An accession of T. timopheevi (PI 290518) used as a female parent, was crossed with an accession of T. monococcum (PI 352267). At 14 days after pollination, hybrid caryopses were harvested and sterilized in 20% bleach (CaCl2), followed by washing four times in distilled water. Young embryos were isolated and plated on MS medium in' darkness at 20C until the embryos germinated and grew roots. Then the embryos were transferred to an incubator with a 12 hour photoperiod and a temperature of 25oC. When the seedlings were about 5 to 10 cm tall, they were planted in pots with a mix of 50% soil, 40% peat moss, and 10% sand.

At the three-tiller stage, the seedlings were removed from soil, and roots of the seedlings were washed and trimmed. The roots and crown of the seedlings were immersed in a solution of 0.1 % colchicine, 10 ppm GA3, 2% dimethyl sulfoxide (DMSO) and 0.01% Tween 20 in a beaker for 5 hours to induce chromosome doubling. After the roots were washed three times in tap water, they were placed in running tap water for 24 hours before transplanting to pots.

Root tips were collected from the F1 seedlings in the pots for cytological observation before the seedlings were treated with colchicine. For the amphiploid of T. timopheevi-T. monococcum, root tips were collected from germinated seeds in petri dishes. The root tips were pretreated for 4 hours in a solution of colchicine (0.05%),8- hydroxyquinoline (0.025%) and DMSO (25 drops per 100 ml), and stained for one week in 2% carmine in 45% acetic acid. The root tips were heated to boiling prior to squashing.