文心蘭之體胚發生與齒舌蘭輪點病毒鞘蛋白基因轉殖

Abstract

文心蘭（Oncidium Louise Elmore ‘Elsa’）幼葉培植體於含0.3與3 mg/l TDZ、3 mg/l BA、0.3、1與3 mg/l zeatin之1/2MS培養基，暗培養3個月後，不經癒傷組織，直接在向軸面形成體胚。較佳的處理是含3 mg/l TDZ之培養基，有55.0％的培植體形成平均3.53個體胚。將體胚培養在未含生長調節劑的培養基，經4個月，可發育成株高4公分的小植株。 以基因槍轉殖法轉殖病毒鞘蛋白基因（ORSV CP gene），探討文心蘭轉殖株對病毒病害之抗性。將文心蘭（Onc. Gower Ramsey）葉片培植體置於含5 mg/L TDZ的體胚誘導培養基，黑暗處理，利用基因槍法進行基因轉殖，於體胚誘導後第10週計算體胚數，之後放入含5 mg/l NAA的培養基培養4-6週，光照處理，最後以含500 mg/l kanamycin的培養基進行篩選。共獲得230株擬轉殖株，隨機挑選21株，進行PCR分析。PCR分析的結果顯示，未偵測到齒舌蘭輪點病毒鞘蛋白基因。

Explants of Oncidium Louise Elmore ‘Elsa’ young leaves produced somatic embryos from adaxial surfaces directly without an intervening callus within 3 months when cultured on a 1/2 MS basal medium supplemented 0.3, 3 mg/l TDZ or 3 mg/l BA or 0.3, 1, 3 mg/l zeatin. The better response was found at 3 mg/l TDZ. In this treatment, 55.0％ of leaf explants formed 3.53 somatic embryos. Somatic embryos developed into plantlets (4 cm in height) with several roots and shoots when cultured on a medium devoid of plant growth regulator after 4 months of culture.

1-cm-long leaf tip segments of Oncidium ‘Gower Ramsey’ were cultured on embryo induction medium (containing 0.5 mg/l TDZ). Until the 8th week of induction, excised leaves were bombarded every week by gene gun with plasmid pKyLx7. At the 10th week, embryos were transplanted to a basal medium supplemented with 0.5 mg/l NAA for 4 to 6 weeks. With light supplement 3 to 4 months, embryos could develop to 1-cm-high plants. Selection for putative transgenic plants were accomplished by using 500 mg/l kanamycin 2 weeks. There were 230 putative transgenic plants survived. Survival transgenic plants were randomly selected 21 plants to conform by PCR. However, the result showed there didn’t exist ORSV coat protein gene in 21 putative transgenic plants.