擴大種原歧異度與育種平台之花卉優勢品種開發

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計畫名稱: 擴大種原歧異度與育種平台之花卉優勢品種開發
計畫主持人: 蔡東明
共同計畫主持人: 金石文;黃雅玲;褚哲維;陳彥銘;徐善德;蔡媦婷;張芝蓉;劉明宗;沈榮壽;陳富永;葉德銘;陳建銘;陳福旗;胡文若;葉育哲;潘怡君;洪惠娟;許雅婷;翁一司;陳錦木;李淑真;黃光亮;黃柄龍;杜宜殷;黃建誌;吳安娜;張元聰;王昭月;詹庭筑;黃鵬林;許嘉錦
計畫編號: 110農科-4.1.5-子-C1
計畫主管機構: 行政院農業委員會
計畫執行機構: 農委會農業試驗所花卉研究中心
全程計畫年: 2021
關鍵字: 櫻花;授粉;孤挺花;催花性;品種選育;秋海棠;彩色海芋;球型花;耐溫性;菊花;雜交;華八仙;需冷量;日均溫;濃度;夏開菊花;八氫番茄紅素脫氫酶;耐候性;萬代蘭;萱草;原生種;觀賞鳳梨;豐花性;文心蘭;肥灌;幼年期;中型室內盆花;垂枝性;菊花;抗病毒;種苗量產;香味;歐拉靈;迷你春石斛;火鶴花;苞舌蘭;蝴蝶蘭;屬間雜交種;紫羅蘭;溫度;薑荷花;馴化;誘變;倍數性;真核轉譯起始因子4E;小萼亞屬;蕙蘭;栽培;麗格秋海棠;栽培介質;光強度;聖誕紅;商業品種;日日春;選拔;種間雜交;盆花;育種;冷凍種子;無菌播種;Prunus spp.;pollination;amaryllis;forcing ability;variety selection;Begonia;calla lily;mophead;temperature tolerance;Chrysanthemum;hybrid;Hydrangea chinensis;Chilling requirment;Mean Daily Temperature;concentration;Summer-flowering Chrysanthemum
摘要: 細部計畫0:文心蘭基因編輯生物技術之開發與應用</strong>   本計畫擬利用群聚且規律性間隔的短回文重複序列 (clustered regularly interspaced short palindromic repeats, CRISPR)/Cas9 系統之基因組定點編輯 (targeted genome editing) 技術,針對文心蘭 <em>EIN2</em> 基因進行編輯,使 <em>EIN2</em> 基因喪失功能,阻斷乙烯訊息傳導,使文心蘭無法感受乙烯,達到延長文心蘭切花花期之目的。為了設計文心蘭 <em>EIN2</em> 基因專一的 CRISPR 序列,首先進行文心蘭 <em>EIN2</em> cDNA 之選殖,並分析可供編輯之不同專一性區域,分別合成寡核苷酸引子,完成基因編輯表現質體之構築。接著採用聚乙二醇 (polyethylene glycol, PEG) 轉殖法,將基因編輯表現質體,進行文心蘭葉片或癒傷組織之原生質體的核轉染,以高解析熔解曲線分析 (high resolution melting curve assay),顯示有鹼基突變,PCR 產物經次選殖後定序分析,顯示編輯位點產生單一核苷酸多型性,編輯效率為 3.3%。另外,農桿菌轉殖基因編輯用表現質體已利用基因槍法及農桿菌媒介法,轉殖至文心蘭癒傷組織,並進行篩選及再生。今年度將以聚合酶反應及南方氏雜交分析,進行基因編輯文心蘭之分子驗證,以 PCR 深度定序進行基因編輯效率分析,並將基因編輯文心蘭培養長大成株,待文心蘭植株出瓶移至溫室後,將進行園藝形狀調查。 <strong>細部計畫0:文心蘭乙烯訊息傳導雙重基因默化轉殖延長花期之研究</strong>本計畫利用 RNA 干擾 (RNA interference, RNAi) 策略,同時默化 <em>EIN2</em> 及其下游蛋白質 <em>EIN3</em> 兩個基因,期望阻斷文心蘭花朵中的乙烯訊息傳導,育成延長花期的文心蘭品種。先前計畫已完成 <em>EIN2</em> 及 <em>EIN3</em> 雙重基因默化農桿菌轉殖用質體之構築,所得到的最終質體經轉型至農桿菌並確認後,利用此農桿菌,進行文心蘭 <em>EIN2</em> 及 <em>EIN3</em> 雙重基因默化質體之基因轉殖,接著持續以含 G418 的培養基進行文心蘭 <em>EIN2</em> 及 <em>EIN3</em> 雙重基因默化質體轉殖細胞之篩選。篩選存活的癒傷組織,經再生得到的文心蘭轉殖植株,本年度將出瓶馴化及溫室栽培,進行園藝性狀調查及遺傳穩定性分析,並進行外源基因的邊界序列分析。預期轉殖株體內可形成髮夾結構之雙股 RNA,達到默化文心蘭內生 <em>EIN2</em> 及 <em>EIN3</em> 基因的效果,以延長文心蘭切花壽命。 <strong>細部計畫0:利用基因編輯技術培育抗病毒菊花品種</strong>菊花是台灣產量最大的切花作物,具有高經濟價值,病毒病是影響菊花產業及外銷的重大因子。自然界中難以找到菊花抗病毒株進行雜交育種,且菊花為異質性高的六倍體,雜交育種或分子標誌輔助育種皆有其困難度,同時難以克服基因連鎖之限制,產業上只能以藥劑控制傳播媒介來預防,無根治的方法。近年來發展的CRISPR (Clustered regularly interspaced palindromic repeats) 基因編輯技術可以打破基因連鎖障礙而快速精準育種,本計劃將建立三種菊花商業品種的再生系統、基因轉殖系統、並利用編輯 PDS (phytoene desaturase) 基因建立菊花的CRISPR基因編輯系統,再利用CRISPR精準編輯菊花的eIF4e (Eukaryotic translation initiation factor 4E) 基因,抑制RNA病毒在菊花中的轉譯作用,育成抗病毒的基因編輯菊花。利用商業品種菊花進行基因編輯將只改變eIF4e基因的少數鹼基,基因編輯菊花將保有所有菊花性狀與商業價值,再利用後代基因分離技術即可快速精準培育出不含外來基因,類似天然突變之菊花商業品種,將有助於台灣菊花產業的發展及外銷市場的擴展。 <strong>細部計畫0:花卉品種選育及育種技術改進</strong>1.垂枝日日春盆花育種    本研究擬進行新花色垂枝日日春盆花的育種,將特殊花色與花型的性狀導入垂枝日日春品種中,透過蒐集垂枝性日日春種原與特殊花色花型之日日春,並測試其雜交組合,從子代中選拔目標花色花型且具有優良生長勢與開花性的垂枝表現株,育成花色更多樣化,創造眼圈顏色對比鮮明、特殊花型的垂枝品種,進行優選株的純化與純系建立。本研究期能育種選拔出臺灣具有商業價值的新品種垂枝日日春F1品種,並試圖建立有效的F1種子生產方法以供產業利用。本研究欲增加垂枝性日日春品種於市場上的多樣性與提供消費者多元的選擇,提升夏季草花種苗生產的國內及國際競爭力有助於市場的開拓,以及建立有效率的日日春育種的規程。 2.仙履蘭Magic Lantern #Chiada 1優選營養繁殖系栽培及開花技術之建立    本計畫擬採本研究室已建立之仙履蘭Magic Lantern #Chiada 1優選營養繁殖系瓶苗,參試其出瓶馴化、盆苗栽培管理及開花條件,並進行新品種命名及權利登記之評估。期望此無性栽培品種之開發,能導引國內仙履蘭種苗生產朝分生模式發展,並加速精準排程性商業生產體系之建立,以促進我國仙履蘭產業的整體發展與全球競爭力。 3.利用秋水仙素創造聖誕紅新品種 (二)    本計畫將收集市場流通之聖誕紅品種,並評估其秋水仙素誘變效率,再透過流式細胞儀評估倍數性,並調查誘變株植物性狀及生理特性,預期創造適應臺灣物候性之且型態優良之聖誕紅新品種。 4.利用種間雜交開發麗格秋海棠新品種(三)    本計畫將收集市場流通之球根秋海棠品種,並評估可用之秋海棠屬植物花粉稔性,再進行種間雜交,並利用胚培養技術以開發適合臺灣物候性、全年開花、分枝性良好之麗格秋海棠新品種。 5.菊花品種選育    進行人工及開放授粉雜交及化學與物理誘變育種,耐熱菊花育成促進台灣菊花品種自有,降低國外品種的依賴,增加新品種促進品種多樣化,提高農民栽培意願促進農民就業。 6.洋桔梗離體採種、種子滲調、造粒及育苗技術之建立(III)     本研究旨在探討洋桔梗授粉時期及瞭解果實離體瓶插採種方式,期能避免夏季高溫採種,減少日後可能之植株簇生化生長;種子進行滲調及造粒處理,以利本地品種商業化。並探討播種後冷藏、苗期溫度、光積值及施肥濃度,對洋桔梗穴盤苗生長及移植後開花之影響,及探討洋桔梗之幼年期,以供業者參考。 7.熱帶地區商業生產迷你春石斛之育種台灣氣候條件適合春斛斛栽培,春石斛有機會形成一新興產業。由於現今市面上的商品多為中大型品種,小型或迷你型品種具有競爭潛力,因此擬藉由本計劃針對迷你型春石斛育種,以應映未來的市場。 8.商業潛力蝴蝶蘭及近緣屬間之雜交品種選育   本計畫目的為利用屬內、屬間雜交,選育出能控制開花之新穎雜交種、具雙(多)梗、多花、蠟質且花型飽滿或兼具香味之新品系。於雜交授粉後進行無菌播種、繼代及馴化出瓶,並以溫度誘導開花,選拔具優良園藝特性單株,再利用花梗節培養增殖一些數量。此外,將以物理切割蝴蝶蘭原種<em>P.</em> <em>micholitzii</em>發芽後之原球體(protocorms),以進行倍數體誘導,優化其園藝特性,並導入其他種原。為利於育種計畫所需特定種原,重要雜交組合的成熟種子將以冷凍保存,並建立冷凍種子無菌播種消毒模式及播種培養基,以提升冷凍種子發芽率。  <strong>細部計畫0:文心蘭與大花蕙蘭之品種選育</strong>台灣地區位居亞熱帶氣候區,四季如春,適宜生產各種蘭花,尤其是蝴蝶蘭與文心 蘭生產佔有世界重要地位,也證明了台灣在亞熱帶蘭花確有其發展潛力,然而目前除蝴蝶蘭外其他花卉作物之自有優良品種極度的缺乏,在植物專利權逐漸受重視的潮流下,專利品種的缺乏將使台灣的花卉生產陷入代工型生產的危機。本計畫主要針對目前國產蘭花中已具市場規模或具發展潛力之花卉作物項目,開發自有及多樣的品種,強化產業競爭力,其主要作物項目包括文心蘭、大花蕙蘭等蘭花之盆、切花新品種開發。 <strong>細部計畫0:火鶴花品種選育與分子遺傳分析研究</strong>台灣的火鶴花生產面積約在160公頃左右,然而大部分的火鶴花品種來自荷蘭,這些在設施溫室育成的品種在亞熱帶地區如台灣的開放環境下栽培,切花品質並不穩定。本計劃即針對上述問題,經由育種及試種選育適合台灣生產環境具市場潛力之新品種,同時透過設施栽培與瓶插技術開發提升品質,並開發分子輔助育種技術協助傳統育種。 <strong>細部計畫0:石竹耐候性品種選育</strong>暖化乃是全球趨勢,每年年均溫越來越高,導致許多花卉產業必須遷移至高冷地,或是利用降溫設備進行栽植,亦或透過進口方式。這種行為之下常伴隨的是國土安全問題及能源問題。若能利用台灣原生種風土適應力佳之特色導入商業品種石竹,則能降低全球氣候變遷帶來暖化的衝擊。本計畫擬以具耐候性佳之原生種石竹及商業品種進行自交選拔優良單株作為花藥培養之試驗品種,並透過花藥培養產生之單倍體植株供做未來一代雜交種親本使用。 <strong>細部計畫0:經濟性蘭花之品種選育</strong>品種多樣是台灣蝴蝶蘭產業特色,但品種耐溫性之差異常造成栽培上之困擾,因此有必要建立耐溫性快篩技術。花粉對溫度相對敏感,本研究擬以電阻抗式流式細胞儀(Impedance Flow Cytometry, IFC)進行蝴蝶蘭品種及原種之花粉耐溫性檢測,建立耐溫性親本種原庫,並建立可提高耐溫性育種及選種效率之技術。萬代蘭已選育出20株以上雙梗小花具芳香味之優良單株,並經多次國際與國內蘭展獲獎肯定,進一步提供更多訊息,亦經萬代蘭業者評估後選定數種品系進行量化增殖。 <strong>細部計畫0:蝴蝶蘭倍數體育種</strong>利用染色體加倍或花藥培養技術,創造蝴蝶蘭不同倍數體的植株,作為雜交親本。 <strong>細部計畫0:繡球花在台灣平地適應性品種選育</strong>在平地栽培的繡球花,夏季 易受高溫影響,冬天易受白粉病為害影響觀賞品質。 先前計畫利用商業流通品種與台灣原生種華八仙作為育種親本,進行雜交後代選育工作,創新的種間雜交後代具耐病及生長勢強等特性,然而花型呈現蕾絲型,不孕花花型單一,較不具市場競爭力。因此擬透過,新穎品種的引進,以及雜交、回交工作,改善花形表現。目標為培育具有亞熱帶特性,且花型表現新穎的繡球花品種,期望雜交後代具有生長強勢、耐病蟲害...等優良特性。可降低商業栽培的困難度 ,提升繡球花的可見度,增加生產者收益。  <strong>細部計畫0:低需冷性櫻花品種選育</strong>本計畫旨在選育低溫需求量低且開花習性佳之觀賞櫻花品種。臺灣山櫻花(<em>Prunus campanulata</em> Maxim)為觀賞櫻花種類中開花之需冷性最低之物種,藉由人工雜交方式將其低需冷性特性導入現有流通之日本櫻花品種中,以選育出重瓣、花色有別於現有商業品種,供臺灣地區都市景觀利用。 <strong>細部計畫0:文心蘭、石斛蘭及夏菊之育種</strong>研發文心蘭切花品種頗具國際市場潛力,育成符合外銷品質的切花品種更顯重要。 本年度培育文心蘭‘Honey Angel’秋水仙素誘變植株、實 生苗瓶內培養及文心蘭無病毒品系繁殖。臺灣夏菊之品種不若秋冬品種之多樣化 ,於高溫逆境下菊花開花延遲之問題需藉由雜交選拔自有品系來克服目前夏菊品種 選擇性少之問題。今年度計畫預定完成授粉雜交組合10組並進行採種,並進行近年雜交子代之品系比較 試驗。春石斛蘭產業受限於品種及因品種而衍生之栽培問題,致使產業規模發展緩慢,本研究採用雜交授粉創育新品種,其育種目標為選育低需冷性之春石斛蘭新品 種,並探討建立高品質種苗繁殖與生產技術,期能協助臺灣春石斛蘭產業 之發展。 <strong>細部計畫0:蕙蘭之育種</strong>蕙蘭育種以雜交方式進行,目標為選育耐熱、花期12月至1月之蕙蘭新品種。本年度進行10個雜交組合,並由雜交後代中初選5個單株,作為後續選拔及育種材料。 <strong>細部計畫0:適合高溫環境栽培之花卉品種選育</strong>為滿足國內外市場對高品質與產品多樣化的要求,並提升產品的競爭能力,臺南區農改場選擇數種具有發展潛力的作物,選育出在臺灣環境下能生長發育良好的品種。重要工作包括:選育具耐熱性適合臺灣中南部栽植之洋桔梗、星辰花、紫羅蘭等優良品系,並進行試驗以建立栽培資料,改善生產品質與產量,符合最大的經濟效益,提高農民的收益。   <strong>細部計畫0:苞舌蘭新品種選育</strong>進行苞舌蘭雜交育種,選出花序集中、開花性好、生育強健的苞舌蘭。利用組織培養大量繁殖苞舌蘭優良單株,加強推廣應用,創造成為新產業。並以無菌播種方式產生大量原生種小苗,配合進行原生地復育。  <strong>細部計畫0:高屏地區熱帶花卉品種選育</strong>本研究之目的為提升熱帶花卉研發能量,加強新品種研發,建構熱帶花卉產業之發展。針對薑科薑荷花、觀賞鳳梨、腎藥蘭及火鶴花等五大類熱帶花卉進行研究,期許發展成為南臺灣地方特色花卉產業。 <strong>細部計畫0:香氣蝴蝶蘭品種選育</strong>本計畫即擬以香氣蝴蝶蘭為育種目標,利用具香氣的蝴蝶蘭優良個體或異屬香氣蘭花為親本雜交。期能選育出具雙梗以上,能讓滿室芬芳的香花蝴蝶 蘭。重要工作包括蒐集具香氣之蘭花原種,建立相關開花性狀資料。並對蝴蝶蘭 種原進行香氣成分調查,評估不同雜交組合後代香氣變化。 <strong>細部計畫0:觀賞用萱草品種選育</strong>萱草為重要觀賞植物,食用品種則稱為金針菜,國內金針產業已逐漸朝休閒方向發展,農民對於品種多樣化之需求日益殷切。本計畫持續進行新品種研發工作,依雜交育種程序,選出優良單株加以培育成營養系,選拔耐銹病與耐褐斑病,表現優良之營養系,再進行新穎性、一致性、穩定性等特性檢定,以培育具有特殊花色、容易栽培之新品種。 <strong>細部計畫0:球根花卉品種選育技術建立</strong>彩色海芋、彩葉芋與孤挺花是台灣具有發展潛力之熱帶或亞熱帶球根花卉。本計畫今年將持續蒐集孤挺花、彩葉芋及彩色海芋品種,藉由相關育種技術,進行雜交育種及篩選具潛力之後裔單株。從彩色海芋、彩葉芋與孤挺花優良雜交後裔中,選出具潛力單株,並量化繁殖評估後,作為未來申請植物品種權準備。 0:Development and Application of Gene Editing Technology in Oncidesa</strong>  In this project, genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was employed to knockout EIN2 gene in Oncidesa to prolong the shelf-life of cut flowers by interrupting ethylene signal transduction. As a first step of this project, EIN2 cDNA was cloned and its sequence was analyzed for candidate target sequences of single-guide RNA (sgRNA). Synthetic oligonucleotides were annealed and constructed to obtain genome editing-expression plasmid. After the expression plasmid was introduced into Oncidesa protoplasts by polyethylene glycol (PEG)-mediated transfection the editing efficacy was investigated by high resolution melting curve assay and PCR-sequencing. Single-nucleotide polymorphisms were detected around the editing site with 3.3% efficiency. On the other hand, both functional sgRNA and Cas9 gene cassettes were constructed in the binary vector for Agrobacterium-mediated transformation. The final construct was transferred into embryogenic calli of Oncidesa via particle bombardment and Agrobacterium method. The embryogenic calli of Ondidesa were selected for the presence of green fluorescent protein gene and further regenerated into Oncidesa seedlings. In this year, genomic DNA will be isolated from these Oncidesa seedlings and NGS-based targeted sequencing will be employed to investigate the CRISPR-induced gene edits. The Oncidesa seedlings will be transfrred from in vitro cultured condition to isolated greenhouse and grown into mature plants and bloom. The horticultural characteristics will be evaluated in greenhouse. <strong>0:Studies on Dual Silencing of Genes Involved in Ethylene Signal Transduction to Prolong Vase Life of Oncidesa Flowers</strong>Two different specific 21 bp from EIN2 and EIN3 genes were constructed together in antisense-loop-sense orientation to obtain hairpin-type siRNA-expression RNAi plasmid. EIN2 and EIN3 RNAi-mediated dual silencing construct was transformed into Oncidesa calli by Agrobacterium tumefaciens previously. Transformed calli were screened on selection medium containing antibiotics. The transformed calli have been regenerated and the transgenic Oncidesa seedlings have been developed. These transgenic Oncidesa plants were further proved by GUS histochemical staining and polymerase chain reaction. In this year, these transgenic Oncidesa plants will be transferred from in vitro environment into isolated greenhouse. During cultivation the horticultural characteristics will be investigated. In this study, the inverse polymerase chain reaction (IPCR) technique will be employed to determine the flanking sequences harboring the transgene in transgenic Oncidesa plants. We expect that expression of EIN2/EIN3 dual gene RNA-mediated silencing construct would block the ethylene signal transduction in Oncidesa and thereby prolong vase life of cut Oncidesa flowers. <strong>0:Breeding of virus-resistant chrysanthemum by genome editing technology</strong>Chrysanthemum is the largest cut flower crop in Taiwan with high economic value. Virus disease is a major factor affecting the chrysanthemum industry and its export. It is difficult in nature to find chrysanthemum antiviral strains for cross breeding because chrysanthemums are hexaploids with high heterogeneity. Hybrid breeding or molecular marker assisted breeding have their difficulties, and it is difficult to overcome the restriction of genetic linkage. The industry can only use medicine to control the media for prevention, no cure method. CRISPR (Clustered regularly interspaced palindromic repeats) gene editing technology developed in recent years can break genetic linkage barriers and quickly and precisely breed. This project will establish the regeneration system and gene transfection system of three commercial chrysanthemum varieties, and use the PDS (phytoene desaturase) gene to establish the CRISPR gene editing system for chrysanthemum, and then use CRISPR to precisely edit the eIF4e (Eukaryotic translation initiation factor 4E) gene of chrysanthemum, and inhibit the translation of RNA virus in chrysanthemums to breed virus-resistant gene editing chrysanthemums. Using commercial chrysanthemum on gene editing will only change a few bases of the eIF4e gene. Gene editing chrysanthemums will retain all chrysanthemum traits and commercial value. Using additional progeny gene segregation can quickly and accurately cultivate foreign genes free, similar to natural mutant, commercial chrysanthemum varieties. This will help the development of Taiwan's chrysanthemum industry and the expansion of the export market. <strong>0:Breeding of new cultivars and improvement of breeding techniques in flower crops</strong>1.Breeding of trailing vinca (Catharanthus roseus (G.) Don.)    This research project is aimed to explore the new color breeding of potted trailing vinca (Catharanthus roseus). In order to breed unique traits such as special corolla colors and shapes with the trailing trait in vinca, we collect trailing parents and parents with special corolla colors or shapes, and cross numerous combinations between trailing ones and others, then select the ones which have successfully combined both the desired corolla colors or shapes and the trailing trait from crossing generations, purify and create purelines, thus to establish a series of multiple colors, special corolla shapes, vigorous and trailing cultivar. This research project expects to produce a valued new trailing F1 vinca cultivar for the use of seed production. It offer a higher diversity of trailing vinca in market and various choice for customers, and enhance the domestic and international competitiveness in summer floral seedling production, and set up an efficient breeding protocol for vinca .   2.Establishment of Cultivation and Flowering Techniques for a Selected Elite Clone of Paphiopedilum Magic Lantern #Chiada 1    Paphiopedilum has been a favorite consumer product due to its attractive color and distinctive shapes of flowers, and has reasonably potential to make it viable large-scale commercial industry. However, commercial paphiopedilum orchids are mainly produced by sexual propagation, and are characterized with diversified flowering behaviors and with long juvenility periods which can last 3 - 6 years or more. Due to differences in genotypes, sexual seedlings have inconsistent growth rates and length of juvenile period. It is difficult to establish standardized cultivation management and flowering regulation systems. Even for population that produce the same pod at the same time, the first flowering time can vary by more than 2 years, making it difficult to establish a precise and scheduled commercial production system. This project intends to use the clonal plantlets of Paphiopedilum Magic Lantern #Chiada 1 that have been established by our research laboratory to test their acclimatization, pot plant cultivation management and flowering conditions, and to conduct the evaluation for new cultivar denomination and right registration. It is hoped that the development of this vegetative cultivar can lead the domestic production of commercial paphiopedilum orchids towards the clonal mode, and accelerate the establishment of a precise scheduled commercial production system, and promote the development of paphiopedilum orchid industry in Taiwan. 3.To develop the novel cultivar of poinsettia by colchicine treatment (II).    This project will collect the commercial cultivars of poinsettia to conduct the colchicine treatment which evaluated the mutation efficiency and ploidy level by flow cytometry, and then obtained the mutants also investigated the plant characteristic and physiological performance for crating the novel cultivars with excellent growth vigor and well desirable traits in Taiwan. 4.To develop the new cultivar of Rieger Begonia by interspecific hybridization (III).    This project will collect the commercial cultivars of tuberous begonia to conduct the interspecfic hybridization with other plant materials of genus begonia which also evaluated the pollen viability, and then obtained the hybrids by embryo culture for creating the new Rieger begonia cultivars with desirable plant characteristics like as excellent growth vigor, blooms all year round, and well branching habits in Taiwan. 5.The breeding of chrysanthemum    Carry out artificial and open pollination hybridization and chemical and physical mutation breeding. The breeding of heat-resistant chrysanthemum promotes the ownership of Taiwan chrysanthemum varieties, reduces dependence on foreign varieties, increases new varieties to promote variety diversification, and increases farmers' willingness to cultivate and promote farmers' employment.  6.Techniques of seed production ex vivo, seed priming, pelleting, and plug production of Lisianthus (III)    Lisianthus [Eustoma grandiflorum (Raf.) Shinn.] is an important cut flower for export trade in Taiwan. Market demand has been increasing with increasing planting area and production. Production during plug stage period of lisianthus is crucial and might be hastened under appropriate environmental conditions and nutritional managements. The objectives of this proproject are to determine pollination timing and to establish the techniques of seed production with cut immature capsule, seed priming and pelleting. The effects of temperature, light integral, and nutrient solution concentration during plug production will be studied on seedling growth and flowering performance of lisianthus. 7.Miniature Nobile type Dendrobium breeding for commercial production in tropical climate.    The beautiful fragrance of nobile Dendrobium is pleasant to be loved. In recent years, due to the maturity of forcing technology, the rise of the European market, market demand increased. As Taiwan's climatic conditions are suitable for the cultivation of nobile Dendrobium, this type Dendrobium has the opportunity to form a new industry. From today's market of commodities are mostly medium and large varieties, small or mini varieties have competitive potential, so it is proposed to use this program for mini-type nobile Dendrobium breeding, in order to meets the future market demand. 8.Breeding of Phalaenopsis and its alliance genera with commercial potential    Phalaenopsis cultivars in the global trade are mostly intra-specific hybrids. In recent years, breeding of commercial cultivars has been a routine by nurseries from some advancing countries, such as the Netherlands, which has affected the export of Phalaenopsis young plants from Taiwan and almost reached the plateau state with very limited market growth.. More severely the situation is the global outbreak of the new coronavirus (COVID-19) pandemic which severely affects the export and consumption of current Phalaenopsis cultivars of flowering plants as well as young plants. To create more market potential post-Covid-19 era, novelty hybrids other than the existing types by cross hybridizing intra-genic Phalaenopsis hybrids or inter-generic hybrids may have the chance for the future trade market. The aims of this project are to make intragenic or intergeneric hybridization, select novelty elite lines with desirable traits such as round shaped flowers with heavy substance, with multi-spikes and multi-flowers in addition to fragrance. After cross pollination the seeds from mature capsule will be harvested and germinated in vitro, seedling grown and transplanted to suitable pot sizes. When the seedlings reach the mature stage they will be transferred to cool room (day/night 18/26℃)to induce flowering and select individual plants with desirable horticultural traits. The selected superior new hybrids will be propagated in vitro by flower stalk node culture and shoot multiplication for a small quantity. In addition, polyploidy plants will be induced by repeated cutting of germinated P. micholitzii protocorms in order to increase its horticultural characteristics so to integrate to other cultivars. In order to utilize certain germplasm during the breeding program, seeds of important cultivars/lines will be frozen stored and used for germination when in need. The disinfection mode for frozen seeds will be established, and germination medium be improved to help increasing the germination rate.  <strong>0:Developing new varieties of Oncidium and Cymbidium</strong>Taiwan, as an island style climate, is a powerful basement for tropical and subtropical flower industry to competition with others county around the world. Look back the trend of industry development in Oncidium and Cymbidium, Taiwan is high potential in orchids. However, most of variety for flower production is from others countries, except Phalaenopsis. Concerning the world trade, plant breader right play a rule in flora Industry, short of patent of new variety, Floriculture Industry in Taiwan may fall into a low level as an agency. Majority of this proposal is to strengthen the competition of flower Industry by varieties improvement, to provide more varieties for cut flower or pot plant grower. We are focus on some market-led item, such as Oncidium and Cymbidium, to develop some new varieties for cutting or pot plant. <strong>0:Breeding and molecular genetic analysis of Anthurium.</strong>The production area of anthurium in Taiwan was more than 160 ha. However, most of anthurium varieties in Taiwan are imported from Holland. The quality of cut flower from these varieties breed under greenhouse in temperate area was unstable while planted under opening field in subtropical area as Taiwan. Patent of varieties and all-year-round quality of cut flower will be the limited factors of anthurium industry in the future. The aims of this study are to improve the cultivation varieties ,techniquesto increase the international trade competition on Anthurium. At the same time, the post harvest technique and greenhouse culture sytem will be development to improved the quality of cut flower, molecular-assisted breeding techniques to assist traditional breeding will be devlopment also. <strong>0:Breeding of Weather Resistance Dianthus</strong>Warming is a global trend. The annual average temperature is getting higher and higher every year. As a result, many flower industries have to move to high-cold areas, or use cooling equipment for planting, or import them. This behavior is often accompanied by homeland security issues and energy issues. If we can use the characteristics of Taiwan's native species with good terroir and adaptability to introduce commercial varieties of Dianthus, it can reduce the impact of global warming brought about by climate change. However, if it can be planted on flat ground, it can also reduce water conservation problems and reduce energy use. The original species of Dianthus, which has weather resistance, and the introduced seed are used for self-purification to improve the heat resistance of the offspring and cross-breed, and to screen whether the offspring is weather-resistant. It should be possible to effectively select individual plants and select those suitable for Taiwan phenology Variety. This project intends to self-select excellent individual plants of Dianthus with good weather resistance and commercial varieties as test varieties for anther culture, and haploid plants produced through anther culture will be used as parents of future generation hybrids. <strong>0:Breeding of economical orchids</strong>A great diversity on varieties is one of the advantage of Phalaenopsis industry in Taiwan, however the differences in temperature tolerance of these varieties have caused problems on cultivation. Therefore, a rapid non-destructive screening technique is urgent needed. Pollen is more sensitive than plant tissue to temperature, and pollen viability tested by Impedance Flow Cytometry (IFC) under various temperature will be used as an indicator for screening temperature tolerance in cultivars and species of Phalaenopsis. A germplasm with temperature tolerance cultivars and technique to enhance breeding and selection of temperature tolerance plants will be established in this study. The results that investigation of specific genetic traits of of Vanda hybrids showed the flowers of C010120-3(Venus series) are fragrant(100%), but 12% and 13%  flowers of C010213-1 and C010405-6 are not fragrant. The flowers of all plants from Cleopatra series are fragrant (100%). However, flowers of Honey series are not fragrant. The flowers of C010619-1(Christine series) are fragrant(100%). <strong>0:ployploid breeding of Phalaenopsis</strong>Using chromosome doubling or anther culture techniques, plants with different ploidy of Phalaenopsis were created as hybrid parents. <strong>0:Selection and Breeding of Hydrangea in adaptability to Taiwan lower altitude area.</strong><strong>Hydrangea has a uniqueflower shape and bright colors. It can be used as potted flowers, cut flowers and landscape cultivation. It is a very popular pot flower. However, hydrangea is a temperate crop. In Taiwan, it needs cold temperature treatments to get high-quality and early flowered hydrangeas. The production relatively costy. Hydrangea cultivated on low land is susceptible to high temperature in summer and powdery mildew in winter which undermine ornamental quality. Commercial varieties and Taiwan native species were used as breeding material in previous projects. Innovative interspecific hybrids have disease resistance and strong growth potential, but the flower type is lace-type which is lack of commercial value in the Taiwan flower market. Therefore, it is planned to improve the flower type performance through the introduction of novel varieties and the work of crossbreeding and backcrossing.</strong><strong>The goal is to cultivate hydrangea varieties with subtropical characteristics and novel flower types. It is expected that hybrid offspring will have excellent characteristics such as strong growth, resistance to pests and diseases. It can reduce the difficulty of commercial cultivation, improve the visibility of hydrangea, and increase the profit of producers.</strong> <strong>0:Selection of low chilling variety on Prunus spp.</strong>The experiment is conducted to select the low chilling requirement varieties of goodshape and color with numerous flowers of Prunus spp.. The chilling requirement of Prunus campanulata Maxim is the lowest one in ornamental sakura species. For gaining the low chilling characteristic with special type of double petal and color, artificial hybridization will be practiced into existing commercial varieties which came from Japan. The superior cultivars selected from hybrids will be evaluated to urban landscape utilization in Taiwan.  <strong>0:Breeding of Oncidium, Dendrobium and Summer Flowering Chrysanthemum</strong>The new varieties of oncidium cut flower possess international market's potency. It is extraordinarily important that breeding cut flower varieties to conform the export quality. We plane to hybridize present long stalk or cut flower varieties with hybrids to obtain new oncidium cut flower varieties. In addition, a problem present in large flower oncidium pot varieties is that they are generally heat-intolerant. Therefore the large flower pot varieties will be hybridized with heat tolerant varieties or hybrid lines bred at low altitude, in order to obtain heat-tolerant large flower varieties The experimental content of this year are: Oncidium ‘Honey Angel’plant cultivation from colchicine mutagenesis, seedlings bottle cultivation and non-virus lines propergation. The cultivars of summer flowering chrysanthemum arefewer than winter flowering cultivars, and current chrysanthemum cultivars has often growth retardant in hot summer. As a result, it is necessary to breed suitable cultivars to adapt to summer climate in Taiwan. The project is to proceed the cross pollination of chrysanthemum lines with 10 cross combinations and collect the seeds. The seeds will be sowed and to cultivate chrysanthemum seedlings in June. In summer, 3 chrysanthemums off springs which are flowering with good characteristics will be selected. ue to the limited of varieties and cultivation problems associated with these varieties, the development of Taiwan nobile-type Dendrobium industries is limited. The purposes of our study is breeding novel nobile-type Dendrobium by hybridization, to create and select new varieties with low chilling requirement, pseudobulbs can mature within one year, higher rate of node-blossom, enhanced flower longevity and quality, better flower color of saturation and contrast, and with medium to small plants size. It is expected that the high-quality propagation techniques of cutting and tissue culture seeding can be established, to promote the nobile-type Dendrobium industries in Taiwan. <strong>0:Breeding of Cymbidium</strong>The method of Cymbidium breeding is hybridization. The objective of Cymbidium breeding is creating new varieties with heat tolerance and flowering during December to January. In this year, the project will cross 10 combinations and select 5 single plants from progeny. <strong>0:Breeding of floral crops adapt to high temperatures.</strong>TNDARES devotes to develop multiple flower varieties with high qualities, good yields and excellent tropical climate adaptations in southern Taiwan. The aims include satisfying cut flower needs for domestic and oversea markets. The project has many majorities which including tropical climate adaptation and/or early flowering breeding of Eustoma, statice and stock. Cultural information of these flowers are also established. The results could improve the qualities and quantities of cut flowers, raise the benefit and earnings of farmers.   <strong>0:The breeding of Spathoglottis.</strong>The hybrid breeding of Spathoglottis will be achieved and compact , more flowers and stronger clongs  will be selected.  The asexual micropropagation techniques will be developed to mass produce clonings.  Seedlings of native species from aseptic sawing will be planted to habitat. <strong>0:Breeding of Kaohsiung and Pingtung Local Tropical Flowers</strong>The purpose of this study is to strengthen the research energy of tropical flowers, especially on the breeding of new varieties for tropical flower industry. Four tropical flowers, Curcuma, bromeliads, Renanthera, and Anthurium were chose for this study and expected to develop into the local characteristics flower industry in southern Taiwan. First, Curcuma breeding: new Curcuma commercial cultivars will be introduced for more new hybridization events. Curcuma hybrid progeny lines will be obtained and preliminary screening. The candidate lines will be selected for the further assessment procedures in the future. Second, bromeliads breeding, besides collection and preservation of Vriesia varieties, seeds of hybridization were obtained and the primary selection of these seedlings will be conducted. Renathera is a new tropical cut orchid in Taiwan. The objective of this study was to develop new cultivars of Renanthera alliance by collection, hybridization, in vitro germination, micropropagation and selection. Anthurium, a member of the Araceae family, which originated from the tropics of Central and South America, stands out amongst most of the tropical cultivated flowers for its elegance, and long vase life. Anthurium cut flowers are produced mainly for export to Japan. Due to the variety rights of most cultivas, the supply of young plants is limited and also dependent on foreign imports, thus the growers will have to pay the high cost of planting materials. The development of new cultivars will rely on efficient micropropagation for mass production of young plants. The purposes of this proposal is to adopt traditional breeding. Besides, the new cultivars will be applied to obtain plant variety rights. It is expected to reduce the cost of young plants for the growers and ultimately enhance the international competitiveness of the industry. <strong>0:Breeding of fragrant Phalaenopsis</strong>Phalaenopsis is the most important economic floral crops in Taiwan. The purpose of Phalaenopsis breeding is not only sought for multiform flower color and shapes, if the flowers have more pleasant scent that will increase the desire to purchase. This research is trying to breed fragrant phalaenopsis by use Phalaenopsis individuals or orchids from other genera that have finefragrance. We expect to breed outstanding Phalaenopsis that got more than two stalks and have fine fragrance. This study began with collect fragrant Phalaenopsis and then establishes the information of flowering data for breeding. Next step we will try to measure the aroma components of Phalaenopsis, evaluate the best method of aroma measurement and detect the fragrant content of different hybrids.  <strong>0:Breeding of ornamental daylily varieties</strong>Daylily is an important ornamental plant, and the edible variety is called ‘Golden Needle vegetable’ in Chinese. The edible daylily industry in Taiwan had been directed from vegetable purpose to leisure agriculture. To fulfill the new trend, the daylily variety should be diversified. We had introduced diversified daylily varieties from abroad to do breeding works, and there were 6 new varieties released in recent years. The new breeding works will be done continuously in this project. This project will follow the hybrid breeding procedure to breed superior progenies. Those superior progenies will be propagated into clones, and than selected with highly resistant to daylily leaf-rust and leaf-streak diseases. The superior clones will be investigated by comparing the characteristics with commercial varieties to perform the DUS(Distinctness, Uniformity, and Stability) tests. New varieties that are special colors and easy cultivation. <strong>0:Techniques development for bulb flowers breeding</strong>Calla lily, caladium and amaryllis are potential tropic or subtropic bulb flowers in Taiwan. The aims of this year will continue to collect commercial cultivars of calla lily, caladium and amaryllis as breeding parents and execute the breeding program by using relative breeding technologies. We also will select and propagate superior progenies of calla lily, caladium and amaryllis and evaluate to apply the plant breeder right.
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