花生胚小葉對卡那霉素的敏感性研究

王鳳歡，何美敬，楊鑫雷，崔順立，穆國俊，侯名語，劉立峰

(教育部華北作物種質(zhì)資源實(shí)驗(yàn)室/河北省作物種質(zhì)資源重點(diǎn)實(shí)驗(yàn)室/河北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，河北 保定 071001)

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花生胚小葉對卡那霉素的敏感性研究

王鳳歡，何美敬，楊鑫雷，崔順立，穆國俊，侯名語，劉立峰*

(教育部華北作物種質(zhì)資源實(shí)驗(yàn)室/河北省作物種質(zhì)資源重點(diǎn)實(shí)驗(yàn)室/河北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，河北 保定 071001)

卡那霉素是植物遺傳轉(zhuǎn)化中常用的一種篩選劑。研究花生胚小葉對卡那霉素的敏感性，對建立利用卡那霉素篩選花生遺傳轉(zhuǎn)化的有效體系具有重要意義。以3個(gè)不同基因型花生弗落蔓生、麻油1-1和濮花23號為試驗(yàn)材料，通過計(jì)算胚小葉黃化率、叢生芽誘導(dǎo)率和生根數(shù)等，并觀察外植體的生長狀態(tài)，研究不同濃度卡那霉素對胚小葉生長發(fā)育、叢生芽分化和叢生芽生根階段的影響，以期確定3個(gè)品種胚小葉各個(gè)分化階段的適宜篩選濃度。結(jié)果表明，不同基因型花生對卡那霉素的敏感性不同，弗落蔓生、麻油1-1和濮花23號胚小葉叢生芽分化篩選濃度依次為： 150mg/L、100mg/L和50mg/L；叢生芽生根篩選濃度分別為：弗落蔓生20mg/L、麻油1-1 20mg/L和濮花23號10mg/L。本研究篩選到不同品種胚小葉叢生芽分化和叢生芽生根階段的適宜卡那霉素濃度，為以花生胚小葉為外植體的花生遺傳轉(zhuǎn)化陽性植株的篩選奠定了基礎(chǔ)。

花生；卡那霉素；基因型；敏感性

花生在我國經(jīng)濟(jì)發(fā)展中占有重要地位，是我國重要的經(jīng)濟(jì)作物和油料作物[1]。隨著基因工程的發(fā)展，利用轉(zhuǎn)基因技術(shù)提高作物產(chǎn)量和品質(zhì)已成為重要的作物育種手段。近幾年在花生遺傳轉(zhuǎn)化中常用的轉(zhuǎn)化方法主要是農(nóng)桿菌介導(dǎo)法[2]。為了快速高效地獲得轉(zhuǎn)化體，轉(zhuǎn)化過程中利用抗生素進(jìn)行篩選是必不可少的。

目前，在植物轉(zhuǎn)基因初步檢測中使用的篩選劑主要有抗生素類和氨基酸類，其中以抗生素類應(yīng)用較多?？敲顾厥悄壳爸参镞z傳轉(zhuǎn)化中應(yīng)用最廣泛的一種篩選劑[3-4]，已用于多種雙子葉植物的遺傳轉(zhuǎn)化[5-11]?？敲顾厥褂玫臐舛纫虿煌贩N或相同品種的不同外植體類型而不同[12-16]。如果卡那霉素濃度太低，則不能充分抑制或殺死未轉(zhuǎn)化的細(xì)胞，從而造成假陽性率過高；若卡那霉素濃度過高，又抑制甚至殺死轉(zhuǎn)化細(xì)胞，導(dǎo)致不能得到足量的轉(zhuǎn)基因植株。為了使卡那霉素對植物材料具有很好的篩選作用，需要在目的基因轉(zhuǎn)化之前，對所研究的材料進(jìn)行選擇性抗生素敏感性試驗(yàn)。

在花生的遺傳轉(zhuǎn)化研究中有關(guān)卡那霉素的敏感性篩選多見于單個(gè)生長階段的研究[12,17-18]，而對卡那霉素在花生外植體整個(gè)生長階段敏感性的系統(tǒng)篩選研究報(bào)道較少[19]。本研究以3個(gè)不同基因型的花生胚小葉為外植體，研究其整個(gè)發(fā)育期階段在添加有不同濃度卡那霉素的培養(yǎng)基中的生長情況及狀態(tài)，明確各個(gè)品種胚小葉外植體在各發(fā)育階段對卡那霉素的適宜篩選濃度，以期為進(jìn)一步花生的遺傳轉(zhuǎn)化和陽性植株的篩選提供試驗(yàn)依據(jù)。

1 材料與方法

1.1 材 料

供試花生(ArachishypogaeaL.)品種為弗落蔓生、麻油1-1和濮花23號，均來自河北農(nóng)業(yè)大學(xué)花生研究所種質(zhì)資源庫。胚小葉萌發(fā)培養(yǎng)基：MSB5+30 mg/L蔗糖+6.95 mg/L瓊脂，pH5.8；叢生芽誘導(dǎo)培養(yǎng)基： MSB5+30 mg/L蔗糖+6.95 mg/L瓊脂+6 mg/L 6-BA+0.2 mg/L NAA，pH5.8；芽伸長培養(yǎng)基：MSB5+30 mg/L蔗糖+6.95 mg/L瓊脂+4 mg/L 6-BA+2 mg/L GA3+0.2 mg/L NAA，pH5.8；生根培養(yǎng)基：1/2 MSB5+30 mg/L蔗糖+6.95 mg/L瓊脂+1 mg/L NAA+0.2 mg/L IAA，pH 5.8。

1.2 方 法

1.2.1 花生無菌苗的培養(yǎng) 選擇大小一致的無病斑且種皮完好的花生種子，用無菌水沖洗1～2次后用吸紙吸干，先后浸于75%酒精中1 min、0.1% HgCl2溶液中15 min， 進(jìn)行表面消毒，再用無菌水漂洗4～5次，剝?nèi)シN皮，浸泡于無菌水中6～7 h之后剝?nèi)∨咝∪~外植體將其接種于萌發(fā)培養(yǎng)基上培養(yǎng)。取培養(yǎng) 8 d 的胚小葉外植體，接種于叢生芽誘導(dǎo)培養(yǎng)基上，誘導(dǎo)叢生芽，待叢生芽長1mm左右，將其轉(zhuǎn)入伸長培養(yǎng)基中生長，每21～25 d繼代一次，直到叢生芽伸長至4～5 cm 后，切下伸長苗轉(zhuǎn)入生根培養(yǎng)基中進(jìn)行生根培養(yǎng)。培養(yǎng)過程中的培養(yǎng)條件均為溫度(26±0.5)℃，光照16 h/d，光照強(qiáng)度18.75～25.00 μmol/(m2·s)。

1.2.2 花生胚小葉分化的卡那霉素選擇壓的確定 取萌發(fā) 8 d 的花生胚小葉作為外植體，接種于含卡那霉素(Kan)的叢生芽誘導(dǎo)培養(yǎng)基上進(jìn)行敏感性試驗(yàn)，卡那霉素的濃度梯度為：0 mg/L、50 mg/L、100 mg/L、150 mg/L和200 mg/L共5個(gè)處理，培養(yǎng)25 d 后調(diào)查生長情況。

1.2.3 花生胚小葉叢生芽伸長的卡那霉素選擇壓的確定 取生長到1mm左右的無菌叢生芽接到含有卡那霉素的伸長培養(yǎng)基中，卡那霉素的濃度梯度分別為：0 mg/L、50 mg/L、100 mg/L、150 mg/L 和200 mg/L 5個(gè)處理，培養(yǎng)25 d后調(diào)查生長情況。

1.2.4 花生胚小葉叢生芽生根的卡那霉素選擇壓的確定 取無菌的待生長到4～5 cm左右的伸長苗接到含有卡那霉素的生根培養(yǎng)基中，卡那霉素的濃度梯度分別為：0 mg/L、10 mg/L、20 mg/L、30 mg/L、40 mg/L和50 mg/L，培養(yǎng)25 d后調(diào)查生根情況。

1.3 數(shù)據(jù)統(tǒng)計(jì)與分析

叢生芽誘導(dǎo)率=(誘導(dǎo)出叢生芽的外植體/接種總外植體數(shù))×100%

黃化率=(誘導(dǎo)出黃化的外植體數(shù)/接種總外植體數(shù))×100%

全部數(shù)據(jù)采用Microsoft Excel 2003與統(tǒng)計(jì)分析軟件SPSS Statistics 17.0進(jìn)行分析。

2 結(jié)果與分析

2.1 卡那霉素對花生胚小葉生長發(fā)育的影響

由圖1和圖2可以看出，花生弗落蔓生胚小葉外植體的三個(gè)生長發(fā)育時(shí)期對卡那霉素的敏感性存在差異，卡那霉素濃度在50 mg/L時(shí)，胚小葉外植體愈傷發(fā)育時(shí)期的黃化率達(dá)到50%，愈傷組織發(fā)白，葉片黃化，后期將未黃化的組織轉(zhuǎn)入低濃度的卡那霉素培養(yǎng)基中也未能分化出芽點(diǎn)，愈傷組織體積沒有增大，且顏色逐漸變?yōu)辄S褐色至死亡。叢生芽分化時(shí)期和伸長苗生長時(shí)期的黃化率分別為37.04%和26.67%，組織正常生長沒有明顯變化?？敲顾貪舛?00 mg/L時(shí)，胚小葉外植體的伸長苗生長對卡那霉素較其他兩個(gè)時(shí)期敏感，生長狀態(tài)表現(xiàn)為整個(gè)伸長苗只有最頂端葉片為淡綠色，葉柄和莖都已黃化至白化?？敲顾貪舛仍?50 mg/L和200 mg/L時(shí)，花生胚小葉外植體的三個(gè)生長時(shí)期的黃化率都達(dá)到100%。該結(jié)果表明,胚小葉外植體在它的不同生長發(fā)育時(shí)期對卡那霉素的敏感程度是存在差異的。因此以卡那霉素作為篩選標(biāo)記時(shí)，在胚小葉培養(yǎng)的不同時(shí)期階段應(yīng)選擇不同的卡那霉素適宜篩選濃度，避免濃度過高或過低而影響篩選效果。

圖 1 卡那霉素對胚小葉生長發(fā)育的影響Fig. 1 The effect of kanamycin on leaflet development

圖 2 卡那霉素對胚小葉生長發(fā)育的影響Fig. 2 The effect of kanamycin on leaflet development注：1～5：卡那霉素的濃度依次為：0 mg/L、50 mg/L、100 mg/L、150 mg/L和200 mg/L；A～C：花生胚小葉不同生長發(fā)育階段依次為：愈傷階段、叢生芽階段和伸長階段。Note: 1～5: The concentration of kanamycin was 0 mg/L, 50 mg/L, 100 mg/L, 150 mg/L and 200 mg/L, successively;A～C: The different growth stages of peanut leaflets were callus stage, clustered shoots stage and elongation stages, successively.

2.2 卡那霉素對不同花生基因型胚小葉分化的影響

附表和圖3表明，不同濃度的卡那霉素對同一品種花生的叢生芽分化的影響具有顯著性差異，隨著卡那霉素濃度的提高，叢生芽率逐漸降低，說明卡那霉素能夠抑制花生叢生芽的生長。不同品種的花生胚小葉對卡那霉素的敏感性差異較大。當(dāng)未添加卡那霉素時(shí)，3個(gè)品種都有較好的叢生芽率；當(dāng)卡那霉素的濃度為50 mg/L時(shí)，濮花23號的胚小葉叢生芽的分化明顯受到抑制，胚小葉的叢生芽率降為53.33%，而弗落蔓生的胚小葉的叢生芽率為87.5%，麻油1-1的胚小葉的叢生芽率為85.83%；當(dāng)卡那霉素的濃度增加為100 mg/L時(shí)，麻油1-1和濮花23號的胚小葉的叢生芽率降為51%以下，未分化叢生芽的胚小葉發(fā)生黃化轉(zhuǎn)為白化最后死亡。弗落蔓生對卡那霉素的敏感性較前2個(gè)品種低，在100 mg/L卡那霉素的選擇壓力下，胚小葉的叢生芽率仍在70%以上；當(dāng)卡那霉素的濃度達(dá)到200 mg/L時(shí)，其抑制效應(yīng)達(dá)到最大，3個(gè)品種的叢生芽率都下降到50%以下。由于卡那霉素隨著濃度的增高對胚小葉的前期生長有明顯的抑制作用，因此在胚小葉生長初期不宜用過高的卡那霉素濃度以免影響后期的轉(zhuǎn)化植株的生長，同時(shí)不同品種對卡那霉素的敏感性不同，因此以叢生芽率50%為標(biāo)準(zhǔn)來確定各品種的初期最適篩選濃度。由此得出，花生胚小葉叢生芽分化的卡那霉素的適宜篩選濃度：弗落蔓生為150 mg/L，麻油1-1為100 mg/L，濮花23號為 50 mg/L。

附表 卡那霉素對不同基因型花生叢生芽誘導(dǎo)率的影響 (%)

注：同列不同大寫字母表示差異顯著水平 (p<0.01)。

Note: The capital letter in the same column indicated the significance of difference at 0.01 level.

圖 3 卡那霉素對不同基因型花生叢生芽的影響Fig. 3 The effect of kanamycin on clustered shoots of different genotypes of peanut注：1～5：卡那霉素的濃度依次為：0 mg/L、50 mg/L、100 mg/L、150 mg/L和200 mg/L；A～C：花生品種依次為：弗落蔓生、麻油1-1和濮花23號。Note: 1～5: The concentration of kanamycin was 0 mg/L, 50 mg/L, 100 mg/L, 150 mg/L and 200 mg/L,successively; A～C: Peanut genotypes were Fuluomansheng, Mayou1-1 and Puhua23, successively.

2.3 對不同花生基因型胚小葉叢生芽生根的影響

當(dāng)花生伸長苗轉(zhuǎn)入含有不同卡那霉素濃度的生根培養(yǎng)基中，經(jīng)過5 d的培養(yǎng)，未添加卡那霉素的伸長苗開始生根，而添加卡那霉素的培養(yǎng)基中的伸長苗無一生根。經(jīng)過10 d的培養(yǎng)之后含有10 mg/L和20 mg/L卡那霉素的培養(yǎng)基中的伸長苗開始生根。隨著培養(yǎng)時(shí)間的增加，生長在未添加卡那霉素的培養(yǎng)基中的根越來越長，平均生根數(shù)量越來越多，而在含有卡那霉素的培養(yǎng)基中的伸長苗隨著培養(yǎng)基中卡那霉素濃度的增加，平均生根數(shù)量和平均根長度顯著下降(圖4～5)。從圖6可以看出，不同的基因型之間存在明顯差異，當(dāng)卡那霉素濃度為0 mg/L時(shí)，三個(gè)品種的根系生長發(fā)達(dá)，植株生長快?？敲顾貪舛仍黾拥?0 mg/L時(shí)，濮花23號的生根數(shù)量明顯下降，而麻油1-1和弗落蔓生變化不明顯；卡那霉素濃度為20 mg/L時(shí)，濮花23號的伸長苗不能生根，弗落蔓生的伸長苗基部有少許的根長出，根細(xì)小而脆弱，麻油1-1的伸長苗能長出明顯根系；卡那霉素濃度為30 mg/L、40 mg/L時(shí)，三個(gè)基因型花生伸長苗不能生根，逐漸變黑，植株枯萎而死亡。由此得出，不同品種花生胚小葉叢生芽生根的卡那霉素的適宜篩選濃度為：弗落蔓生為20mg/L，麻油1-1為20 mg/L ，濮花23號為 10 mg/L。

圖 4 卡那霉素對不同基因型根系長度的影響

圖 6 卡那霉素對不同基因型花生叢生芽生根的影響Fig. 6 The effect of kanamycin on rooting of clustered shoots of different genotypes of peanut注：1～5：卡那霉素的濃度依次為：0 mg/L、10 mg/L、20 mg/L、30 mg/L和40mg/L；A～C：花生品種依次為：麻油1-1、弗落蔓生和濮花23號。Note: 1～5: The concentration of kanamycin was 0 mg/L, 10 mg/L, 20 mg/L, 30 mg/L and 40 mg/L, successively.A～C: Peanut genotypes were Mayou1-1, Fuluomansheng and Puhua23, successively.

3 討 論

適宜的抗生素篩選濃度是提高花生遺傳轉(zhuǎn)化效率的影響因素之一，篩選濃度過高或過低均會(huì)影響轉(zhuǎn)化體的篩選效果。卡那霉素是植物遺傳轉(zhuǎn)化中常見的一種篩選劑，其使用的濃度因不同植物類型或相同品種的不同外植體類型而不同。張春濤等[5]研究表明，不同的大豆品種有其適宜的卡那霉素篩選濃度，子葉節(jié)分化時(shí)，墾農(nóng)18為 40 mg/L、綏農(nóng)14 和墾農(nóng)4為 60 mg/L；張靜妮等[11]發(fā)現(xiàn)不同的紫花苜蓿品種在下胚軸分化階段，不同品種選擇壓分別為秘魯50 mg/L、富平和甘農(nóng)3號60 mg/L；不同的煙草品種在葉片分化和生根階段，不同品種適宜的卡那霉素篩選濃度也有所不同[10]?？梢姡煌参锘蛲恢参锊煌贩N以及同一品種外植體不同生長階段之間對卡那霉素的敏感性均表現(xiàn)出顯著差異，導(dǎo)致卡那霉素在使用濃度的篩選時(shí)相差很多。因此對試驗(yàn)中將要用于轉(zhuǎn)化的外植體進(jìn)行系統(tǒng)篩選在植物轉(zhuǎn)基因研究中是非常必要的。

張甲佳等[12]以花生胚小葉外植體作為材料，對不同卡那霉素濃度下的不同花生品種的敏感性研究表明，在愈傷發(fā)育時(shí)期，D16、花育22和魯花11三個(gè)品種的卡那霉素的臨界濃度分別為100 mg/L、200 mg/L和150 mg/L。胡曉君[19]以花生胚小葉外植體為材料，研究卡那霉素對再生芽叢的影響，結(jié)果表明，對于花生品種豐花1號和豐花2號，400 mg/L的卡那霉素濃度是轉(zhuǎn)基因再生芽叢和非轉(zhuǎn)基因再生芽叢篩選的臨界濃度。本試驗(yàn)研究了花生胚小葉對卡那霉素的敏感性，表明不同花生基因型之間存在明顯差異結(jié)果，與以上研究相一致，但本研究系統(tǒng)地從胚小葉外植體的整個(gè)生長階段進(jìn)行研究，通過對生長狀態(tài)的觀察綜合考慮后確定不同生長階段所使用的卡那霉素的適宜濃度。研究結(jié)果為，胚小葉叢生芽分化的卡那霉素篩選濃度：弗落蔓生為150 mg/L，麻油1-1為100 mg/L，濮花23號為50 mg/L；叢生芽生根的卡那霉素篩選濃度：弗落蔓生為20 mg/L，麻油1-1為20 mg/L，濮花23號為10 mg/L。

根據(jù)本試驗(yàn)，通過觀察外植體的生長狀況，在用卡那霉素進(jìn)行篩選轉(zhuǎn)化時(shí)，適宜的繼代時(shí)間應(yīng)盡量控制在25 d，若培養(yǎng)時(shí)間過長，由于培養(yǎng)基的營養(yǎng)消耗，再生芽會(huì)出現(xiàn)生長不正常；同時(shí)由于卡那霉素長期處于培養(yǎng)基中，活性會(huì)下降，從而使非轉(zhuǎn)化芽也生長起來，因此在轉(zhuǎn)化研究中應(yīng)在適當(dāng)?shù)暮Y選時(shí)間內(nèi)進(jìn)行外植體及轉(zhuǎn)化芽的轉(zhuǎn)移，這不僅可以使轉(zhuǎn)化芽正常生長，同時(shí)也可避免在繼代過程中有非轉(zhuǎn)化芽出現(xiàn)，影響篩選效果[20]。

本研究篩選到不同花生品種胚小葉叢生芽分化和叢生芽生根階段的適宜卡那霉素濃度，為以花生胚小葉為外植體的花生遺傳轉(zhuǎn)化提供理論基礎(chǔ)，應(yīng)用于花生遺傳轉(zhuǎn)化中可以有效提高轉(zhuǎn)化效率。

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DOI：10.14001/j.issn.1002-4093.2016.02.004

收稿日期：2016-1-13

基金項(xiàng)目：國家花生產(chǎn)業(yè)技術(shù)體系(CARS-14)；山東省農(nóng)業(yè)科學(xué)院科技創(chuàng)新重點(diǎn)項(xiàng)目(2014CGPY09)；青島市民生計(jì)劃(14-2-3-34-nsh)

作者簡介：張青云(1989-)，女，河北承德人，吉林農(nóng)業(yè)大學(xué)碩士研究生，主要從事花生種用品質(zhì)研究。

*通訊作者：王傳堂(1968-)，研究員，博士，主要從事高油酸花生育種研究。E-mail: chinapeanut@126.com

Abstract: As high in oil, common peanuts may quickly deteriorate and lose seed vigor under ambient conditions. That is the reason why only seeds harvested in previous season/year can be used as seeds in north China, and only the fall crops producd seeds can be chosen for next spring's crop in south regions of China. The present study revealed, for the first time, that high-oleic (HO) peanuts after an extended period of storage at ambient temperature (19 months), were still as good as those harvested from previous year in most of the seed quality characteristics. For each of the 3 HO peanut cultivars used, seeds of 2013 and 2014 did not differ significantly in standard seed germination on the 7th day and field emergence. Use of HO peanuts may therefore sustain the vigor of seed, in addition to health benefits for humans and longer shelf life of food products.

Key words: peanut; electric conductivity; field emergence; germination; high-oleic; seed vigor

摘要：普通花生含油量高，在自然溫度下易快速劣變喪失種子活力。這是我國北方僅上年或上一季花生而我國南方僅秋花生來年可做種的原因。本研究首次證實(shí)，高油酸花生經(jīng)過19個(gè)月自然條件下貯藏，在多數(shù)種用特性上不差于上年收獲的花生。所有參試的3個(gè)高油酸品種，其2013年種子與2014年種子在第7天的發(fā)芽率和田間出苗率均無顯著差異。由此證明，高油酸品種不僅有利于健康，能延長制品貨架期，而且可保持種子活力。

關(guān)鍵詞：花生；電導(dǎo)率；田間出苗率；萌發(fā)；高油酸；種子活力

1 Introduction

Undoubtedly, high oleate has become and will continue to be one of the most important breeding objectives of peanut. Earlier studies have showed that peanuts high in oleate are advantageous over their normal-oleic counterparts. Food products made from high-oleic (HO) peanuts have longer shelf life and are heart-healthier[1]. Research concerning seed storability of peanuts has been concentrated on normal-oleic (NO) genotypes. Perez and Arguello (1995) studied deterioration in peanut (ArachishypogaeaL. cv. Florman) seeds under natural and accelerated ageing, and concluded that while germination percentage was not a sensitive assay for detecting the degree of deterioration, changes in membrane integrity associated with seed deterioration occurred first in the embryonic axes, which could best be monitored by the conductivity seed vigor test[2]. Promchote et al. (2005) used hull-scrape method to divide NO peanut seeds into three different maturity groups to study the influence of maturity on seed storability[3]. They found that artificial and natural ageing of immature peanut seeds deteriorated faster than intermediate and mature seeds[3]. Using relative germination as an indicator for accelerated ageing tolerance (AAT), Shen et al. (2014) noted that AAT was correlated positively to oleate content, and negatively to linoleate in some treatments, without mentioning if HO peanut genoptypes were used in their study[4]. Up to now, no attempts have been made to ascertain if HO peanuts, after a longer duration of storage, is still usable as seeds without compromise in field emergence and productivity.

The aim of the present study is to make it clear if natural ageing affects seed germination and field emergence of HO peanut cultivars.

2 Materials and methods

3 HO peanut cultivars bred by Shandong Peanut Research Institute (SPRI) were used in this study (Table 1). Among them, Huayu661 and Huayu662 are small-seeded varieties, and Huayu961 is a large-seeded cultivar.

Table 1 Some quality characteristics of the 3 HO peanut cultivars used in the study

Note: *Based on a report from Supervising and Testing Center for Oilseeds and Their Products (Wuhan), Ministry of Agriculture, China.

Peanut seeds used in the study were either from the 2014 crop or from the 2013 crop. Peanuts were sown in spring (early May), and harvested in fall (before mid-September). At the end of September of the same year, after sundried, pods or seeds (shells removed by hands) were stored at ambient temperature on the SPRI Experimental Farm in Laixi, Qingdao, China. All seeds used were sound mature kernels (SMKs).

In-house seed test began on May 8, 2015, when the seed dormancy of the entries vanished. For each entry, a total of 60 seeds were used for analysis (2 replications). For the samples stored as pods, shells were removed by hands just before initiation of the seed test. The roll towel method (between paper) as depicted by Upadhyaya and Gowda (2009)[5]and an incubation temperature of 28℃was used in the test. Only seeds with extruding hypocotyl and radicle no shorter than the length of the individual single seeds were counted as sprouts. Germination was recorded daily, and germination index (GI), vigor index (VI) and simplified vigor index (SVI)[6]were calculated using the following formulas:

GI= ∑(Gt/Dt)

Gt= No. of new sprouts counted on a specific day (Dt)

VI=GI×(Average radicle length in cm)

SVI=G3×(Length of radicle and hypocotyl in cm)

G3= No. of total sprouts by the 3rdday

Electric conductivity (EC) analysis was conducted according to the protocol described by Zhang et al. (2012)[7]with minor modifications.ECwas measured with a METTLER TOLEDO's FiveEasyTMConductivity Meter, model FE30 (Mettler-Toledo, LLC, Columbus, OH, USA). Two 30-seed subsamples were weighted to an accuracy of 0.01 g (W) and placed in 200 mL of de-ionized water in Erlenmeyer flasks, and the initial EC (d1) was measured. Flasks were covered to avoid loss of water and interference of dust and held at 20℃ for 24h, and conductivity (d2) was then measured. Absolute conductivity of seed leachate post boiling (d3) was also recorded. Electric conductivity of seed leachate (ECsl), reported as μS·cm-1·g-1, and relative electric conductivity (ECr) were calculated using the following formulas:

ECs l= (d2-d1)/W

ECr= (d2-d1)/(d3-d1)×100%

To test field performance, peanuts were sown with an expected population of 141176 hills per ha (one seed per hill) under polythene mulch

(Herbicide was sprayed prior to the placement of

the polythene film) on the same day with 1 replication in Experiment I (60 seeds/entry) and 4 replications in Experiment II and III (Totally 240 seeds/entry, randomized block design). Field emergence was counted 20 days after sowing.

Statistical analysis were performed with the DPS package (version 14.50)[8]. Data transformation was exploited where appropriate. Multiple comparison was conducted using Duncan's Multiple Range Test.

3 Results and analysis

3.1 In-house standard seed germination test

For seed germination (%) on the 3rdday (G3), significant difference was only detected in In-house Experiment II (x2=5.00234,df=1,p=0.02531<0.05), whereG3of Huayu662 seeds harvested in 2014 more than doubled that of naturally aged Huayu662 seeds of 2013 harvest (Table 2). Seed germination (%) on the 7thday (G7) ranged from 96.67%～100.00%, with no significant difference in all the 3 in-house experiments (Table 2). ForGI,VIandSVI, significant difference was solely reported from In-house Experiment II, where seeds of the 2013 crop had a much lessSVIthan the 2014 seeds (Table 2).

Table 2 Seed germination (%) on the 3rd(G3) and the 7thday (G7), germination Index (GI),

Note: *Figures marked with different letters were statistically differed at 0.05 probability level.

3.2 Electric conductivity analysis

Relative electric conductivity (ECr) of the entries within individual in-house experiment did not differ significantly (Table 2). Only electric conductivity of seed leachate (ECsl) from In-house Experiment III was statistically different (p= 0.0448<0.05) (Table 2). 2013 seeds of Huayu661 stored as seeds had anECslvalue significantly greater than that of 2013 seeds stored as pods or 2014 seeds of the same variety (Table 2).

3.3 Field studies

Field emergence in the 3 experiments was listed in Table 3. No significant difference in field emergence in Field Experiment I was detected as analyzed withx2test (x2=0.00444,df=1,p=0.94687). No significant difference in field emergence in Field Experiment II and III was detected as analyzed with ANOVA (Using arsine square root data transformation and Duncan's Multiple Range Test) orx2test (In Field Experiment II,x2=0.01093,df=1,p=0.91674. In Field Experiment III,x2=0.12087,df=2,p=0.94135). Field survey showed that plants grown from different seed lots of each HO peanut cultivar developed equally well (Fig.).

Table 3 Field emergence (FE) of the entries

Fig. Plants grown from two seed lots of Huayu661 (75 days after sowing)Left row: 2014 seeds. Right row: 2013 pods.

3.4 Correlation analysis of seed quality characteristics

As shown in Table 4,G3was positively correlated withSVIandECr, whileG7was negatively related toECsl. A significant positive correlation also existed betweenGIandVIand betweenGIandSVI. Generally speaking, a HO peanut cultivar with a higherSVIorECralso has a greaterG3. Likewise, a lowECslmay be, however, an indicator of higherG7. We failed to establish a relationship between field emergence and any of the rest parameters. Please note that NO peanut seeds were not included in the present study. Inclusion of NO peanuts will possibly reveal relationships unidentified in the study.

Table 4 Pearson's correlation between seed quality characteristics of HO peanut cultivar

Note: Figures above the diagonal were probability levels, and figures below the diagonal were correlation coefficients. *Significant at 0.05 level. ** Significant at 0.01 level.

4 Conclusions and discussion

This communication reported, for the first time, the advantage of HO peanuts as seeds over common peanuts. On SPRI Experimental Farm (N36°48'40.44", E120°29'59.65"), the seeds from the 3 HO peanut cultivars, after an extended period of storage (19 months) under ambient conditions, viz., the seeds harvested in fall 2013, were still usable as seeds in spring 2015. For these seeds, according to the results from in-house seed test, only one of the 3 cultivars germinated slowly at early stage (Day 3), but at later stage (Day 7), germination percentage of the cultivar was roughly the same as that of seeds harvested in fall 2014. As expected, in field studies, for a specific HO peanut cultivar, the emergence ofoldandnewseeds lots was almost equivalent. It is interesting to find if theoldpeanuts may achieve yields as high as thenewones.

As reviewed by Sun et al. (2007)[9], lipid peroxidation, chromosome/gene aberrance, and embryo protein degradation are among the reasons causing seed vigor losses, seed ageing and deterioration. Peanut seeds contain about 50% oil and 26% protein. As such, ageing may adversely affect seed oil and protein. Sung and Jeng (1994) demonstrated that accelerated aging (AA) stimulated lipid peroxidation, and inhibited the activity of radical- and peroxide-scavenging enzymes[10]. Vasudevan et al. (2012) observed alteration in band number/intensity of protein/ peroxidase profiles in naturally and artificially aged peanut seeds[11]. As compared to linoleate, oleate is less prone to oxidization. NO peanuts generally have an oleate to linoleate ratio (O/L) of less than 2.5, with lower than 60% oleate, whereas linoleate may be as high as 50%. In contrast, HO peanuts have an O/L of no lower than 9[12], with more than 72% oleate, and linoleate may be as low as around 3%. The good storability of HO peanut seeds in the present study may be largely ascribed to their high oleate and low linoleate content. In addition, it is believed that other components, such as tocopherols and non-tocopherol antioxidants, may also have some roles[13]. But this has not been validated.

Anyway, the results from the present study is good news to peanut seed industry. In north regions in China (cooler areas), only peanuts harvested from the previous year/season can be used as seeds; NO peanuts harvested in the year before last year, when used as seeds, will encounter marked reduction in field emergence, incurring large yield losses. In south peanut production regions of China (warmer areas), peanuts may be sown in spring, fall, and even in winter. But, in general, merely the low-yielding fall peanuts can be used as seeds for next year's spring crop[14], in spite of their highly variable seed size, which is a stumbling block for mechanized sowing. Spring peanuts, though well developed and high yielding, subjected to high humidity coupled with high ambient temperature after harvest, will quickly lose their seed vigor under ordinary storage conditions, rendering them unusable as seeds for next year's crop. In China, HO peanuts provide a good opportunity to regulate seed supplies between years in north regions and may be of some help to find a solution to use spring peanuts as seeds for the subsequent year in south regions, with minimal storage measures taken. Similar needs also exist in other peanut producing countries worldwide. Hopefully, with the application of HO peanuts in seed industry, sufficient seed supply will result in reduced seed costs, eventually benefiting the whole peanut industry and consumers.

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Study on Sensitivity of Peanut Leaflet to Kanamycin

WANG Feng-huan, HE Mei-jing, YANG Xin-lei, CUI Shun-li, MU Guo-jun, HOU Ming-yu, LIU Li-feng*

(NorthChinaLaboratoryofCropGermplasmResourcesofEducationMinistry/KeyLab.forCropGermplasmResourcesofHebei/CollegeofAgronomy,Agr.Univ.ofHebei,Baoding071001,China)

Kanamycin is a screening agent commonly used in plant genetic transformation. The study of susceptibility of peanut leaflet to kanamycin is important to the genetic transformation of peanut. 3 different genotypes of peanut (Fuluomansheng, Mayou1-1 and Puhua23) were used to study the impact of different concentration of kanamycinon on the development, differentiation and rooting of clustered shoots of peanut leaflet by calculating their yellowing rate, clustered shoots induction rate, rooting percentage and observing the growing status of explants, so as to confirm the suitable screening concentration. The results showed that different genotypes of peanut had different degrees of susceptibility to kanamycin. The suitable concentration of kanamycin for the differentiation of leaflet clustered shoots was 150 mg/L for Fuluomansheng, 100 mg/L for Mayou1-1 and 50 mg/L for Puhua 23; while for rooting of clustered shoots, the suitable concentration of kanamycin was 20 mg/L for Fuluomansheng, 20 mg/L for Mayou1-1 and 10 mg/L for Puhua 23. In this study, suitable screening concentration of kanamycin in stages of differentiation and rooting of clustered shoots was obtained, which laid foundation for the screening of positive plant in genetic transformation of peanut leaflet.

peanut (ArachishypogaeaL.); kanamycin; genotype; susceptibility

Effect of Natural Ageing on Seed Quality of High-Oleic Peanut

ZHANG Qing-yun1, WANG Chuan-tang1,2*, TANG Yue-yi2, WANG Xiu-zhen2, WU Qi2, SUN Quan-xi2, ZHANG Jian-cheng2, HU Dong-qing3, YU Shu-tao4, CHEN Ao5

(1.CollegeofAgronomy,JilinAgriculturalUniversity,Changchun130118,China; 2.ShandongPeanutResearchInstitute,Qingdao266100,China; 3.QingdaoEntry-ExitInspectionandQuarantineBureau,Qingdao266001,China; 4.LiaoningPeanutResearchInstitute,LiaoningAcademyofAgriculturalSciences,Fuxin123000,China; 5.InstituteofPeanut,ZhanjiangAcademyofAgriculturalSciences,Zhanjiang524094,China)

自然老化對高油酸花生種用品質(zhì)的影響

張青云1，王傳堂1,2*，唐月異2，王秀貞2，吳 琪2，孫全喜2，張建成2，胡東青3，于樹濤4，陳 傲5

(1. 吉林農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，吉林 長春 130118； 2. 山東省花生研究所，山東 青島 266100； 3. 青島出入境檢驗(yàn)檢疫局，山東 青島 266001； 4. 遼寧省農(nóng)業(yè)科學(xué)院花生研究所， 遼寧 阜新 123000； 5. 湛江市農(nóng)業(yè)科學(xué)院花生研究所，廣東 湛江 524094)

10.14001/j.issn.1002-4093.2016.02.003

2016-04-18

國家自然科學(xué)基金(31471523)；農(nóng)業(yè)部引進(jìn)國際先進(jìn)農(nóng)業(yè)科學(xué)技術(shù)計(jì)劃(“948”計(jì)劃)(2013-Z65)；高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金(2012130211002)；河北省高等院?？茖W(xué)技術(shù)研究重點(diǎn)項(xiàng)目(ZH2011209)

王鳳歡(1991-)，女，河北滄縣人，河北農(nóng)業(yè)大學(xué)在讀碩士，研究方向?yàn)榧?xì)胞、分子遺傳及其育種應(yīng)用。

*通訊作者：劉立峰，教授，博士，主要從事花生基因組學(xué)與分子育種研究。E-mail:lifengliucau@126.com

S565.2;Q

A

565.2; S330.3+1 文獻(xiàn)標(biāo)識(shí)碼：A