr. Marek Mayka and Mrs. Katarzyna Milczek for their technical support in performing the experiments. References Alikamanoğlu, S., Yaycılı, O., Atak, Ç., Rzakoulieva, A., 2007. Effect of magnetic field and gamma radiation on Paulownia tomentosa tissue culture. Biotechnol. Biotechnol. Equip. 21 (1), 49–53. http://dx.doi.org/10.1080/13102818.2007.10817412. Atak, Ç., Emiroglu, Ö., Alikamanoglu, S., Rzakoulieva, A., 2003. Stimulation of regeneration by magnetic field in soybean (Glycine max L. Merrill) tissue cultures. J. Cell Mol. Biol. 54 (4), 703–706. Betekhtin, A., Rojek, M., Jaskowiak, J., Milewska-Hendel, A., Kwasniewska, J., Kostyukova, Y., Kurczynska, E., Rumyantseva, N., Hasterok, R., 2017. Nuclear genome stability in long-term cultivated callus lines of Fagopyrum tataricum (L.) Gaertn. PLoS One 12 (3), e0173537. http://dx.doi.org/10.1371/journal.pone. 0173537. Burbulis, N., Kupriene, R., Zilenaite, L., 2004. Embryogenesis, callogenesis and plant regeneration from anther cultures of spring rape (Brassica napus L.). Acta Univ. Latv. Biol. 676, 153–158. Chattarjee, J., Mandal, A.K.A., Ranade, S.A., Teixeira da Silva, J.A., Datta, S.K., 2006. Molecular systematic in Chrysanthemum × grandiflorum (Ramat.) Kitamura. Sci. Hortic. 110, 373–378. http://dx.doi.org/10.1016/j.scienta.2006.06.004. Chen, Y.P., 2006. Microwave treatment of eight seconds protects cells of Isatis indigotica from enhanced UV-B radiation lesions. Photochem. Photobiol. 82 (2), 503–507. http://dx.doi.org/10.1562/2005-06-29-RA-595. Cretescu, I., Rodica, C., Velicevici, G., Ropciuc, S., Buzamat, G., 2013. Response of barley seedlings to microwaves at 2.45 GHz. Anim. Sci. Biotechnol. 46 (1), 185–191. Diprose, M.F., 2001. Some considerations when using a microwave oven as a laboratory research tool. Plant Soil 229, 271–280. Grover, A., Sharma, P.C., 2016. Development and use of molecular markers: past and present. Critic. Rev. Biotechnol. 36 (2), 290–302. http://dx.doi.org/10.3109/ 07388551.2014.959891. Halmagyi, A., Surducan, E., Surducan, V., 2017. The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage. J. Biol. Phys. 43 (3), 367–379. http://dx.doi.org/10.1007/s10867-0179457-4. Jakubowski, T., 2015. Evaluation of the impact of pre-sowing microwave stimulation of bean seeds on the germination process. Agric. Eng. 19 (2), 45–56. http://dx.doi.org/
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