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Effect of Different Genotype and Hormonal Combination on in Vitro Callus During Anther Culture of Hybrid Mustard

M.A. Halim
Department of Biotechnology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka- 1207, Bangladesh

M.S. Islam
Student, Department of Biotechnology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh

M.E. Hoque
Department of Biotechnology, Sher-e-Bangla Agricultural University, Sher-e-BanglaNagar,Dhaka-1207,Bangladesh

F. Mahmud
Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh

M.S.R. Bhuiyan
Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh

Abstract/ Executive Summary:

The experimental materials were F1 regeneration of SAU sharisha 1×Tori 7, BARI sharisha 6×Tori 7 & BARI sharisha 15×Tori 7. The experiment was conducted in the Biotechnology Laboratory of the Department of Biotechnology, Sher-E-Bangla Agricultural University, Dhaka-1207 during the period of August 2011 to March 2012. The experiment was conducted to study the effect of different genotype and hormonal combination on in vitro callus of hybrid mustard (Brassica rapa L.). The maximum (0.7020 and 1.302 g at 14 and 35 DAI respectively) and the minimum (0.0003 and 0.30 g at 14 and 35 DAI respectively) callus weight was observed in combination T₇(6.0 mg/L 2, 4-D+2.0 mg/L BAP) and the genotype of F1 generation of SAU Sharisha 1 x Tori 7 and T₃(2.0 mg/L 2, 4-D+6.0 mg/L BAP) and the genotype of F1 generation of BARI Sharisha 15 x Tori 7, respectively. At 14, 21, 28 and 35 the maximum size (0.922, 1.438, 1.640 and 1.720cm respectively) of callus was observed at T₇ hormonal treatment ( 6.0 mg/L 2, 4-D +2.0mg/L BAP) with the genotype of F1 generation of SAU sharisha 1 × Tori 7 and the minimum size (0.466, 0.7160, 0.9160 and 1.014 cm at 14, 21, 28 and 35 DAI respectively) of callus at T₃ ( 2.0 mg/L 2, 4-D + 6.0 mg/L BAP) hormonal concentrations with the genotype of F1 generation of BARI sharisha 15×Tori 7. The best hormonal treatment is T₇ ( 6.0 mg/L 2, 4-D +2.0mg/L BAP) with the genotype of F1 generation of SAU sharisha 1 × Tori 7 for considering the maximum weight and size of callus at different days after anther inoculation in the culture medium containing different combinations of growth hormone.

Keywords: Anther, Hormone, Ggenotype, Generation

Location: Biotechnology Laboratory of the Department of Biotechnology, Sher-E-Bangla Agricultural University, Dhaka-1207

Start Date: 00-08-2011

End Date: 00-03-2012

Program Area: Variety and Species

Commodity: Mustard

Objectives:

1. To study the effect of different genotype and hormonal combination on in-vitro callus of hybrid mustard (Brassica rapa L.).

Methodology:

The experimental materials were F1 regeneration of SAU sharisha 1×Tori 7, BARI sharisha 6×Tori 7 & BARI sharisha 15×Tori 7.The experiment was conducted in the Biotechnology Laboratory of the Department of Biotechnology , Sher-E-Bangla Agricultural University, Dhaka-1207 during the period of August 2011 to March 2012. Seeds were germinated and grown under controlled environment in the net house. Unopened flower buds were collected from the donor plants having the different nucleated stages (observed using 1%accetocarmine under compound microscope).The 3.01-3.50 mm size of buds were selected and wrapped with aluminum foil and kept at 4?C chamber 4 days. Full green and healthy flower buds were used for culture. The following MS tissue culture medium supplemented with following concentrations of 2,4-D and BAP were used as phytohormone: T₁=2.0 2,4-D + 2.0 BAP, T₂=2.0 2,4-D + 4.0 BAP, T₃=2.0 2,4-D + 6.0 BAP, T₄=4.02,4-D + 2.0 BAP, T₅=4.0 2,4-D + 4.0 BAP, T₆=4.0 2,4-D + 6.0 BAP, T₇=6.02,4-D + 2.0 BAP, T₈=6.0 2,4-D + 4.0 BAP and T₉=6.0 2,4-D + 6.0 BAP. Sterilization : Sterilization of flower bud was practiced by sterile water then 70% ethyl alcohol for 2 minute, again washed by sterile water and then 0.1% w/v HgCl₂ for 1 minute and finally washed by DDH₂O twice. Different phytohorme were sterilized by using filter (Sieve size 0.45μm). Growth medium and required glassware and small instrument were sterilized with autoclave (at121?C, 15psi and for 20 minute). Measurement of flower Bud: By using digital slide calipers flower bud size was measured and expressed in millimeter (mm).It is well established that uninucleated stage is most responsive to regeneration. The uninucleated satge was identified by 1% accetocarmine test of microspore under compound microscope. Growth of vegetative nucleus in microsporogenesis should be retarded as it were pollen grain can not formed during anther culture. This is important for development of haploid. Duration of cold pretreatment (4?C) was counted for 4 days. Inoculation of anthers: Anthers were removed by using sterilized fine Tweezers (forceps) and inoculated on the sterile culture vials containing medium and supplemented with growth hormone. The inoculated vials were cultured at 32?C growth chamber for 2 weeks in complete dark condition and next 1-2 weeks in 5000 lux light intensity with 70% relative humidity and 14/10 hour light-dark condition. Sixty anthers of each flower bud group were inoculated. The culture vials showing signs of contamination were discarded. Day to day observations was carried out to record the responses on callus induction.

Source of Information: BANGLADESH RESEARCH PUBLICATIONS JOURNAL; ISSN: 1998-2003, Volume: 8, Issue: 4, Page: 247-255, July - August, 2013

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The maximum(0.325 and 0.8544g at 14 DAI and 35 DAI, respectively) and the minimum (0.2796 and 0.8151 g at 14 and 35 DAI, respectively) weight of callus was observed in the genotype of F1 generation of BARI Sharisha 6x Tori7 and BARI Sharisha 15 x Tori 7, respectively. The maximum (0.6793 and 1.229 g at 14 and 35 DAI, respectively) callus weight and the minimum (0.0006 and 0.3g at 14 & 35 DAI, respectively) callus weight at T₇ (6.0 mg/L 2, 4-D+2.0 mg/L BAP) and T3(2.0 mg/L 2, 4-D+6.0 mg/L BAP) treatment respectively. The maximum (0.7020 and 1.302 g at 14 and 35 DAI respectively) and the minimum (0.0003 and 0.30 g at 14 and 35 DAI respectively) callus weight was observed in combination T₇(6.0 mg/L 2, 4-D+2.0 mg/L BAP) and the genotype of F1 generation of BARI Sharisha 6 x Tori 7 and T₃ (2.0 mg/L 2, 4-D+6.0 mg/L BAP) and the genotype of F1 generation of BARI Sharisha 15 x Tori 7, respectively. The maximum size (1.311 cm at 35 DAI) and the minimum size (1.286cm at 35 DAI) was observed in the genotype of F1 generation of SAU Sharisha 1 x Tori 7 and BARI Sharisha 15x Tori 7. Treatment T₇ (6.0 mg/L 2, 4-D+2.0 mg/L BAP) and T3 (2.0 mg/L 2, 4-D +6.0 mg/L BAP) showed the maximum size (1.475cm at 35 DAI) and minimum size (1.310cm at 35 DAI) respectively of callus. The maximum size (1.720cm at 35 DAI) and the minimum size (1.014 cm at 35 DAI) as observed in combination of T₇ (6.0 mg/L 2, 4-D+2.0 mg/L BAP) and the genotype F1 generation SAU Sharisha 1 x Tori7 and T3 (2.0 mg/L 2, 4-D+6.0 mg/L BAP) and BARI Sharisha 15 x Tori 7 respectively.

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