蘭花花粉塊組織培養下小孢子發育的研究

Studies on the development of orchid microspores in vitro culture of pollinium

鄭伊婷(Yi-Ting Cheng)

指導教授 ： 盧虎生

共同指導教授 ： 張松彬(Song-Bin Chang)

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01102

摘要

花藥培養產生之單倍體植株經染色體加倍後獲得的純合同質雙單倍體，可廣泛應用於作物品種改良、基因圖譜、基因轉殖、突變遺傳和細胞遺傳等方面的研究。本論文使用臺灣蝴蝶蘭、姬蝴蝶蘭等兩種臺灣原生種蘭花和滿山紅蝴蝶蘭、迷文文心蘭等兩種啇業品種蘭花的花粉塊作為材料進行培養，探討不同因子對花粉塊誘導之影響，並觀察小孢子的發育形態，以嘗試建立起初步的蘭花花藥培養系統。
姬蝴蝶蘭6 mm以上花苞內的花粉塊經50 ppm GA溶液處理一天後，置於蔗糖濃度為6%的培養基內，並於黑暗環境下進行培養時，會具有較高的誘導率。迷你文心蘭的花苞長度為6-7 mm，此即小孢子處於單核期至雙核期階段時最適合用來進行培養，且培養基含糖量為6%時誘導表現會最佳，20%濃度下則無法形成針狀構造。從臺灣蝴蝶蘭的花苞及所有蘭花的盛開花朵中所取出的花粉塊，無論在何種處理下皆無法被誘導。
從外觀上來看，成功被誘導的花粉塊表面會產生許多透明的針狀結構，而顯微鏡下觀察到的小孢子細胞也有向外延伸出長管狀構造的情況出現。部分小孢子的外層物質會遭到瓦解，因此造成小孢子間會相互交疊或彼此游離分散，也有些小孢子的外層物質雖然仍存在，但內含的小孢子數量會多於4個而形成一個形狀不規則的小孢子團。
將產生透明針狀結構的花粉塊移至含有3%蔗糖、1 mg/l NAA、4 mg/l kinetin和0.2%(w/v)活性碳的MS固態培養基中誘導其進行器官分化，培養基內的花粉塊會逐漸白化，且形成的針狀構造會變的粗短、稀疏或完全消失，但沒有任何芽體或不定根的形成。

關鍵字

蘭花 ； 花藥培養 ； 小孢子發育 ； 植物激素 ； 蔗糖濃度

並列摘要

Homozygous double haploids (DHs) generated from haploid plants through chromosome doubling in anther culture, are widely used in crop breeding programs. It has been useful in such research areas as gene mapping, genetic transformation, mutation studies, and cytogenetic studies. In order to investigate effects of different factors on induction of pollinia and development of microspores, and to establish primary protocols of anther culture of orchids, pollinia from two Taiwan native species orchids Phalaenopsis aphrodite sub. formosana Shimadzu, Phalaenopsis equestris (Schauer) Reichb. f. and two commercial cultivars Tolumnia hybrid, Phalaenopsis spp. hybrid ‘Manshanhung’ were used as materials in this thesis.
The percentages of induction were higher when pollinia from the ca. 6 mm length of buds of P. equestris (Schauer) Reichb. f. were treated with 50 ppm GA solution for one day and cultured on the medium with 6% sucrose in the dark.
The results indicated that buds of Tolumnia hybrid about 6-7 mm in length was appropriate for culture, and the microspores at that bud size were at uninucleate to binucleate stage. Besides, the best response was obtained when medium containing 6% sucrose but pollinia can’t differentiate in the 20% sucrose medium. Pollinia from premature buds of Phalaenopsis aphrodite sub. formosana and from open flowers of four orchids did not have any reaction in all treatments.
There are a lot of crystalloid needle-like structures on the surface of differentiated pollinia and long tube-like structures could be observed on microspores. Substances that hold microspores together disappeared in some case, so microspores might be on top of another or away from others. Some microspores divided repeatedly and resulted in syncytia of more than 4 nuclei.
The color of pollinia turned white and the needle-like structure became shortened or disappeared when the pollinia were subcultured in the differentiation medium on MS salts, 3% sucrose, 1 mg/l NAA, 4 mg/l kinetin, 0.2% (w/v) charcoal, and 0.8% (w/v) agar. Shoots and roots could not be generated in the tested differentiation medium.

並列關鍵字

Orchids ； Anther culture ； Microspore development ； Plant hormones ； Sucrose concentration

參考文獻

張有明、劉邦基、蕭吉雄。1996。甘藍及青花菜的花藥培養 Ⅰ.品系、發育期及培養基與再生之關係。中華農業研究 45(1): 35-46。

曾銀位、王瑞章、葉茂生。2004。蔗糖對花生花藥癒合組織誘導及分化的影響。台南區農業改良場研究彙報 43: 18-27。

曾銀位、葉茂生。2000。花生花藥培養的研究 Ⅲ.ABA 及不同種類澱粉對花藥癒合組織分化之影響。農林學報49(3) : 25 – 38。

莊茗翔。2003。南西之雄不孕品系與黃葉瘤唇蘭花部形態發育及小孢子發生之比較。國立成功大學生物學研究所碩士論文。臺灣 臺南。125pp。

Arzate-Fernandez AM, Nakazaki T, Yamagata H, Tanisaka T (1997) Productionof doubled-haploid plants from Lilium longiflorum Thunb. Anther culture. Plant Sci 123:179–187