Patent ID: 7230166

Claim:
A method of breeding a new variety of four leaflet clover from a wild-type three-leaflet clover, Trifolium repens L ., the method comprising the steps of: (A) providing stem segments containing leaves of the wild-type three leaflet clover, which includes the sub-steps of: (a) cutting each of the leaved stems of the wild-type three-leaflet clover at a length of about 5 cm; (b) removing the leaves from the stem and cutting each of the stems including a growing point at a length of 3-4 cm; (c) transferring the segments into a conical flask and treating them with a solution of ethyl alcohol of 70% for 20 to 30 seconds to sterilize the segments and washing the segments with sterile water three times; (d) treating the segments with a solution of NaOCl of 1.0% for 15 minutes to sterilize the segments and washing the segments with sterile water three times; (B) cutting each of the sterilized segments from step (A)(d) at a length of 2-3 mm in a clean bench, wherein each of the cut segments includes a growing point allowing shoots to grow from the segments; (C) treating the shoots obtained from step (B) with cold water at a temperature of 5° C. for 20 seconds; treating the shoots with Ethyl Methane Sulfonate of 0.1-0.3 μg/L for 3 seconds; washing the shoots with sterilized water; and then incubating the sterilized shoots in a MS basal medium supplemented with 6 Benzylaminopurine of 0.5-2.0 mg/L, Indol-3-Acetic Acid of 0.5 mg/L and α-Naphthalene Acetic Acid of 0.5 mg/L under 500 lux illumination at 20-22° C.; and (D) obtaining a new variety of four-leaflet clover from the shoots obtained from step (C), including the sub-steps of: (a) transplanting the shoots to soil comprising a 1:1:1 mixture of perlite, peat moss and sterile sand in a greenhouse under the environment of humidity of 90%, temperature of 18-20° C. and illumination intensity of 70%, when a bud and roots are generated in the supplemented MS basal medium; (b) selecting shoots of the four-leaflet clover obtained from step (D)(a) and propagating the shoots asexually by repeated tissue-culture of the segments throughout 5 generations; and (c) subjecting the stems and roots of the four-leaflet clover to dividing/transplanting or cuttage to obtain a new variety of four-leaflet clover.