The size and appearance of floral buds prior to tripping and pollen release (full anthesis) were recorded by daily monitoring of tagged buds. Crosses were performed into emasculated buds corresponding to 1, 2, and 3 days prior to full anthesis (d.p.a.). We determined that buds 2 d.p.a. were most amenable to cross pollination, giving a high degree of cross pollination and relatively low (< 20%) self pollination. by comparison, seed set in buds 3 d.p.a. was inefficient (<25%), although self pollination was never observed. buds 1 d.p.a. often had mature anthers and consequently gave a high degree of self pollination, despite emasculation. prior to hybridization, flowers were suction emasculated using vacuum applied through a 1 ml micropipette tip. to facilitate crossing we use a 2.5x binocular headset with a 10 inch working distance. pollen is obtained from freshly tripped flowers and applied to the stigma of emasculated flowers using extra fine forceps. in cases where unambiguous (no false positives) crossing is required, we use a male sterile floral homeotic mutant of M. truncatula cv. Jemalong. In this male sterile line, flowers corresponding to the day of anthesis or one day later are used, and self pollination is never observed. In all cases the resulting pod are wrapped in surgical gauze and tied to the stem at 2-3 weeks after crossing; this prevents mixing of pod upon abscission.
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REFERENCE:
An Improved Method for Artificial Hybridization In Annual Medicago Species.
W. Pathipanawat, R. A. C. Jones, and K. Sivasithamparam. Aust. J. Agric. Res., 1994, 45: 1329-1335.
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