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Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3336-3339 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 06 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.706.390 Regeneration of Haploids in Oil Seed through Embryonic Microspore Shahina A. Nagoo1*, GulZaffar2, Sabina Naseer4, N.A. Dar3, Sabia Bashir4, M. Altaf Wani4 and Shabina Majid4 1 Division of GPB FoAWadura, SKUAST-K, India 2 DARS-Budgam SKUAST-K, India 3 Saffron Research Station, SKUAST-K, India 4 SKUAST-K, India *Corresponding author ABSTRACT Keywords Haploids, Brassicarapa, Shoot/ Root regeneration Article Info Accepted: 22 May 2018 Available Online: 10 June 2018 Anther Culture or microspore culture is a very important and useful tool in plant breeding for efficient production of haploids and subsequent doubled haploid plants in many species, limited research has been conducted with Brassica –rapaas it is more recalcitrant in cell and tissue culture than other Brassicas. In order to access the effect of genotype on microspore embryogenesis in B –rapa, two high yielding brown Sarsoon cultivars (KS-101 and KOS-1) were studied for their response to haploid plant regeneration through Anther culture. Flower buds of 2.2 mm to 2.7 mm size representing late uninucleate stage of anther microspores were subjected to low temperature treatment 4 0C for 7days and anthers from these buds were cultured on MS medium having 13% sucrose. Anthers were then sub cultured in MS media supplemented with various concentrations of auxins and cytokinins. The best callus development and proliferation was achieved in MS media supplemented with 2,4-D 1mg/l and 0.5 mg/l NAA. Sucrose concentration, cold pre treatment and incubation time influenced embryogenesis. For regeneration of haploid plantlets anther derived calli were transferred to MS full strength medium supplemented with kn92.0mg/l) fallowed by the use of BA (2.0mg/l) and incubated at 22 + 20C in light and gave maximum regeneration of 75%. The regeneration shoots (at three leaf stage with length of 5cm) were subculture in rooting media and maximum rooting (30.07%) was achieved when MS medium was supplemented with IBA (0.4mg/l). Root tip mitosis chromosome counts revealed percentage of haploid frequency of KOS-1(41.85) followed by KS101(38.84%).After 5 days of rooting root samples were taken for confirmation of haploid nature of the plant, Haploid frequency was greater than 65%. Introduction Brown sarson is the major oilseed crop of valley grown on about 50 thousand hacters and fits in rotation with Rice crop. No significant breakthrough in enhancing the existing productivity levels (7-8 q/ha) and oil quality has been possible due to lack of variability in the available germplasm (Singh et al 2007). Operation of self incompatibility 3336 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3336-3339 limits production of homozygous inbred lines through conventional breeding procedures, necessitating biotechnological interventions for accelerating breeding progress and generating genetic variability useful for developing high yielding varieties.The efficient production of haploid and double haploid (DH) plants from anther or microspore culture has become an important new tool for Brassicabreeders. But limited research has been conducted with Brown Sarson (Brassicarapa) as it is more recalcitrant in cell and tissue culture than other Brassicas. The present investigation was undertaken to develop a protocol for production of DH Plants in B.rapa from anderogenic embryos. Materials and Methods Flowered buds 2.5 to 2.7 mm long representing late uninucleate stage were collected from field grown plants of 2Brassica compestris varieties namely KOS-1 and KS101, subjected to low temperature pretreatment and surface sterilized with 1% sodium hypochloride for 8 to 10 minuts (Gu et al 2004). Anthers were picked from buds and cultured on MS medium suplimented with various concentrations of 2,4-D and NAA for embryogenic callus induction.For regenerations from embryogenic callus MS media suplimented with various concentrations of auxins and cytokinens were used. Root induction in regenerated shoots was tried using various concentrations of IBA. The cultures were incubated at 22+- 20 C. Root tip mitotic chromosome counts were used to confirm haploid nature of Anther derived regenerates and double haploidy level induced through use of various concentrations of colchicines was detected cytologically using pollen mother cell (PMC). Results and Discussion Maximum percentage of aseptic culture was achieved in KS-101followed by KOS1genotypes.Callus initiation was maximum (32.62) when anthers were given pretreatment (chilling treatment) temperature of 40C for seven days (Zhang et al 2006,Kumari and Singh 2014). Maximum percentage of callus initiation (22.48) was observed in KS-101 genotype followed by KOS-1(Gu etal 2003)[Table 1]. Maximum number of anthers per 100 buds forming callus was observed in Murashige and Skoog medium 1962, supplemented with 2,4-D (1.0 mg/lit) and NAA(0.5 mg/lit) (36.60). Days taken to initiate callusing was minimum (22.48 days) when induction medium (MS medium) was supplemented with BA (1.5 mg/lit) and 2,4-D (1.0 mg/lit) (Li etal., 2005). Table.1 Effect of pre treatment of anthers on callus initiation Genotype T1 T2 T3 T4 T5 T6 Mean KOS-I 23.86 (29.22) 21.88 (27.87) 22.88 (28.55) 30.25 (33.35) 23.02 (28.65) 21.01 (27.26) 23.81 (29.15) KS-IOI 22.03 (27.97) 20.98 (27.24) 21.98 (27.94) 45.78 (42.57) 23.34 (28.88) 19.23 (25.99) 25.55 (30.10) 3337 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3336-3339 Table.2 Influence of growth regulators on number of plants regenerated Growth regulatormg/l BA (0.0) BA(2.0) BA(3.0) Kn(0.0) Kn(2.0) Kn(3.0) Mean Main effect Genotype (GT) = Temperature (T) = Interaction effect GT x T LSD < (p = 0.01) 1.44 1.10 = 3.12 KS-101 32.00 69.00 48.00 44.00 86.00 44.00 53.83 T1=10C T2 = 2Oc T3 =30c S.E 0.60 0.49 T4 = 1.20 KOS-1 48.00 63.00 46.00 42.00 72.00 51.00 53.66 0 4 c T4= 5 0 c T6 = 6 0 Table.3 Influence of growth regulators on per cent in vitro rooting of micro shoots Growth Regulators IBA(0.0) IBA(0.2) IBA(0.4) IBA(0.6) IBA(0.8) Mean LSD (p < 0.01) S.E + Main effect Growth regulator (GR) Genotype (GT) Interaction effect KOS-1 24.01(29.33) 25.14(30.09) 26.16(30.75) 21.11(27.35) 19.20(25.98) 23.12(28.70) = 0.88 = 0.78 GR x GT KS-101 18.06(25.14) 20.09(26.62) 23.08(28.71) 19.39(26.12) 18.66(25.58) 19.89(26.44) 0.32 0.36 = 1.95 0.72 Table.4 Influence of different treatment combinations and media formulations on haploid frequency (Per cent) Growth regulators mg l-1 Number of plants regenerated KOS-1 24.04 (29.36) 30.22 (50.89) 40.21 (39.35) 30.24 (33.36) 56.22 (48.57) 40.22 (39.35) BA (0.0) BA (2.0) BS (3.0) Kn (0.0) Kn (2.0) Kn (3.0) Mean Effect LSD < (p = 0.01) Main effects Growth regulator (GR) = Genotype (GT) = Interaction effect GR x GT = * Data in parenthesis are transformed values (Sin-1 √p) KS-101 18.44 (25.42) 58.25 (49.74) 37.25 (37.61) 25.24 (30.15) 54.22 (47.42) 39.66 (39.03) 38.84 (38.23) 41.85 (40.15) S.E + 0.25 0.20 0.12 0.10 0.50 0.24 3338 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3336-3339 Highest number of shoots per explants of proliferated cultures (86.00) was achieved (Natalajietal 2006) [Table 2] when MS full strength medium was supplemented with Kn (2.0 mg/lit) followed by the use of BA(2.0 mg/lit)(69.00), highest number of shoots was regenerated in KS-101(53.83)followed by KOS1(53.66).These shoots devoid of any callus, were sub-cultured on rooting medium containing various concentrations of auxin (IBA). Maximum rooting (30.75%) was achieved when MS medium was supplemented with 0.4mg/lit IBA[Table 3]. Root tip mitosis chromosome counts revealed maximum percentage of haploid frequency in KOS-1(41.85%) followed by KS-101(38.84%) (Natalijaetal 2004)[Table 4]. Diploidization of haploid plants was carried out by using different concentrations of colchicines for different time durations (Dwarkeshetal 2006).Maximum doubling efficiency was obtained when roots of plantlets were submerged in 20mg/lit colchicines for 2 hours and about 40-45 per cent diploidization was achieved. In conclusion, protocol developed for the production of double haploid plants through endrogenesis offers a viable alternative to develop homozygous lines for further use in the development of hybrid/synthetic varieties having higher yield, better quality and tolerance to stresses. References Dwarkesh, S., Parihar.,Sanjit. A. 2006. 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Int.J.Curr.Microbiol.App.Sci. 7(06): 3336-3339. doi: https://doi.org/10.20546/ijcmas.2018.706.390 3339