Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/37071
標題: 甘藷體胚與器官形成及植株再生之研究
Studies on in vitro somatic embryogenesis, organogenesis and plant regeneration of sweet potato ( Ipomoea batatas L.)
作者: 鄭皓鴻
Cheng, Hao-Hung
關鍵字: sweet potato;甘藷;somatic embryogenesis;organogeesis;plant regeneration;plant tissue culture;plant growth regulator;ehtylene inhioitor;體胚形成;器官形成;植株再生;植物組織培養;植物生長調節劑;乙烯抑制劑
出版社: 農藝學系
摘要: 
甘藷體胚與芽體形成及植株再生之研究
中 文 摘 要
在甘藷體胚形成之部分,為了瞭解不同種類、濃度之auxins及基礎培養基對甘藷體胚形成之影響,本研究以栽培種甘藷台農67號(TNG67)及台農68號(TNG68)頂芽與側芽之莖頂做為培植體,接種於以MS及修飾之MS為基礎培養基,分別添加不同濃度之2,4-D、與4-FA(0.1、0.5、1 mg/l)之培養基中,暗處理6~8週進行胚性癒合組織之誘導,而後再繼代於原來之培養基或含有0.5 mg/l ABA之MS培養基中進行體胚之分化;在器官形成方面,以前述二品種不同葉片位之葉片與葉柄以及根做為培植體,為了瞭解不同種類、濃度的auxins(IAA(0.1, 0.5, 1, 2 mg/l)、IBA(0.05, 0.1, 0.5, 1 mg/l)、NAA(0.05, 0.1, 0.5, 1 mg/l)及picolinic acid(0.1, 0.5, 1, 2 mg/l))、不同培養日數之培植體(培養20、30及60日之無菌苗)、不同扦插培養基(MS + 30 g/l sucrose、MS + 30 g/l sucrose + 1 mg/l IAA、MS + 60 g/l sucrose、LS + 30 g/l sucrose + 1 mg/l IAA及MS + 60 g/l sucrose + 1 mg/l IAA)及不同濃度乙烯抑制劑(AgNO3(5 mg/l and 10 mg/l) and CoCl2(0.65 mg/l and 1.3 mg/l))對甘藷芽體再生的影響;以甘藷台農68號之葉片、葉柄及根做為培植體,以探討不同醣類(15 g/l, 30 g/l, 45 g/l 蔗糖或果糖及 15 g/l 蔗糖 + 15 g/l 果糖)、鉀離子(添加 KH2PO4及KCl 使N: K=1:2 或1:3)及GA3濃度(0.1, 0.5 and 1 mg/l),對甘藷芽體再生的影響;另外並以石蠟切片及掃描式電子顯微鏡觀察甘藷之體胚形成及芽體再生,結果摘要如下:
1. 甘藷台農67號頂芽與側芽莖頂之胚性癒合組織誘導率低於台農68號;在含有低濃度(0.1 mg/l、0.5 mg/l) auxin之培養基中,二品種中或上節位之胚性癒合組織誘導率,顯著優於下節位;對於胚性癒合組織誘導,以培養基中添加4-FA之效果優於2,4-D,其中以0.5 mg/l 4-FA之誘導效果最佳;以修飾MS基礎培養基對胚性癒合組織誘導未必優於以MS基礎培養基;將胚性癒合組織繼代於含有0.5 mg/l ABA之培養基中有利於體胚之分化。
2. 整體而言台農67號的芽體再生率低於台農68號;根的芽體再生率較高,葉片次之,葉柄最低;不同葉位之間,以中、上葉位之芽體再生率較高;不同種類之auxins,最適於芽體再生之濃度不盡相同,但auxins濃度太高,不利於芽體再生;在培養初期,若葉片無法維持綠色,則即使有根之形成,仍然無法有芽體之再生。
3. 取自培養20日無菌苗之葉片培植體,對台農67號芽體再生率之增加確實有助益,但對於台農68號而言,則反而有抑制作用;無菌苗之株齡愈高愈不利於芽體之再生;扦插培養基中添加IAA,僅有利於台農67號葉片培植體之芽體再生;培養基中添加乙烯抑制劑有助於葉片綠化之維持,但整體而言不利於芽體再生;添加乙烯於培養基中導致芽體再生率降低,由此可知適當乙烯含量對於芽體之誘導是必要的。
4. 培養基中添加KCl,使氮鉀比為1:2之MSBKC1(MS + 0.1 mg/l IBA + 0.94g/l KCl)培養基芽體形成率高於對照組;培養基中低醣類濃度不利於芽體之再生,但提高醣類濃度則不利於葉片培植體發根;隨著醣類濃度提高,葉片培植體之芽體再生率有增加的情形,但根培植體之芽體再生率則反而有下降的趨勢;培養基中添加蔗糖之芽體再生率,高於添加果糖之芽體再生率;高濃度之醣類,除了做為能量及碳源外,亦有滲透調節的作用;添加GA3不利於芽體再生,但有利於根培植體之膨大及澱粉之累積。
5. 各個處理中葉柄之芽體再生率,均低於葉片之芽體再生率,顯示葉片身的存在有利於芽體再生;各葉片位之芽體再生以中、上葉片位葉片培植體較佳,顯示培植體的年齡愈年輕,對芽體再生愈有利,但由不同培養日數及添加乙烯抑制劑的試驗中得知,培植體的成熟度及適當的乙烯對於芽體再生是必要的。由影響芽體再生之不同因子中,可以更瞭解甘藷台農67號及台農68號的差異。
6. 甘藷芽體再生的方式,是由葉柄基部分化出根,而後由根上形成芽體或葉柄產生芽體;前者利用石蠟切片與掃瞄式電子顯微鏡之觀察,發現芽體是由根上之突起分化產生;品種、培植體之培養日數、扦插培養基之成分、誘導培養基中auxins及營養成分之種類與濃度、培養基中乙烯抑制劑與鉀離子來源之種類及其濃度,均會影響葉片培植體芽體再生部位之改變。

Studies on in vitro somatic embryogenesis, organogenesis and plant regeneration of sweet potato ( Ipomoea batatas L.)
Abstract
In order to studies the effects of different types and concentration of auxins and basal media for somatic embryogenesis of sweet potato, the plantlets in vitro of sweet potato of cultivars TNG67 and TNG68 were used as material source. The shoot tips of apical and lateral buds from plantlets mentioned above were cultured on MS and modified MS basal media contained with different concentration of 2,4-D and 4-FA, respectively. After culture 6~8 weeks in dark, the induced embryogenic callus were subcultured on the same fresh media or the medium supplemented with 0.5 mg/l ABA for differentiation of embryogenic callus. To understand the effects of different types and concentration of auxins, sucrose and ethylene inhibitors on organogenesis, explants leaf, petiole and root of TNG67 and TNG68 were cultured on these media contained with some agents or treatments including(1) different types of auxin were added to MS basal medium(e.g. IAA(0.1, 0.5, 1, 2 mg/l), IBA(0.05, 0.1, 0.5, 1 mg/l), NAA(0.05, 0.1, 0.5, 1 mg/l) and picolinic acid(0.1, 0.5, 1, 2 mg/l)); (2) explant from different culture time of plantlet(after cultured for 20, 30 and 60 days); (3) exlants from different media for plantlet(e.g. MS + 30 g/l sucrose, MS + 30 g/l sucrose + 1 mg/l IAA, MS + 60 g/l sucrose, LS + 30 g/l sucrose + 1 mg/l IAA, MS + 60 g/l sucrose + 1 mg/l IAA); (4) ethylene inhibitors AgNO3(5 mg/l and 10 mg/l) and CoCl2(0.65 mg/l and 1.3 mg/l) were added to media contained 0.1 mg/l NAA or 1 mg/l piciolinic acisd, respectively. The explants leaf, petiole and root of TNG68 were also cultured on the media contained with different types and concentration of sugar(15 g/l, 30 g/l, 45 g/l sucrose or fructose and 15 g/l sucrose + 15 g/l fructose added in MS basal medium), K+(adding KH2PO4 and/or KCl to MS basal medium and making N: K=1:2 or 1:3) or GA3(0.1, 0.5 and 1 mg/l). The somatic embryogenesis and organogenesis of sweet potato were observed by ways of the paraffin section and scanning electron microscope. Results were summarized as follows:
1. The induction rate of embryogenic callus from explants of TNG67 was lower than that of TNG68. For both cultivars, the induction rates of embryogenic callus from upper and middle node explants were higher than those from the lower node explants cultured on media contained with low concentration of auxins(0.1 mg/l、0.5 mg/l). The rates of embryogenic callus induction on media contained with 4-FA were better than those contained with 2,4-D. For advanced effect of embryogenic callus induction, it is not necessary to modify the composition of MS basal medium. For somatic embryo differentiation, it is better to subculture the embryogenic callus to the medium contained with 0.5 mg/l ABA.
2. The rates of shoot regeneration of TNG67 were lower than those of TNG68 generally. Among the three explants, the shoot regeneration rate of root was followed with those of leaf and petiole. The shoot regeneration rates from upper and middle leaves were higher than the other. Among different auxins, the appropriate concentration for shoot induction are different. With increasing concentration of auxins, shoot regeneration showed a tendency to decline. At early culture stage, if the leaf explant could not keep green, there was no shoot regeneration even the roots had been induced.
3. The leaf explant from plantlet after cultured 20 days was good for shoot regeneration of TNG67, but was bad for TNG68. The older the plantlet was, the lower the shoot regeneration rate was. It was only useful for shoot regeneration from leaf explant of TNG67 that the media for plantlet growth was supplemented with IAA. Media contained with different ethylene inhibitors were advantage to keep leaf green, but no advantage to induce shoot regeneration. The shoot regeneration rate declined with the concentration increase of ethylene. It was suggested that appropriate concentration of ethylene was useful for shoot regeneration.
4. The ratio of N/K attained to 1 : 2 by adding KCl into medium was noted BKC1 medium(MS + 0.1 mg/l IBA + 0.94g/l KCl). The shoot regeneration rate and shoot mean was higher than that on the control. Low sugar concentration was disadvantage to induce shoot regeneration and high sugar concentration was bad for leaf rooting. The shoot regeneration rates on the media contained with sucrose were higher than those on the media contained with fructose. The shoot regeneration rates of leaf increased with sugar concentration increase but those are opposite for root explant. Except for energy and carbon source, high concentration of sugar also had osmotic effect. GA was disadvantage for shoot regeneration but was advantage for root swelling and starch accumulation.
5. Among treatments,it was found that the regeneration rates of petiole were much lower than those of leaf. It was suggested that the exist of lameria was advantage to shoot regeneration. The shoot regeneration rates of upper and middle leaf position were higher than that of lower leaf position. Although younger explants seemed to shoot regeneration, appropriate mature of explant was more important for shoot regeneration. On different factors for shoot regeneration between TNG67 and TNG68, the differences of two cultivars became more distinguished.
6. There were two ways for shoot regeneration of sweet potato. One was from the node of leaf-derived root, and the other was from leaf base. The shoot regeneration way of the former was from the node of leaf-derived root by the observation of the paraffin section and scanner electron microscope. The factors involved shoot regeneration way included cultivar, the types and concentration of auxin, source plant age of explant, the contents of medium for source plant growth, the types and concentration of ethylene inhibitor, the types and
URI: http://hdl.handle.net/11455/37071
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