盤固草組織培養與基因鎗轉殖系統之建立

Abstract

盤固草A254 (Digitaria decumbens)為台灣地區栽培最廣的牧草品種，由於花粉不稔，造成傳統育種之困境，為加速傳統育種效率，期利用生物技術提供遺傳改良的另一個途徑。因此本研究之首要目標為建立盤固草基因轉殖技術及植株再生系統。 以盤固草未成熟花穗為培殖體，接種於含有2,4-D與BA、KN及CPPU不同濃度組合之MS培養基，探討細胞分裂素對盤固草癒合組織形態、癒合組織誘導率及植株再生率之影響。在含有2,4-D 2.0 mg/l及不同細胞分裂素的MS培養基，白色緊密癒合組織的誘導率KN 0.5 mg/l 為 100.0 %、BA 0.5 mg/l為95.0 %、CPPU 0.5 mg/l為 90.0 %，差異不顯著。將不同細胞分裂素誘導的癒合組織，接種至含有TDZ 0.05 mg/l 的MS培養基，其中KN 0.5 mg/l 誘導的白色緊密癒合組織有68.9 % 具植株分化率，BA 0.5 mg/l 為 96.7 %，CPPU 0.5 mg/l為80.0 %，而透明鬆軟的癒合組織甚少植株的分化。根據本試驗結果，以2,4-D 2.0 mg/l 與BA 0.5 mg/l 誘導的癒合組織，具有很高的植株再生率，此植株再生系統之建立將有助於後續細胞懸浮培養與基因轉殖之研究。 盤固草細胞懸浮培養方法之建立，以盤固草A254未成熟花穗，接種於含有2,4-D 2 mg/l的MS固體培養基，誘導透明鬆軟的癒合組織，可持續繼代培養於含有2,4-D 2 mg/l的MS液體培養基，至少六個月以上。一旦需要植株再生時，可於原懸浮細胞培養基添加BA 0.5 mg/l ，培養一週後，再移至含有2,4-D 2 mg/l及BA 0.5 mg/l 的固體培養基，以誘導白色緊密的胚性癒合組織。將這些胚性癒合組織移至含有BA 0.5 mg/l 或TDZ 0.05 mg/l 的MS培養基，分別有88.9 % 或87.7 % 的癒合組織可再生為完整植株。本研究結果，建立一個可促進細胞持續增殖，並保有高頻度植株再生能力的細胞懸浮培養方法。 利用盤固草組織培養系統，以基因鎗粒子轟擊的方式，將帶有CaMV35S 起動子、GUS或GFP報導基因及HPT基因篩選標示基因的載體，轉殖至盤固草幼穗誘導的癒合組織。以含有50 mg/l hygromycin 培養基進行癒合組織及再生植株抗性篩選。表現抗hygromycin 的癒合組織，在基因鎗轟擊後二週經組織染色分析，仍可穩定表現出GUS蛋白質活性。再生植株經由長達四個月的hygromycin篩選，經PCR分析部份植株帶有報導基因片段。目前已由497個經基因鎗轟擊後的癒合組織，篩選出16株抗hygromycin 的綠色正常植株，平均基因轉殖效率為3.2 %。本研究為盤固草以基因鎗基因轉殖後獲再生植株之首篇報告，可作為將來多倍體牧草基因轉殖之參考。

Pangolagrass A254 (Digitaria decumbens) is a forage species and has a wide adaption in Taiwan. Male sterility is a major limiting factor in traditional breeding program of pangolagrass. The objective of this study was to develop an efficient plant regeneration system from immature inflorescences and cell suspension culture of pangolagrass to accelerate crop improvement of this species by genetic transformation. Explants were cultured in MS medium supplemented with different concentrations of 2,4-D, KN, BA and CPPU. The effects of cytokinins on callus induction frequency and morphology were examined. The frequency of white and compact callus induced were KN 0.5 mg/l (100.0 %)，BA 0.5 mg/l (95 %), CPPU 0.5 mg/l (90.0 %) with 2,4-D 2 mg/l was supplied. Calli induced with different cytokinins and cultured on MS medium supplemented with TDZ 0.05 mg/l. The plant regenerated from white and compact callus with 2,4-D 2.0 mg/l and BA 0.5 mg/l were 96.7 %, KN 0.5 mg/l (68.9 %) and CPPU 0.5 mg/l (80.0 %) respectively. There was no plant regeneration from friable and transparent callus. The optimal medium for callus induction was 2,4-D 2 mg/l with BA 0.5 mg/l and plant regenerated with BA 0.5 mg/l. The high frequency of plant regeneration was very essential and beneficial for cell suspension cultures and genetic transformation studies. The transparent and friable calli were used to establish and maintain the suspension cultures with 2,4-D 2 mg/l for at least 6 months. For plant regeneration, the embryogenic calli were induced by adding BA 0.5 mg/l in suspension medium for one week and then transferring to solid medium with 2,4-D 2 mg/l and BA 0.5 mg/l. The addition of BA in the suspension medium enhanced embryogenic callus formation and the ability of plant regeneration. The plant regeneration frequency of the embryogenic calli derived from cell suspension system reached up to 88.9 % and 87.8 % cultured with BA 0.5 mg/l and TDZ 0.05 mg/l, respectively. The results showed that the calli could keep proliferating and regenerating into the plantlets with high frequency under control. Embryogenic callus, initiated from immature inflorescence of pangolagrass, was bombarded with a vector mainly containing the CaMV35S promoter, GUS or GFP reporter gene and HPT (hygromycin phosphotransferase) selectable marker gene. Stable calli with GUS stained were visualized for 2 weeks after bombardment. Plantlets regenerated from callus with hygromycin selection medium for four months were analyzed by PCR, and indicate the reporter gene integration into the plant genome. The transformation efficiency was 3.2 % which was estimated with 16 plantlets regenerated with hygromycin selection medium from 496 calli after particle bombardment. These results would be played a very important base of polyploid grass and it is the first report on transgenic plants production of triploid pangolagrass using particle bombardment technique.