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Seema Singh et al., IJSID 2011, 1 (2), 247-254RESULTS AND DISCUSSIONThe multiple shoot form cotyledonary node and hypocotyl explants were observed after 21 daysin different combinations of hormone used. The percentage of responsive cotyledonary nodes andhypocotyls varied from 44 to85% and 28 to 61% respectively. A combination of growth regulatorscontaining BAP (1mg/L)+ Kn(1mg/L)+ IAA (1mg/L)(K) was found most suitable for multiple shootinduction in both explants. The maximum number of shoots per explant from cotyledonary node was15.40± 0.70 (Fig. 5A) and from hypocotyl explant (Fig 5B) was 5.40± 1.08 (Table-1). Our results indicatehighly significant (P

Seema Singh et al., IJSID 2011, 1 (2), 247-254Table 1. Effect of different combinations of hormones on multiple shoot regeneration from cotyledonary and hypocotylexplants in Brassica comprastis (l.) var. sawarnaMedia (MS media+phytohormone)Cotyledonary node% ofresponsiveexplants Shoots/explant Shoot length% ofresponsiveexplantsHypocotylShoots/explantShootlengthBAP (0.5 mg/l)- a 55 1.90 ± 0.57 3.57 ± 0.41 36 1.30 ± 0.48 2.12 ± 0.68BAP (1.0 mg/l)- b 64 2.00 ± 0.82 3.29 ± 0.71 28 1.40 ± 0.52 2.25 ± 0.79BAP (1.5 mg/l)- c 48 2.60 ± 0.70 2.34 ± 0.61 40 1.90 ± 0.74 2.42 ± 0.75Kn (0.5 mg/l)- d44 1.40 ± 0.52 2.65 ± 0.59 38 0.90 ± 0.57 1.08 ± 0.33Kn (1.0 mg/l)- e 56 2.10 ± 0.57 1.73 ± 0.37 41 1.70 ± 0.48 1.00 ± 0.36Kn (1.5 mg/l)- f 51 1.30 ± 0.48 1.50 ± 0.31 58 1.20 ± 0.42 1.14 ± 0.30BAP+Kn (0.5 mg/l + 1.0 mg/l)- g72 3.10 ± 0.88 3.41 ± 0.63 55 1.80 ± 0.79 2.40 ± 0.64BAP+Kn (1.0 mg/l + 1.0 mg/l)- h 74 4.40 ± 0.84 2.31 ± 0.66 57 2.00 ± 0.67 2.81 ± 0.76BAP+Kn (1.0 mg/l + 0.5 mg/l)- i 58 3.00 ± 0.82 2.95 ± 0.76 61 1.80 ± 0.79 2.10 ± 0.67BAP+Kn+NAA (1.0 mg/l+1.0mg/l + 1.0 mg/l)- j 68 2.70 ± 0.48 3.58 ± 0.87 51 1.40 ± 0.52 3.26 ± 0.56BAP+Kn+IAA (1.0 mg/l + 1.0mg/l + 1.0 mg/l)- k 85 15.40 ± 0.70 4.46 ± 0.85 61 5.50 ± 1.08 2.35 ± 0.85BAP+Kn+IAA (2.0 mg/l + 1.0mg/l + 1.0 mg/l)- l 80 5.80 ± 0.79 3.08 ± 0.52 56 3.90 ± 0.88 2.06 ± 0.54BAP+Kn+IAA (3.0 mg/l + 1.0mg/l + 1.0 mg/l)-m 74 5.40 ± 0.70 2.22 ± 0.43 48 5.40 ± 1.07 2.01 ± 0.68BAP+Kn+IAA (1.0 mg/l + 1.0mg/l + 2.0 mg/l)- n 63 3.90 ± 0.88 5.58 ± 0.89 49 3.10 ± 0.74 3.39 ± 0.77BAP+Kn+IAA (1.0 mg/l + 1.0mg/l + 3.0 mg/l)- o 73 3.30 ± 0.82 5.22 ± 0.84 43 1.60 ± 0.70 4.65 ± 0.73BAP+Kn+IAA (1.0 mg/l + 2.0mg/l + 1.0 mg/l)- p 69 5.30 ± 0.67 2.94 ± 0.59 50 3.00 ± 0.82 2.51 ± 0.60BAP+Kn+IAA (1.0 mg/l + 3.0mg/l + 1.0 mg/l)- q 81 2.70 ± 0.48 2.83 ± 0.55 49 2.70 ± 1.06 2.27 ± 0.58ANOVA F (DF=16,153) - 215.45 ** 29.44 ** - 34.11 ** 19.17 ****- p0.05**- p

Seema Singh et al., IJSID 2011, 1 (2), 247-254Fig. 1. Surface plot showing relative association of shoot length with % of responsive explants and shoots/explantin cotyledonary node.Fig. 2. Surface plot showing relative association of shoot length with % of responsive explants and shoots/explantin hypocotyl.International Journal of Science Innovations and Discoveries VOL1, Issue 2, September-October 2011251

Seema Singh et al., IJSID 2011, 1 (2), 247-254Fig.3. Cluster analysis showing tree diagram for 17 treatments on the basis of % of responsive explants, meanshoots/explant and mean shoot length of Cotyledonary node.Fig.4. Cluster analysis showing tree diagram for 17 treatments on the basis of % of responsive explants, meanshoots/explant and mean shoot length of Hypocotyl.International Journal of Science Innovations and Discoveries VOL1, Issue 2, September-October 2011252

Seema Singh et al., IJSID 2011, 1 (2), 247-254Fig.5. Multiple shoot initiation in Brassica campestris (L.): (A) Multiple shoots from cotyledonary node explant;(B) Multiple shoots from hypocotyls explant; (C) Rooting in in-vitro regenerated shoot.A highly significant and positive correlation (P

Seema Singh et al., IJSID 2011, 1 (2), 247-254and full strength media, supplemented with IBA (0.1mg/L) and NAA (1.0 mg/L), produced roots andconsequently the plantlets could be developed.CONCLUSIONIt is apparent from these results that MS media supplemented with BAP(1mg/L)+ Kn(1mg/L)+IAA(1mg/L) is more efficient for multiple shoot regeneration and it may be utilized in in-vitroimprovement program of Brassica campestris(L.) var. sawarna because in Brassica species organogenesisresponse are species and genotype specific 13 .REFERENCES1. Ali H, Ali Z, Ali HA, Mehmood S, Ali W (2007). In-vitro regeneration of Brassica napus L., cultivars (Star, Cyclone andWestar) from hypocotyls and cotyledonary leaves. Pakistan Journal of Botany 39(4), 1251-1256.2. Glemelius K., 1984. High growth rate and regeneration capacity of hypocotyl protoplast in some Brassicaceae, PhysiologyPlant 61:38-44.3. Lazzeri, P.A., Dunwell J.M., 1984 a. In-vitro shoot regeneration from seedling roots segments of Brassica oleracea var.Italica .Annuls. Botany 54, 351-361.4. Lazzeri, P.A., Dunwell J.M., 1984 b. Establishment of isolated root culture of Brassica species and regeneration fromcultivated roots segments of Brassica oleracea var Italica Annuls Botany 54, 351-361.5. George, L., Rao, P.S., 1980. In-vitro regeneration of mustard plant (Brassica juncea var.RAI-5) on cotyledon explants fromnon-irradiated, irradiated and mutagen treated seeds. Annuls Botany46,107-112.6. Chi GL, Pua E C, 1991. Role of ethylene on de-nova shoot regeneration from cotyledonary explants of Brassica campestrisspp. pekensis (Lour) oisson in –vitro. Plant Physiology 96,178-183.7. Dunwell, J.M., 1981. In-vitro regeneration from excised leaf disc of Brassica species. Journal of Experimental Botany32,789-799.8. Burbulis N, Blinstrubiene A, Kupriene R, Jonytiene V, Rugienius R, Staniene G, (2009). In- vitro regeneration of Brassicanapus L. shoots from hypocotyls and stem segments. Zemdirbyste- Agriculture 3, 176-1859. Murashige, T., Skoog. F., 1962. A revised medium for rapid growth and bioassay with Tobacco tissue cultures. PlantPhysiology 15, 473-497.10. Stewart, C.N., Adang, M.J., Ali, J.A., Wetal. 1996. Insect control and dosage effect in transgenic canola containing a syntheticBacillus thuringenensis cry IAC gene. Plant Physiology 112, 115-120.11. Kamal, G.B., Asadollah, A., 2007. Effect of genotype explant type and nutrient medium components on canola (Brassicanapus L.) shoot in –vitro organogenesis. African Journal of Biotechnology.67, 861-897.12. Koh, W.L., Loh, C.S., 2000. Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid cycling Brassicanapus. Plant Cell Report 19, 1177-1183.13. Dietert, M.F., Barron, S.A., Ocvodar, 1982. Effect of genotype on in-vitro culture in genus Brassica. Plant Science Letters26,233-240.International Journal of Science Innovations and Discoveries VOL1, Issue 2, September-October 2011254