|J. Chin. Soc. Anim. Sci. 37(Suppl.)|
林德育 賴永裕 黃鈺嘉 許進德 蕭素碧 吳明哲
為瞭解畜產植物-牧草種子對於超低溫保存的耐受性及逐步降溫是否可以改善超低溫保存後的發芽率，選擇8種牧草(青割玉米 forage corn、綠肥大豆台南4號 Green manure in soybean crops of Tainan No.4.、綠肥大豆台南7號 Green manure in soybean crops of Tainan No.7.、苕子 Hairy vetch、田菁 sesbania、泰樂豆 stylo、中東苜蓿 Far east Alafa、澳洲大豆 Australian soybean)各分成4組，探討牧草種子可否經由控制降溫速度改善超低溫凍存後的發芽率。試驗材料以50顆種子為單位，先以錫箔紙包裝，減少空氣直接接觸，再置於密封的塑膠袋內，每一處理中每一個物種計有2重複，合計3200棵種子(50棵*2重複*8物種*4處理)，A組與B組利用微電腦程式降溫儀分別以每分鐘0.5 ℃與1 ℃逐步降溫，從室溫降到 –40℃後，再快速置入液態氮桶；C組為直接快速置入液態氮桶中。D組為對照組是以非超低溫的傳統 4℃冰箱保存。A、B及C組之種子於超低溫凍存21天後，以40℃溫水浸泡10分鐘進行解凍，比較各組種子發芽試驗結果顯示，除苕子及玉米在沒有超低溫凍存的對照組最好( P < 0.05)外，其餘處理組間差異均不顯著，且均有種子發芽，但物種間差異顯著(P <0.05)，其中以中東苜蓿的發芽率最高，可達90%以上，而青割玉米與澳洲大豆則較差，發芽率在50%以下，顯示兩種逐步降溫法，對此8種牧草，並無法改善發芽率，由8個物種經超低溫凍存後均有種子發芽，顯示錫箔紙與塑膠袋包裝的簡便快速凍存法，可作為牧草種原的超低溫長期保存的一種選項。
EFFECT OF FREEZING RATES ON CRYOPRESERVATION OF EIGHT LIVESTOCK FORAGES
D.Y. Lin, Y.Y. Lai, Y.C. Huang, C. T. Hsu, S. P. Shaug and M C. Wu
Livestock Research Institute, Council of Agriculture
To explore whether the freezing rate was the critical success factor for forage seed cryopreservation, eight species livestock forage seeds, forage corn, Green manure in soybean crops of Tainan No.4., Green manure in soybean crops of Tainan No.7., Hairy vetch, sesbania, stylo, Far east Alafa, Australian soybean, were stored into liquid nitrogen(LN) by different freezing rates. Each species had four treatments, two repeats and 50 seeds per repeat. Total was 3200 seeds (50 seeds *2 repeats *4 treatments *8 species). Seeds were sealed by aluminum foil first and then zipped in zipper plastic bag. Before putting into LN tank, treatment A and treatment B were chilled by 0.5 ℃ per minute and 1 ℃ per minute freezing rates to -40 ℃ by a programmable cryopreser, respectively. Treatment C was direct plunging sample into liquid nitrogen vapor and treatment D was stored in a 4 ℃ refrigerator. After 21 days storage in LN, treatment A, B and C were thawed at 40℃water for 10 minutes. Germination rate was the cumulated three days counts in percentage for all treatments. Difference of germination rate was not detected for three treatments, A, B and C. However, treatment D, non-cryopreserved group, was better than others’ for hairy vetch and forage corn. Eight forage seeds in all four treatments had seeds Germinated. Species effect was significant for the germination rates(P<0.05), and Far east Alafa with higher than 90% was the best, and forage corn and Australian soybean with less than 50% were worse than average. Two freezing rate controlled methods were not better than the instant freeze method(Treatment C) for these 8 forage seeds. Because all 8 species had seed germinated by the instant method, results suggested direct plunging the aluminum foil sealed and plastic bag zipped seeds into liquid nitrogen vapor was one of choice for long term preservation of genetic material of forage crops.
Key words: Cryopreservation, Livestock forage, Seed