劉柿良， 楊秀利， 馬明東， 但 方， 楊 君， 胡 菊， 鄔夢(mèng)希

(四川農(nóng)業(yè)大學(xué)風(fēng)景園林學(xué)院， 四川成都 611130)

曼地亞紅豆杉(Taxusmediacv“Hicksii”)栽培基質(zhì)中適宜的錳、鋅、銅濃度及采收時(shí)期研究

劉柿良， 楊秀利， 馬明東*， 但 方， 楊 君， 胡 菊， 鄔夢(mèng)希

(四川農(nóng)業(yè)大學(xué)風(fēng)景園林學(xué)院， 四川成都 611130)

曼地亞紅豆杉； 紫杉醇； 錳； 鋅； 銅; 生長發(fā)育； 采收季節(jié)； 采收年齡

本課題組已采用“Hicksii”帶芽莖段外植體進(jìn)行腋芽增殖再生, 成功實(shí)現(xiàn)其高效快速繁殖[12-13]。然而, 對(duì)獲得組培苗最佳移植生長環(huán)境, 以產(chǎn)生更高含量紫杉醇的研究還未開展。藥用植物的生長環(huán)境所含微量元素不僅對(duì)其生長有著直接的影響[14]，還與其療效密切相關(guān)[15]。因此, 本研究選用3年和5年生“Hicksii”樹苗為對(duì)象, 研究錳、鋅和銅對(duì)兩種樹齡植株的生長及紫杉醇含量的影響, 為曼地亞紅豆杉藥用原料林培育、采收以及合理施用微肥確定提供借鑒。

1 材料和方法

1.1 研究材料與試驗(yàn)地概況

1.2 試驗(yàn)方法

盆栽基質(zhì)為河砂和蛭石。河砂消毒后洗凈、晾干；蛭石用稀鹽酸(HCl)溶液浸泡 72 h 后用水反復(fù)沖洗(6次以上)后晾干。將河砂和蛭石各15 kg均勻裝入盆中, 盆底墊鋪尼龍紗。將樹苗根系洗凈，于2012年2月上旬移植到內(nèi)徑45 cm、高35 cm的花盆中(帶托盤), 每盆栽植樹苗2株, 幼苗間互不遮蔭。試驗(yàn)大棚透光率為 80%; 大棚內(nèi)外溫度接近, 為20±3℃；相對(duì)濕度為70%。所有處理均用去離子水。

營養(yǎng)液為改進(jìn)Hoagland (霍格蘭氏)完全營養(yǎng)液[16]: KNO35 mmol/L、 Ca(NO3)25 mmol/L、 KH2PO41 mmol/L、 MgSO42 mmol/L、 NaFeEDTA 0.1 mmol/L、 HBO346 μmol/L、 NaMoO40.l μmol/L、 MnSO49 μmol/L、 ZnSO40.8 μmol/L、 CuSO40.3 μmol/L。鐵(Fe)、硼(B)、鉬(Mo)鹽濃縮到原標(biāo)準(zhǔn)的1000倍混合放置, Mn、Zn、Cu鹽濃縮1000倍單獨(dú)放置; 其余的大量元素濃縮到100倍低溫保存。施用時(shí), 當(dāng)添加1種不同濃度的微量元素時(shí), 其他2種微量元素以改進(jìn)的營養(yǎng)液為基礎(chǔ)濃度作為基礎(chǔ)溶液[10]，其他按配制的濃縮液量取、稀釋。2012年2月13日起, 每盆植株澆施營養(yǎng)液500 mL, 每周更換1次營養(yǎng)液。

1.3 HPLC測(cè)定條件和參數(shù)

1.3.1 流動(dòng)相選擇 HPLC為島津 LC-20AB 高效液相色譜儀(LC-20AB, Shimadzu Inc., Japan)。色譜柱為phenomenex Luna C 18 (4.6 mm × 250 mm, 5 μm), 流動(dòng)相為乙腈 ∶水 ∶異丙醇=45 ∶ 55 ∶ 1 (v ∶ v ∶v), 檢測(cè)波長為227 nm, 流速1.0 mL/min, 柱溫30℃, 進(jìn)樣量為10 μL時(shí), 保留時(shí)間30 min。

1.3.2 標(biāo)準(zhǔn)液制備 準(zhǔn)確稱取紫杉醇標(biāo)準(zhǔn)品10.0000 mg, 置于25 mL容量瓶中加甲醇溶解至刻度, 得到0.4 mg/mL 標(biāo)準(zhǔn)品儲(chǔ)備液。

取標(biāo)準(zhǔn)品制備測(cè)定液，峰面積測(cè)定值相對(duì)標(biāo)準(zhǔn)偏差(RSD)值為0.57%, 表明試驗(yàn)儀器精密度良好。將標(biāo)準(zhǔn)品溶液于4℃保存, 于0、 1、 2、 4、 8、 16 h取樣測(cè)定, 峰面積RSD為0.56%, 表明標(biāo)準(zhǔn)品溶液在16 h內(nèi)穩(wěn)定性良好。

精確稱取3年生曼地亞紅豆杉樣品粉末5份, 各1.000 g, 按樣品溶液的制備方法制備待測(cè)溶液進(jìn)行測(cè)定，5份樣品峰面積相對(duì)標(biāo)準(zhǔn)偏差為0.84%, 表明試驗(yàn)重復(fù)性良好。

1.3.4 加樣回收考察 取已施加Hoagland標(biāo)準(zhǔn)溶液的3年生曼地亞紅豆杉樣品粉末9份, 各1.0 g, 每3份為一組, 各加入紫杉醇標(biāo)準(zhǔn)品粉末, 按樣品溶液的配制方法制取待測(cè)液10 μL, 進(jìn)樣分析并計(jì)算回收率。高、中、低三種水平的加樣回收率平均值分別為99.69%、 99.02%和99.00%,RSD分別為1.22%、 0.24%和0.52%(n=3)，均在允許范圍內(nèi), 說明該法測(cè)定曼地亞紅豆杉枝葉紫杉醇含量結(jié)果可靠。

1.4 植株采樣與測(cè)定

紫杉醇(%)=(S樣×m標(biāo)×V樣×標(biāo)準(zhǔn)品純度)/(S樣×m標(biāo)×V樣) ×100

式中: S 指色譜圖峰面積； m 指樣品或標(biāo)準(zhǔn)品質(zhì)量； V 指溶液體積。

1.5 生長指標(biāo)測(cè)定

1.6 數(shù)據(jù)處理

采用SPSS 17.0 (SPSS, Chicago, USA)軟件進(jìn)行單因素方差分析(one-way ANOVA), 并用最小顯著差數(shù)法(LSD)檢驗(yàn)差異性。采用生長綜合指標(biāo)法(Overall Desirability, OD)對(duì)生長進(jìn)行綜合評(píng)定, OD即為同處理各生長指標(biāo)測(cè)定值與參照測(cè)定值(本試驗(yàn)為Mn0、Zn0、Cu0處理)的比值之和[18]。

2 結(jié)果與分析

2.1 微量元素對(duì)曼地亞紅豆杉“Hicksii”樹苗生長的影響

移栽后未經(jīng)處理的健康3年和5年生曼地亞紅豆杉“Hicksii”樹苗的基礎(chǔ)生長值為: 3年生植株株高28.13 cm, 地徑5.02 cm, 新梢長度1.38 cm, 枝葉鮮重24.67 g/plant, 根系鮮重10.28 g/plant, 枝葉干重2.36 g/plant, 根系干重1.24 g/plant; 5年生植株株高31.08 cm, 地徑6.11 cm, 新梢長度0.74 cm, 枝葉鮮重31.27 g/plant, 根系鮮重22.32 g/plant, 枝葉干重3.15 g/plant, 根系干重2.28 g/plant。

表1 不同施錳量對(duì)曼地亞紅豆杉“Hicksii”樹苗生長的影響

注(Note): OD—綜合指標(biāo) Overall desirability. 數(shù)值后不同大寫字母表示相同處理下3年和5年生樹苗間差異顯著(P<0.05); 不同小寫字母表示3年和5年生樹苗在不同處理間差異顯著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

2.1.2 鋅 隨著Zn施加量的增大, 兩種樹苗的生長指標(biāo)值均呈先升高后降低的趨勢(shì)。3年生“Hicksii”樹苗的株高和地徑在Zn 0.8 μmol/L最大, 而新梢長度、枝葉鮮重(干重)和根系鮮重(干重)在Zn 0.4 μmol/L最大, 且OD值為8.93 (表2)。5年生樹苗的株高、新梢長度、根系鮮重(干重)在Zn 0.4 μmol/L處理時(shí)最大, OD值達(dá)到最高(8.71)。比較3年和5年生樹苗可知, 5年生樹苗的株高、地徑及鮮重(干重)均顯著高于3年生樹苗(P<0.05), 而新梢長度卻相反。

表2 不同施鋅量對(duì)曼地亞紅豆杉“Hicksii”樹苗生長的影響

注(Note): OD—綜合指標(biāo) Overall desirability. 數(shù)值后不同大寫字母表示相同處理下3年和5年生樹苗間差異顯著(P<0.05); 不同小寫字母表示3年和5年生樹苗在不同處理間差異顯著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

2.2 微量元素對(duì)曼地亞紅豆杉“Hicksii”樹苗紫杉醇含量的影響

移栽后未經(jīng)處理的健康 3 年和5年生曼地亞紅豆杉“Hicksii”樹苗的紫杉醇基礎(chǔ)值為: 3 年生植株枝葉紫杉醇含量為 2.16%, 5 年生植株枝葉紫杉醇含量為 3.02%。

表3 不同施Cu量對(duì)曼地亞紅豆杉“Hicksii”樹苗生長的影響

注(Note): OD—綜合指標(biāo) Overall desirability. 數(shù)值后不同大寫字母表示相同處理下3年和5年生樹苗間差異顯著(P<0.05); 不同小寫字母表示3年和5年生樹苗在不同處理間差異顯著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

圖1 不同施錳量曼地亞紅豆杉“Hicksii” 3年(a) 和5年(b) 生樹苗紫杉醇含量Fig.1 Taxol content in 3-year (a) and 5-year (b) “Hicksii” scalps dependent on Mn application rates

圖2 不同施鋅量曼地亞紅豆杉“Hicksii” 3年(a)和5年(b)生樹苗紫杉醇含量Fig.2 Taxol content in three-year (a) and five-year old (b) Hicksii scalpels dependent on Zn application rates

圖3 不同施銅量對(duì)曼地亞紅豆杉“Hicksii” 3年(a) 和5年(b)生樹苗紫杉醇含量Fig.3 Taxol content in three-year (a) and five-year old (b) scaples of T. media cv “Hicksii” dependent on Cu application rate

2.2.3 銅 銅處理紫杉醇積累量6月最低值3年生幼樹為 1.953.43，5年生為2.463.68。3年和5年生樹苗枝葉紫杉醇積累量6月份比3月份顯著下降39.02%68.69% 和 32.79%44.97% (P<0.05)。

3 討論與結(jié)論

3.1 適量微量元素能顯著提高曼地亞紅豆杉“Hicksii”樹苗的生長

3.2 適量微量元素能顯著提高樹苗紫杉醇的積累

紅豆杉細(xì)胞合成紫杉醇的能力除了受遺傳特性影響之外, 外界逆境信號(hào)激活細(xì)胞的防御反應(yīng)是改變細(xì)胞合成紫杉醇速率和積累的重要途徑[24]。本試驗(yàn)中, 3種微量元素對(duì)樹苗紫杉醇含量影響效果的順序?yàn)镸n>Zn>Cu，與王建安等[31]的研究結(jié)論相似, 盾葉薯蕷(Dioscoreazingiberensis)薯蕷皂苷元對(duì)施Mn肥的敏感性高于Zn?？赡茉蚴荕n對(duì)代謝合成酶具有高度專一性, 促進(jìn)植物吸收土壤中氮和磷, 高M(jìn)n可抑制吲哚乙酸(IAA)氧化酶活性, 從而促進(jìn)產(chǎn)量增加[32]。Zn通過參與葉綠素合成, 改變光能利用率, 影響有機(jī)物合成[14,17]。Liu[20]指出, Zn2+可能首先與細(xì)胞膜上受體的結(jié)合, 改變膜離子通道, 促使誘導(dǎo)過程迅速完成。Zn2+與其受體高度親合, 引起位于原生質(zhì)膜上的離子通道改變, 引起 Ca2+內(nèi)流并迅速發(fā)生 H2O2, 胞內(nèi)依賴 Ca2+的蛋白質(zhì)磷酸化作用, 激活核內(nèi)防御基因(如TCH基因), 引起防御反應(yīng), 誘導(dǎo)合成植保素酶合成植保素而完成信號(hào)傳遞作用。當(dāng)氧迸發(fā)(OXB)達(dá)到最大時(shí), 胞內(nèi)苯丙氨酸鮮氨酶(PAL)活性、Taxol 和酚的合成開始激活[20-21,23]。因此, 防御應(yīng)答的強(qiáng)弱可能與 Taxol 的合成有密切的正相關(guān)性。而Cu影響紫杉醇積累主要是通過與過氧化氫酶(Catalase, CAT)活性中心結(jié)合, 影響酶與底物的結(jié)合能力[33]。同時(shí), 高濃度的 Cu2+可能會(huì)導(dǎo)致細(xì)胞活性氧(ROS)的積累。ROS積累和次生代謝產(chǎn)物都是植物在響應(yīng)外界刺激過程中強(qiáng)有力的化學(xué)武器, Cu2+誘導(dǎo)次生代謝產(chǎn)物的合成而抗氧化劑的加入則阻止次生代謝物的生成[15,23]。ROS可能作為第二信使調(diào)控防衛(wèi)基因的表達(dá)和啟動(dòng)與植保素合成基因相關(guān)的基因轉(zhuǎn)錄, 因此推斷ROS 對(duì)植保素的調(diào)控作用可能發(fā)生在轉(zhuǎn)錄水平上, 也可能使 mRNA 穩(wěn)定性有所提高[14]。

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Suitable Mn, Zn and Cu concentrations in the culture media and proper harvest time forTaxusmediacv“Hicksii”

LIU Shi-liang, YANG Xiu-li, MA Ming-dong*, DAN Fang, YANG Jun, HU Jü, WU Meng-xi

(CollegeofLandscapeArchitecture,SichuanAgriculturalUniversity,Chengdu,Sichuan611130,P.R.China)

【Objectives】 Paclitaxel (Taxol?) shows excellent antitumor activity against breast cancer, ovarian cancer and non-small cell lung cancer, is one of the best chemotherapeutic agents developed from plant sources. Taxus production has

attention as an important natural material for extracting taxol. The purpose of this study was to study the effects of mircoalements (Mn, Zn and Cu) on the growth and paclitaxel accumulation in seedlings of Taxus, providing a basis for the optimum harvest period and the rational application of micronutrient fertilizer.【Methods】 A sand culture experiment was conducted and the seedlings of 3-year-old and 5-year-oldTaxusmediacv“Hicksii” were growen. Five concentration of Mn 0, 4.50, 9.00, 13.50 and 18.00 μmol/L, Zn 0, 0.15, 0.30, 0.45 and 0.60 μmol/L, and Cu: 0, 0.40, 0.80, 1.20 and 1.60 μmol/L were designed, each treatment replicated three times. Branches and leaves of plants were collected monthly from March 15, 2012 to October 15, 2012. The paclitaxel accumulation contents was determined with high-performance liquid chromatography (HPLC) method, the seedling growth were observed in November 15, 2012, the biomass were weighed at the same time. 【Result】 1) Mn, Zn and Cu treatment levels significantly affect the growth and paclitaxel contents, increase the plants heights, crossing diameters and shoot length, increase the weights of branches, leaves and roots of the saplings. For the growth of seedlings, the optimum treatment is Cu 0.15 μmol/L for 5-year-old seedlings and 0.30 μmol/L for 3-year-old seedlings, Mn 9.00 μmol/L and Zn 0.40 μmol/L for the two ages of seedlings. For paclitaxel accumulation, the optimum level is Zn 0.40 μmol/L and Cu 0.30 μmol/L for the both ages, and Mn 9.00 μmol/L for the 3-year-old and 4.50 μmol/L for 5-year-old saplings. When the micronutrient treatment levels exceeded the optimum one, the paclitaxel accumulation will be inhibited and the higher the treatment levels, the stronge the inhibition. 2) The paclitaxel content in 5-year-old plants are significantly higher than in 3-year-old ones under all the treatments, and the differences are significant at harvest. 3) The paclitaxel content start to decrease from March to June, and is lowest in June, then increase and keep relatively stabile in September and October. The effects of Mn on the paclitaxel accumulation is higher than Zn and higher than Cu, no matter their treatment concentrations.【Conclusions】 The paclitaxel content in 5-year-old saplings is significantly higher than the 3-year-old ones, the proper harvest time for obtaining high paclitaxel content of saplings is September and October. The optimum treatment level is Mn 9.00 μmol/L, Zn 0.40 μmol/L and Cu 0.15-0.30 μmol/L.

Taxusmediacv “Hicksii”; paclitaxel; Mn; Zn; Cu; ontogenetic development; gathering season; gathering year

2014-01-06 接受日期： 2014-02-25

四川省教育廳重點(diǎn)攻關(guān)項(xiàng)目“曼地亞紅豆杉快繁技術(shù)研究”(2003A023)資助。

劉柿良(1986—)，男，四川南充人，博士研究生，主要從事植物營養(yǎng)及生理生態(tài)等方面研究。E-mail： liushiliang9@163.com * 通信作者 E-mail： mmingdong1958@gmail.com

S791.49; S725.5

A

1008-505X(2015)02-0439-10