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Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 01 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.701.332 Standardization of in vitro Culture Establishment and Proliferation of Micro-Shoots in African and French Marigold Genotypes K. Ravindra Kumar1*, Kanwar Pal Singh2, D.V.S. Raju3, Sapna Panwar2, Reeta Bhatia4, Surendra Kumar2 and Pavanesh Kumar Verma2 1 Dr.YSRHU, HRS-Kovvur, West Godavari Dist, Andhra Pradesh, India 2 ICAR-Indian Agricultural Research Institute, New Delhi, India 3 ICAR-Directorate of Floriculture Research, Pune, India 4 ICAR-IARI Regional Station, Katrain, Himachal Pradesh, India *Corresponding author ABSTRACT Keywords African marigold, French marigold, Nodal segment, Micropropagation, Culture establishment, Vitrification, Proliferation Article Info Accepted: 20 December 2017 Available Online: 10 January 2018 Marigold is native to Mexico and one of the commercial loose flower crops in India. In general it is commonly propagated through seeds, but some ornamentally high valued petaloid and gynomonoecious lines can only be maintained through vegetative propagation. Initial in vitro axenic culture establishment, poor multiplication rates, excess callusing and vitrified cultures are the major hindrances in its commercial micro-propagation. Therefore, the objective of the present investigation was to develop efficient in vitro protocol for mass multiplication of commercially popular African and French marigold cultivars Pusa Basanti Gainda (PBG) and Pusa Arpita (PA) respectively. Nodal segments were chosen as explant of these two open field cultivars. Explants were pre-treated with carbendazim (0.2%) + metalaxyl (0.2%) + 8-hydroxy quinoline citrate (200 mg/l) for 60 minutes followed by surface sterilization with 0.1% HgCl 2 for 4 minutes to eliminate the microbial contamination. Highest culture establishment (69.44%) and earliest bud emergence (4.45 days) was recorded in Murashige and Skoog (MS) medium supplemented with BAP (2.0 mg/l) and NAA (0.05 mg/l). Among the different proliferation treatments, 100% proliferation was recorded in MS medium devoid of any growth regulators, MS + 0.5 mg/l Kinetin + 0.1 mg/l NAA and 0.5 mg/l BAP + 0.1 mg/l NAA + 2.5 mg/l AgNO3 supplemented media. The maximum numbers of quality shoots (4.3, 18.8, 64.2 and 208.2 shoots/explant) were obtained on MS medium supplemented with 0.5 mg/l BAP + 0.1 mg/l NAA + 2.5 mg/l AgNO3 in 30, 60, 90 and 120 days after culture respectively. This protocol is highly useful for mass multiplication of true-to-type, disease free planting material as well as helpful in long term maintenance of germplasm lines. Introduction 2768 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 reproducible in nature. Marigold is a member of the Asteraceae family and popular for commercial loose flower cultivation. It is a native of Mexico and naturalised in India about 350 years ago. Marigold is one of the high valued ornamental crop in India on account of its easy cultivation, short duration, vast adaptability, wide spectrum of shape, size and good keeping quality. Among the floriculture crops, it is cultivated in an area of 56.04 thousand ha. with 501.87 thousand MT production and occupied first in area and production (Anonymous, 2015). Apart from loose flower cultivation, it is also widely grown for extraction pigments (lutein) added to poultry feed for intensification yellow colour of egg yolk (Hojnik et al., 2008). It is also endowed with other properties like insecticide (pyrethrins), antibiotic, nematicide and fungicides (thiophenes). Marigold is sexually propagated through seeds. But, seed propagation has limited application in some of the popular petaloid commercial varieties, due to poor seed set, low viability and genetic segregation of progeny. These varieties are being propagated asexually through herbaceous shoot-tip cuttings for commercial cultivation. Tejaswini et al., (2016) reported the vegetative propagation of marigold petaloid and gynomonoecious lines in different breeding programmes. However, vegetative multiplication is cumbersome, slow, season dependent and one of the prime causes for spread of diseases like phyllody which is caused by phytoplasma. Plant tissue culture has the potential for rapid multiplication of a large number of diseasefree, true-to-type quality plants in the shortest possible time and can be employed as an alternative tool. Earlier, few workers demonstrated techniques of multiplication of marigold through shoot tip and axillary bud proliferation (Misra and Datta 2000, Kumar et al., 2003, Gupta et al., 2013 and Majumder et al., 2014). However, these results were not Therefore, a study was conducted to develop an efficient and reproducible protocol for rapid in vitro propagation of commercially important African and French marigold cultivars. Materials and Methods The present experimentation was carried out at the Central Tissue Culture Laboratory, National Research Centre on Plant Biotechnology, New Delhi during 2014-2017. African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita were used for the study (Fig. 1a & b). In this research work, axillary shoots containing dormant buds were selected as explants. The explants were collected in early hours from the actively growing mother plants before the commencement of reproductive phase. The availability and quality of explants were observed to be low during flowering stage. Nodal segments of 2.0-2.5 cm length were excised and the leaf primordia removed with a sterile scalpel blade. Well prepared nodal segments were washed with Teepol® (0.1%) solution for 5 minutes followed by washing under running tap water for 10 minutes to remove the residue of the detergent. The explants were pre-treated with carbendazim (0.2%) + metalaxyl (0.2%) + 8-hydroxy quinoline citrate (200 mg/l) on a horizontal shaker (100 rpm) for 60 minutes followed by surface sterilization using HgCl2 (0.1%) for 4 minutes under laminar air-hood. The sterilised nodal segments were thoroughly washed with sterile double distilled water for 3 to 4 times to remove the chemical residues. The above treatments were used on the basis of initial experiments conducted by using different pretreatment and surface sterilisation combinations. The nodal segment was inoculated in each test tube (150 mm × 25 mm) with 15 ml of modified Murashige and 2769 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Skoog (MS) medium, supplemented with 3% sucrose, 0.8% agar and various concentrations of BAP (0 - 3.0 mg/l) with NAA (0.05 mg/l) for culture initiation. Thereafter, the microshoots were excised from aseptic cultures and subculture at 30 days interval on proliferation media containing BAP (0 (T0), 0.5 (T1), 1.0 (T2), 1.5 (T3), 2.0 (T4) and 3.0 (T5) mg/l), kinetin (0.5 (T6) and 1.0 (T7) mg/l) individually and in combination (0.5 + 0.5 (T8), 1.0 + 0.5 (T9) mg/l) with NAA (0.1 mg/l). On the basis of initial experiment results silver nitrate (2.5 mg/l) was tested with 0.5 mg/l BAP and 0.1 mg/l NAA as one of the proliferation treatment (T10). As AgNO3 is a thermolabile compound it was added to autoclaved medium after filter sterilisation with 0.22 µM filters. To test the efficiency of different proliferation media and to determine the rate of proliferation the experiment was continued up to 120 days. The cultures were maintained at 24 ± 2°C under fluorescent white light (47 mol/m2/s) at a photoperiod of 16/8 hours light and dark cycles. All cultures were examined periodically and observations on any morphological changes were recorded. Twenty-five explants were inoculated per treatment and each treatment was replicated thrice and the reported data are mean of three replications. The data was statistically analysed employing completely randomised design. The percentage data were subjected to angular transformation before analysis. Results and Discussion Pre-treatments Aseptic culture establishment is first and foremost step for the successful development of micro-propagation protocol on a commercial scale. In this study, various fungicides and bactericides were tried in different combinations and durations to eliminate the microbial contamination from the nodal explants. Among the different fungicidal treatments tried, explants agitation in carbendazim (0.2%) + metalaxyl (0.2%) + 8-hydroxy quinoline citrate (200 mg/l) for 60 minutes gave significantly higher survival (66.67%) over other treatments (Table 1). In comparison between the two genotypes, percent survival was significantly highest in Pusa Arpita (32.06%) over Pusa Basanti Gainda (27.14%). The two-way interaction between the pre-treatment and genotype was found to be non-significant. Under our experimental conditions, significantly lowest contamination (26.67%) was observed in explant treated with carbendazim (0.2%) + metalaxyl (0.2%) + 8-hydroxy quinoline citrate (200 mg/l) for 90 minutes, which was followed by 60 minutes duration (30.00) of treatment. However, the survival percentage (8.89%) was significantly low when explants were treated for 90 minutes. This might be due to the toxic effect of chemicals under prolonged duration of treatment (Table 1). All pre-treatments gave significantly better response compared to control, where 98.33 percent contamination was noted. Microbes such as bacteria and fungi were responsible for culture contamination and can completely spoil the cultures. Among the different pretreatments, highest explant toxicity (64.44%) was recorded with highest fungicide dosage and prolonged (90 min) treatment duration (Table 1). These findings are in close confirmation with earlier results reported by Singh et al., (2011) in grape, Verma et al., (2012) in chrysanthemum and Sen et al., (2013) in Achyranthes aspera L. Most of these findings proved the usefulness of carbendazim (0.1 - 3.0%) and metalaxyl (0.1 - 3.0%) as effective fungicides. Fungicide dosage and treatment duration depend on the type and tenderness of explant. But higher concentrations of these disinfectants and prolonged durations of treatment became toxic 2770 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 and were responsible for poor growth and low establishment of cultures particularly in herbaceous crops. Surface sterilization Standardisation of surface sterilisation treatment followed by efficient pre-treatment is a vital process for axenic culture establishment. It is clear from the Table 2 that significantly higher survival (73.3%) was recorded when the explants were pre-treated with carbendazim (0.2%) + metalaxyl (0.2%) + 8hydroxy quinoline citrate (200 mg/l) for 60 minutes followed by 4 minutes HgCl2 (0.1%) treatment over all other treatments. It was also observed that explants were killed when treatment duration was increased beyond 4 minutes in HgCl2 (0.1%). This might be due to the toxic effect of surface sterilant on explants (Table 2). It was clearly evident from the data, NaOCl (4%) treatment for 15 and 20 minutes was less efficient than HgCl2 (0.1%) for 4 minutes in controlling the microbial contamination. Among the two genotypes, per cent survival was significantly highest in Pusa Arpita (41.70%) over Pusa Basanti Gainda (37.20%). The two-way interaction between the surface sterilant and genotype was found to be nonsignificant. Our research finding revealed that explants treated with HgCl2 (0.1%) for short duration (< 3 minutes) failed to kill the microbes effectively, whereas longer durations (5 to 8 minutes) resulted in complete or partial tissue killing in both the species of marigold. Treating the explants with HgCl2 (0.1%) for 4 minutes resulted in higher survival of explants with low contamination (24.4%). Our results are in tantamount to Singh et al., (2011) in grape and Verma et al., (2012) in chrysanthemum. But these results are in contrary with Majumder et al., (2014), where they reported only 2 minutes treatment with HgCl2 (0.1%) resulted in highest culture establishment in Pusa Narangi Gainda and the variation might be due to change in the genotype. Culture initiation Different BAP concentrations (0, 0.5, 1.0, 2.0 and 3.0 mg/l) were tried along with NAA (0.05 mg/l) for culture establishment (Table 3). Under our experimental conditions, among the different growth regulators tested, the highest culture establishment (69.44%) was noted with 2.0 mg/l BAP + 0.05 mg/l NAA, followed by 1.0 mg/l BAP + 0.05 mg/l NAA (56.11%), which were significantly different (Fig 2 a & b). The culture establishment was higher in the genotype Pusa Arpita (49.33%) followed by Pusa Basanti Gainda (46.89%) both are at par with each other. The interaction between treatment and genotype was also insignificant. Early (4.45 days) bud sprouting was observed on MS medium supplemented with 2.0 mg/l BAP + 0.05 mg/l NAA, followed by 3.0 mg/l BAP + 0.05 mg/l NAA (4.82 days), which were statistically significant with each other. Explants cultured on MS medium devoid of any growth regulators took longer duration (11.55 days) for axillary bud sprouting. Among the genotypes, significantly earlier axillary bud sprouting (6.77 days) was recorded in Pusa Arpita compared to Pusa Basanti Gainda (7.55 days). The interaction between growth regulator and genotype was also found significant. Duration for bud sprouting was the earlier in Pusa Arpita (4.07 days) than Pusa Basanti Gainda (4.83 days) when they were cultured on MS medium supplemented with 2.0 mg/l BAP + 0.05 mg/l NAA treatment (Table 3). 2771 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Table.1 Effect of different pre-treatments in the sterilization of nodal explants in African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita (PA) Treatment Treatment details Duration (minutes) Survival (%) PBG PA Mean Contamination (%) PBG PA Mean Toxicity (%) PBG PA Mean T0 Control (Distilled water shake) 60 1.11 (3.50)* 2.22 (7.01)* 1.67 98.89 (85.25)* 97.78 (82.35)* 98.33 0.00 (0.00)* 0.00 (0.00)* 0.00 T1 Carbendazim (0.1%) + Metalaxyl (0.1%) + 8-HQC (200 mg/l) 30 27.78 (31.79) 31.11 (33.88) 29.44 71.11 (57.49) 66.67 (54.73) 68.89 1.11 (3.50) 2.22 (7.01) 1.67 T2 Carbendazim (0.1%) + Metalaxyl (0.1%) + 8-HQC (200 mg/l) 60 37.78 (37.88) 46.67 (43.06) 42.22 60.00 (50.77) 50.00 (44.99) 55.00 2.22 (7.01) 3.33 (8.49) 2.78 T3 Carbendazim (0.1%) + Metalaxyl (0.1%) + 8-HQC (200 mg/l) 90 15.56 (23.02) 22.22 (28.01) 18.89 41.11 (39.82) 30.00 (33.18) 35.56 43.33 (41.09) 47.78 (43.69) 45.56 T4 Carbendazim (0.2%) + Metalaxyl (0.2%) + 8-HQC (200 mg/l) 30 38.89 (38.55) 40.00 (39.20) 39.44 57.78 (49.48) 56.67 (48.82) 57.22 3.33 (8.49) 3.33 (8.49) 3.33 T5 Carbendazim (0.2%) + Metalaxyl (0.2%) + 8-HQC (200 mg/l) 60 61.11 (51.44) 72.22 (58.33) 66.67 34.44 (35.89) 25.56 (30.28) 30.00 4.44 (11.99) 2.22 (14.96) 3.33 T6 Carbendazim (0.2%) + Metalaxyl (0.2%) + 8-HQC (200 mg/l) 90 7.78 (15.63) 10.00 (18.00) 8.89 31.11 (33.84) 22.22 (28.01) 26.67 61.11 (51.43) 67.78 (55.55) 64.44 27.14 32.06 56.35 49.84 16.51 18.10 CD (p<0.05) SEm± CD (p<0.05) SEm± CD (p<0.05) SEm± Treatments 4.88 1.69 6.37 2.20 6.37 2.20 Genotype 2.61 0.9012 NS 1.18 NS 1.18 T×G NS 2.38 NS 3.11 NS 3.11 Mean *Figures given in parentheses are angular transformed values 2772 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Table.2 Effect of different surface sterilisation treatments of nodal explants in African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita (PA) Treatment Treatment details Survival (%) T0 Control (Distilled water shake) T1 0.1 % HgCl2 for 3 min. PBG 2.2 (7.0)* 60.0 (50.8) T2 0.1 % HgCl2 for 4 min. 71.1 (57.6) T3 0.1 % HgCl2 for 5 min. 52.2 (46.3) T4 0.1 % HgCl2 for 6 min. 23.3 (28.8) T5 0.1 % HgCl2 for 7 min. T6 0.1 % HgCl2 for 8 min. T7 4.0 % NaOCl for 15 min. 8.9 (17.1) 3.3 (8.5) 51.1 (45.6) T8 4.0 % NaOCl for 20 min. 62.2 (52.1) Mean Treatments Genotype T×G 37.2 CD (p<0.05) 5.33 2.51 NS PA 3.3 (8.5)* 68.9 (56.1) 75.6 (60.4) 58.9 (50.1) 27.8 (31.8) 17.8 (24.9) 5.6 (13.1) 52.2 (46.3) 65.6 (54.1) 41.7 SEm± 1.86 0.90 2.60 Mean Contamination (%) 2.8 PBG 97.8 (82.8)* 64.4 40.0 (39.2) 73.3 27.8 (31.7) 55.6 22.2 (28.0) 25.6 17.8 (24.9) 13.3 14.4 (22.3) 4.4 5.6 (13.5) 51.7 46.7 (43.0) 63.9 32.2 (34.6) 33.8 CD (p<0.05) 4.87 2.29 NS *Figures given in parentheses are angular transformed values 2773 PA 96.7 (81.3)* 31.1 (33.9) 21.1 (27.3) 17.8 (24.9) 17.8 (24.8) 3.3 (8.5) 1.1 (3.5) 44.4 (41.8) 27.8 (31.8) 29.0 SEm± 1.69 0.80 2.4 Mean Toxicity (%) 97.2 PBG 0.0 (0.00)* 35.6 0.0 (0.00) 24.4 20.0 1.1 (3.5) 25.6 (30.1) 17.8 58.9 (50.1) 8.9 76.7 (61.1) 3.3 91.1 (73.2) 45.6 2.2 (7.0) 5.6 (13.1) 30.0 29.0 CD (p<0.05) 5.58 NS NS PA 0.0 (0.00)* 0.0 (0.00) 3.3 (8.5) 23.3 (28.6) 54.4 (47.5) 78.9 (62.7) 93.3 (75.8) 3.3 (8.5) 6.7 (14.6) 29.3 SEm± 1.95 0.90 2.80 Mean 0.0 0.0 2.2 24.4 56.7 77.8 92.2 2.8 6.1 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Table.3 Effect of BAP and NAA on in vitro culture establishment (%), days to bud sprouting, no. of shoots per explants, avg. shoot length (cm)and callusing after 25 days after culture initiation in African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita (PA) Treat ment Growth regulators (mg/l) Culture establishment (%) NA A 0.00 PBG PA T0 BA P 0.0 T1 0.5 0.05 T2 1.0 0.05 T3 2.0 0.05 T4 3.0 0.05 30.00 (33.18 )* 42.22 (40.46 ) 52.22 (46.27 ) 72.22 (58.30 ) 37.78 (37.90 ) 46.89 CD (p<0.0 5) 4.28 NS NS 27.78 (31.73 )* 46.67 (43.06 ) 60.00 (50.75 ) 66.67 (54.78 ) 45.56 (42.42 ) 49.33 SEm± Mean Treatments Genotype TXG 1.441 0.911 2.038 Mean Days to bud sprouting PBG PA 28.89 12.13 10.97 44.44 8.30 56.11 Mean Shoots per explant PBG PA 11.55 1.00 1.00 7.30 7.80 1.00 7.56 6.80 7.18 69.44 4.83 4.07 41.67 4.90 4.73 7.55 CD (p<0.05 ) 0.857 0.542 NS 6.77 SEm ± 0.29 0.18 0.41 Mean Av. shoot length (cm) PBG PA 1.00 0.73 0.67 1.00 1.00 1.13 1.55 1.30 1.43 4.45 1.92 1.83 4.82 1.63 1.54 1.42 CD (p<0.05 ) 0.12 0.077 NS 1.33 SEm± *Figures given in parentheses are angular transformed values 2774 0.04 0.026 0.058 Mean Establishment Index Mean PBG PA 0.70 30.0 27.70 28.8 0.88 1.01 42.2 46.60 44.4 1.47 1.40 1.43 80.8 77.60 79.2 1.88 2.17 2.00 2.08 138.0 122.3 130.2 1.58 1.63 1.57 1.60 62.0 69.90 66.0 1.43 CD (p<0.0 5) 0.22 NS NS 1.30 SE m± 70.6 CD (p<0.05 ) 10.54 NS NS 68.88 SEm± 0.08 0.05 0.11 3.57 2.25 5.05 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Table.4 Effect of BAP, Kinetin, NAA and AgNO3 on micro-shoot proliferation in African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita (PA) Treat ment Proliferation (%) Treatment details (mg/l) Average shoot length Mean Mean PBG PA PBG PA BAP Kinetin NAA AgNO3 T0 0 0 0 0 100.0 100.0 100.0 3.7 4.5 4.1 T1 0.5 0 0.1 0 100.0 96.7 98.3 2.3 2.0 2.2 T2 1.0 0 0.1 0 96.7 83.3 90.0 0.6 0.9 0.7 T3 1.5 0 0.1 0 70.0 56.7 63.3 0.0 0.0 0.0 T4 2.0 0 0.1 0 36.7 30.0 33.3 0.0 0.0 0.0 T5 3.0 0 0.1 0 23.3 16.7 20.0 0.0 0.0 0.0 T6 0 0.5 0.1 0 100.0 100.0 100.0 1.5 2.0 1.7 T7 0 1.0 0.1 0 63.3 56.7 60.0 0.8 1.0 0.9 T8 0.5 0.5 0.1 0 43.3 33.3 38.3 0.4 0.8 0.6 T9 1.0 0.5 0.1 0 23.3 13.3 18.3 0.0 0.0 0.0 T10 0.5 0 0.1 2.5 100.0 100.0 100.0 2.8 2.6 2.7 68.8 62.4 1.1 1.2 CD (p<0.05) SEm± CD (p<0.05) SEm± Treatments 5.870 2.060 0.508 0.178 Genotype 2.500 0.878 NS 0.076 NS 2.910 NS 0.251 Mean T XG *Figures given in parentheses are angular transformed values 2775 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 Table. 5 Effect of BAP, Kinetin, NAA and AgNO3 on number of micro-shoots per explant after 30, 60, 90 and 120 days of proliferation in African marigold cv. Pusa Basanti Gainda (PBG) and French marigold cv. Pusa Arpita (PA) Treat ment Treatment details (mg/l) BAP No. of shoots after 30 days NAA AgNO PBG PA 0 0 1.7 2.7 Mean No. of shoots after 60 days PBG PA 2.2 4.0 4.7 Mean No. of shoots Mean No. of shoots Mean after 90 days after 120 days T0 0 Kineti n 0 PBG PA 4.3 18.7 15.0 T1 0.5 0 0.1 0 2.7 3.3 3.0 5.7 11.7 8.7 23.3 30.0 T2 T3 T4 T5 T6 1.0 1.5 2.0 3.0 0 0 0 0 0 0.5 0.1 0.1 0.1 0.1 0.1 0 0 0 0 0 2.0 1.7 1.0 1.0 1.7 1.7 1.0 1.0 1.0 2.3 1.8 1.3 1.0 1.0 2.0 6.7 0.0 0.0 0.0 4.7 5.3 0.0 0.0 0.0 7.0 6.0 0.0 0.0 0.0 5.8 18.3 0.0 0.0 0.0 17.7 0.0 0.0 0.0 0.0 20.7 T7 0 1.0 0.1 0 2.3 2.7 2.5 6.0 7.7 6.8 21.0 20.7 T8 T9 T10 0.5 1.0 0.5 0.5 0.5 0 0.1 0.1 0.1 0 0 2.5 1.0 1.0 4.3 2.3 1.0 4.3 1.7 1.0 4.3 1.7 0.0 18.3 3.3 0.0 19.3 2.5 0.0 18.8 3.0 0.0 68.7 0.0 0.0 59.7 Mean 1.8 CD (p<0.05) 2.1 SEm± 4.3 CD (p<0.05) 5.4 SEm± 13.3 SEm ± Treatments Genotype T XG 0.57 0.24 0.81 0.20 0.09 0.28 2.04 0.87 NS 0.72 0.31 1.01 15.5 CD (p<0.05 ) 4.45 1.90 6.30 PBG PA 61.7 36.3 49.0 94.7 70.0 82.3 56.0 0.0 0.0 0.0 67.3 0.0 0.0 0.0 0.0 55.0 28.0 0.0 0.0 0.0 61.2 62.7 50.0 56.3 9.0 0.0 237.7 0.0 0.0 178. 7 35.5 SEm ± 4.5 0.0 208.2 3 2776 1.60 0.67 2.21 16. 8 26. 7 9.2 0.0 0.0 0.0 19. 2 20. 8 1.5 0.0 64. 2 53.5 CD (p<0.05 ) 13.50 5.77 19.10 4.70 2.02 6.70 Int.J.Curr.Microbiol.App.Sci (2018) 7(1): 2768-2781 2777