結球白菜和不結球白菜花粉超低溫冷凍保存流程之探討

張心怡

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結球白菜和不結球白菜花粉超低溫冷凍保存流程之探討

Investigation on Pollen Cryopreservation Protocols of Brassica campestris L. subup. pekinensis and Brassica campetris L. subsp. chinensis

張心怡 , Masters　　Advisor：廖松淵　　

繁體中文

結球白菜；不結球白菜；花粉；超低溫冷凍保存；花粉活性；含水量； Brassica campestris L. subup. pekinensis ； Brassica campetris L. subsp. chinensis ； Pollen ； Cryopreservation ； Pollen viability ； water content

Abstract │ Reference (123) │ Altmetrics

Abstract 〈TOP〉

本試驗以不同品系結球白菜 (Brassica campestris L. subup. pekinensis) 和不結球白菜 (Brassica campetris L. subsp. chinensis) 的花粉為材料，進行乾燥法超低溫冷凍保存，探討不同含水量對花粉體外和活體萌發率影響。
    體外萌發結果顯示，自然乾燥一小時的各品系花粉在21 %蔗糖濃度的B&K培養基具萌發率較高。取出新鮮花苞內花藥，放在直徑2.2 cm的塑膠小盒，利用矽膠乾燥劑脫水處理，再經液態氮保存至少一週，回溫後塗抹於21%蔗糖濃度培養基，測試體外萌發率。結果顯示，各品系脫水40 ~ 60 min後，經液態氮保存至少一週，花粉開始具有體外萌發力。結球白菜’201’、’801’和’玉豐’保存至少一週的高萌發率組別，介於乾燥處理80 ~ 140 min，萌發率為11.5 ~ 13.4 %，此時花藥含水量介於4.7 ~ 18.2 % 之間。不結球白菜’三鳳’和’鳳珍’冷凍保存至少一週的高萌發率組別，介於乾燥處理60 ~ 100 min，萌發率為7.5 ~ 11.0 %，此時花藥含水量介於15.4 ~ 27.7 %之間。冷凍保存至少一年與一週的花粉體外萌發率無顯著差異(P<0.05)。
    活體萌發則藉由授粉觀察結莢率、結籽數與種子萌發率。結球白菜’201’、’801’和’玉豐’各品系自交，於乾燥脫水處理80 min時具有較高結莢率，此時花藥含水量分別為21.2、15.2和23.0 %。雜交結果則因品系有差異，以’201’、’801’和’玉豐’為父本，分別和’玉豐’、’玉豐’和’801’雜交。活體萌發最佳處理組分別為脫水時間100、120和120 min，花藥含水量分別為13.1、7.5和5.4 %。不結球白菜’三鳳’和’鳳珍’最佳活體萌發處理組脫水時間80和60 min，花藥含水量介於18.2和27.7 %。各最佳處理組均具有一定結籽數與種子萌發率。冷凍保存至少一年乾燥處理60 ~ 100 min的組別，冷凍保存後，回溫授粉具結莢率結籽數和種子萌發率。
    總結以上，結球白菜最佳處理組為花粉乾燥處理80 ~ 120 min，花藥含水量介於8.6 ~ 18.2 %之間；不結球白菜最佳處理組為花粉乾燥處理60 ~ 80 min，花藥含水量介於18.3 ~ 27.7 %之間。冷凍保存一週後，可達最佳體外萌發率和活體萌發率。利用乾燥法超低溫冷凍保存流程可有效長期保存結球白菜和不結球白菜花粉，並配合體外萌發率可快速檢驗花粉活性。使用上簡單方便，具實用價值，可應用於育種及種原保存。

Parallel Abstract 〈TOP〉

In this study the pollen of Brassica campestris L. subup. pekinensis and Brassica campetris L. subsp. chinensis are used for drying protocols of cryopreservation, and investigated for the effect of different water content on pollen viability with in vitro and in vivo test.
    In vitro test showed the pollen of different lines treated with one hour air drying have highest pollen germination rate on B&K medium with 21% sucrose concentration. The test process removed the anther from fresh buds, and then placed it into small plastic bowl whith the diameter of 2.2 cm. The anther was then dehydrated by silica desiccant and storage in liquid nitrogen (-196°C) at least one week for cryopreservation. Afterwards, the pollen was rewarmed and then smeared on B&K medium with 21% sucrose concentration to exmine its in vitro germination rate. The result shows, after 40 ~ 60 minutes of dehydration and one week of nitrogen storage, lines of pollens produced the ability to germinate in the pollen tube. Brassica campestris L. subup. pekinensis ‘201’、’801’ and ‘Yu-Fong’ anther, which were dehydrated 80 ~ 140 min, produced a high pollen germination rate of 11.5 ~ 13.4 % while having 4.7 ~ 18.2 % water content. Brassica campetris L. subsp. chinensis ‘San-Fon’ and ‘Fon-Jan’ anther, which were dehydrated 60 ~ 100 minutes, also produced a high pollen germination rate of 7.5 ~ 11.0 %, while having 15.4 ~ 27.7 % water content. Pollens cryopreserved over one year maintain similar trends of in vivo germination rate compared with cryopreservation over one week.
    On the other hand, In vivo test shows Brassica campestris L. subup. pekinensis ‘201’,’801’ and ‘Yu-Fong’ with self-pollination have highest podding rate when it has been dehydrated for 80 minutes, with the water content of 21.2, 15.2 and 23.0 %; with cross-pollination on ‘Yu-Fong’, ‘Yu-Fong’ and ‘801’, the optimization treatment was dehydrated for 100,120 and 120 minutes, with the water content of 13.1, 7.5 and 5.4 %. Brassica campetris L. subsp. chinensis ‘San-Fon’ and ‘Fon-Jan’ with the optimization treatment was dehydrated 80 and 60 minutes, with the water content of 18.2 and 27.7 %.Generally speaking, the best group of different pollen lines produces a distinctive amount of seed yield and seed germination. Pollen for dehydrated 60 ~ 100 minutes and cryoperserved for one year stilling possess both podding and seeding ability. 
    Based on these results, the research samples of Brassica campestris L. subup. pekinensis dehydrated 80 ~ 120 min, with the water content of 8.6 ~ 18.2 % and Brassica campetris L. subsp. chinensis dehydrated 60 ~ 80 min, with the water content of 18.3 ~ 27.7 %, both cryopreserved in liquid nitrogen for at least one week, reflects the best in vitro and in vivo germination rate. The drying protocol of cryopreservation is an effective long term storage option for Brassica campestris L. subup. pekinensis and Brassica campetris L. subsp. chinensis pollen. When combined with in vivo examination the pollen germination rate can rapidly tested. This protocol is simple to execute and carries practical usability on the breeding and germplasm conservation of Brassica campestris L. subup. pekinensis and Brassica campetris L. subsp. chinensis.

Reference ( 123 ) 〈TOP〉

江徽之。(2012)。臺灣白花蝴蝶蘭莖頂玻璃化法超低溫冷凍保存流程之探討。中興大學生命科學系所學位論文。
連結：
李坤紘。（2008）。臺灣藥用植物食茱萸與牛樟玻璃化法超低溫冷凍保存前處理流程之探討。國立中興大學生命科學系碩士論文。
連結：
林經剴。(2009)。臺灣原生藥用植物石薺薴和六角草玻璃化法超低溫冷凍保存流程之探討。中興大學生命科學系所學位論文.
連結：
林維熙。(2008)。臺灣原生藥用植物高氏柴胡玻璃質化法超低溫冷凍保存流程之探討。中興大學生命科學系所學位論文。
連結：
邱智聖。(2011)。青花菜和臺灣蝴蝶蘭花粉乾燥法超低溫冷凍保存流程之探討。中興大學生命科學系所學位論文。
連結：

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