二倍體香蕉胚乳組織程序性細胞凋零及其體外培養與再生

Abstract

參試兩倍體香蕉2n = 22，包括拔蕉(Musa balbisiana)及台灣芭蕉(M. formosana)，其中以拔蕉作為胚乳程序性細胞死亡( programmed cell death, PCD)時程之試驗材料，並取用二品種之胚乳於體外培養誘導癒合組織、建立懸浮系統及再生植株。探討胚乳發育過程中細胞膜通透性變化及核基因組段節之標誌，與程序性細胞死亡及體外培養再生能力之相關性。同時測試影響胚乳癒合組織發生之相關因子，並於胚乳衍生之懸浮細胞平板前藉由再生培養基及pH緩衝液之預處理提升平板後大量同步化體胚發生，並且利用流式細胞儀檢測不同培養階段由胚乳衍生之癒合組織所獲得之體胚苗之相對DNA含量變化。 拔蕉胚乳發育過程依胚乳形態可分為四個時期，即為液狀期、膠狀期、固化期及粉狀期。初生胚乳呈游離核型發育，因此早期液狀胚乳期其胚乳形態呈游離胚乳核，九月授粉之拔蕉胚乳以0.1% Evans blue染色觀察細胞膜通透性變化，於授粉後50天內之液狀期胚乳，僅胚乳外圍組織呈色，液狀胚乳因無細胞膜無法染色。授粉50天後因胚乳組織由外向中央細胞化，繞著中腔之最內層細胞膜分化仍未完全，故可被染色並可觀察到有一藍色環形成，此與細胞死亡無關。胚乳細胞化後之膠狀期無法被染色並於進入固化期，則再度由外向心呈色。DNA電泳分析結果顯示當胚乳發育至固化期(約授粉後72天後)有DNA降解條帶出現，而與Evans blue染色結果相呼應，推測為程序性細胞死亡所致。 拔蕉膠狀期胚乳接種含有2,4-D、cytokinins之 MS培養基能被導向逆分化形成癒合組織，以含有2,4-D 1mg/L、BA 0.5 mg/L、TDZ 2 mg/L及GA 0.5 mg/L之生長調節劑組合獲得37.5%癒合組織發生率較佳。以九月開花授粉之拔蕉參試不同發育期胚乳組織當培植體，結果顯示液狀期並無逆分化能力，癒合組織發生頻率隨膠狀期生長而增加，至中期約53%發生率( 授粉後64天)，固化期約授粉後75天則發生率急降為2-12 %，此乃與胚乳細胞凋亡有關。同配方培養基添加麥芽粹取物200mg/L能促進癒合組織發生，但添加檸檬酸抗氧化物1-5 mg/L則無促進癒合組織形成效果。台灣芭蕉膠狀中期胚乳接種MS培養基，添加2,4-D 1.0mg/L、BA 0.5 mg/L、TDZ 0.1 mg/L、GA 1.0 mg/L培養30天觀察，癒合組織發生率為35.0%。GA濃度增加會延遲胚性癒合組織的形成，且台灣芭蕉胚乳培養獲得胚性癒合組織時間較拔蕉長，約需三個月。 台灣芭蕉胚乳癒合組織接種於SH3 ( SH salt + 2ip 0.2mg/L+ kinetin 0.1mg/L+ zeatin 0.05mg/L+α-biotin 1mg/L+glutamine 100mg/L + Proline 230mg/L+ Lactose 10mg/L+ Malt-extreat 100mg/L+ sucrose 45g/L+ gelrite 2.5g/L) 再生培養基，培養45天會有體胚再生。而拔蕉再生之體胚則已發育成芽並有根的伸長。 懸浮細胞建立，以拔蕉胚乳衍生之胚性癒合組織接種於TB5培養基，其配方為2,4-D 1.0 mg/L、glutamine 100 mg/L、biotin 1.0 mg/L、malt extract 100 mg/L以及sucrose 45 g/L，胚性細胞增生情況良好，繼代增生培養約3個月可得較均質懸浮細胞。台灣芭蕉胚乳胚性癒合組織之懸浮細胞培養系統建立，初期培養以TB5培養液，二星期繼代一次有較多細胞團釋放，但增殖速率較同期培養之拔蕉胚乳細胞慢。 取均質拔蕉胚乳懸浮細胞液，將細胞大小分為<60, 60-30 及>30目之細胞團，稀釋成30倍取1c.c.PCV平板培養於修正SH3培養基，培養60天後，接種 30-60目之細胞團，平均可再生1285體胚，且<60目細胞團則有1533體胚。而且在水晶洋菜介質培養下，其平板效率高於濾紙棉花墊。 拔蕉胚乳衍生懸浮胚性細胞平板前，以TB5在振盪培養14天中，SH3處理胞外PH值雖呈波動性，大都維持在PH 4.55-5.48，而SH3 + MES 10g/L，其胞外pH值維持穩定5.35-5.63範圍，兩者處理皆可使前胚性細胞導向前胚發生，並進一步發育為球胚期。TB5培養液其胞外pH則漸調降至pH 3.95-4.43，補充MES初期雖能維持在5.67-4.58，但後期則調降4.16-4.45，兩種培養皆未有正常前胚發生。以SH3及SH3添加MES 10 g/L有較多體胚且體胚發育迅速，其平板效率較TB5預處理佳。 以乳白色大約0.8mm大小者之拔蕉胚乳再生體胚進行胚苗轉換，接種於1/2MS + NAA 0.1 mg/L下，可有較好之發根率及發芽率為81.0 %及75.9 %。添加低濃度生長素亦有利體胚苗、葉片生長及形成優良根系。添加GA 0.5mg/L並無助胚苗轉換及小植株建立，且易使地上部莖葉發生異常脫色及突長。取台灣芭蕉胚乳再生已發芽之體胚，接種於濾紙橋添加1/2MS+ NAA 0.1 mg/L液態培養基中培養一個月，發根較固態培養基快且正常。接種於同配方液態培養基懸浮培養一個月，能誘導發根但有部份褐化及畸形根產生。 流式細胞儀分析胚乳衍生之癒合組織、體胚、懸浮細胞及小植株其核DNA含量，經與二倍體植株葉片比較，知拔蕉胚乳衍生細胞系相對基因組大小比值為1.74-1.01之間，以比值為1.53之細胞系與雞血核( DNA content=2.5 pg)比對，可換算出DNA含量約為 1.58 pg。由於胚乳衍生癒合組織及再生小植株核型表現不穩定，因此未來需進一步篩選出同質之三倍體株。

Diploid bananas (2n=22) Musa balbisiana and M. formosana were used, for studying programmed cell death (PCD), rescuing culture, and regeneration in endosperm tissue. The developmental stages of endosperm in M. balbisiana could be categoried into liquid stage(1-50 days after pollination, DAP), glutinous stage(62-70 DAP), solid stage(74-83 DAP) and starchy stage (>83DAP). The initiation of PCD in developing endosperm was studied using Evans blue staining method and DNA fragmentation markers. We demonstrated that dense blue staining took place in late glutinous stage about 72 DAP, that was also coincided with the timing of genomic DNA degradation by electrophoresis, about 74 DAP during fall season. The various development stage endosperm were excised and cultured on MS medium supplemented containing 1mg/L 2,4-D, 0.5 mg/L BA, 2 mg/L TDZ and 0.5 mg/L GA. Callus formation rate in glutinous stage 64 DAP was 53%. Callus formation decreased in solid stage( 74 DAP) due to PCD. The addition of 200 mg/L malt extract increased callus formation. Glutinous endosperm of M. formosana cultured on MS medium containing 1.0mg/L 2,4-D, 0.5 mg/L BA, 0.1 mg/L TDZ, 1.0 mg/L GA showed higher callus formation rate, about 35.0 %. Increasing GA concentration delayed the embryogenic callus formation. The endosperm-derived embryogenic callus of M. formosana were recorded 3 months after cultivation, which is longer than what is recorded in M. balbisiana. The endosperm-derived callus of M. formosana formed adventitious bud and embryoids on SH3 medium. The endosperm-derived calli of M. balbisiana and M. formosana were inoculated on TB5 liquid medium containing 1.0 mg/L 2,4-D, 100 mg/L glutamine, 1.0 mg/L biotin and 100 mg/L malt extract with110rpm shaking. Homogenous cell population of M. balbisiana were obtained after 3 months in suspension cell culture, while homogenous suspension cell of M. formosana were obtained after 4 months in suspension culture. The size of suspension cell cluster was separated with <60, 60-30 and >30 meshes and were diluted 1/30 c.c PCV proceeding to plat on SH3 modified medium. The size classes cell clusters of 30-60 and <60 mesh respectively generated 1285 and 1533 embryoids/0.033 ml PCV in 60 DAC. Cell clusters on gelrite semisolid medium had greater plating efficiency than that on paper cushion enriched with liquid medium. Before plating, the suspension prembryogenic cells of M. balbisiana were subcultured on TB5, SH3 medium with or without was consistent 10 g/L MES. That the extracellular pH of SH3 was remaining in the rage of 4.55-5.48 and SH3 + MES solution between 5.35 and 5.56 during 14 subculture period. Furthermore, two pretreated cells regenerated larger and higher quality of somatic embryos on plating medium than those on TB5 or TB5+MES pretreated. Somatic embryos inoculated with 1/2MS solid medium adding 0.1 mg/L NAA induced 81.0% and 75.9% of rooting and shooting respectively. The plantlets grew well in medium added auxin. However the plantlets had exceptional discoloration and elongation on medium adding GA. Endosperm-derived adventitious buds of M. formosana inoculated on paper bridge adding 1/2MS and 0.1 mg/L NAA liquid medium induce rooting sooner than that in agar medium. The plantlets were transplanted into soilless mix, and obtain more than 90% survival. Endosperm- derived calli, adventitious buds, suspension cells, and plantlets of M. balbisiana were analyzed by flow cell cytometry for measurement of nuclear DNA content which were range 1.74-1.01 times of diploid bananas. The endosperm-derived calli or plantlets exhibit karyotype instability. It is required to select the homogenous triploids in future.