三倍體香蕉懸浮細胞培養及體胚發生

Abstract

香蕉AAA基因組群華蕉系栽培種北蕉及寶島蕉之幼雄花序，切段培殖體，接種於含IAA 1㎎/L及NAA 1㎎/L之逆分化培養基，另參試組合2,4-D（2、4 ㎎/L）及Picloram（2㎎/L），可獲得具擬胚之顆粒狀癒合組織。含2,4-D之培養基，可誘導顏色較深黃、顆粒較大之癒合組織，且發生率較高。Picloram 2㎎/L則可誘導出顏色淺黃、顆粒較小且數量較多之癒合組織，褐化機率也較低。細胞懸浮培養則採用TB5培養基，北蕉可於懸浮培養4-6月後形成均質之細胞系。寶島蕉之癒合組織則易於懸浮培養過程中出現細胞增大與液胞化，不易釋放出胚性細胞，經6個月培養後，仍無法建立均質細胞系。 懸浮細胞胞外pH測定及細胞極化誘導，參試材料為AAB cv. Raja細胞系，細胞系維持繼代培養於TB5增生培養基，添加NaH2PO4及MES（2-N-morpholino-ethanesulfonic acid）處理14天，細胞自調生長至特定位階，而無法朝極化生長。外控pH 5.0-5.3及再生培養基添加MES培養，隨極化時間延長，第一次不對稱分裂細胞比率增加，以SH3 + MES 10g/L處理之不對稱分裂提升時間點較早，處理期間pH穩定維持於5.22 ± 0.24，但以外控pH 5.0-5.3 之不對稱分裂雙胞比值較高，約為1.22。經極化誘導，可使細胞同步朝向極化生長，於第7天形成原胚或球胚。AAA基因組以北蕉懸浮細胞更換為再生培養基及再生培養基添加MES 10g/L，兩處理皆可誘導細胞朝向極化，形成具完整原表皮之球胚。添加MES 10g/L於處理期間胞外pH維持於5.19 ± 0.26，並具促進前胚發生，提早發育為增大之球胚期。 胚性細胞平板培養體胚誘導試驗，參試預培養包括培養基組成、及MES添加對體胚形成之影響，並測試再生培養液用量及細胞團大小分及對體胚發生的影響。Raja及北蕉胚性細胞於平板前以再生培養基添加MES預處理，以SH3 + 10 g/L MES處理2週，細胞可朝極化生長、同步誘導進入球胚期，平板後可得生長較同步、早熟性且形態正常之體胚。Raja胚性細胞於平板前進行大小分級，較大之細胞團易產生叢生胚，60目以下之細胞團可產生個別之體胚。

Banana Pei-Chia and Formosana（Musa spp. Sub-group Cavendish AAA）cultured in MS medium with complex auxin（IAA 1㎎/L、NAA 1㎎L）and 2,4-D、Picloram addition, could induce embryogenic calli with embryoids. The callus formation rate was higher when the mediums were added 2,4-D, and the calli induced was granular、dark yellow. When use Picloram 2㎎/L as the sole strong auxin, explant browning rate was lower, and calli formed with light yellow、smaller granule and more callus amount. During the suspension culture, Pei-Chiao could formed more uniform cell line after 4-6 month culture, while calli of Formosana turned to enlarge instead of release embryogenic cells. After 6 months of culture, still could not develop the cell line of Formosana. AAB cv. Raja embryogenic cells cultured in proliferation medium TB5 with NaH2PO4 and MES (2-N-morpholino-ethanesulfonic acid) for 14 days, cells auto-regulated to steady state and could not turn to polar growth. Controlling extracellular pH at 5.0-5.3 and cultured in SH3、SH3 with MES 10g/L led up to the asymmetric division. Moreover, the cells cultured in SH3 with MES 10 g/L were polarized earlierly, and kept steady extracellular pH at 5.22 ± 0.24 .Those cell lines treated by controlling extracellular pH 5.0-5.3 possess the higher axis ratio at 1.22 . After polarization treatment for 7 days, the embryogenic cells could formed proembryo or globular bodies.The embryogenic cells of AAA cv. Pei-Chiao could also be induced to polarization and formed globular bodies with complete protoderm when cultured in SH3 and SH3 with MES 10 ㎎/L. The extracellular pH of Pei-Chiao could be kept at 5.19 ± 0.26 in SH3 with MES 10 ㎎/L. Raja and Pei-Chiao pretreated by SH3 with MES 10 g/L for 2 weeks, cells tend too form globular bodies synchronously, and could produce synchronous、separate and normal somatic embryos after plating culture. Embryogenic cells of AAB cv. Raja classified according to size before plating, the larger cell mass tended to form aggregated embyos, since the cell clusters under 60 mesh could form separate ones.