唐菖蒲花藥培養之研究

Abstract

本研究採用唐菖蒲六個商業栽培品種、射干及射干菖蒲為試驗材料進行花藥培養。結果顯示唐菖蒲及射干菖蒲花藥之最適培養時期為單核期，射干則為四分子期；六個唐菖蒲品種癒合組織及擬胚誘導率以 ‘Princess Margaret Rose’最佳(分別為45.6%和11.1%)；蔗糖濃度以6%，生長調節劑以2mg/l NAA及1mg/l Kinetin之組合對唐菖蒲及射干菖蒲花藥培養癒合組織誘導最好，而2mg/l NAA及1mg/l BA組合則有利於射干癒合組織之誘導。唐菖蒲 ‘Princess Margaret Rose’低溫(5℃)前處理花蕾24小時，可促進唐菖蒲花藥培養癒合組織的形成，擬胚之誘導則以處理12小時最佳。 將唐菖蒲花藥培養所誘導之癒合組織移植於含植物生長調節劑0.2 mg/l NAA,0.2 mg/l BA及3%蔗糖之分化培養基，90天後可獲得擬胚體與再生植株。液體培養癒合組織的增殖率較固體培養佳，鮮重較重，但分化再生植株則以固體培養為佳。唐菖蒲 ‘Princess Margaret Rose’品種花藥培養誘導之癒傷組織經繼代培養後之切片觀察，可明顯看到橢圓狀的癒合組織細胞，分化成小而密、排列整齊的薄壁細胞，進而分裂成芽原體(shoot primordia)，大部份的癒合組織會形成排列緊密的薄壁細胞，有些則形成巢狀(nest-like)、繩索狀(strand-like)或排列狀結構的管細胞(tracheary elements)，並有根及維管束的形成。 射干菖蒲在各培養基中皆能增殖，但外觀形態上1mg/l Kinetin所增殖之癒合組織呈根狀體，含BA之培養基則呈圓球胚狀體；將射干及射干菖蒲繼代增殖之癒合組織轉移至分化培養基，經培養90天後仍然沒有分化植株產生；經切片觀察，癒合組織外層細胞小而排列緊密，內層細胞則為排列鬆散之不規則細胞。 唐菖蒲 ‘Princess Margaret Rose’及 ‘Mascagni’ 染色體數為60條(2n = 60 = 4X)，再生分化之植株經根尖壓片觀察，染色體數為30條(2n = 30 = 2X)，因此確定再生分化植株為半倍體植株，但唐菖蒲之再生分化半倍體植株移出種植後，易產生根部褐化現象，因此瓶苗馴化問題仍有待進一步的探討與研究。

Six commercial Gladiolus cultivars, one Belamcanda chinensis and one Tritonia ×crocosmaeflora were used to investiage callus induction and plant regeneration via anther cultures. Results showed that the best developed anthers of Gladiolus and Belamcanda chinensis for culturing in vitro were at uninucleate microspore stage, but that of Tritonia was at tetrads microspore stage. It was found that the highest rate of callus(45.6%) and embryoids(11.1%) induction was the anther culture of ‘Princess Margaret Rose' among six Gladiolus cultivars. Half strength medium of Murashige and Skoog''s (1962) basal salts except iron modified with 6% sucrose, 2mg/l NAA and 1 mg/l kinetin was the best for culturing Gladiolus and Belamcanda anthers, but for culturing Tritonia modified with 6% sucrose, 2mg/l NAA and 1 mg/l BA instead. The highest increase in anther-derived callus and anther-derived embryoids were low-temperature(5℃) pretreatment 24 hours and 12 hours of Gladiolus ‘Princess Margaret Rose' . The produced embryogenic callus was able to differentiate into embryoids and plantlets after having been transferred to medium containing 0.2 mg/l NAA, 0.2 mg/l BA and 3% sucrose 90 days. The proliferation rate and the fresh weight in the liquid culture were better than that on the solid culture . But the regeneration of plantlets on the solid culture would raise more significantly. Observation of paraffin section of callus from subculture of Gladiolus ‘Princess Margaret Rose' showed the small and dense parenchyma development from callus. Some showed nest-like tracheary elements or strand-like tracheary elements. Shoot primodium and root were developed from parenchyma and thus originated vascular bundle. It could be proliferated on every medium of Tritonia. But that callus subcultured mediums containing 1 mg/l Kinetin or BA that callus showed Rhyzome type or embryo. The callus wasn't able to differentiate into embryoids and plantlet after having been transfered to differentiation medium of Belamcanda and Tritonia 90 days. Observation of paraffin section of callus from subculture of Tritonia showed that the outer cell of callus were small and dense , but the inter cell of callus were loose. Chromosome numbers of root - tip cells of Gladiolus ‘Princess Margaret Rose' and ‘Mascagni' were diploids (2n =60 = 4X). Chromosome numbers of root - tip cells of regeneration of haploid plants from anther culture of Gladiolus ‘ Princess Margaret Rose' and ‘ Mascagni' were thirty(2n = 30 =2X). Besides, the roots were brown and low survival rate after transplanted, so further studies in acclimatization of in vitro culture still left a lot to be desired.