Gynogenesis

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0-32.6% of cultured ovary pieces and 0-18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gametederived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.

The objectives of the present study was to determine the best age of the zygote for gynogenesis of seurukan fish (Osteochilus vittatus) using heat shock treatment. The completely randomized design was utilized in this study. Five levels of zygote age were tested, namely: 3 minutes, 5 minutes, 7 minutes, 9 minutes and 11 minutes after fertilization and one control treatment (without heat shock). The zygotes were shocked at the temperature of 38 o C for 60 seconds and every treatment was done at three replications. The ANOVA test showed that the zygote age gave the significant effect on the triploidy level and growth performance of the seurukan fish (P<0.05). The highest percentage of the triploid fish were found at zygote age of 3 minutes, but this value was not different significantly with 5 min, 7 min and 9 min. The highes t weight gain was found at zygote age of 3 minutes, but this value was not different significantly with 5 min, 7 min and 9 min. In addition, the highest of length gain was also recorded at zygote at zygote age of 3 minutes, but this value was not different significantly with 5 minutes of the zygote (P>0.05). It is concluded that the best zygote age of the seurukan fish for gynogenes is using heat shock treatment was 3 minutes after fertilization. ABSTRAK Penelitian ini bertujuan untuk mendapatkan umur zigot terbaik untuk proses ginogenes is ikan seurukan (Osteochilus vittatus) dengan menggunakan kejutan suhu panas. Metode yang digunakan adalah eksperimen dengan model Rancangan Acak Lengkap faktor tunggal dengan enam taraf perlakuan, masing-masing perlakuan dengan tiga kali ulangan. Kejutan suhu yang diberikan yaitu 38 o C dengan lama kejutan 60 detik, dengan perlakuan yaitu: kontrol (tanpa perlakuan kejutan panas), umur zigot 3 menit setelah pembuahan, umur zigot 7 menit setelah pembuahan, umur zigot 9 menit setelah pembuahan dan 11 menit setelah pembuahan. Uji ANOVA menunjukkan bahwa pemberian kejutan suhu panas pada umur zigot yang berbeda berpengaruh nyata terhadap triploidy dan pertumbuhan ikan seurukan (P<0,05). Persentase ikan triploid tertinggi dijumpai pada perlakuan 3 menit setelah pembuahan, nilai tidak berbeda nyata dengan perlakuan 5 menit, 7 menit dan 9 menit. Pertambahan bobot tertinggi dijumpai pada umur zigot 3 menit setelah pembuahan, nilai ini tidak berbeda nyata dengan perlakuan 5 menit, 7 menit dan 9 menit. Pertambahan panjang tertinggi dijumpai pada perlakuan 3 menit setelah pembuahan, nilai ini tidak berbeda nyata dengan perlakuan 5 menit (P<0,05). Berdasarkan hasil tersebut dapat disimpulkan bahwa umur zigot yang berbeda berpengaruh terhadap triploidy, dan umur zigot terbaik adalah 3 menit setelah pembuahan.

"Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.
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