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研究生：	黃子容 Huang, Tzu-Jung
論文名稱：	花藥培養生產辣椒雙單倍體方法的初探 Preliminary studies on producing pepper (Capsicum annuum ) double haploid by anther culture.
指導教授：	張松彬 Chang, Song-Bin
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	45
中文關鍵詞：	辣椒、雙單倍體、花藥培養、胚發生
外文關鍵詞：	Peppers, Double haploid, Anther culture, Embryogenesis
相關次數：	點閱：68 下載：8
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雙單倍體是將特定的單倍體植物器官（如胚珠、花藥或花粉）透過組織培養的方式產生。在誘導癒傷組織形成或是胚發生後，它們在特定的分化及誘導培養條件之下將染色體複製並形成同型合子（純系）。不同於傳統利用多次自交的方式獲得純系，雙單倍體技術更節省時間也更有效率。雙單倍體的產生對於學術和農業研究發展有相當大的貢獻，例如作物育種、基因型研究、突變研究等。然而，建立針對不同物種甚至是不同栽培種的花藥培養和器官分化的培養條件相當困難。近年來已經有許多關於不同植物與栽培種培養雙單倍體的相關研究。在這篇論文中，我們針對亞蔬中心的兩個辣椒栽培種SP112-2-3 與“Susan's Joy”（AVPP9905）發現了一些不同的花藥培養方法。
首先，由於花苞的大小與花粉發育階段密切相關，我們測量了不同大小的花苞，並鑑定內部花粉的小孢子發育階段，以尋找最佳的花藥培養材料。接著，我們將適合的花藥（其小孢子處於單核期）種在不同的花藥培養基中誘導癒傷組織的產生。之後，將癒傷組織轉移到其他培養基中以誘導產生出芽和根。
兩個辣椒品種在花藥培養的條件方面有所不同：針對SP112-2-3（甜椒與辣椒的雜交種），我們使用長度5mm以下的花苞作為花藥培養的材料。其花藥培養基的最佳條件是含有5% (w/v)蔗糖、2 μM玉米素（zeatin）、1μM NAA和1％ (w/v) 活性碳的MS固態培養基。值得一提的是，胚發生在這樣培養基中能夠被觀察到。另一方面，針對“Susan's Joy”（AVPP9905），我們使用長度低於 3.1 mm的花苞作為花藥培養的材料。其花藥培養基的最佳條件是含有5% (w/v)麥芽糖、2 μM玉米素（zeatin）、1 μM NAA和1％ (w/v)活性炭的B5固態培養基。然而，在誘導分化的培養基研究方面，沒有任何癒傷組織被誘導產生芽或根。
在這項研究中，我們發現了不同於先前研究的花藥培養基成分。此外，我們發現在花藥培養中，提高培養基中糖的濃度可能會促進這兩個品種的癒傷組織誘導率。而某些不同濃度的生長調節劑也可能影響花藥培養的癒傷組織誘導率。因此，我們在本論文中改進了一些辣椒的花藥培養條件。

Double haploids are generated from tissue culture of specific haploid plant organs such as ovules, anthers or pollens. After inducing callus or embryogenesis, they are cultured in differentiation inducing and chromosome doubling conditions to become homozygous. Different from the traditional self-breeding method to get pure lines, the double haploid technique requires less time and more effective. Additionally, double haploids are useful for academical and agricultural contributions such as crop breeding, genotype researches, mutation studies, etc. However, it is difficult to create protocols suitable to anther culture and organ differentiation of different species. Therefore, there were many researches about different plants published in recent years. In this study, we found some different anther culture protocols for culturing two different pepper cultivars, SP112-2-3 and ‘Susan’s Joy’ (AVPP9905), in World Vegetable Center (AVRDC).
First, because the size of buds is closely related to the pollen development stage, we measured different size of buds and checked the inside microspore developmental stage to find the best culturing material for anther culture. Then, we planted the appropriate anthers, whose microspores undergo uninucleate stage, in different anther culture medium to grow callus. After that, the callus is transferred to other medium for inducing shoots and roots.
For the two cultivars, there are different conditions for anther culture. For SP112-2-3, the hybrid of sweet pepper and hot pepper, we used the buds under 5 mm in length for anther culture. The best condition for the anther culture medium was the MS basal medium containing 5% (w/v) sucrose, 2 μM zeatin, 1 μM NAA, 1% (w/v) activated carbon, and 0.8% (w/v) agar. It is worth mentioning that the embryogenesis happened in this medium. On the other hand, for ‘Susan’s Joy’ (AVPP9905), we used the buds under 3.1 mm in length for anther culture. The best condition for the anther culture medium was the B5 basal medium containing 5% (w/v) maltose, 2 μM zeatin, 1 μM NAA, 1％ (w/v) activated carbon, and 0.8% (w/v) agar. However, for the differentiation medium, there was no any shoots or roots generated from callus.
In this study, we found different compositions of anther culture medium. Moreover, we found that the increasing concentration of sugar may promote the callus induction rate for both cultivars. The different concentrations of some growth regulator may also influence the callus induction of anther culture. Therefore, we improved the anther culture condition for peppers in this thesis.

摘要  I
Abstract  III
Contents  V
List of Tables  VII
List of Figures  Ⅷ
Chapter 1  Introduction	1
1  Peppers (Capsicum annuum)  1
2  Breeding pure lines  1
3  Double haploid	2
4  Methods for culturing double haploid  3
4.1  Androgenesis  3
4.1.1  Anther culture	4
4.1.2  Microspore culture  4
4.2  Parthenogenesis	5
5  Possible ways for microspores to develop into haploids  5
5.1  Embryogenesis	5
5.2  Organogenesis	5
6  Factors affecting the efficiency of anther culture	6
6.1  Plant materials	6
6.2  Microspore development stage  8
6.3  Pretreatment	10
6.4  Heat shock pretreatment	10
6.5  Cold shock	11
6.6  Colchicine treatment	11
6.7  Anther culture medium composition  12
6.7.1  Basal medium	12
6.7.2  Carbohydrates	13
6.7.3  Plant growth regulators  14
6.7.4  Active carbon (active charcoal)  15
6.8  Culture condition  16
6.8.1  Temperature  16
6.8.2  Light	17
7  Purpose  17
Chapter 2  Material and method   19
1  Pepper materials  19
2  Pollen developmental stage confirmation  19
3  Cold shock	20
4  Anther culture medium  20
5  Anther culture  20
6  Roots and shoots induction	21
Chapter 3  Results  22
1  Pollen developmental stage confirmation  22
2  Anther culture  22
2.1  The anther culture of SP112-2-3	23
2.2  Anther culture of ‘Susan’s Joy’ (AVPP9905)  23
3  Roots and shoots induction	24
Chapter 4  Discussion	25
1  Microspore development stage  25
2  Anther culture conditions	25
2.1  Basal medium  26
2.2  Plant growth regulators	26
2.3  Carbohydrate concentration  27
2.4  Cold shock pretreatment	27
2.5  Light  28
3  Differentiation induction	28
4  Future research directions	29
Chapter 5  Reference	30
Chapter 6  Tables  36
Table 1  The anther culture medium  36
Table 2  Differentiation medium for growing shoots or roots from callus.  37
Table 3  The confirmation of the bud size related to the inner microspore developmental stage.	39
Chapter 7  Figures  40
Figure 1  Pollen developmental stage confirmation  40
Figure 2  Anther culture of SP112-2-3.	41
Figure 3  The callus induction rate of SP112-2-3 anther culture.  42
Figure 4  Anther culture of ‘Susan’s Joy’ (AVPP9905).  43
Figure 5  The callus induction rate of ‘Susan’s Joy’ (AVPP9905) anther culture.  44
                                    

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