In Vitro Selection and Biochemical Characterization of Carnation (Diathus Caryophyllus L.) Callus Culture Tolerant to Alternaria Dianthi
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Indian J. Plant Physiol., Vol. 12, No. 2, (N.S.) pp. 120-126 (April-June, 2007) IN VITRO SELECTION AND BIOCHEMICAL CHARACTERIZATION OF CARNATION (DIATHUS CARYOPHYLLUS L.) CALLUS CULTURE TOLERANT TO ALTERNARIA DIANTHI RUPALI MEHTA, SARITA SHARMA AND AMARJIT K. NATH* Department of Biotechnology, Dr. Y.S. Parmar University of Horticulture & Forestry, Nauni Solan, Himachal Pradesh Received on 16 April, 2007, Revised on 8 June, 2007 SUMMARY For the establishment of callus cultures of carnation (Dianthus caryophyllus L.), internodal segments were used as explants. Maximum percentage of callus induction was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l naphthalene acetic acid (NAA) and 2.0 mg/l kinetin. Whereas, maximum shoot regeneration was obtained on medium supplemented with 2.0 mg/l 6-benzyladenine (BA) and 0.5 mg/l NAA. Well developed root system was obtained on liquid MS medium supplemented with 2.0 mg/l indole–3-butyric acid (IBA) and 0.2 per cent activated charcoal. Calli were subjected to different concentrations of culture filtrate of Alternaria dianthi and cell survival at 15 per cent selective dose of culture filtrate was 11.67 per cent. Selected calli showed significantly higher levels of biochemical constituents viz., total sugars, phenols, reducing sugars and proteins as compared to non-selected calli. Banding patterns of isozymes esterase, peroxidase and polyphenol oxidase were different in selected and non selected calli. In vitro shoot regeneration from selected calli was obtained on MS medium supplemented with 2.0 mg/l thidiazuron (TDZ). Key words: Biochemical constituents, callus culture, carnation, culture filtrate, isozyme, organogenesis INTRODUCTION of carnation, which initially produces purple small circular and later light brown to black spots on carnation leaves, Carnation (Dianthus caryophyllus L.) is one of the stem, petioles including flowers giving blightened most important cut flower of the world and ranks next appearance (Fig. 2A). In Solan district, A.dianthi causes to rose (Laurie et al. 1968). In India, the specific 28 % loss in the yield and quality of flowers (Chandel climatic requirements of the crop are met in Himachal and Bhardwaj 2001). Fungicides, herbicides and Pradesh, West Bengal, Kashmir and Karnataka in one pesticides used commercially for the management of the season or other. With increase in its cultivation, the diseases are expensive and are causing environmental limiting factors for the production of quality flowers are hazards. In vitro culture is the promising tool for the also increasing simultaneously. Carnation is attacked by selection of resistant mutants. The use of resistant a number of pathogens viz. fungi, bacteria, viruses and cultivars may help to reduce the incidence of the diseases among the various pathogens attacking this crop, F. or pathogens attack. A selection criterion used widely oxysporum, F. roseum and Alternaria dianthi are till date is the growth of the callus in the presence of known to cause heavy losses in yield and quality of culture filtrate or toxin of pathogen (Sacristan 1982) flowers. A. dianthi is known to cause leaf blight disease * Corresponding author 120 Indian J. Plant Physiol., Vol. 12, No. 2, (N.S.) pp. 120-126 (April-June, 2007) IN VITRO CELL SELECTION IN CARNATION MATERIAL AND METHODS preparation for the selection of calli resistant to A. dianthi. The present investigations were carried out in the Department of Biotechnology, Dr. Y S Parmar, UHF, The small pieces of macerated callus (approx 1-3 Nauni, Solan with the objective to select the cell lines mm) were cultured on selective medium prepared by resistant to A. dianthi in carnation cv. Tempo using mixing pure culture filtrate of A. dianthi with sterilized internodal segments of size 0.2 to 0.5 cm from stem molten MS medium (v/v) to obtain the concentrations of cutting as explants. These explants were thoroughly 5, 7.5, 10, 12.5, 15, 17.5, 20, 25 % respectively and washed under running tap water and surface sterilized monitored for the growth of callus. A control containing with 0.1 % mercuric chloride solution for 2 min and were MS medium without culture filtrate was also prepared. washed with sterilized distilled water 2 to 3 times to Calli showing resistance to culture filtrate was isolated remove the traces of mercuric chloride. MS salts (macro and sub-cultured on fresh MS medium devoid of culture and micro), vitamins supplemented with 100 mg/l, filtrate for 4 weeks to check the stability for resistance. mesoinositol, 3 % sucrose and 0.8 % agar-agar were Selected calli showing resistance to culture filtrate was used as basal medium (Murashige and Skoog 1962). multiplied and used for biochemical characterization. Surface sterilized internodal segments were cultured Biochemical characterization of selected and non on MS medium containing various concentrations and selected calli: For biochemical characterization, ethanol combinations of plant growth regulators viz., NAA (1.0- extraction was carried out by boiling 1 gm of selected 2.0 mg/l), kinetin (1.0-2.0 mg/l) and 2, 4-D (0.5-2.0 mg/ and non selected callus in 80 % ethanol for 10 min, at l) and incubated under 16-h photoperiod at 26+2 oC for room temperature. Supernatant was evaporated; residue the induction and establishment of callus. After the was dissolved in 20 % ethanol and centrifuged. The clear establishment of callus culture, fresh green callus was supernatant was used for estimations. Total soluble cut off into small pieces and sub-cultured on the same sugars were estimated according to the method described nutrient medium for further multiplication. After sufficient by Dubios et al. (1956). Reducing sugars were estimated multiplication of callus, it was transferred to shoot according to Nelson (1944). Total phenols were regeneration medium. In vitro regenerated shoots were estimated according to method described by Bray and transferred to rooting medium supplemented with Thorpe (1954) and total soluble proteins were estimated activated charcoal and different concentrations of IAA after trichloro acetic acid (TCA) precipitation as and IBA. In vitro raised plantlets were hardened and described by Lowry et al. (1951) further maintained in glass- house. For isozyme studies, three enzymes i.e. esterase (EC In vitro selection and biochemical characterization: 3.1.1.2), peroxidase (EC 1.11.1.7) and polyphenol Pure culture of A. dianthi was isolated from infected oxidase (EC 1.10.3.2) were investigated. For extraction leaves of carnation plants, maintained and multiplied on of enzyme, 1 g of selected and non selected calli were Potato Dextrose Agar (PDA) medium (Fig. 2B). To crushed in pre-chilled pestle and mortar using extraction carry out pathogenicity test, mycelial suspension of buffer as described by Parafitt et al. (1986) .The fungus was applied on leaves of carnation plants for homogenate was spun at 10,000 rpm for 20 min at 4 0C development of disease symptoms under high humid to remove cellular debris. The clear supernatant was conditions in glass- house. For preparation of culture used for separation of enzyme by polyacrylamide gel filtrate, small bits (5 mm) of mycelium were inoculated electrophoresis at 4 0C using bromo phenol blue (0.2 %) in liquid Richard’s medium, incubated at 25 oC for 15- as a tracking dye. Gel buffer and tray buffer were 20 days. The crude culture filtrate was purified following prepared as per the method described by Scandalios filtration through ordinary filter paper, Whatman filter (1969). The electrophoresis was carried out at constant paper, sintered glass filter (G-S grade) and finally the current of 50 mA till the tracking dye reached the end pure culture filtrate was used for selective medium of slab gel and gels were removed. Esterase and Indian J. Plant Physiol., Vol. 12, No. 2, (N.S.) pp. 120-126 (April-June, 2007) 121 RUPALI MEHTA et al. polyphenol oxidase were stained as described by Kuhns AB and Fretz (1978). Polyphenol oxidase isozymes were stained as described by Kaushal Kanika (1997), using catechol and sulphanilic acid as staining reagents. Locations of isozymes were specified by relative mobility (Rm) values. In vitro plant regeneration from selected calli: Selected calli were sub-cultured on different CD concentrations and combinations of BAP, IAA and TDZ. Shoot regeneration from callus was obtained only on MS medium supplemented with 2.0 mg/l TDZ (Fig. 2D). Statistical analysis: The data recorded for different parameters was subjected to completely randomized design (Cochran and Cox 1963). For biochemical constituents, experiments were done in three replicates EF with duplicates. RESULTS AND DISCUSSION Callus initiation on cultured internodal segments started within 7-10 days from cut ends of the explants. All the media showed some degree of callus initiation. Maximum callus induction (92 %) was obtained in MS Fig. 1. (A-F). In vitro plant regeneration in carnation (Dianthus medium supplemented with 2.0 mg/l NAA and 2.0 mg/ caryophyllus L. cv. Tempo): A) Callus induction from l kinetin (Fig. 1A). For callus multiplication, callus was internodal segment explants, B) Callus multiplication, C) In vitro shoot regeneration from callus on shoot regeneration cut into small pieces and sub cultured on fresh MS medium, D) In vitro shoot multiplication, E) Root regeneration medium .The callus multiplication was also obtained on in in vitro developed shoot and formation of complete callus induction medium (Fig. 1B). Callus induced on MS plantlets, F) Hardening of in vitro raised carnation plants medium supplemented with 2, 4-D alone or in combination with NAA was friable, light green and less For shoot regeneration, calli pieces (0.5 cm approx. in amount as obtained on best callus induction medium. in dia) were cultured on MS medium supplemented with Garg (1995) also reported callus formation from leaf 9 different combinations of BAP and NAA.