張樂樂,陳 翔,柯媛媛,柳彬彬,Muhammad Ahmad Hassan,張 妍,許 輝,李金才,2**
冬小麥抗倒春寒性能鑒定方法和指標的比較*
張樂樂1,陳 翔1,柯媛媛1,柳彬彬1,Muhammad Ahmad Hassan1,張 妍1,許 輝1,李金才1,2**
(1.安徽農業大學農學院/農業農村部華東地區作物栽培科學觀測站,合肥 230036;2.江蘇省現代作物生產協同創新中心,南京 210095)
倒春寒是限制黃淮冬麥區小麥穩產和豐產的主要因素之一,選育抗倒春寒小麥新品種是降低倒春寒危害的重要手段,建立簡單易行、易推廣且全面系統的小麥抗倒春寒性能鑒定方法和指標體系對保障糧食安全有著重要意義。本文系統總結了冬小麥抗倒春寒鑒定的方法和指標,進行了比較和評價,并提出鑒定原則和程序。(1)鑒定方法方面,主要包括直接鑒定法、間接鑒定法和綜合鑒定法等,直接鑒定法有田間自然鑒定法(引種栽培應用最廣,但周期長、工作量大)、人工模擬氣候室法(周期短、可重復性強,但對設備與技術要求較高)和生長恢復法(冬前凍害鑒定較多,抗倒春寒鑒定方面較少,一般與其它鑒定方法配合使用)等;間接鑒定法有助倒春寒危害溫度閾值的研究,但目前指標與抗寒性關系還未有明確定論,應用受到限制;綜合鑒定法鑒定結果全面而準確,但對技術、成本要求較高且工作量較大。因此,在鑒定過程中要結合實際情況合理搭配、綜合運用。(2)鑒定指標方面,主要包括形態與農藝、生理生化、氣象與生態指標等,形態與農藝指標一般用于田間自然鑒定法,常以數量、質量、顏色和形態變化等作為參考項;生理生化指標一般用于間接鑒定法,包括活性氧、滲透調節物質、內源激素含量、抗氧化酶活性以及光合與呼吸特性等;氣象與生態指標常應用于各種鑒定方法,氣溫、地溫、冠層溫度、半致死溫度和恢復生長最低溫度常作為鑒定指標,而光照、CO2、濕度和肥力等因素會影響結果準確性。(3)關于鑒定原則與程序,本文提出了鑒定結果的地域性、指標選用的針對性、鑒定方法與指標的綜合性和實用性等鑒定原則,并以易災期、品種敏感期、敏感器官、鑒定方法和指標、低溫程度、田間/盆栽試驗、數學分析方法、抗寒性能評價與分級為主要流程的鑒定程序。最后,從抗寒生理研究、新興技術鑒定研究與應用和鑒定產品的研發等方面做了展望。
冬小麥;倒春寒;鑒定;指標;方法
小麥(L)是世界三大糧食作物之一,全球約60%的人口以小麥為主食[1]。2050年世界人口預計達到97.3億,小麥產量年均增長率要達到2.4%才能滿足全球人口增長對糧食的需求,但目前全球小麥產量年均增長率僅為0.9%[2?3]。同時隨著全球氣候變暖,極端低溫災害事件頻發重發,嚴重影響了小麥的穩產、豐產[4?5]。低溫災害給中國、美國、澳大利亞以及部分歐洲國家的小麥生產造成了極大的損失[6?11]。據報道,1995?2010年美國堪薩斯州的低溫災害事件發生次數多達41次,導致小麥減產超過538kg·hm?2[6]。澳大利亞昆士蘭和新南威爾士北部每年因低溫災害導致小麥減產10%[7],年均經濟損失達1億澳元[8]。1980?2020年中國小麥主產區發生近20次大規模低溫災害事件,其中尤以黃淮冬麥區的倒春寒(Late Spring Coldness,LSC)危害最為嚴重,該地區發生頻率可達40%,在2009、2013、2015、2018和2020年均有發生[9?11]。因此,倒春寒已成為限制中國小麥穩產豐產與優質的主要農業氣象災害。
小麥倒春寒災害主要指在拔節?孕穗期遭遇突然降溫天氣,造成幼穗受傷或死亡,部分小穗不結實甚至全穗不結實,從而導致小麥減產的一種農業氣象災害[12]。前人從光合作用[13]、呼吸作用[14]、活性氧代謝[15]、滲透調節[16]、內源激素代謝[17]以及蛋白質組學[18]和基因標記技術[19]等方面對倒春寒危害機理[20]進行了大量研究,但其致災機理尚未明確。同時由于倒春寒的發生具有隨機性與不可預見性,且受害癥狀具有隱蔽性,從而導致災前預防和災后減災措施難以及時到位,因此,小麥生產中應對倒春寒的防災減災能力不強。選用抗倒春寒品種是小麥生產中降低倒春寒危害的重要手段,但由于缺少準確有效的鑒定方法和指標體系以及相關標準,使得抗倒春寒品種的選育面臨重大難題。因此,本研究通過總結和綜合評價前人研究的冬小麥品種抗倒春寒性能(Tolerance against Late Spring Coldness,TLSC)鑒定方法和指標,提出相關鑒定原則和程序,并對未來的研究方向進行展望,旨在為抗倒春寒小麥新品種選育和減災保產提供理論參考。
目前,冬小麥TLSC鑒定可以參考小麥品種抗寒性鑒定的一般方法,主要有三種,分別是直接鑒定法(DIM)、間接鑒定法(IIM)和綜合鑒定法(CIM),這些方法通常與各種數學分析方法(MAM)組合使用。各種方法的具體分類見圖1。

圖1 冬小麥品種TLSC鑒定方法分類
1.2.1 直接鑒定法
(1)田間自然鑒定法(FIM)
FIM是在倒春寒發生后對田間受害植株的器官或組織以一定的標準進行評價和比較,根據作物受災表現評價其抗寒性的一種直觀鑒定方法。該方法是引種栽培應用最廣泛的一種鑒定方法,也是公認最早、最有效的鑒定方法之一[21]。農業農村部標準《NY/T 1307?2007》[22]中提出的小麥抗寒性分級就是利用這一方法將冬麥區春季階段的小麥抗寒性分為5級,即1級(無凍害表征)、2級(葉尖受凍發黃)、3級(葉片凍死一半)、4級(葉片全枯)和5級(植株或大部分分蘗凍死)。喬玉強等[23]在倒春寒發生年份對同一品種進行不同播期試驗,調查了田間異型穗率、殘缺穗率和無籽穗率,結合其它相關指標通過聚類分析得出同一品種在不同播期和不同地區的TLSC強弱表現均有差異,表明田間栽培管理措施和不同地區易災期以及凍(冷)害敏感期等因素影響著品種的TLSC評價結果。FIM雖然能較好地鑒定冬小麥TLSC,但由于鑒定周期漫長且工作量較大,一定程度上影響了抗倒春寒新品種選育和推廣效率。
(2)人工模擬氣候室法(ACCM)
ACCM是通過人工模擬大田低溫環境來進行小麥抗寒性鑒定的一種常用方法。目前,利用該方法研究小麥抗寒生理機制和鑒定小麥抗寒性已成為主流。Liu等[24]利用ACCM在小麥拔節期和孕穗期進行低溫脅迫模擬試驗,發現低溫脅迫程度和持續時間顯著影響小麥的光合生產能力,進而影響產量的形成。Zhang等[25]利用ACCM發現孕穗期低溫會誘導穗部脫落酸的積累,同時提升蔗糖合成酶的活性,進而增加穗部蔗糖含量來提升抗寒能力,但會影響幼穗的正常發育。ACCM鑒定周期較FIM短,可重復性強,同時減少了環境與品種基因型互作的影響[26]。但該方法對設備要求較高,且對各種變量也有嚴格要求,尤其是對溫度參數的設置,需要對大量氣象數據進行分析確定合理的降溫模式和水平。
(3)生長恢復法(GRM)
GRM是通過觀察和測定小麥植株或組織受低溫脅迫后各項指標的恢復水平來評判其抗寒性的一種鑒定方法。半致死溫度(LT50)是該方法中最常用到的指標,多用于小麥冬前凍害的鑒定[27?28],但單獨利用GRM鑒定冬小麥品種TLSC的情況較少。GRM常與ACCM、IIM等鑒定方法配合使用,通過小麥各項指標的恢復水平對其TLSC進行鑒定。
1.2.2 間接鑒定法(IIM)
IIM是采用化學和生物技術研究作物群體、個體、組織器官與細胞的生理生態變化,通過分析各項指標與抗寒性之間的關系來鑒定小麥抗寒性的一種方法。倒春寒發生后,通過測定可溶性糖等滲透調節物質的含量、活性氧含量與抗氧化酶活性、含水量與電導率、光合特性、呼吸特性等相關指標[29?39],以及對小麥受凍細胞的顯微與超微結構觀察[40]等來鑒定小麥抗寒性的方法均屬于IIM。該方法通過量化各項指標準確反映冬小麥的TLSC,有助于倒春寒危害的溫度閾值研究。但由于機理復雜,目前部分測定指標與抗寒性關系還未有明確定論,該方法的應用受到限制。
1.2.3 綜合鑒定法(CIM)
CIM是將多種鑒定方法與鑒定指標相結合,利用統計分析方法,綜合鑒定或評價小麥抗寒性的一種方法。李桐等[41]利用了ACCM、FIM和IIM綜合鑒定冬小麥品種,通過各種方法相互驗證研究發現室內快速降溫處理結合超氧化物歧化酶(SOD)活性及抗壞血酸、過氧化氫和丙二醛(MDA)含量的分析,能有效鑒定出小麥品種抗寒性的強弱。因此,利用ACCM結合GRM、FIM和IIM等多種方法和多種指標綜合鑒定冬小麥TLSC,其鑒定結果可能更加全面而準確,但該方法對技術、成本要求較高且工作量較大。
1.2.4 數學分析方法
主成分分析法(Principal Component Analysis Method)[42]、聚類分析法(Cluster Analysis Method)[43]、極點排序法(Polar Ordination Method)、隸屬函數法(Membership Function Method)[44?45]、加權法(Weighting Method)以及模糊評判法(Fuzzy Evaluation Method)[42]等是TLSC鑒定常用的數學分析方法。主成分分析法通過降維和去噪,加快對樣本有價值信息的處理速度,常被用來篩選TLSC鑒定的關鍵性指標。馬德栗等[46]利用主成分分析法對湖北省1951?2010年的倒春寒災害事件建立了評估模型,并對其危害程度做了等級劃分。隸屬函數法、加權法和模糊評判法在小麥TLSC鑒定的使用過程中需設定各指標的權重值,相對于主成分分析法,其主觀性較強。此外,鑒定中也常用到一些數學模型,例如累計低溫度日(Accumulated Cooling Degree Days,ACDD,℃·d)也能作為數學分析方法較好地量化不同低溫脅迫強度和持續時間的綜合效應[1,47]。
鑒定冬小麥品種的TLSC,不僅要選擇合適的鑒定方法,還要選用合適的鑒定指標。小麥遭受倒春寒危害后,植株內部會發生一系列復雜的生理生化變化,外部形態也會表現出不同的受害特征,從而影響產量和品質。目前,國內外學者根據冬小麥植株的內外變化及產量等相關特征,從形態與農藝、生理生化以及氣象與生態等方面來研究和鑒定冬小麥TLSC(圖2)。
(1)形態與農藝指標
形態與農藝指標一般用于田間自然鑒定法,它包括形態、穗部結實特性以及產量等指標,且常以數量、質量、顏色和形態變化等作為參考項。研究表明[48?49],倒春寒會導致小麥葉片發黃、皺縮甚至枯萎,農業農村部標準《NY/T 1307?2007》[22]將小麥葉片受害癥狀作為TLSC分級的重要指標。小麥遭遇倒春寒后,莖的生長點受到損傷會導致節間變色、粗糙、裂開、塌陷或節擴大等癥狀[50]。大量研究表明[51?55]倒春寒對小麥穗部危害更大,在拔節后期至孕穗期主要影響幼穗小花的發育[51],導致結實率大幅降低,從而造成減產。由于同一麥穗不同穗位的小花發育順序不同[52],如果該時期遭遇倒春寒,會導致退化、不孕小花和小穗數增加[53?54],出現異形穗、殘缺穗或無效穗[23]等癥狀。胡新等[55]利用加權平均法構建殘穗指數來評價冬小麥TLSC,同樣發現殘穗指數越大,危害發生越嚴重。

圖2 冬小麥品種TLSC鑒定指標分類
因此,在選擇TLSC鑒定指標時,要充分考慮小麥品種在多年田間試驗中的形態與農藝指標變化,進行田間調查并統計出現頻率最高、影響最大的指標作為鑒定關鍵指標。此外,鑒定時還要選擇受倒春寒危害最嚴重器官的相關指標作為參考。
(2)生理生化指標
活性氧、滲透調節物質、內源激素含量、抗氧化酶活性以及光合與呼吸特性常被作為TLSC鑒定的生理生化指標,該類指標一般用于間接鑒定法。研究表明,幼穗、葉片與根系的可溶性糖、游離脯氨酸、可溶性蛋白、MDA含量及過氧化物酶、SOD等抗氧化酶活性與相關內源激素含量等均可作為小麥TLSC的鑒定生理生化指標[27,29?32]。此外,自由水/束縛水比值、電解質滲透率和電導率等也常被用作TLSC鑒定的生理生化指標[33?34]。光合和呼吸特性方面,光合速率、蒸騰速率、葉片氣孔導度、胞間CO2濃度及最大光化學效率(Fv/Fm)也可作為鑒定冬小麥品種TLSC的指標[35?38]。Kurimoto等[39]研究認為,低溫下植物呼吸穩態的保持和較高的ATP生產效率有助于維持正常的生長發育,表明呼吸特性相關指標用于冬小麥品種TLSC鑒定也具有可行性。
由于倒春寒分為0℃以上的冷害和0℃以下的凍害兩種類型,利用生理生化指標鑒定時要考慮這兩種脅迫水平生理生化響應機制的差異,從而篩選出適宜的鑒定指標。倒春寒發生后,小麥生理生化指標的變化比農藝指標更為顯著,對測定環節的技術要求更高,都可能造成結果的準確度和實用性降低。因此,鑒定時利用相對受害值作為評價指標,其結果更具代表性。
(3)氣象與生態指標
前人研究常以氣溫作為冬小麥品種TLSC鑒定的指標,但實際上地溫會補償低溫天氣所帶來的負面影響,致使小麥生長的環境溫度要比氣溫高。因此,冠層溫度更能代表小麥生長的環境溫度[56],將冠層溫度作為鑒定的氣象指標更為切合實際。在具體的鑒定應用中,如GRM還常利用LT50和恢復生長最低溫度作為冬小麥品種TLSC的鑒定指標。
鑒定冬小麥品種TLSC時,還易受光照強度、田間CO2濃度、空氣濕度和土壤肥力等因素的影響,導致結果的準確性降低。此外,鑒定中還需考慮群體與個體及不同器官之間的差異,明確三者與氣象和生態指標之間的關系,對大田生產上鑒定冬小麥品種TLSC更具實際意義。
(1)鑒定結果具有地域性。由于不同地區倒春寒易災期不同,同一品種在不同地區可能表現出不同的TLSC,小麥的敏感期與易災期重合將對小麥造成更為嚴重的危害。
(2)指標選用具有針對性。小麥主莖與分蘗的生育時期并不完全同步,抗倒春寒能力也不相同,因此,鑒定時要注意區分是主莖還是分蘗帶來的差異,還要統一鑒定的生育時期。此外,由于冬小麥不同生育時期各個器官的抗寒能力不同,在鑒定時還應重點考慮不同器官敏感性的差異。
(3)鑒定方法和指標具有綜合性。冬小麥品種TLSC是一個復雜的生物學數量性狀,其鑒定方法和指標之間并不是相對獨立的,單獨使用其中任何一種單項指標或單個方法,所得的結果都具有片面性[57?58]。鑒定時還需結合相關氣象指標同步進行,注重地溫和冠層溫度對鑒定結果的影響[59]。
(4)鑒定時需排除交叉逆境脅迫的干擾。在鑒定冬小麥品種TLSC過程中要嚴格控制干旱等交叉逆境脅迫的干擾,以免影響鑒定結果的準確性。
(5)鑒定方法和指標要具有實用性。鑒定指標和方法的研究和選用,要服務于冬小麥抗倒春寒新品種選育,符合“簡單易行、易推廣、低成本”原則。
鑒定冬小麥品種TLSC時,需要結合各地氣候條件和栽培管理方式,選用合適的鑒定方法和指標,針對不同氣候的小麥產區建立不同的鑒定方法和指標體系,以便選育出適合當地的抗倒春寒新品種。目前,小麥育種中鮮有TLSC鑒定標準程序,因此,結合前人研究和鑒定原則,總結出一套冬小麥品種TLSC鑒定程序如圖3。該鑒定程序通過氣象易災期和品種敏感期分析,結合適宜的鑒定方法和指標對冬小麥品種TLSC進行綜合鑒定,對國家和地方推廣品種均有一定參考價值。

圖3 冬小麥品種TLSC鑒定程序
近年來,農業遙感技術、表型組學、作物模型、蛋白質組學以及基因標記技術飛速發展,對農業生產的快速發展起到了巨大的推動作用[60?73]。遙感技術[60?61]被認為是大范圍探測作物物候狀況的可靠工具,利用智能手機、無人機或者相機對冬小麥進行近地表遙感圖像監測,評估極端天氣事件對作物的影響,將遙感探測到的植被指數與氣象信息相結合,可作為天氣變化對作物產量影響的評價和鑒定方法,成本低且快速高效,可及時做出預防或應對。還有學者[62?63]將高時空分辨率遙感數據與作物模型相結合,獲取農田作物生長發育實時狀況的信息。作物模型方面,國際上小麥生理模型的研究發展迅速[64?65],其中CERES-Wheat[66]、WOFOST[67]、FROSTOL[68]等模型可進行低溫模擬并已在生產上推廣應用。蛋白質組學方面,低溫脅迫下植物體內大量基因表達,誘導合成低溫脅迫響應蛋白以抵御低溫對植物的破壞[69]。Janmohammadi等[70]研究表明,低溫脅迫初期光合作用代謝通路中的放氧增強蛋白、NADH脫氫酶和脫氫抗壞血酸還原酶等蛋白下調?;驑擞浄矫妫赏ㄟ^QTL等分子標記技術[71]結合傳統育種技術,使育種家無需等待田間試驗中倒春寒的發生就可選擇耐寒關鍵基因[72],從而加快小麥抗倒春寒新品種的選育。薛輝等[73]研究發現,1B染色體上的AX-109492586、3A染色體上的AX-110064042具有較好的耐倒春寒特性,可用于開發CAPS標記。但目前來看,新興技術在冬小麥品種TLSC上的應用還較少,且與小麥抗寒性能方面的交叉研究不足。
冬小麥抗寒生理研究是完善TLSC鑒定方法與指標的重要基礎,這些研究包括冬小麥倒春寒發生的致災溫度閾值、敏感生育時期、敏感部位以及生理、生化和生態變化等。今后需繼續深入小麥倒春寒災害的災變過程和致災機理的研究,從而加快推進國家和地方冬小麥品種TLSC鑒定標準的制定和推廣。
隨著現代信息技術的快速發展,大數據技術與作物模型、表型組學、遙感技術及分子生物技術的結合是冬小麥品種TLSC鑒定研究的創新突破點。CRISPR/Cas9和基因堆積等分子生物技術在育種工作中的應用已逐漸成熟,可對小麥抗倒春寒基因進行定位和編輯[74]。目前,國內外學者已經鑒定出一批抗寒基因和抗寒蛋白,未來應該對已鑒定出的抗倒春寒基因或蛋白質進行重點篩選,驗證其作為冬小麥品種TLSC鑒定指標的可靠性,推進分子鑒定指標體系的構建,加快研究成果轉化到實際生產應用和抗倒春寒新品種選育工作中。
目前,育種機構常需采用多年復雜的傳統方法來鑒定小麥新品種的TLSC,成本大,周期長,效率低,使新品種的推廣受到阻礙,亟需一種既能提升鑒定效率又能降低成本的TLSC鑒定產品。這種產品既可以是鑒定儀器,也可以是鑒定服務,這對推進抗倒春寒小麥新品種的推廣種植意義非凡。此外,鑒定產品的研究和開發既要服務于新品種選育,還要服務于生產實際。對農民來說,倒春寒災前預防,災后及時采取減災措施,選用簡便易行、快速和成本低廉的方法和產品更具實際意義。如應用抗寒劑提升小麥TLSC也是有效的防災減災手段,利用現有的研究基礎開發成本低廉的抗寒劑與配套的農機裝備應是今后研究的重點方向之一。因此,加強研究并開發服務于育種工作的抗寒性鑒定高端技術產品和服務于生產中防災減災的實用新型技術產品,對提高中國小麥生產的防災、減災能力,促進小麥生產提質減損增效,保障國家糧食安全具有重要意義。
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An Inclusive Comparison of Identification Methods and Indices of Winter Wheat Tolerance against Late Spring Coldness
ZHANG Le-le1, CHEN Xiang1, KE Yuan-yuan1, LIU Bin-bin1, MUHAMMAD Ahmad Hassan1, ZHANG Yan1, XU Hui1, LI Jin-cai1,2
(1.College of Agronomy,Anhui Agricultural University/Crop Cultivation Science Observatory in East China of the Ministry of Agriculture and Rurad Affairs,Hefei 230036,China;2.Collaborative Innovation Center of Modern Crop Production in Jiangsu,Nanjing 210095)
Late spring coldness is an important factor limiting the stable and high yield of wheat in Huang-huai winter wheat region. New wheat varieties with tolerance to late spring coldness is an important strategy to reduce the damage of late spring coldness. It is of great significance to establish a simple, easy to popularize and comprehensive evaluation methods and indices system of wheat tolerance to late spring coldness for ensuring food security. In this paper, the methods and indices for identification of winter wheat tolerance to late spring coldness were summarized, compared and evaluated, and the identification principles and procedures were also put forward. (1) The identification methods of winter wheat tolerance to late spring coldness mainly consisted of direct identification method, indirect identification method and comprehensive identification method. Direct identification methods consisted of field natural identification method, artificial climate chamber method and growth recovery method. The field natural identification method had the characteristics of most widely used in the introduction and cultivation, but it had long cycle and heavy workload. Artificial climate chamber method had the characteristics of short cycle and strong repeatability, but it had high requirements for equipment and technology. The growth recovery method was widely used in the identification of freezing injury before winter, but it was less used in the identification of tolerance to late spring coldness. The indirect identification method was helpful to study the temperature threshold of late spring coldness, but the relationship between the indices and cold resistance had not been clear, so its application was limited. The comprehensive identification method had the characteristics of comprehensive and accurate appraisal results, but it had high requirements for technology, cost and workload. Therefore, in the identification process, it was necessary to combine the actual situation, reasonable collocation and comprehensive use of a variety of methods. (2) The identification indices mainly consisted of morphological and agronomic, physiological and biochemical, meteorological and ecological indices. Morphological and agronomic indices were generally used in field natural identification, and the quantity, quality, color and morphological changes were often used as reference items. Physiological and biochemical indices were generally used for indirect identification, including reactive oxygen species, osmotic adjustment substances, endogenous hormone content, antioxidant enzyme activity, photosynthetic and respiratory characteristics, etc. Meteorological and ecological indices were often used in various identification methods. Air temperature, ground temperature, canopy temperature, half lethal temperature and minimum temperature of restoration growth were often used as identification indices, while factors such as light, CO2, humidity and fertility could affect the accuracy of results. (3) Appraisal principles and procedures. This paper put forward the identification principles, such as the regionality of identification results, the pertinence of indices selection, the comprehensiveness and practicability of identification methods and indices, etc. The identification procedures were mainly composed of disaster prone period, variety sensitive period, sensitive organs, identification methods and indices, low temperature degree, field or pot experiment, evaluation and classification of cold resistance by mathematical analysis method and writing report. Finally, the prospects of cold resistance physiology research, identification research and application of emerging technologies and identification product development were made.
Winterwheat; Late spring coldness; Identification; Index; Method
10.3969/j.issn.1000-6362.2021.02.006
張樂樂,陳翔,柯媛媛,等.冬小麥抗倒春寒性能鑒定方法和指標的比較[J].中國農業氣象,2021,42(2):146-157
2020?12?02
“十三五”國家重點研發計劃課題(2017YFD0300408);安徽省自然科學基金(2008085QC122);安徽省科技重大專項(202003b06020021)
李金才,教授,博導,主要從事作物生理生態研究,E-mail:ljc5122423@126.com
張樂樂,E-mail:zhanglele912@163.com