LA與LO型雜交種百合組織培養之癒合組織形成與植株再生

Abstract

試驗結果顯示將培植體鱗片進行分切處理，’Gel × PS’之鱗片基端癒合組織再生頻率最高，可達40％。完整鱗片之再生部位亦為靠近基盤位置。’Gel × PS’癒合組織再生位置多位於培植體基端，而’LD × CA’培植體頂端之形態發育多為不定芽及不定根之再生。將鱗片培植體四分切，由於培植體過小，誘導反應多為培植體稍微膨大，生長緩慢以及切口處褐化等現象，無法進行逆分化或誘導再生。 單一種類生長素之處理結果：Picloram對於癒合組織之誘導效果最佳，且隨著施用濃度增加而提高，’Gel × PS’、 ’LD × CA’、 ’Casa Blanca’最佳再生頻率分別為54％、70％、98％。2,4-D處理隨濃度提高，培植體褐化與畸形情形越嚴重，’Gel × PS’、 ’LD × CA’以2,4-D 5.0 mg．L-1處理分別產生88％、42％之褐化率。施用NAA容易誘導培植體分化多量不定根。處理Dicamba 1.0 – 2.0 mg．L-1對於癒合組織再生頻率較高，但培植體隨培養時間朝定向分化或褐化，導致癒合組織發生頻率降低。 處理組合以Picloram 0.5 mg．L-1、2,4-D 2.0 mg．L-1、以及kinetin 1.0 mg．L-1三種組合對於 ‘LD × CA’與’Casa Blanca’誘導癒合組織頻率較高，分別達38％與22％之誘導再生率。另以1.0 mg．L-1之Picloram與2,4-D組合zeatin 0.1 – 1.0 mg．L-1培養基進行誘導，三雜交品種對於2,4-D與zeatin之組合誘導不定芽再生頻率高，皆可達80％。2,4-D 1.0 mg．L-1組合zeatin 0.1 mg．L-1易誘導培植體形成不抽葉之白化叢生芽體，提高zeatin濃度會減少小鱗莖再生數目，但會抽葉。培植體對於Picloram與zeatin組合處理，較易朝向癒合組織之形成，但褐化與畸形情形也較嚴重。 添加不同濃度ABA於2,4-D 1.0 mg．L-1培養基，對‘LD × CA’誘導癒合組織再生頻率皆很低，培植體大多朝向不定芽與不定根之器官發生。低濃度ABA可促進鱗片葉之再生，而高濃度ABA處理誘導之小鱗莖較為膨大，但無抽葉現象。單獨施用ABA 2.5 mg．L-1之培植體之發根數與不定芽再生數皆降低，且生長緩慢。 在切片組織形態觀察方面，’Gel × PS’以Picloram組合2,4-D繼代培養，癒合組織表面呈現細胞小且細胞質稠密，相似於分生組織之構造，靠近中心之癒合組織則為液胞化之空心構造。

Explants of ‘Gel × PS’ from proximal ends showed the highest rate of callus regeneration, and the frequency was 40％. Regeneration positions of intact mini-scale cultures were also approach to the basal plates. Oppositely, explants of ‘LD × CA’ from distal ends formed adventitious roots or shoots. As a result of explants cut into four were too small to induce responses towards swelling slightly, growth slowly, and browning in cut sites. Explants couldn’t be induced to dedifferentiation or regeneration neither. Picloram was more effective for inducing callus than other kinds of auxin. The callus inducing efficiency went along with the concentrations. The highest frequency of callus formation respectively in ‘Gel × PS’, ‘LD × CA’, ‘Casa Blanca’ were 54％, 70％, and 98％. Explants with 2,4-D treatment showed an increased portion of brownish and malformed structures, and the browning frequency in ‘Gel × PS’ and ‘LD × CA’ were 88％ and 42％ in medium containing 5.0 mg．L-1 2,4-D. Multiple adventitious roots were stimulated by NAA. Explants treated with 2.0 mg．L-1 Dicamba were suitable for callus formation, but the frequency declined when cultured time passed. The explants could result in determinated differentiation or browning. More calli were formed well on MS medium supplemented with 0.5 mg．L-1 Picloram, 2.0 mg．L-1 2,4-D and 1 mg．L-1 kinetin for explants of ‘LD × CA’ and ‘Casa Blanca’, and the regeneration rates were 38％ and 22％. Shoots formation efficiencies were high on MS medium supplemented with 2,4-D (1.0 mg．L-1) and zeatin （0.1 – 1.0 mg．L-1）for three hybrid lilies. The frequencies could be above 80％. Multiple albino shoots without leaves were induced on MS medium supplemented with 1.0 mg．L-1 2,4-D and 0.1 mg．L-1 zeatin. Raising the concentrations of zeatin would decline regeneration numbers of bulblets with leaves emergence. Explants cultured in Picloram and zeatin combination developed towards callus formation, but the brownish and malformed cases were as well as more serious. Explants of ‘LD × CA’ cultured on MS medium containing ABA (0.1 – 2.5 mg．L-1) and 2,4-D (1.0 mg．L-1) induced towards organogenesis, but callus induction frequency was low. Low concentration of ABA increased scale number, while high concentration of ABA promoted non-leaving bulbing. Addition 2.5 mg．L-1 ABA to medium showed the decline in number of roots and shoots regeneration and tardy growth for explants. Morphological and anatomic observations in ‘Gel × PS’ sub-cultured with Picloram and 2.4-D showed the surface of the callus cells were small and dense, and they resembled meristemic structures. Cells inside the callus observed vacuolated and hollow structures.