日日春體胚發生與植株再生

Somatic Embryogenesis and Plantlet Regeneration from Embryogenic Callus Culture of ＂Catharanthus roseus＂

日日春利用莖段、胚軸、葉柄及小花蕾為培植體，於MS培養基添加0.1 mg•L-1 kinetin組合0.5-2.0 mg•L-1 2,4-D可誘導癒傷組織形成率達100%。以0.5 mg•L-1 2,4-D之處理，莖段所誘導癒傷組織生成面積最多（158.3 mm2），下胚軸培植體誘導之癒傷組織為114.8 mm2。細胞懸浮培養結果顯示，起始培養密度為25 mg•mL-1和50 mg•mL-1，細胞皆為典型S型曲線（sigmoid curve）生長，適當的懸浮培養繼代週期，分別為18-24天與14-20天為佳，其細胞分裂能力均能夠維持並增殖。此外藉由細胞懸浮培養觀察日日春體胚發生顯示，其早期胚分化從極化、極性軸選擇至不對稱細胞分裂約於2-4天內完成，並形成2胞期胚體細胞。培養後4-6天進入4胞期，8-10天達到8胞期的原胚發育，進而於培養後20天進入圓球胚發育階段。觀察顯示，從下胚軸或莖段培植體衍生之硬實轉綠的癒傷組織，為具胚性潛能之癒傷組織。另外，以BA處理胚性之癒傷組織團塊體胚，經八週培養，癒傷組織皆由淡黃色漸轉為淺綠色外觀，1.5mg•L^(-1)BA處理之體胚形成率可達70%，獲得體胚數達96個為最多，其中發育至魚雷胚和子葉胚之成熟體胚數也最多。顯示BA參與並影響日日春體胚增殖和成熟發育。藉由體胚發育解剖觀察，次級胚起源自初代癒傷組織團塊周邊細胞，且具有典型雙子葉植物體胚發育之形態構造，並進而在原培養基培養獲得胚苗轉換成小植株，而可建立日日春經由體組織培植體衍生胚性癒傷組織之體胚發生再生體系。 The effects of the stem segments, hypocotyls, petioles and young flower buds as explants cultured were studied on Murashige and Skoog (MS) medium supplemented with 0.1 mg•L^(-1) kinetin combining 0.5-2.0 mg•L^(-1) 2,4-D for callus induction of periwinkle [Catharanthus roseus (L) G. Don]. After 8-week culture, all treatments had the 100% callus formation. Medium containing 0.5 mg•L^(-1) 2,4-D resulted in maximum (158.3 mm2) callogenesis in the stem segment explant, followed by hypocotyl explant of 114.8 mm2. Suspension culture was constructed with initial densities of 25 mg•mL^(-1) and 50 mg•mL^(-1), the cell exhibited a typical sigmoid curve, and appropriate subculture cycles were 18-24 d and 14-20 d, respectively. Cell division and further proliferation maintained during subculture. Embryogenesis started with cell polarization, polar axis selection to asymmetric cell division, and then the formation of two-cellular embryonic cell within 2-4 d. The two-cell stage subsequently reached 4-cell stage after 4-6 d culture, and octant pro-embryoid stage after 8-10 d, and then the globular-embryoid stage after 20 d. The green hard callus derived from stem segments and hypocotyl explant showed the embryogenic competence. The effect of BA treatment on the proliferation and maturation of embryoids was studied using embryogenic clumps as explant, and all calli gradually changed color from pale yellowish to light green after eight weeks of culture. The 1.5 mg•L^(-1) BA treatment induced the highest embryo formation percentage (70%), and maximum somatic embryos (96), which reached torpedo- and cotyledonary stages. This result suggested that BA participated in the proliferation and maturation of somatic embryos. Anatomical observations of embryo development revealed that secondary embryoids formed from the surrounding cells of primary embryogenic callus mass. These embryoids showed a typical morphology and histological structure of dicot embryoid, and could develop to plantlets. A regeneration system through somatic embryogenesis was established by using embryogenic callus derived from somatic tissue explants of periwinkle.