PROCEDURE

Induction of calli ( 34 weeks ) 1- Dehusk mature healthy seeds with a rice husker and surface-sterilize in 70% ethanol for 30 s, then in 1.5% sodium hypochlorite for 30 min with vigorous shaking, followed by five rinses in sterile water. 2- Place ten sterilized seeds per plate on the surface of N6D medium and seal the plate with surgical tape. 3- Incubate the seeds on the medium at 29.5 1C at about 3.5 klux for 34 weeks. Calli are formed from the scutella after 34 weeks in culture. Preparation of calli and Agrobacterium ( 3 days ) 4- Collect the actively growing calli (yellowish white, relatively dry and about 13 mm in diameter and subculture on fresh N6D medium at 29.5 1C at about 3.5 klux for 3 days. 5- Streak a single colony of Agrobacterium harboring the gene of interest in a binary vector on AB medium, supplemented with the appropriate antibiotics for binary vector selection. Incubate at 28 1C for 3 days. Infection of callus with Agrobacterium ( 3 days ) 6- Collect Agrobacterium cells on a microspatula and suspend in 30 ml AAM medium containing 30 ml of 1,000_ AS (OD6000.050.1). Gently but completely resuspend the cells with a disposable sterilized pipette. 7- Collect subcultured calli (from Step 4) in sterilized stainless-steel sieves held over plastic Petri plates. 8- Pour the Agrobacterium cell suspension prepared in Step 6 into the plate from Step 7. Immerse calli in the Agrobacterium solution for 90 s, briefly shaking the sieve. 9- Blot the sieve containing calli dry on sterilized paper towels to remove excess Agrobacterium. 10- Drip 1 ml AAM containing 30 ml 1,000_ AS onto a sheet of filter paper on 2N6-AS medium and transfer calli onto the filter paper. 11- Wrap the plate with aluminum foil and incubate at 28 1C in the dark for 4860 h. Washing of infected calli, selection and regeneration of transgenic plants ( 68 weeks) 12- Collect the infected calli in a 50 ml sterile tube and wash with sterile water by shaking the tube. Repeat washes until the rinse water is clear. 13- Rinse the calli with sterile water, adding 1,000_ Car at a concentration of 500 mg l_1 to kill Agrobacterium cells. Pour the callus suspension through sterilized stainless-steel sieves held over plastic Petri plates. 14- Place stainless-steel sieves containing calli onto paper towels and dry well.

15- Transfer calli onto N6D-S medium. Up to 16 calli can be placed on one plate. 16- Seal plates with surgical tape. Incubate at 29.5 1C at about 3.5 klux for 34 weeks. 17- Transfer the growing calli to MS-NK medium for shoot regeneration. 18- Seal plates with surgical tape. Incubate at 29.5 1C at about 4.5 klux for 34 weeks. 19- Transfer shoots of 34 cm in length to MS-HF medium in plant boxes . TIMING The above procedure will take approximately 1013 weeks to complete. Induction of calli: 34 weeks Preculture of calli and Agrobacterium: 3 days Inoculation of Agrobacterium and co-cultivation: 3 days Selection of transformed cells: 34 weeks Regeneration of transgenic plants: 34 weeks