選育耐候抗病之花卉新品種

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計畫名稱: 選育耐候抗病之花卉新品種
計畫主持人: 莊耿彰
共同計畫主持人: 洪惠娟;許雅婷;蘇俊峰;張芝蓉;陳富永;杜宜殷;許嘉錦;陳錦木;黃柄龍;陳彥銘;翁一司;李淑真;王昭月;王美琴;曹進義;吳安娜;陳淑佩;詹庭筑;蔡東明;劉明宗;金石文;黃光亮;陳建銘;黃雅玲;胡文若;葉德銘;徐善德;潘怡君;吳容儀;沈榮壽;謝廷芳;鍾淨惠;葉育哲;褚哲維;黃鵬林;陳福旗
計畫編號: 109農科-7.5.1-子-C1
計畫主管機構: 行政院農業委員會
計畫執行機構: 行政院農業委員會農業試驗所
全程計畫年: 2020
關鍵字: 誘變;細胞膜熱穩定技術;貯運;屬間雜交;連作障礙;真核轉譯起始因子4E;分子輔助育種;病害管理;倍數性;催花性;遠緣雜交;蟲害防治;需冷量;基因編輯;秋水仙素;屬間雜交;遺傳力;多梗性;病毒檢測;種子滲調;胚培養;發芽勢;香味;開花誘導;種子貯藏;雙梗;種間雜交;八氫番茄紅素脫氫酶;Mutation;Thermostability of Cell Membranes;transportation;intergeneric crosses;continuous cropping obstacle;Eukaryotic translation initiation factor 4E;MAS;disease management;Ploidy level;forcing ability;distant hybridization;pest control;Chilling requirm
摘要: 1.利用秋水仙素創造聖誕紅新品種     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。2.利用種間雜交開發麗格秋海棠新品種(二)     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。3.夏菊品種選育     收集及調查分析台灣流通菊花品種,建立育種資料,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。4.洋桔梗離體採種、種子滲調、造粒及育苗技術之建立(II)    洋桔梗苗期生長速度緩慢,加速穴盤苗育成時間及提高品質之方法亟待建立。 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。5.蝴蝶蘭商業品種育種技術探討    利用蝴蝶蘭蠟質花品種、國內外重要商業品種、自行選育優良品系或種原為親本進行雜交授粉,選育出具光澤、橘黃或綠色花、多梗、多花性且花型飽滿之蠟質花新品系選拔具優良園藝特性單株,並利用花梗節培養增殖選拔品系,以利後續調查其生長及園藝特性。以物理方式持續切割蝴蝶蘭原種Phal. pulchra原球體 (protocorms)以誘導倍數體,利用於雜交育種。測試冷凍保存種子以次氯酸鈉消毒之濃度及消毒時間對於發芽率之影響,並利用TTC檢測種子活力,針對冷凍種子發芽率進行評估。6.熱帶地區商業生產迷你春石斛之育種     春石斛花型優美香味宜人為人喜愛。近年由於花期調節技術成熟,歐洲市場崛起,市場需求增加。由於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。7.芭菲爾鞋蘭屬內雜交育種及開花調節技術之研究     芭菲爾鞋蘭引人注目的花色與獨特的花形確實深受消費者喜愛,臺灣芭菲爾鞋蘭產業的突破性進展,預期亦仰賴新品種之選育及高效率的微體繁殖技術然而,芭菲爾鞋蘭幼年性長,通常可達3-6年,甚至更長,且其開花習性獨特又分岐。再者,其開花模式之資訊鮮少揭露,也已阻礙了新品種之育成。本研究旨在,進行芭菲爾鞋蘭屬之種原蒐集,藉由屬內雜交育成具有優良理想特性的新穎個體,同時建立芭菲爾鞋蘭屬雜交種之開花調節技術,以期增進育種效率。生物技術於花卉作物育種之開發應用 文心蘭乙烯訊息傳導雙重基因默化轉殖延長花期之研究     文心蘭 (Oncidesa) 是臺灣外銷第一大切花作物,其切花經過繁複的採後處理及分級篩選,花藥蓋容易脫落,導致乙烯大量產生,使花朵壽命減短,造成嚴重的經濟損失。本計畫利用RNA干擾 (RNA interference, RNAi) 策略,同時默化 EIN2 及其下游蛋白質 EIN3 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。前一年度計畫已完成 EIN2 及 EIN3 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,完成文心蘭 EIN2 及 EIN3 雙重基因默化質體之基因轉殖;另一方面,持續以含 G418 的培養基進行文心蘭 EIN2 及 EIN3 默化質體轉殖細胞之篩選。 文心蘭基因編輯生物技術之開發與應用  本計畫擬利用群聚且規律性間隔的短回文重複序列CRISPR)/Cas9 系統之基因組定點編輯 (targeted genome editing) 技術,針對文心蘭 EIN2 基因進行編輯,阻斷乙烯訊息傳導,達到延長文心蘭切花花期之目的。上一年度完成之基因編輯農桿菌轉殖用表現質體,已利用基因槍法及農桿菌媒介法,轉殖至文心蘭癒傷組織,今年度將進行基因編輯文心蘭細胞之篩選及再生,期望取得基因編輯成功之細胞,經增殖及再生為延長花期的文心蘭。利用基因編輯技術培育抗病毒菊花品種    近年來發展的CRISPR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文心蘭與大花蕙蘭盆花之品種選育 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、蕙蘭等蘭花之盆、切花新品種開發。經濟性蘭花之品種選育 比較藍紫色蝴蝶蘭 Phal.Siam Treasure 2倍體與秋水仙素誘導所得之4倍體植株的園藝性狀,結果顯示4倍體 P . Siam Treasure花徑雖未較2倍體大,但花瓣質地較佳且植株與花梗型態則顯示出明顯差異,花梗較粗矮,葉片較寬短、厚。有關4倍體之雜交親和性尚需進一步測試。本研究以屬間雜交種的花梗芽為培植體進行增殖,結果以stage1花梗頂芽與stage 2-3花梗頂芽兩種培植體進行誘導時,培植體存活率最高,芽體誘導率也最高,誘導效率較其它節位為佳。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。在萬代蘭特定性狀分析方面,Venus 系列編號 C010120-3 之後代花朵均具有芳香味(香味比例 100%)。埃及豔后系列之後代花朵均具有芳香味(香味比例 100%)。 Honey 系列之後代花朵則不具有香味。克莉絲汀系列雜交編號 C010619-1 之後代花朵均具有芳香味(香味比例 100%) 。火鶴花品種選育與分子遺傳分析研究 台灣的火鶴花生產面積已成長至200公頃以上,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,並開發分子輔助育種技術協助傳統育種。降低生物因子引起蝴蝶蘭貯運耗損之關鍵技術開發 針對目前影響台灣蝴蝶蘭拓展外銷通關與品質之幾項問題,探討改善之技術,包括病害、蟲害與遠距海運等問題之改善。蝴蝶蘭黃葉病嚴重影響蝴蝶蘭的生產與出口,因此本研究擬研擬病害綜合管理策略以提供花農栽培參考。蟲害改善上,本計畫將於主要外銷新興市場之蝴蝶蘭之栽培區進行蟲害調查及針對重要之檢防疫害蟲擬定防治策略。遠距海運的探討,將探討貯運溫度或是貯運後栽培溫度之影響,進而探討改善之方法,包括貯前施肥改變,含鈣藥劑處理,貯前高溫處理,貯後高溫處理等,尋求實用方法,進行實際出口測試。石竹耐候性品種選育 以具耐熱特性的巴陵石竹跟引 進之種原進行自交純化,以便提高後代的耐熱特性並雜交育種,並篩選其後代是否 具有耐熱性,應可有效選拔單株及挑選適合台灣物候性之品種。本計畫擬以具耐熱 性之特有種石竹及商業品種進行自交選拔優良耐熱單株作為親本。利用選拔之 優良單株進行誘變育種擴展性狀及生產四倍體植株以利後續三倍體後代育成。然而將特有種花粉進行儲藏以便後續育種使用,也進行特有種基因庫之保護,並透過種子催芽處理提高種子發芽勢。耐熱具香味蝴蝶蘭品種選育及根節蘭繁殖與栽培技術之改進 選育具有香味之中小型且多花蝴蝶蘭優良單株與具耐熱性且香的中小型蝴蝶蘭品系。確立長距白鶴蘭最佳分株繁殖季節及白鶴蘭提早1-2個月花期調節技術。繡球花景觀抗病品種選育 繡球花為溫帶作物,在臺灣需要栽培於冷涼地區或是透過上山進行花期調節以培養高品質的繡球花,生產成本較高。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。 本計畫工作為收集商業流通品種與台灣原生種華八仙,作為育種親本,進行華八仙與商業流通品種雜交及品種選育工作。本年度完成正反交工作,選出10株優良雜交後代,創新的種間雜交後代具耐病及生長勢強等特性益。耐熱杜鵑花及耐蟲害茶花品種選育 杜鵑花及茶花為國內北部重要景觀花卉產業,栽培歷史悠久且育種資源豐富 。然而杜鵑花景觀品種變化性少,盆花用品種生長衰弱,而茶花缺乏不同季節開放 之商業品種 ,為符合市場需求、增進花卉的觀賞性,將具有耐熱、抗蟲潛力及良好開化特性的杜鵑花及茶花親本進行雜交育種,並進行杜鵑花耐熱性篩選,及茶花的抗蟲性篩選。低需冷性櫻花品種選育 臺灣山櫻花(Prunus campanulata 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。文心蘭、石斛蘭及夏菊之育種    文心蘭大花品種存有不耐熱問題,收集大花品種與平地培育成之子代或耐熱品種雜交,以獲得耐熱大花盆花品種。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。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。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,選育低需冷性之春石斛蘭新品種,具備穩定一年開花、開花節位率高、觀賞壽命長、花色飽和且對比佳及中至小型株等特性,並探討建立高品質種苗繁殖與生產技術。蕙蘭與百合之育種 蕙蘭選育耐熱、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。篩選台灣百合適合作為切花育種親本的個體供未來育種應用。適合高溫環境栽培之花卉品種選育 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,選育出在臺灣環境下能生長發育良好的品種,並針對現有栽培技術進行改進以節省成本並提昇品質。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。苞舌蘭育種及新品種(系)量化繁殖之研究 本計畫擬進行苞舌蘭雜交育種工作,選出3個苞舌蘭優良單株並進行其性狀觀察。開發所育成新品種大量繁殖技術,以微體繁殖方式量化生產種苗,以利後續品種授權及推廣工作之進行。高屏地區花卉品種改良 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、腎藥蘭及火鶴花進行研究,發展南臺灣特色花卉。包括薑荷花之雜交育種,持續引進新的薑荷花種原以進行雜交組合,推出新品種,以擴增薑荷花品種多樣性;觀賞鳳梨方面Vriesia之品種蒐集和保存及自交和雜交育種工作,由獲得之後代苗株中進行性狀調查及篩選;腎藥蘭方面,進行種原蒐集與雜交育種、並進行無菌播種與胚拯救技術開發,選育新的腎藥蘭栽培品種。火鶴用傳統育種技術,選育適合本地種植的火鶴花品種。觀賞用萱草品種選育 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。國際上萱草品種花型與花色極多樣化,近年來已育成5個觀賞用品種並技轉予業者推廣中。本計畫持續依雜交育種程序,選出優良單株加以培育成營養系,選拔耐銹病與耐褐斑病者,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,且適合於休閒農場利用之新品種。香氣蝴蝶蘭品種選育 蝴蝶蘭為台灣目前最重要的經濟花卉作物,育種上除了花形花色力求多變外,若花朵具怡人氣味更能提高蝴蝶蘭產品買氣。本計畫即欲以香氣蝴蝶蘭為育種目標,利用具香氣優良個體為親本雜交。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蝴蝶蘭原種,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,評估不同萃取條件下之萃取效率並調查外在環境對蝴蝶蘭香氣產生之影響。蘭花及球根花卉品種選育及關鍵技術之研發 萬代蘭為近年來臺灣發展之熱帶切花潛力項目,生產切花數逐年增加中,唯品種多來自泰國,育成適應臺灣當地環境之優良品種將有助產業提昇,在種苗生產體系方面,建立穩定、優良之量產體系。目前國內仙履蘭缺乏大量繁殖體系及後續種苗栽培管理技術,期能藉由本試驗開發相關體系,以提升種苗品質。花期調節方面,利用藥劑及環境開發春石斛周年生產體系,並調整GA應用於仙履蘭催花之品質,提早開花維持優良性狀。彩色海芋與孤挺花是台灣具有發展潛力之球根花卉,藉由育種將會從彩色海芋與孤挺花優良後裔中,選出具潛力單株,在繁殖體系方面,將以組織培養方式選擇較最適合之組培量化繁殖進行種球量化肥培管理試驗,建立繁殖系統。Breeding of new cultivars and improvement of breeding techniques in flower crops 1.To develop the novel cultivar of poinsettia by colchicine treatment.     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 e excellent growth vigor and well desirable traits in Taiwan. 2.To develop the new cultivar of Rieger Begonia by interspecific hybridization (II).    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. 3.The breeding of summer chrysanthemum.    Collect and investigate and analyze the chrysanthemum varieties in circulation in Taiwan, establish breeding materials, and breed heat-resistant chrysanthemums to promote Taiwan's own chrysanthemum varieties and reduce dependence on foreign varieties.Add new varieties to promote variety diversification, increase farmers 'willingness to cultivate and promote farmers' employment. 4.Techniques of seed production ex vivo, seed priming, pelleting, and plug production of Lisianthus.(II)    Lisianthus [<em>Eustoma grandiflorum</em> (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 nutrional managements. The objectives of this proproject are to determine pollination timing and to establish the techniques of seed production wuth 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. 5.Exploitation of breeding techniques for commercial cultivars of Phalaenopsis.    The aim of this project is using waxy flower, important commercial cultivars of either domestic or foreign markets, our own selected elite lines and important germplasms of Phalaenopsis orchids for cross hybridization, and select novel cultivars such as shiny, orange flower, green flowers. Round shaped flowers with heavy substance with multi-spikes and multi-flowers will be the target for selection. After pollination the mature capsule will be harvested and seeds germinated in vitro, the hybrid seedlings in flasks will be transplanted with sphagnum moss until mature stage, which will be transferred to cool room to induce flowering. The selected superior new hybrids will be propagated in vitro by flower stalk node culture and shoot multiplication for a small quantity. Subsequent investigation of growth and horticultural traits will be performed in these propagated plants after transplanting and growth in the greenhouse. In addition, polyploidy plants will be induced by repeated cutting of <em>P</em>. <em>pulchra</em> protocorms, due to its glossy flower trait be incorporated into desirable cultivars. Finally, the disinfection effect on in vitro germination of frozen stored seeds by use of sodium hypochlorite at different concentration and time will be examined. The seed viability will be evaluated by suing TTC staining and subsequent germination rate. 6.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. 7.Intrageneric cross-breeding and investigation of flowering regulation techniques in <em>Paphiopedilum.</em><em>    Paphiopedilum</em> 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. Since, diversified excellent varieties and well-established systems of micropropagation have become the main driving force in the development of Taiwan's phalaenopsis industry. The breakthrough of the paphiopedilum orchid industry in Taiwan is also expected to rely on the breeding of new cultivars and efficient micropropagation techniques, being the one of the important limitations of <em>Paphiopedilum</em> orchid industry in Taiwan. However, <em>Paphiopedilum</em> orchids are characterized with long juvenility periods which can last 3 - 6 years or more, and with distinct flowering behaviors. Little understanding of flowering patterns in these crops hindered the breeding of new cultivars. The purpose of this study aimed to carry out the collection of <em>Paphiopedilum</em> species, to create excellent individuals with novel and desirable characteristics by intrageneric crossings, and to establish the flowering regulation technique for improving breeding efficiency.    Development and Application of Biotechnology in Flower Crops  Studies on Dual Silencing of Genes Involved in Ethylene Signal Transduction to Prolong Vase Life of Transgenic<em> Oncidesa</em>     In Taiwan <em>Oncidesa</em> orchids are grown for export as cut flowers, amounting 80% of its total production. The operation of harvest and classification of cut flowers after harvest result in ethylene production induced by pollinia cap dislodgment and petal senescence. Therefore, it is important to delay <em>Oncidesa</em> flower senescence. To prolong vase life of cut <em>Oncidesa</em> flowers, two specific 21 bp from <em>EIN2</em> and <em>EIN3</em> genes were constructed together in antisense-loop-sense orientation to obtain hairpin-type siRNA-expression RNAi plasmid. To test the efficiency of the construct under study, reporter plasmids containing the <em>EIN2</em> and <em>EIN3</em> attaching to the gene encoding green fluorescent protein (GFP) were constructed. Transient expression of RNAi plasmid were performed in <em>Arabidopsis</em> protoplasts by polyethylene glycol (PEG) transformation and analyzed by flow cytometry to establish a fast and reliable quantitative analysis for gene silencing by assaying the inactivation of GFP expression. <em>EIN3</em> siRNA reduced 50% endogenous <em>EIN3</em> mRNA. <em>EIN2</em> and <em>EIN3</em> RNAi-mediated dual silencing construct was transformed into <em>Oncidesa</em> calli by <em>Agrobacterium tumefaciens</em> last year. Transformed calli were screened on selection medium containing antibiotics. In this year, the transformed calli will be regenerated and the transgenic<em> Oncidesa</em> seedlings will be developed. These transgenic <em>Oncidesa</em> plants will be further proved by polymerase chain reaction and Southern analysis. We expect that expression of <em>EIN2/EIN3</em> dual gene RNA-mediated silencing construct would block the ethylene signal transduction in <em>Oncidesa</em> and thereby prolong vase life of cut <em>Oncidesa</em> flowers. Development and Application of Gene Editing Technology in <em>Oncidesa</em>   <em>Oncidesa</em> orchid one of the most importnat cut flowers in Taiawn. Currently, about 80% of <em>Oncidesa</em> cut flowers are exported, with the biggest export value in cut flower industry. During harvest and postharvest ethylene production is induced by pollinia cap dislodgment and makes petals wilt. The effect of fumigation by fresh-keeping agent is still not good enough. In this project, genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) were employed to knockout <em>EIN2</em> gene in Oncidesa to prolong the shelf-life of Oncidesa cut flowers by interrupting ethylene signal transduction. As a first step of this project, <em>EIN2</em> 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, T7 endonuclease I 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<em> Agrobacterium</em>-mediated transformation last year. The final construct was transferred into embryogenic calli of Oncidesa via particle bombardment and <em>Agrobacterium</em> method. In this year, the embryogenic calli of <em>Ondidesa</em> will be selected for the presence of green fluorescent protein gene and further regenerated into <em>Oncidesa </em>seedlings. Hopefully, cut flowers of edited <em>Oncidesa</em> could last longer in a vase. Virus-resistance chrysanthemum breeding by genome editing technology    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.  Developing new varieties of <em>Oncidium </em>and <em>Cymbidium</em>  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.  Study on the hybridization of economical orchids  The CYT177 and CYT197 crosscombinations of blue-purplePhalaenopsiswere successful to get 3-101 seedlings to select blue-purple flowers. Seedlings of these two crosscombinations had short juvenile phase for flowering ability after culturing four months from flask stage. In this study, the flower stalk nodal buds ofRhynchonopsisTariflor Blue Kid were used as explants for proliferation. The results showed that the highest survival percentage , the highest induction percentage of buds and induction rate were induced by two apical shoot at stage1 and stage 2-3. Comparison horticultural traits of diploid and tetraploid (colchicine tretment) plants ofPhalaenopsis pulcherrimafma.coeruleashowed that tetraploid with lager flowers and seed capsules, thicker diameter of stalk and ovary, shorter length of leaf and stalk, and thicker texture of petal were observed. Although tetraploid plants show better horticultural traits than that of diploid plants, their hybridization affinity needs more cross investigations for further information.This experiment was applied to Rhynchonopsis Tariflor Blue Kid mericlones by photoperiod treatment B, which promoted flowering one month earlier than natural daylength.  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%).    Breeding and molecular genetic analysis of Anthurium.  The production area of anthurium in Taiwan was more than 200 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 and develop molecular-assisted breeding techniques to assist traditional breeding.  Development of key techniques for reducing the losses of Phalaenopsis caused by biological factors during transportation  It is known that the pathogen <em>Fusarium solani </em>caused yellow leaf of <em>Phalaenopsis</em> and had seriously affected the production and export of <em>Phalaenopsis</em>. The purpose of this study is to develop an Integrated Pest Management (IPM) for farmers in cultivated reference. Another, this project will take pests surveys of exporting <em>Phalaenopsis </em>nurseries, make identification of pests categories from those nurseries, and develop control strategies. After long distance marine transportation, the other some problems of <em>Phalaenopsis</em> will be studied, for example chilling injury. The methods for reducing chilling injury including fertilization,temperature conditioning before and after transportation will also be investigated.  Breeding of Weather Resistance Dianthus  The production periods and altitude in many flower industries have become shorter and highter due to highter average temperature in Taiwan. Use Taiwan's native Diathus species that have weather tolerance for breeding can increase the growth rate and yield in low altitude field, and reduce the damage of hillsides. The self breeding purification line of heat tolerate Dianthus palinensis and introduced Dianthus species can use for improve heat tolerance of later generations. Use these two self breeding purification line for cross breeding and select the heat tolerant generations can get the line that tolerant of Taiwan weather efficiently. We use heat tolerant endemic and commercial Dianthus for self breeding purify and select the excellent line as the cross breeding parents in this plan. We use excellent later generations for mutation breeding and produce tetraploid plants that can use for triploid breeding. We also stored the pollen of endemic species for breeding later and protect the gene pool and through seed germination treatment to improve seed germination potential.  Breeding of Heat-resistant and Scented of Phalaenopsis Varieties and Improvement of Propagation and Cultural of Calanthe    In order to develop hybrid breeding of <em>Phalaenopsis</em> spp. and small-sized and multi-flowered <em>Phal. </em>excellent plants and heat-resistant and fragrant small and medium-sized <em>Phal.</em> strains were selected. Establish the best propagation season of <em>Calanthe </em>Rollissonii and adjust the flowering period of 1-2 months earlier in <em>Cal. triplicata</em>.  Breeding and Selection of Landscape Hydrangea  Hydrangea (Hydrangea macrophylla Thunb.) is famous for its distinct flower type and delightful colors. There are pot flower ,cut flower and landscape hydrangeas. Most of the hydrangeas have better floral quality in temperate area. In Taiwan, flower regulation was needed for better flower quality and hight price. However, the costs will be higher. Mites and powdery mildew may damage visual quality and growth of hydrangea. The main purpose of this work is to do cross breeding with Hydrangea macrophylla Thunb. and Hydrangea chinensis Maxim as parental materials. This year, back cross and off spring selection will be completed. New hybrid offspring may have stronger growth potential and increase farmers' income.  Breeding of Heat Tolerant Azalea and Camellia  Azalea and camellia are important ornamental flower in north of Taiwan. However, azalea variety had little choice in landscape and pot flowers were not able to survive over hot summer. Camellia had little flower in summer. In order to meet consumers’ demand and increase flower traits, we continue doing hybrid and offspring selection. In order to evaluate heat tolerance of camellia and azalea, cell membrane thermostability of mature leaves and field culture will be done this year.  Selection of Low Chilling Variety on <em>Prunus</em> spp.  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.   Breeding of Oncidium, Dendrobium and Summer Flowering Chrysanthemum      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. Due 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.  Breeding of Cymbidium and Lily  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. Selecting the single plant of <em>Lilium </em><em>formosanum</em> for cut flower breeding.  The flower breeding of high temperature adaptability  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 and improving planting practices. 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.  Studies on breeding and mass propagation of <em>Spathoglottis</em> new cultivars.  Three superior selected plants of Spathoglottis will be selected and cultivar comparison for five clones of superior plants of Spathoglottis will be tested.  The hybrid breeding of Spathoglottis had achievements. The asexual micropropagation techniques will be developed to mass produce clonings.  Kaohsiung and Pingtung Local Flowers Breeding  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 hybrids, 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 for new varieties publishing 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 flower for cut flower in Taiwan. The objective of this study was to develop new cultivars of Renanthera alliance by collection, intergeneric hybridization, <em>in vitro</em> propagation and embryo rescue techniques. 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.  Breeding of ornamental daylily varieties  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 5 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 with special flower colors and easier to grow characters will be released afterwards.  Breeding of fragrant Phalaenopsis  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 that have finefragrance to hybrid. 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 Phalaenopsis under different external environments.  Breeding and key techniques research on orchids and bulb flowers  Vanda orchids has become more and more popular as a cut flower in Taiwan recent years. Production of Vanda cut flowers is increasing year by year and it was not only for domestic market but also exported to Japan and other countries. However, most varieties cultivated in Taiwan were breeded by Thailand which adapt to tropical climate. Kaohsiung and Pingtung counties are the major production area of Vanda in Taiwan. High-temperature often causes poor quality of cut flowers and shorter vase life. It will be very helpful to improve Vanda industry by breeding varieties adapt to climate of Taiwan. Meanwhile, stable production system of Vanda cut flowers is also very critical. As regards setting up flowering regulation system, different forcing treatment are going to be conducted to evaluate the effects on maturity and quality of induced flowering of commercial varieties of <em>Dendrobium</em> . Meanwhile, application of GA combinded with cytokinins will also be conducted to improve the flowering rate and quality of Paphiopedilum. Calla lily 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 and amaryllis and to execute the breeding program. We also will select superior progenies to apply the plant breeder right of calla and amaryllis. For studying on important bulb flowers propagation system, we will test and select suitable propagation system for calla lily and amaryllis by tissue culture and fertilizer test methods to set up the best mass propagation system.
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