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Research Detail

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P. Saha
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

M. Afrin
Department of Biotechnology and Genetic Engineering,

A.K.M Mohiuddin
Department of Biotechnology and Genetic Engineering, Mawlana Bhasani Science and Technology University, Tangail-1902, Bangladesh

A.M Shohael*
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

Black Gram (Vigna mungo L.), widely known as Mashkalai in Bangladesh is an important protein source used as human food as well as fodder. BARI Mash 2 is a popular black gram variety released by Bangladesh Agriculture Research Institute (BARI) which is cultivated throughout the country and very popular especially in the char areas. Establishment of a reliable regeneration system for BARI Mash 2 has been tried for further genetic improvement. A rapid, reproducible and efficient in vitro regeneration method was developed using hypocotyl and young leaf explants through callus formation. The frequency of callus formation was highest (75%) on Murashige and Skoog (MS) medium supplemented with a high concentration (31.66 μM) of 2,4-Dichlorophenoxyacetic Acid (2,4-D) using the young leaf as explants’ source. Callus induction rate was less in hypocotyls in the same medium.  No further progress was observed from those calluses. MS medium containing 16.11μM of αNaphthalene acetic acid (NAA) showed the 70% calli induction from hypocotyls segment.  These calli were amenable to produce multiple shoots (5-6 shoot) in the medium containing 17.75 μM of 6 Benzyl aminopurine (BAP) alone and the combination of BAP (17.75 μM  ) and NAA (2.68 μM). Shoots were rooted most effectively (55%) in half strength MS basal medium containing 7.38 μM of Indole-butyric Acid (IBA). Well rooted plantlets were successfully acclimatized, transferred to the soil and found to produce flowers and fruits. The efficient and reproducible regeneration protocol described here allows for successful in vitro regeneration of BARI Mash 2 that is vital for future genetic manipulation.

  Callus, Control environment, Growth regulator, Hypocotyls, Indole-butyric Acid (IBA).
  Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh
  
  
  Crop-Soil-Water Management
  Black gram

The present investigation was attempted to develop an efficient and reproducible protocol for successful in vitro regeneration and multiple shoot formation through callus using different explants such as hypocotyls and leaf segments of BARI Mash 2.  

Plant material: Seeds of BARI Mash 2 for this study were collected from Bangladesh Agricultural Research Institute (BARI) through the courtesy of Bangladesh Livestock Research Institute (BLRI). Young leaves and hypocotyl were used as explants’ source from in vitro grown seedlings. In vitro seed germination: The seeds of BARI Mash 2 were surface sterilized with 70% ethanol (v/v) for 2 min followed by washing twice with sterilized distilled water for 4 times. For in vitro germination the surface of BARI Mash 2 seeds were sterilized using different concentration and incubation time on commercial Clorox (1% Sodium Hypochlorite) and Tween 20. Seeds were treated with 10, 20, 30, 40, 50, 60, 70 and 80% of commercial bleach Clorox (1% Sodium Hypochlorite) and incubated for 15- 45 mins. Few drops of Tween 20 were added to enhance the effect of Clorox. The seeds were then rinsed 3-4 times with sterile distilled water to remove all of the Clorox. After that, the seeds were placed in sterilized tissue paper with the help of forceps to remove excess water before inoculation in the medium. The sterilized seeds were placed on a solidified agar medium containing no growth regulators in sterile jars and incubated in the dark room for germination. The sterilized seeds were germinated in the dark room within 2-3 days and reached up to 7-8 cm long within 5-6 days. Young leaves and hypocotyls segment were ready for further experiments. Nutrient medium and growth regulators: Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) was used for callus induction and plant regeneration. For the induction of callus, different concentrations (0.0, 2.26 µM, 4.52 µM, 9.04 µM, 13.57µM, 18.10 µM, 22.62 µM, 27.14 µM , 31.66µM and 36.19 µM) of 2, 4Dichlorophenoxyacetic Acid (2, 4-D)  and (0.0, 2.68 µM, 5.37µM, 10.74 µM, 16.11 µM, 21.48 µM) Naphthalene Acetic Acid (NAA) alone were used.  6-Benzyl Aminopurine (BAP) alone (0.0, 4.44 µM, 8.88 µM, 13.31 µM, 17.75 µM, and 22.20 µM) and different combinations of BAP and NAA (13.31 µM, +1.34 µM, 13.31 µM +2.68 µM, 13.31 µM + 5.37 µM, 17.75 µM +1.34 µM, 17.75 µM +2.68 µM, 17.75 µM +5.37) were added to the MS medium for shoot initiation and plant regeneration. For root induction different concentrations (2.46 µM, 4.92 µM, 7.38 µM and 9.84 µM) of Indole Butyric Acid (IBA) and (1.34 µM, 2.68 µM, 5.37 µM) NAA were added in to the ½ strength of MS medium. Media sterilization and culture condition: All the culture media were sterilized in an autoclave machine at 121°c for 20 minutes at 15psi. The explants were cultured in each media combination under aseptic conditions and incubated at 24±1oC under dark conditions for germination and 16/8h light/dark regime for callus initiation and plant regeneration. Callus induction and regeneration: For callus induction, young leaves and hypocotyl were collected from 5-6 days old in vitro grown seedlings. In the laminar hood leaves and hypocotyl were cut into small pieces (1.0cm) in a sterile glass Petri dish and placed into the other Petri dish with sterile distilled water, not for drying. After that small pieces were placed into the appropriate semi solid medium into the glass jar having different concentration of growth regulators. 4-6 explants were placed in one jar and sealed properly before inoculation in the growth chamber. Rooting and Acclimatization: Regenerated shoots were separated and sub cultured into the half strength of fresh MS medium with rooting hormone and incubated further for rooting. Well rooted plants were selected and taken out from the glass jar, washed with running tap water to remove the agar in the roots and planted in sterile soil mix and kept in a humid chamber for acclimation. Data recording: Data were recorded for callus induction and growth in the different time period and observed the callusing responses daily. Shoot regeneration data was recorded after five weeks of culture in regeneration medium. Data for root initiation was taken after 28 days of culture. Statistical Analysis: The data collected in different parameters like callus initiation, callus frequency, regeneration percentage, the number of shoots per callus, percentage of root initiation were calculated on the basis of percentage, average, and standard deviation respectively.

  Jahangirnagar University J. Biol. Sci. 6(1): 23-33, 2017 (June)
  
Funding Source:
1.   Budget:  
  

Several attempts were taken to enhance the black gram tolerant against different abiotic and biotic stresses through classical breeding with very limited success. However, breeding of black gram is difficult due to the fact that it is self-pollinating crop and the genetic variation among its varieties is narrow. So there is an urgent need to use transgenic approach for the improvement of black gram. Grain legumes are comparatively recalcitrant to regeneration and transformation. In grain legumes, tissue culture has been repeatedly described as difficult. In our experiment, an efficient regeneration protocol was successfully established for in vitro culture of BARI Mash 2, which will be useful in future genetic improvement of plants through biotechnology and genetic engineering. This will definitely help in developing novel cultivars suitable for growth in different stress affected areas in Bangladesh.

  Journal
  


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