熱處理及激勃素打破番木瓜種子休眠之研究

Abstract

番木瓜（Carica papaya L.）為台灣重要的經濟果樹，繁殖方式有實生苗、嫁接、扦插與組織培養，但目前仍以種子播種培養而得之實生苗種植為主，因此其種子發芽能力對番木瓜苗木生產影響甚鉅。番木瓜種子有發芽時間長、發芽不整齊的特性，雖已有多位學者提出各種促進發芽方法，但促進效果會因品種與環境之不同而有所差異，且不同報告結果也互有矛盾之處，至今番木瓜種子播種後發芽時間冗長與整齊度差等問題仍舊存在，因此，尋求有效促進發芽方法為番木瓜種苗生產上重要的課題。 由‘台農2號’完熟果採收、去假種皮、清洗的新鮮番木瓜種子具休眠性，刻傷與濃硫酸處理未能促進發芽；將內、外種皮剝除可得65%發芽率，顯示種皮在番木瓜種子休眠所扮演的角色主要是胚根突出的物理障礙，而非阻止水分或氣體進入種子。新鮮番木瓜種子在20℃經乾燥後熟4週至種子含水量5.31~12.24%，仍無法順利發芽。外施GA3與GA4+7處理可促進番木瓜種子發芽，GA4+7效果較GA3佳，尤以GA4+7 100 mg/L效果最好，顯示其種子發芽所需之生物活性GAs型式可能是GA4和/或GA7，番木瓜後熟14天之種子經100 mg/L GA4+7浸種24小時後，移至Paclobutrazol和Ancymidol溶液浸潤的濾紙上進行發芽試驗，兩種藥劑對胚根突出的抑制效果隨著濃度提高而增加，間接證明持續生成GA在番木瓜種子發芽之重要性，而番木瓜種子需要外加GAs才能順利發芽，表示其胚活力不足，加上種皮為機械限制，因此推測番木瓜種子應屬於「生理休眠（physiological dormancy）」。GAs處理雖然可提高發芽率和整齊度，但會產生30%畸形苗，較不利於應用在番木瓜實生苗木生產。 番木瓜種子休眠可藉由熱水浴處理而促進發芽。將經後熟種子浸潤1天後，以34、36、38、40℃熱水浴處理3~6小時皆有促進發芽之效果，其中36℃熱水浴5小時處理為最佳條件，發芽率58.67%；種子浸潤5天再行熱水浴處理之打破休眠效果更好，發芽率提高至74%，但延長種子浸潤時間超過5天以上則效果與浸潤5天者無顯著差異。比較不同種子熱休克處理方法，熱水浴處理促進發芽效果較熱風處理佳，且經後熟種子對熱休克處理較新鮮種子敏感，而熱休克處理誘導發芽的幼苗沒有GAs處理畸型苗高的副作用，健壯植株，可供番木瓜種苗生產時，種子播種前打破休眠之處理技術。 為進一步釐清熱休克處理促進番木瓜種子發芽的機制，番木瓜種子經36℃熱水浴處理5小時後，移至Paclobutrazol和Ancymidol溶液浸潤之濾紙培養，10 μM Ancymidol即可完全逆轉熱水浴促進發芽之效果；10~250μM Paclobutrazol 拮抗熱水浴處理的效果則隨著濃度提高而增強，暗示熱水浴處理促進番木瓜種子發芽必須經過GAs重新合成的步驟。直接分析熱水浴處理番木瓜種子內生GA1、GA3、GA4、GA7、GA20及ABA含量結果顯示，熱處理之後GA4含量為未處理組的1.4倍，且經熱處理之種子內生GA4含量有隨著培養天數持續上升的現象，但熱處理與否對GA3含量無顯著影響，顯示番木瓜種子發芽之主要型式可能為GA4。而番木瓜種子ABA含量低於偵測範圍，推測ABA可能並非影響番木瓜種子發芽之限制因子，此與本試驗用10~100μM Fluridone無促進番木瓜種子發芽的結論相符。綜合上述結果，熱休克處理促進番木瓜種子發芽的機制，本試驗提出的假說模式為番木瓜種子浸潤恢復種子生理代謝後，經熱休克（36℃ 5小時）刺激，直接或間接誘導番木瓜種子內生GAs生合成速率增加或抑制GAs降解速度，使種子GA4含量因而增加，經過GA訊息傳導啟動種子貯藏物質分解運移和降低胚根前方胚乳蓋阻力與種皮細胞壁的水解酵素合成，因而使胚根向外推擠的生長潛勢大於胚乳蓋與種殼細胞壁的物理阻力，而順利完成發芽。

Papaya (Carica papaya L.), an important economical fruit crop in Taiwan, can be propagated by seeds, grafting, cutting and tissue culture, but mainly from seeds. Therefore, the germinability of seeds are vital to the industry. The characteristics of papaya seed germination are slow and asynchronous. Numerous researchers had proposed different kinds of methods to improve papaya seed germination, but the efficiency is significant different and contradictory depends upon cultivars and environment. The problem of poor and asynchronous germination of papaya seeds still exists. Thus, it is important for papaya nursery industry to look for effective methods to increase germination. Fresh papaya seeds harvested from‘Tainung no. 2’fruits at yellow-ripen stage are dormant after removing the sacrotesta and washing. Scarification and H2SO4 treatment have no effect on improving germination. The germination percentage can be achieved to 65% after removing the testa and tegmen, suggested that testa is the main physical constraint to radicle protrusion, instead of limiting the water and air permeability of seed. Fresh papaya seeds were after-ripened at 20℃ for 4 weeks and the water content of seeds decreased to 5.31~12.24%, but couldn’t germinate as well. Papaya seeds can improve germination by treated GA3 and GA4+7 solutions, and the most effective treatment is GA4+7 100 mg/L, implies that the main bioactive GAs for papaya seed germination may be GA4 and/or GA7. Seeds that were after-ripened for 14 days was soaked in 100 mg/L GA4+7 for 24 hours and incubated in Paclobutrazol and Ancymidol solutions. The inhibitory effects of these two inhibitors enhance as the concentration increases, demonstrates indirectly that the ability of papaya seeds to synthesize GAs continuously is quite important. Papaya seeds can’t germinate without adding GAs, implies that the embryo of papaya seed is weak. In addition to the weak embryo and the mechanical constraint of testa, papaya seeds are belonged to physiological dormancy. Although GAs treatment can improve the germination and synchronicity, it’s improper to be put in use due to the 30% abnormal seedlings occurrence. Papaya seed dormancy can be alleviated by hot water immersion treatment. 34, 36, 38, 40℃ hot water immersion treatment can enhance the germination of after-ripened seeds imbibed for 1 day. The best condition is 36℃ treated for 5 hours, and the germination percentage is 58.67%. The efficiency will be better as the imbibition duration before hot water immersion treatment increases to more than 5 days, the germination can be increased to 74%, but there’s no significant difference when the imbibition duration is more than 5 days. Compare the effects of different heat shock treatment, the positive improvement of hot water immersion (36℃, 5h) is better than hot air treatment (36℃, 5h), and after-ripened seeds are more sensitive to heat response than fresh seeds. Seedlings after heat shock treatment are healthy, have no side effects, unlike GAs treatment which will produce abnormal seedlings, and the method can be regarded as a dormancy breaking technology in papaya seedling propagation. In order to figure out the mechanism of heat shock treatment to improve papaya seed germination, we incubated seeds in Paclobutrazol and Ancymidol solutions after 36℃ hot water-immersion treated for 5 hours. 10 μM Ancymidol completely reverse the promoting effect of hot water immersion treatment. Counteracting effects of 10~250μM Paclobutrazol enhance as the concentration increases, implies that hot water immersion treatment should improve papaya seed germination via GAs de novo synthesis. According to the results of endogenous GA1, GA3, GA4, GA7, GA20 and ABA analysis, the GA4 content of hot water immersion treated seeds increases 1.14 times and enhances continuously as incubation time increases, but the heat treatment has no significant effect on endogenous GA3 content, implies that the bioactive form of papaya seed germination is GA4. The ABA contents of papaya seeds is not detected, suggested that ABA may not be the main factor influencing papaya seed dormancy and germination. This is conformed that 10~100 μM Fluridone treatment has no effect on improving germination. In conclusion, we proposed a hypothesis of mechanism how heat shock treatment can improve papaya seed germination. Seeds can restore physiological metabolism during the imbibition period. After heat stimulated, seeds were directly or indirectly induced increasing the rate of endogenous GAs synthesis or inhibiting the rate of GAs breakdown, and the GA4 content increases. Through GA signal transduction to start the degradation and mobilization of seed reserves and weaken the physical constraint of the cell wall of endosperm cap and testa, and then seeds can germinate successfully.