流蘇之胚、幼花與胚乳組織之培養及體胚發生

Abstract

流蘇 ( Chionanthus retusus Lindl.)屬於木犀科 ( Oleaceae)，為台灣之原生稀有木本植物，盛花時一片雪白，樹型及花型極為優美，是具有發展潛力的觀賞花木，並且是具有多種療效的藥用植物。其種子具有上胚軸休眠性，扦插繁殖時難發根，因此被列為難再生植物。本研究利用流蘇的胚乳組織、幼花與細胞懸浮培養以及胚組織探討流蘇的胚性細胞增生與體胚發生，期能提供流蘇不同之培養途徑，可供微體繁殖之參考。 流蘇之胚乳組織生長期中，在膠狀期以前組織成水狀，無法固定在培養基上，接種後也未分化。取固化期的流蘇胚乳組織以MS 培養基搭配2,4-D 2 mg/L或5 mg/L處理，先與胚共培養 10天後再將胚剝離，可在胚乳組織上獲得 16.7%的逆分化頻率。 以 WPM配方搭配不同的生長素與BA組合，可使流蘇小花基部切口處或花萼與花房之間的位置產生透明的癒合組織。以picloram搭配 BA處理，則癒合組織的增生較多，且發生率高，其中以picloram 2 mg/L搭配 BA 1 mg/L 為高，有100%，而癒合組織較為鬆軟，較不具胚性。 以pH自動控制系統進行流蘇花序衍生細胞懸浮培養，持續控制胞外 pH在 4.4~5.0之間可影響細胞之狀態，使細胞進入游離細胞期而不易轉入體胚發生。相較之下控制胞外 pH在 5.7-6.1的細胞狀態比較快轉入體胚發生。以再生培養基(SH + NAA 0.4 mg/L)進行細胞懸浮培養，20天後可將細胞轉為原球胚期，細胞幾乎全為圓形、細胞質濃、富含澱粉粒的胚性細胞。 以 MS培養基培養流蘇貯藏六個月的胚體組織，以強活性生長素picloram或 dicamba 1-2 mg/L誘導，可使子葉的背軸面逆分化得到胚性癒合組織，再出現間接體胚發生。picloram 2 mg/L 處理得到的癒合組織增生量比2,4-D與 dicamba大。一個月後，可見未成熟的小型體胚從向軸面的黃色或白色半透明癒合組織間接發生。培養兩個月後，picloram 2 mg/L的處理可以得到35%的體胚發生率，平均每一個培植體可得到 2.35個體胚。以 MS培養基加入dicamba 1 mg/L培養之，培養兩個月可以得到 30%的體胚發生率與4.45個體胚(平均每一個培殖體)。 0.1 mg/L與 0.2 mg/L的ABA會使流蘇小型體胚(<3 mm)的發育停滯，並反而與癒合組織開始同時膨大，可發育成熟的體胚頻率為33.8%與38.89％。ABA 0.5 mg/L則有慢慢成熟轉綠的現象，1 mg/L使體胚型態變為淡綠卻細小，仍然會抑制體胚成熟或導致體胚不正常。2 mg/L與 ５ mg/L的ABA造成次生子葉的發生，體胚快速生長，並且抑制體胚的轉綠，使之呈白色透明。體胚成熟率可達81.82%與100%。 流蘇體胚在 MS基本培養基中成熟發芽情形較好，顏色很快即開始轉為深綠，且開始發根。以少量的強生長素如2,4-D 0.1 mg/L處理之，則可使較成熟的體胚快速發育，同時造成未成熟的體胚或連接體胚的癒合組織再次逆分化，形成具胚性的癒合組織，可從其上再發生體胚。

The fringe tree (Chionanthus retusus Lindl.) is a graceful flowering tree which tree crown is beautiful. It would like snowing on tree when fringe is flowing. Fringe tree is considered as a rare species and limited population in Taiwan. There is epicotyl dormancy on the seed and hard to rooting through cutting. The purpose of this research is to induce callus formation from endosperm, young floret, derived embryogenic cell for suspension culture, and embryo tissue, to study embryonic tissue cultures and somatic embryogenesis. Endosperm culture before glutinous stage was hard to success because the endosperm tissue is like liquid. It could not be affixed on the medium and differentiate on it. There was dedifferentiation and reproliferation in the solid stage endospermic tissue ( intacted with embryo culture for 10 days) on MS medium supplemented with 2,4-D 5 mg/L. The dedifferentiation frequency of endospermic tissue was 16.7%. There was transparent callus formation in calyx and ovary on WPM medium supplemented with different auxins(2,4-D or picloram) and BA. Callus formation was more and frequency of callus induced is higher by picloram and BA. Frequency of callus induced was 100% by picloram 2 mg/L and BA 1 mg/L but the callus was friable. Giving control extracellular pH 4.4-5.0 level, could made the young floret-derived cell suspension culture of fringe tree change the phase. The cell masses would be induced to free cells and cell cluster stage and had less somatic embryogenesis. Giving control extracellular pH 5.5-6.1 level could made it somatic embryogenesis faster. The cell suspension culture of fringe tree under SH (added NAA 0.4 mg/L) would appeared dense cell mass and some globular structures by 20 days after suspension. The callus formation from abaxial side of cotyledon and hypocotyl surface from zygote embryo tissue stored 6 month and then indirectly regenerate somatic embryogenesis on MS medium supplemented with picloram and dicamba 1-2 mg/L.The callus formation was more from zygote embryo tissue of fringe tree by MS medium and picloram 2 mg/L. Indirect somatic embryogenesis were from the yellow or white embryonic callus after 1 month. The frequency of somatic embryogenesis was 35% and average of somatic embryogenesis was 2.35 (somatic embryo/per explent) by picloram 2 mg/L after 2 month. The frequency of somatic embryogenesis was 30% and average of somatic embryogenesis was 24.45 (somatic embryo/per explent) by dicamba 1 mg/L. Small somatic embryo (< 3 mm) of fringe tree would be suppressed by ABA 0.1-0.2 mg/L on normal development and enlarge with callus. 33.8% and 38.89% of somatic embryo could be maturation. ABA 0.5 mg/L made embryo to turn green slowly and ABA 1.0 mg/L would suppress development of somatic embryos or made them to become abnormal. ABA 2 mg/L and 5 mg/L would induce secondary cotyledon formation, somatic embryo growing fast, maintain the color of cotyledon in white and transparent. 81.82% and 100% of somatic embryos were maturation. Somatic embryo development of fringe tree was better in MS medium without PGR. They began to turn green and rooting. Strong auxin like 2,4-D 0.1 mg/L could let bigger somatic embryo development but others would dedifferentiation to become embryonic callus. The embryonic callus could have somatic embryo again.