In Vitro Mutation Induction and Selection of Chrysanthemum (Dendranthema Grandiflora Tzelev) Lines with Improved Resistance to Septoria

This paper describes in vitro mutation selection technique for improved resistance in chrysanthemum (Dendranthema grandiflora Tzelev) cv. Snow Ball against culture filtrate of Septoria obesa, a leaf spot pathogen. The callus was initiated from leaf exp lant on MS medium supplemented with 10 mg/ l kinetin and 1 mg/l NAA. Opt imal doses of gamma radiation and culture filtrate on the per cent survival of calli were standardized. The optimal dose of gamma radiation was 20 Gy and that of culture filtrate was 15%. The selection of the resistant calli was made at 15% cu lture filtrate concentration and the calli were subjected to two more cycles of selection (30 day cycle) to obtain resistant cell lines. 100% of the plants raised from cuttings had acquired resistance against the pathogen in the pot in greenhouse. The plants were maintained for four years without any symptoms with regular inoculation of fungal spore suspension. No phenotypic variation was observed in the resistant plants.


Introduction
Chrysanthemum is one of the most important commercial cut flowers of the world and is the second largest in demand after rose, among the ornamental plants traded in the global flower market [1]. Economic returns fro m cut flowers main ly depend upon the quality of the flowers [2]. Garden chrysanthemu ms are the number one herbaceous perennial in United States with whole sale farmgate value of $141.8 million in 2005 [3]. Diseases caused by fungi, bacteria, mycoplasma, and viruses are responsible for poor quality of flowers and reduction in yield [4].
Septoria obesa Syd is one of the most important pathogens of chrysanthemu m. This pathogen causes leaf spots, resulting in 15-20% yield loss. The control of leaf spot pathogen by fungicides is difficult because fungicides are often ineffective as the pathogen spreads rapidly under favourab le conditions. On the other hand, major destructive fungi are developing resistance to most classes of fungicides and environ mental pollution caused by these chemicals is a serious problem [ 5,6]. Genetic resistance in D. grandiflora is therefore of considerable importance. Mutation techniques have been widely applied to improve crop yield, quality, disease and pest resistance [7]. About 2335 variet ies were released through mutagenesis in the world, in which ornamental crops and decorative crops are 552 [8]. In chrysanthemum mutant cultivars can account for almost 40% of the Dutch flower market and about 236 officially announced commercial mutants have been obtained through X-or γ-ray mutation breeding [9]. A large nu mber o f germp lasm with novel, desired traits have been produced through mutation techniques [10][11][12][13]. Co mb ining mutation technique with in v itro selection, allow the selection of traits controlled by their recessive or dominant genes, can improve recovery of the desired mutant. In vitro mutation and selection has advantages over traditional breeding methods as these are mo re efficient and cost effect ive. In order to screen target characters in a large nu mber o f mutagenised cells, it is essential to have efficient selection agents [7]. To xic cu lture filtrate and purified toxins have been used in in vitro selection of disease resistant plants [14][15][16]. It has been attempted at the plant level where a large population of plants, raised through in vitro callus cultures, have been screened for resistance in the field capacity or v ia a mo re targeted approach of regeneration of d isease resistant plants through resistant callus culture selected against fungal toxins [17]. Ku mar et al. [16] reported resistant cell lines of chrysanthemu m against Septoria obesa. In this paper the development chrysanthemum cv. Snow Ball with an improved resistance to Septoria obesa, is described.

Plant Materials
Chrysanthemum (Dendranthema grandiflora Tzelev) cv. Snow Ball, gro wing in the Depart ment of Biotechnology, University of Ho rticulture and Forestry, So lan, Himachal Pradesh, India was propagated from cuttings and maintained under greenhouse at 24±2℃ with 80% relative humidity.. Young leaves (upper 3-4 leaves)isolated from the one-yearold plants washed thoroughly under running water, cut into small seg ments (0.2-0.5 cm) and were used as explants. The explants were surface sterilized with 5% sodium hypochlorite and washed 3-4 times with sterile water prior to culturing. The callus cultures were initiated by culturing the leaf explants on MS [18] med iu m containing 3% sucrose (w/v), 10 mg/l kinetin and 1 mg/ l NAA. The p H of the mediu m was adjusted to 5.8 before autoclaving. The med iu m was solidified with 0.8% (w/v) agar and autoclaved at 121℃ for 15 min. Erlen meyer conical flasks (100 ml) were cast with 30 ml med ia and sealed with cotton plugs made fro m non-absorbent cotton. The cultures were incubated under cool fluorescent lamps at an light intensity of 50-60 µmo l m -2 s -1 with a light/dark cycle of 16/8 h at 24±2℃. All the cultures were transferred to the fresh medium at 30 days interval.

Determination of Opti mal Dose of Gamma Ray
To determine the suitable dose of gamma ray calli (50 mg fresh weight) were cu ltured on MS mediu m and exposed to gamma irradiat ion of 0, 10, 20 and 30 Gy (cobalt 60). About 50 calli pieces were tested for each dose. The surviving calli were subcultured onto fresh MS mediu m supplemented with growth regulators used for callus induction and growth. (After calli irrad iation, d id you allow to grow on the same med iu m or transferred to the fresh mediu m fo r further growth). The calli were observed for their growth after 30 days recovery period, after which surviving calli were counted, weighed and LD50 (half lethal dose) of gamma rays was estimated. (What is the parameter you used for the determination of LD50 dose) IAEA, Vienna has been carrying out work on induced mutations. You have not quoted.

Maintenance and Multi plicati on of the Pathogen Cultures
The pure cultures of S. obesa were obtained from the department of Mycology and Plant Pathology, Un iversity of Horticulture and Forestry, Solan (H.P.), India. The isolate was multip lied on PDA mediu m at 25℃ and maintained at 4℃ on the same mediu m. The pH of the PDA med iu m was adjusted to 6.0-6.5. After inoculation, the cultures were incubated at 25℃ in the dark for 21 days until a uniform fluffy mycelial growth was obtained.

Preparati on of Culture Filtrate
The culture filtrate was prepared by inoculating I mm 2 . of the fungal myceliu m in 100 ml liquid Czapek do x medium.The culture was incubated in a BOD incubator shaker at 25℃ in dark. After 21 days the fungal cultures were fil-tered through Whatman filter paper no. 1 and the filtrate centrifuged at 20, 000 rp m for 30 min. The supernatant was then filtered through sintered glass filter (G-5 grade, 0.25 µm pore size) to produce transparent culture filt rate.

Determination of Opti mal Concentrati on of Culture Filtrate for Screening
To determine the effects of culture filtrate on survival and growth of calli, one hundred pieces of mutated calli (20 mg fresh weight) per cu lture filt rate treatment, in ten flasks (100 ml), each having 10 p ieces, were inoculated on MS mediu m containing 0%, 2.5%, 5.0%, 7.5%, 10%, 12.5%, 15%, 17.5% and 20% (v/v) culture filt rate and the growth regulators used for callus induction and the mediu m was solid ified with 0.8% agar. In control cultures an equal volume of Czapek dox med iu m rep laced with culture filtrate. The cu ltures were maintained at light intensity of 50-60 µmo l m -2 s -1 under light/dark cycle of 16/8 h at 24±2℃ fo r four weeks. The calli obtained from the selection med iu m containing 15% cu lture filtrate, the highest concentration of culture filtrate at which only few calli survived, were subcultured on the same medium for two mo re cycles (30 day cycle) to select resistant calli. The surviving calli were cultured on MS med iu m supplemented with 10 mg/l kinetin and 1 mg/l NAA for mu ltiplication. The p H of the mediu m was adjusted to 5.8.

Plant Regeneration from Calli
After determin ing the optimal concentration of culture filtrate, the surviving calli were induced to regenerate plantlets. For shoot regeneration, calli were transferred onto MS mediu m supplemented with 0.2 mg/ l BA, 0.2 mg/l NAA and 1 mg/l GA 3 without culture filtrate. After shoot formation, they were separated from the callus mass and cultured on MS mediu m containing 0.3 mg/ l IBA and 0.2% act ivated charcoal for rooting. The p lantlets were maintained at 24±2℃ under 16/8 h (light/dark) photoperiod. The plants with well developed root systems were transferred to pots containing soil:sand (1:1) mixture and grown in the g reenhouse. The average number of shoots and roots produced were recorded to examine the effect of cu lture filtrate concentration on plant regeneration.

Leaf Spot Resistance of Selected Regenerated Plants
Hundred plants (one plant in each pot) were tested for resistance against leaf spot pathogen. The plants were subjected to in fection by spraying spore suspension (40 spores/ml) of S. obesa and were observed daily for the development of symptoms. Disease severity was recorded according to 0-4 rating scale [16]. Two hundred cuttings (5-6 cm long) were collected fro m one-year-o ld resistant plant and treated for 24 h with 100 mg/ l IBA solution for rooting. The rooted cuttings were transferred to pots containing soil: sand (1:1) and maintained in greenhouse for three months before spraying with spore suspension (40 spores/ml) of S. obesa. The plants were observed daily fo r the appearance of symptoms. The resistant plants were maintained for four years in the greenhouse and inoculated regularly by spraying spore suspension of the pathogen and observed for the development of sympto ms.

Statistical Analysis
The experiment was repeated three times with similar trends of results using completely randomized design [19]. The significance of treat ment effects on various parameters was determined using analysis of variance (ANOVA).

Results and Discussion
To determine the optimal dose of gamma rays (optimal dose not concentration), the calli were exposed to 10, 20 and 30 Gy gamma ray doses. The percentage of surviving calli decreased with the increasing gamma ray doses (Figure 1). There was no calli recovered at gamma ray dose 30 Gy, about 22.2% of the calli survived at 20 Gy dose of gamma irradiation. Therefore subsequent physical mutation was carried out only with 20 Gy of gamma irrad iation. To determine the effects of culture filtrate, survival of the calli decreased with the increase in the culture filtrate concentration, reaching a 0% survival at 17.5% culture filtrate ( Figure  2). A significant negative correlation was found between culture filtrate concentration and average survival of calli. The cell selection was therefore carried out at the 15% culture filtrate level to exert maximu m selection pressure that allo wed the recovery of resistant lines. The selected calli were subcultured on culture med iu m in the absence of culture filtrate for three subcultures to get rid of physiological adaptations and again cultured at 15% culture filtrate for t wo more cycles (30 day cycle) before the plants were regenerated.  The results presented in this paper describe the development of protocol for selecting resistance to leaf spot using in vitro mutation and selection. Earlier [16] it has been reported that about 70 to 80% of the plants raised from resistant cell lines showed resistance to S. obesa. Radiation mutation breeding with tissue culture technique have made a significant contribution to plant breeding. They have introduced new techniques for inducing genetic variat ion by improving selection technology and by accelerating breeding time. This method can produce large population of calli for in vitro mutation-selection, which increases the probability of obtaining mutants with the desired t raits. Liu et al. [7] reported that in vitro mutation-selection method with calli prove especially useful with genotypes for which it is difficult to obtain large number of emb ryos from microspore culture.
The present study revealed the production of chrysanthemu m plants with greater resistance to leaf spot disease than the lines produced by other methods [17,20,21]. The improvement in leaf spot disease resistance using this protocol over a period of four years was more than achieved earlier [16]. Sy mpto ms were observed in the control plants (Figure 3a). 100% resistance to leaf spot pathogen was observed in all the plants raised through cuttings fro m resistant cell lines under greenhouse (Figure 3b ). About 150 resistant plants were maintained in the greenhouse. No morphological variations were observed in the plants. Liu et al.[7] also observed improved resistance to Sclerotinia sclerotiorum in doubled-haploid Brassica napus.
The variations arising fro m in vitro mutation-selection may be the result of calli developed fro m a variety of diplo id and polyploids [22]. The develop ment of resistance in vitro in callus cultures against the phytotoxins has been correlated with the secretion of extacellular p roteins by the host in culture, which may be antifungal hydrolases or pathogene- sis-related proteins [23]. The callus tissue offers opportunities for a variety of selection, including tolerance to phytotoxins and may allow recovery of desired variants, related to recessive or dominant genetic changes. The present investigation describes the development of a protocol for in vitro mutations and selection of resistant plants of chrysanthemum to culture filt rate of Septoria obesa, a leaf spot pathogen.
The genetic stability of selected resistant plants is not shown in the subsequent breeding lines.