Caucasian clover (Trifolium ambiguum Bieb.) × white clover (T. repens L.)-interspecific hybrids developed through tissue culture

In order to create new forage variety, caucasian clover (Trifolium ambiguum Bieb.) with strong tolerance to drought and cold introduced from New Zealand as the female parent was hybridized with a white clover (T repens L.) collected from northeast China, which has strong nitrogen fixation capacity. Hybrid embryos of caucasian clover × white clover were successfully raised using embryo rescue. 13.8% of these embryos grew to maturity. The successfully grown plants were transferred to sterilized vermiculite after 5–6 weeks and then to field after 3–4 months. The hybrid plant showed intermediate morphological features and a chromosomal number of 2n=5X=40. In order to obtain more plants for backcross, a tissue culture system was established for the hybrids, using MS+0. 1mg l¹ 2, 4-D+2mg l¹ 6-BA, MS +0.5mg l¹ NAA+1mg l¹ 6-BA+1mg l¹ KT and 1/2MS as the medium for bud-induction, differentiation and root-induction respectively. 80% of the cultured plantlets survived these growing conditions. Hybridity of the plant was also confirmed by ISSR markers using 16 primers.