Institute Of Biotechnology And Genetic Engineering

Published Date: 02 Nov 2017

The experiment was conducted at the Institute of Biotechnology and Genetic Engineering, University of Agriculture Peshawar during 2012. In the present work callus formation and transformation efficiency of 5 local rice varieties was analyzed under different MS media compositions.

Rice Varieties

1) Shadab 31

2) Dokri basmati

3) TN-1

4) Swat-1

5) Muskan

The entire experiment was divided into two main steps:

Agrobacterium tumefaciens preparation

Rice transformation

3.1 Agrobacterium tumefaciens preparation:

3.1.1 Media used:

The type of media used for the growth of Agrobacterium was LB medium. The components of this medium are as follow:

Trypton 1%

Yeast extract 0.5%

Nacl 1%

Bacterial agar 1%

The above mentioned components were poured in a beaker on stirrer and water was added to dissolve all the components.

The required volume of the media was 500 ml that was adjusted.

Then the PH of the media was adjusted that is 7.

The agar was added and the media was autoclaved and poured into the plates under aseptic conditions.

3.1.2 Growth conditions for Agrobacterium:

The Agrobacterium requires a specific temperature for its growth that is 28 oC in dark. The Agrobacterium was cultured on solid LB medium and incubated in static incubator for 2 days.

3.1.3 Competent Cells Preparation for Agrobacterium (Jyothishwaran, 2007)

A single colony of Agrobacterium strain EHA101 was taken from agar plate and grown in 30-40 ml LB medium without antibiotics at 28°C.

Centrifuged at 5000 rpm, for 10 min at 4 oC.

Washed pellet two times with 10 ml pre-cooled sterile TE buffer on ice.

Resuspended the pellet in 1.5 ml fresh LB and in 1.5 ml sterile glycerol.

Divided above solution in about 200 µl in each eppendorf tube.

Chilled in freezer and stored at -80 oC.

3.1.4 Agrobacterium tumefaciens transformation. (Dityatkin et al., 1972)

Agrobacterium competent cells were melted on ice.

4 µl of recombinant plasmid DNA was added to the Agrobacterium competent cells on ice.

Incubated on ice for 5 min.

Incubated at -85 oC for 10 min.

Finally incubated or gave heat shock at 37oC for 5 min.

1 ml LB medium was added.

For 2-3 hours Incubation was done at 28 oC at 120 rpm.

Cells will be centrifuged at 12000 rpm for 3min at 20 oC.

Supernatant of 500 µl was discarded.

Pellet will be resuspended in about 200 µl LB medium.

Streaking was done on LB plate containing 50 mg/l kanamycin and the Plates were incubated at 28oC and colonies were observed after 2-3 days.

3.2 Rice Transformation

Rice transformation can be done through the following steps:

Callus induction

Callus transformation

3.2.1 Callus induction:

3.2.2 Media preparation for callus:

The type of media used for callus induction was half MS medium. The components and amounts of this medium are as follows:

3.2.2.1 Preparation of stock solutions

Stock solutions of different nutrients of MS medium are divided in five groups. These groups comprised of different combination of nutrients required seed germination and plant regeneration. These stock solutions are named as MS-A. MS-B, MS-C, MS-D and MS organics. Concentrations and components of these groups are listed below:

 

COMPONENTS

CONCENTRATION

MS-Micro (1000 ml)

 

 

 

KI

830 mg

 

CuSO4.5H2O

1000 mg

 

CoCl2.6H2O

1000 mg

 

H3BO

6.2 g

 

ZnSO4.7H2O

0.6 g

 

MnSO4.5H2O

24.1 g

 

NaMoO4.2H2O

250 mg

MS-organics 200x (1000 ml)

 

 

 

Myoinositol

20 g

 

Nicotinic acid

0.1 g

 

Thiamine HCl

20 mg

 

Glycine

0.4 g

 

Pyridoxine HCl

0.1 g

MS-A 50x (1000 ml)

 

 

 

KNO3

95 g

 

KH4NO3

82.5 g

 

KH2PO4

8.5 g

 

Micro stock

50 ml

MS-B 100x (1000 ml)

 

 

 

CaCl2.2H20

44 g

MS-C 100x(1000 ml)

 

 

 

MgSO4.7H2O

37 g

MS-D 100x (1000 ml)

 

 

 

Na2EDTA

37.25 g

 

FeSO4.7H2O

2.78 g

3.2.3 Half strength MS Medium

Steps involved in preparation of half strength MS medium are:

1.5-2% sucrose was weighed and added to distilled water in a beaker.

Then all the MS stock solution were added:

MS-A 1% (v/v)

MS-B 0.5% (v/v)

MS-C 0.5% (v/v)

MS-D 0.5% (v/v)

MS-organics 0.5% (v/v)

5mg/l 2,4-D solution was added.

Then pH (5.8) was adjusted. Then final required volume was adjusted and the media was autoclaved and poured into autoclaved plates afterwards.

3.2.4 Seed Sterilization

15 to 18 seeds from each variety were taken. Then the seeds were sterilized using the following protocol:

Seeds were de-husked and washed with autoclaved water

Then the seeds were rewashed with 70% ethanol.

The seeds were rewashed with 30% bleach plus 1 drop of tween-80 and kept on shaking for 15 minutes.

Step c was repeated.

In the end seeds were rewashed with autoclaved water.

Finally the seeds were dried on a sterilized tissue paper and stored in dry autoclaved petri plate.

3.2.5 Seed culturing for callus induction:

Seeds already sterilized were transferred to petriplates having half MS media under aseptic conditions.

The plates were properly sealed and placed in dark at 25°C.

The callus was observed every day.

3.2.6 Callus efficiency (Aditya and Baker 2006)

Data for callus formation efficiency was taken about 3 weeks after culturing. The data was taken in percentage using the following formula:

Callus frequency = No. of callus formed/total No. of seeds cultured* 100

3.2.7 Callus transformation (Toki et al., 2005)

Agrobacterium strains were grown for 2 days at 28 °C on LB medium supplemented with 200 mM acetosyringone and suitable antibiotics.

Bacterial cells were scooped up from the plate, re-suspended in 20 ml of LB medium and shaken for 1 h at 28 °C.

Culture plates containing callus were flooded with bacterial suspension OD = 0.75 (600 nm) for 5 min.

Liquid culture was removed, and each callus was picked up and blotted onto sterile filter paper before being placed onto co-cultivation medium for 2 days in the dark at 25 °C.

After co-culturing, callus was put onto selection medium containing 150 mg l–1 timentin plus appropriate antibiotics in the dark at 28 oC.

3.3 Assay of GUS activity (Jefferson Oefferson 1987):

Expression of GUS in rice cells was assayed using the following procedure.

The segments of rice calli were incubated in X-gluc solution containing 50 mM phosphatebuffer, 1 mg/ml X-gluc (5-bromo-4-chloro-3-indolyl-~-D-glucuronide), 0.5%Triton X-100 and 20% methanol.

The reaction mixture was placed under a mild vacuum for a few minutes and then incubated overnight at 37 °C.

The stained calli were scored for blue spots, their number and intensity of colour under stereomicroscope.

3.3.1 Transformation efficiency (Sarangi et al., 2010)

Transformation efficiency was observed in percentage using the following formula:

Transformation efficiency= No. of stained calli/total No. of calli* 100