Fertilization of ovules in test tube was reported first in Papavea somniferum
by Kanta et al. (1962). In tobacco, crosses of distantly related species were
made using this technique (Yeh et al. 1980). For test-tube fertilization,
sterilization of the flowers of both the maternal and pollen parents is
necessary.
Panicles of a rice variety (Erjiu-quing) were emasculated by the hot-water treatment method (45degreesC, 5 min), and the spikelets ready to bloom were sterilized and put in test-tubes. For sterilization, they were wiped with a piece of ethanol-moistened gauge several times, soaked in 70% ethanol for five seconds, and were washed with sterile water three times. A spikelet with a piece of peduncle was placed upright on solid medium of type N6 containing 4% sucrose (pH 5.8).
Cultivar Zidao with purple color of foliage was used as male parent. Its panicles were sterilized as follows: 1) exposure to ultra-violet rays (30 min. under a 30W lamp, at a 33 cm distance), 2) presoaking in 70% ethanol for 5 seconds, soaking in 0.1% HgCl\2\ solution for 5 minutes, and thorough washing, and 3) wiping 8-10 times with a piece of ethanol-moistened gauze. Then, the anthers were drawn out of the spikelet with a set of sterilized forceps and each was inserted in the maternal spikelet in a test tube.
The test tubes were kept in darkness at room temperature. The ovaries started swelling four days after pollination and continued to grow for 15 days. The seeds obtained were generally small in size, being about 1/3 of a normal seed. All the seeds had the embryo and endosperm, and germinated 20-25 days after pollination. The seedlings showed purple coloration indicating that they were hybrids.
Wiping the spikelets of pollen parent with a piece of ethanol-moistened gauge gave the best results, the rate of successful fertilization being 5.8%, and the rate of bacterial contamination was about 7%. When the pollen from sterilized spikelets was used for pollination of emasculated spikelets of potted plants, the rate of seccess was 27%. On the other hand, when non- emusculated and sterilized spikelets were self-pollinated in test tubes, the rate of seed set was 7.8%. Sterilization by soaking in HgCl\2\ solution gave a much lower rate of success as it affected the viability of pollen grains. The exposure to ultra-violet rays gave a high rate of contamination (80%). It was found further that when the stigma and style were cut off, pollination was totally unsucessful.
References
Kanta, K., N.S. Rangaswamy and P. Maheshwari, 1962. Test-tube fertilization in a flowering plant. Nature 194: 1214-1217.
Yeh, Shu-Mo, Xu-Mai Yang and Sho-Wu Zhang, 1980. A preliminary report on test- tube fertilization in tobacco and wheat. Acta Genetica Sinica 7: 261-266.