Mechanisms generating the numerous hues of chrysanthemum petals can be traced to in vitro γ-ray-induced mutagenesis.

In terms of global cut ower production, chrysanthemum (Dendranthemum grandiorum kitam.) ranks among the top ten. When a plant species possesses colour characteristics associated with those of ancestral species, that colour is described as being white, yellow, or pink, with white coloration representing an absence of pigments, and yellow and pink coloration each represents the presence of carotenoids and anthocyanins, respectively. Pigments, or a combination of pigments, have been improved upon to provide an expansive rainbow of oral colours like orange, dark red, purplish-red, and red. Recent green-owered varieties have developed a reputation for containing chlorophylls in their ray petals. Additionally, transgenic interventions have been used to breed violet/blue owers. Flower colour is a highly regarded characteristic of any ower cultivar, particularly chrysanthemum. Breeders' ultimate goal is to create newer chrysanthemum cultivars with innovative characteristics, such as new ower colours, using a time and input-ecient approach. Investigating the molecular mechanisms that regulate ower pigmentation may yield critical insights into the rational manipulation of oral colour. To generate a diverse array of ower colour mutants in chrysanthemum cv. “Candid” through mutagenesis, in vitro grown micro shoots were exposed to 10, 20, 30, and 40 Gy gamma irradiation at 100 Gy per minute and were evaluated for different parameters. The rhizogenesis parameters declined with the increase in irradiation dose from 0 Gy to 40 Gy, while as, 10 Gy dose proved to record minimum decline in contrast to the control. Survival, leaf size, and the number of leaves plant -1 after the 8 th -week interval exhibited a downward trend with the increasing trend of gamma irradiation dose but recorded a least drop in plants raised from shoots irradiated with 10 Gy gamma irradiation dose with respect to the control. Apparently, the minimum delay in the number of days to oral bud appearance took under 10 Gy compared to control. The highest number of ower colour mutants was recorded under 10 Gy (light pink, orange-pink, white and yellow). Demountable mutation frequency based on ower colour was desirable in plants irradiated


Introduction
Chrysanthemum is a widely grown and indispensable cut ower crop in China, Japan, France, the United States of America, India, and the United Kingdom. It is a signi cant oricultural crop, ranking second in terms of cut owers, after rose, among oricultural crops traded on the international ower market (Kumar et al. 2006). The complex genetic hetrozygosity makes the cultivated chrysanthemum an unlimited source of new ower form and cultivars. The common garden chrysanthemum is hexaploid with 54 chromosomes (Wolff 1996). It is propagated vegetatively and has a robust self-incompatibility system (Richards 1986); therefore, new varieties are complicated to acquire by crossing. Usually, new varieties have been developed from spontaneous mutations in vegetative reproduction, sports, being some variations more stable than others (Miñano et al., 2009). In recent years induced mutations along with the somaclonal variations resulting from the tissue culture practices have been in work as an innovative source of variability (Schum 2003, Datta et al. 2005, Jain et al. 2006, Zalewskam and Miller 2007, Chatterjee et al. 2006, Barakat et al. 2010) Even though comprehensive efforts have been undertaken to expand uniqueness in chrysanthemum cultivars by induced mutations employing physical and chemical mutagens (Broertjes and Van-Harten 1978), there is a constant need to investigate the chances of a new cultivars for horticulture trade. Mutation breeding by radiation has been extensively used to improve well-established plant cultivars and develop novel variants within superior consumer characteristics. While the majority of cultivated chrysanthemum varieties are polyploids with huge genetic heterogeneity, mutants with allied ower shape, oral size, and colour, are frequently recovered.
Associated ower colours with chimeric tissue can be simply stimulated by radiation and isolated with in vitro apparatus (Kumar et al., 2006). Chrysanthemum is a hexaploid plant, and vegetative propagation (rather than sexual propagation) makes it di cult to carry out the hybridization, hence mutation processes are utilized (Broertjes and Van-Harten 1978). In any plant breeding programme, genetic variability is crucial for crop enhancement. In ower breeding, particularly for colour, mutation breeding is an e cient method of producing heritable change.
To create novel Chrysanthemum ower color/shape mutants, radiation has been used successfully (Misra et al. 2003;Dwimahyani and Widiarsih 2011). As a result, radiation-induced mutagenesis has become a critical approach for ower breeding. When observable characteristics such as ower colour, shape, and size, or leaf structure and growth habit are considered, the selection of ornamentals is quite simple. By 2005, 2,335 varieties were released through mutagenesis in the world, in which ornamental and decorative crops were 552 (IAEA 2005). However, the main bottleneck is formation of chimera, which appears after physical mutagen treatment. The uses of in vitro mutagenesis for development of solid mutant were previously reported by Latado et al. (2004) and Chakrabarty et al. (1999). The mutant sector can range in size from a thin streak on a petal to the entire ower in chimeric tissues. When a petal or an entire ower is mutated, the mutant tissue can be extracted and studied. On the other hand, using normal replication techniques, a small segment of a mutant blossom cannot be separated. As a result, many novel ower colour/shape mutants caused by mutagens are lost due to the absence of a system for regeneration from small-mutated sectors in vivo or in vitro. Thus, the ability to regenerate plants from single oret cells would be an advantageous strategy for establishing mutants in pure form from chimaeras and for facilitating the generation of a diverse range of unique cultivars with innovative ower colours and shapes.
The assumption was made for a good mutation breeding programme; there are numerous considerations to bear in mind. These include the sort of genetic material, the traits to be enhanced, the type of mutagens, and their dosage to be utilised, as well as an experimental procedure to choose. The hypothesis goes further, asserting that this method for inducing variation, called mutation breeding, makes it possible to produce genetic diversity that is both statistically and qualitatively meaningful in the programme. In the study of mutation and variability, researchers were able to come up with a novel chrysanthemum cultivar with a highly anticipated customer appeal. As a result of the foregoing, the current study was undertaken with the goal of generating a diverse array of ower colour mutants via mutagenesis.

Materials And Methods
The current study was carried out in plant tissue culture laboratory at the Department of Floriculture and Landscape Architecture SKUAST-Kashmir. Tissue culture developed micro shootlets of chrysanthemum cv. 'Candid' (parent cultivar, Figure-1) were exposed to Cobalt 60 gamma irradiation doses of 0, 10, 20, 30, and 40 Gy at 100 Gy per minute and were allowed to raise vegetatively mutated generations rst and second at 5-week intervals. Finally, shoots obtained from vegetatively mutated generation 2 were allowed for rooting and consequent acclimatization. Rooted shoots were allowed to grow in pots in the eld to obtain new enviable colour mutants, and rooting parameters were recorded in terms of percentage rooting and the number of roots per shoot. Survival (%), leaf area plant -1 (cm 2 ), number of leaves plant -1 were recorded at 4 th and 8 th weeks growth in the eld. Days to ower bud appearance were recorded at the initiation of ower bud appearance. Plant height was noted at the end of full ower bloom. Flower colour was recorded in terms of the difference between the parent ower and mutants obtained. The frequency of mutation was calculated based on ower colour, as the ratio between such desired or undesired colour mutant and whole plants irradiated with each gamma irradiation dose.
The investigation used a completely randomised design with four replications, and the data were analysed using analysis of variance after being compiled from various trail observations (ANOVA). To ensure that the model assumptions for ANOVA were met, percent age data were transformed using the square root method recommended by Steel and Torrie (1980), and the average data were classi ed using Duncan's multiple range test. (Gomez & Gomez, 1983).

Effect of gamma irradiation on rhizogenesis
Data pertaining to the effect of 60 Co gamma irradiation on rooting per cent and root number of chrysanthemum (Dendranthemum morifolium L.) cv. "Candid" have been presented in Table 1. It is quite vivid from the data that irradiation doses had enervating effect on all the parameters of rooting in comparison to control ( Fig.6a and 6b). Perusal of data reveal that there was a signi cant decrease in mean rooting per cent in all the irradiation treatments in comparison to control shoots (89.28%).
Among irradiation treatments maximum (85.71%) rooting was recorded under 10 Gy dose, representing only 1.90 per cent decrease in rooting in comparison to control. This treatment dose was followed by 20 Gy (79.76%) and 30 Gy (74.99%), corresponding to 5.40 and 8.26 per cent decrease over control, respectively. Both the treatments varied signi cantly different from each other. Further perusal of data reveal that highest dose 40 Gy of 60 Co gamma irradiation resulted signi cantly minimum 73.80 per cent rooting, corresponding to a heavy decrease (9.00%) in rooting over control (Figure 2a and 2b). There was signi cant differences in mean root number 7.75 and 15.75 shoot -1 recorded under 10 Gy dose and control, respectively. Under higher doses of 20, 30 and 40 Gy, root number signi cantly dropped to 5.50, 3.00 and 2.75 shoot -1 , respectively. Correlation analysis of number of root per shoot showed a signi cant relation with the induction of rooting percentage ( Figure 3).

Effect of gamma irradiation on survival of rooted plantlets under polyhouse conditions
Data recorded on survival of rooted plantlets of chrysanthemum (Dendranthemum morifolium L.) cv.
"Candid" at the end of 4 and 8 weeks under polyhouse conditions have been presented in Table 2.Perusal of data reveal that survival of rooted plantlets declined with each increment of gamma dose in both the intervals of 4 th and 8 th week. In cv. "Candid" survival of rooted shoots was signi cantly minimum (85.71%) with 10 Gy dose as against 95.23 per cent in control at the end of the 4 weeks ( Fig. 6c). It further decreased to 76.18, 61.90 and 53.56 per cent, respectively with 20, 30 and 40 Gy radiation doses. Latter treatment corresponded to 25.10 per cent decrease of survival over control. In second interval (i.e., after 8 weeks) survival of 82.14 per cent recorded under 10 Gy dose was signi cantly decreased in comparison to 95.23 per cent in control (Fig. 6d). This treatment was followed by 20 Gy (67.85%) and 30 Gy (53.56%) doses. Microshoots exposed to 40 Gy dose signi cantly decreased survival to 40.47 per cent, corresponding to 35.04 per cent decline in comparison to control. Also strong correlation was found between survival percentage and dosage ( Figure. 4).

Effect of gamma irradiation on leaf number and leaf size
Data regarding the effect of 60 Co gamma irradiation on leaf number and leaf size of chrysanthemum (Dendranthemum morifolium L.) cv. "Candid" at 4 and 8 week intervals are presented in Tables 3. In cv.   (Figure 6i).

Effect of gamma irradiation on ower colour and mutation frequency
Regarding the effect of gamma irradiation on ower colour, desired colour mutants were selected only from the plants irradiated with 10 Gy dose, which evolved 60 per cent of light pink, 15 per cent of orangepink, 10 per cent white, 5 per cent light yellow (5%) and remaining 10 per cent were as same as control i.e., showing original red colour (Fig.5 a, 5b, 5c and 5d). Higher doses of 20, 30 or 40 Gy produced either distorted red bud or distorted red oret ( Fig.5e and 5f).Colour mutants under 20, 30 and 40 Gy were undesirable. Data regarding the mutation frequency in chrysanthemum owers on the basis of ower colour, there was a highly desired mutation frequency amounting to 90 per cent when the plants were irradiated with 10 Gy dose. Whereas, under 20, 30 and 40 Gy doses ower mutation frequency although recorded cent per cent, but produced undesirable mutants.

Effect of gamma irradiation on rhizogenesis
It is quite vivid from the data (Table 1), that irradiation doses had enervating effect on all the parameters of rooting in comparison to control. Perusal of data revealed that there was a signi cant decrease in mean rooting per cent and number of roots shoot -1 in all the irradiation treatments in comparison to control. Among irradiation treatments maximum rooting and number of roots were recorded under 10 Gy dose, followed by 20 and 30 Gy irradiation doses. Minimum rooting and number of roots were registered with 40 Gy dose. Rhizogenesis in vitro involves development of roots de novo. It is a process of dedifferentiation of speci c pre-determined cells near the vascular bundles. Any damage to cell division ability will have a negative effect on dedifferentiation of cells and subsequent reorganization into root primordia. This may result in failure of rooting or delayed emergence of roots. Singh et al. (1999), also reported that increased doses of gamma irradiation (from 20 to 50 Gy) decreased rooting percentage of carnation cv. 'Espana'. Radiation treatments also delayed root initiation signi cantly in comparison to control. Sooch et al. (2000), observed delayed root initiation of carnation shoots of cv. 'Scania' under 1.00, 1.50 and 2.00 K-rads gamma irradiation doses. The deleterious effects of radiations were also re ected in signi cant decline in root number shoot -1 under 10 to 30 Gy treatments. El-sharnouby and El-Khateeb (2005), also reported that most of the gamma irradiation treatments (10,20,30 and 40 Gy) without or with NAA in the rooting medium decreased the number and the length of roots in the carnation cultivars "Medea", "Candela" and "Picaro". All the above quoted studies are in close conformity with the observations recorded in the present study.

4.2.
Effect of gamma irradiation on survival of rooted plantlets Perusal of data ( Table 2), reveal that survival of rooted shoots at the end of 4 week was signi cantly minimum by the shoots treated with 40 Gy dose as against control, followed by 30 and 20 Gy dose. Under minimum dose of 10 Gy, there was a minimum decline in survival of shoots at the end of the 4 week over control. At the end of 8 week, shoots treated with 10 Gy dose recorded maximum survival, followed by 20 and 30 Gy dose. While as, lowest survival per cent was recorded in shoots treated with 40 Gy dose corresponding to heavy decline in comparison to control. Broertjes and De-Jong (1984), obtained 100% survival when chrysanthemum plantlets transferred to soil were irradiated with 2.5 or 5 kGy. Major reason for mortality in post irradiation propagation generations is due to deleterious chimera load carried by the plants. The impaired cells or tissue sectors on coming to occupy signi cant portions of growing point lead to death or debilitated plants which do not survive more exacting environmental demands in the eld. Any impairment in the epidermal skin of the plants gets quickly exposed in the eld leading to mortality. Another reason might be formation of low or reduced wax component on the post irradiation plants. As the wax component determines the rate of water loss through the cuticle and the susceptibility of tissue-cultured plants to desiccation attributed to a reduction or absence of wax acting as antitranspirant. The epicuticular wax is reduced or absent on the carnation leaves of in vitro cultured plants compared to glasshouse or eld-grown plants (Sutter and Langhans, 1979), but during acclimatization, the density of waxes increases as the humidity is lowered (Wardle et al., 1983). Since the irradiation impairs the epidermal skin of the plants leading to low wax formation even during the acclimatization process, hence any impairment in the epidermal surface of the plants gets quickly exposed in the eld or polyhouse leading to mortality. This nding is in accordance to the results in present investigation.

4.3.
Effect of gamma irradiation on leaf number and leaf size In cv. "Candid", perusal of data (Table 3) reveal that gamma irradiation treatments signi cantly recorded a decline in leaf number plant -1 and leaf size in both the intervals i.e., 4 and 8 week as compared to control. At the end of 4 week, signi cantly minimum leaf number plant -1 and size was registered under highest dose of 40 Gy, followed by 30 and 20 Gy and the lowest gamma irradiation dose 10 Gy recorded a minimum decrease in leaf number and size, as compared to the control. At the end of 8 week, both leaf number as well as leaf size improved in all the gamma irradiation doses including the control plants but recorded the similar trend of decline in both the parameters as in the 4 week interval with the successive gamma irradiation doses. Increase in leaf area is a function of the expansion of leaf cells which depends upon the acid growth driven elasticity of cell walls mainly controlled by auxins. Irradiation is known to disturb auxin synthesis and distribution patterns (Gordon, 1956). This may be the reason for poor expansion of leaf surface and hence lower leaf area due to high irradiation exposure. Several workers have reported decline in leaf area in many ower species as a result of irradiation (Gupta et al., 1974 in tuberose;Misra and Bajpai, 1983 in gladiolus;Gupta et al., 1982 in costus;Siranut et al., 2000 in chrysanthemum). Kahrizi et al. (2011), in rose and Srivastava et al. (2007), in gladiolus also reported the decrease in number of leaves with the increase in dosage of gamma irradiation. They further reported that there was no signi cant effect of 10 Gy dose on leaf size explant -1 , however 20 to 40 Gy irradiation signi cantly reduced leaf size plant -1 recorded at the end of two intervals. Inhibitory effect of gamma rays on vegetative characters of carnation at higher doses has also been observed by Acharya and Tiwari (1996). In another study Simard et al. (1992) and Cassels et al. (1993), recorded biological damage in carnation on increasing the dose of radiation. Misra et al. (2009), in chrysanthemum have also reported decline in leaf area as a result of irradiation whereas, Kumari et al. (2013), reported reduction in leaf size in terms of length and width of plants treated with higher doses of gamma rays in variety "Otome Pink". They found that petiole length was shorter with increasing dose of mutagenic agents. All mutagenic agents decreased the leaf area over the control, except 0.5 gamma rays. Reduction in leaf area was observed with increasing dose of mutagens. Mahure et al. (2010), recorded that lower doses like 10 and 20 Gy increased leaf area but 30 Gy decreased leaf area over control. In yet another study by Dilta et al. (2006), rreduction in leaf number was reported in Dandranthema morifolium cv. "Gulmohar" under 1.0, 1.5, 2.0, 2.5 and 3 kR of gamma irradiation. All the above quoted studies are in concurrence with the ndings recorded in the present study.

4.4.
Days to oral bud appearance and Plant height (cm) at owering.
In cv. "Candid" regarding days to bud appearance, perusal of data (  Datta and Banerji (1993), who observed delayed owering behaviour after irradiating rooted cutting of small decorative type chrysanthemum cv. "Kalyani Mauve". In another study Dilta et al. (2003), also observed signi cant delay in days to bud formation, buds showing colour and days for full bloom in the treated plants of ten chrysanthemum cultivar as compared to control. Similar were the results obtained by Misra et al. (2009)  gamma rays treated plants depends on the nature and extend of chromosomal damage or due to physiological, morphological and cytological disturbance caused by irradiation. Also due to the reduction of internal auxin production which leads to reduction of growth of plant (Singh et al., 2011). Also due to the inactivation and decrease in auxin synthesis and nature and extent of chromosomal aberration (Dilta et al., 2003). The results in the present study are in concurrence with the ndings of Datta (1992, 2002), who observed decrease in plant height with increased dose of gamma irradiation in rooted cutting of chrysanthemum cv. "Jaya" and "Lalima". Kole and Meher (2005), also reported decreasing effects in zinnia might be due to physiological damage caused by mutagen at higher doses.

Flower colour and Mutation Frequency
Regarding the colour of owers after irradiation, desired colour mutants were selected only from the plants irradiated with 10 Gy dose, which evolved 60 per cent of pink, 15 per cent of orange pink, 10 per cent white, 5 per cent light yellow (5%) and remaining 10 per cent were as same as control i.e., showing original red colour. Higher doses of 20, 30 or 40 Gy produced either distorted red buds or distorted red.
Colour mutants under 20, 30 and 40 Gy were undesirable. The results in the present study may be due to physiological changes which occur in plant, hence, delayed owering occur at higher doses due to inhibitory effect. This can be attributed to the fact that no chimeric growth was developed in shoot as result of mutagenesis. Shoot or tissue without chimeric growth lead to non-formation, different colour variation in petals reported by Longton (1980) in chrysanthemum. This quoted observation is in close conformity to the present study Data regarding the mutation frequency in chrysanthemum owers on the basis of ower colour, there was a highly desired mutation frequency amounting to 90 per cent when the plants were irradiated with 10 Gy dose. Whereas, under 20, 30 and 40 Gy doses ower mutation frequency although recorded cent per cent, but produced undesirable mutants. The results obtained in the present study are in accordance to the nding of Siavash et al. (2009), who reported increase in mutation frequency when plants were UV irradiated.

Conclusion
The study found that a 40-Gy dose of irradiation resulted in a signi cant decrease in the percentage of rooting, which led to a subsequent decrease in the survival rate of the plant in the eld. It also found that plants didn't grow as high as they normally would, that the period of time between bud emergence and owering was delayed, and that mutation frequencies were also lower. With regard to ower colour, Light pink, Orange-Pink, White, and yellow were the most preferred mutants, while their mutation frequency was highest when the sample was irradiated with 10 Gy. Additionally, the treatment of 10 Gy with gamma rays is suitable for mutagenesis in Chrysanthemum Cv. "Candid".  In uence of γ-rays on a) rooting percentage b) root number per shoot c) survival percentage of rooted shoots at 4th week d) survival percentage of rooted shoots at 8th week e) leaf number per plant at 4th week f) leaf number per plant at 8th week g) leaf size per plant at 4th week h) leaf size per plant at 8th

Declarations
week i) plant height at owering.