根癌農(nóng)桿菌介導(dǎo)的橡膠樹花藥愈傷組織遺傳轉(zhuǎn)化體系的建立_百度文

1、作物學(xué)報 ACTA AGRONOMICA SINICA 2010, 36(10: 16911697/zwxb/ISSN 0496-3490; CODEN TSHPA9E-mail: xbzw本研究由現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(nycytx-34, 國家自然科學(xué)基金項目(30960322, 中國熱帶農(nóng)業(yè)科學(xué)院橡膠研究所基本科研業(yè)務(wù)費專項資金(XJSYWFZX2008-01和國際科技合作項目(2008DFA32020資助。*通訊作者(Corresponding author: 黃華孫, E-mail: xjshhs第一作者聯(lián)系方式: E-mail:

2、 tdhuang1986Received(收稿日期: 2010-02-05; Accepted(接受日期: 2010-04-20.DOI: 10.3724/SP.J.1006.2010.01691根癌農(nóng)桿菌介導(dǎo)的橡膠樹花藥愈傷組織遺傳轉(zhuǎn)化體系的建立黃天帶 李 哲 孫愛花 周權(quán)男 華玉偉 黃華孫*中國熱帶農(nóng)業(yè)科學(xué)院橡膠研究所 / 國家橡膠樹育種中心 / 農(nóng)業(yè)部橡膠樹生物學(xué)重點開放實驗室 / 省部共建國家重點實驗室培育基地 / 海南省熱帶作物栽培生理學(xué)重點實驗室, 海南儋州 571737摘 要: 為建立根癌農(nóng)桿菌介導(dǎo)的橡膠樹遺傳轉(zhuǎn)化體系, 以花藥愈傷組織為受體, 研究了農(nóng)桿菌菌株、共培養(yǎng)溫度、共培

3、養(yǎng)時間、乙酰丁香酮(AS等因素對遺傳轉(zhuǎn)化效率的影響。結(jié)果表明, 農(nóng)桿菌菌株、共培養(yǎng)溫度和共培養(yǎng)時間對轉(zhuǎn)化效率具有顯著影響, AS不影響轉(zhuǎn)化效率。2.2萬個花藥愈傷組織經(jīng)EHA105菌株侵染后, 轉(zhuǎn)入未添加AS 的培養(yǎng)基, 22共培養(yǎng)6 d, 通過50 mg L1卡那霉素抗性篩選、葉片GUS 染色、uid A 和Npt II 基因PCR 特異擴增、PCR 產(chǎn)物測序及Npt II 基因Southern 檢測, 鑒定出11株轉(zhuǎn)基因植株, 并通過嫁接和次生體胚發(fā)生, 獲得來自8個轉(zhuǎn)基因株系的681株轉(zhuǎn)基因植株, 移栽成活253株。關(guān)鍵詞: 橡膠樹; 根癌農(nóng)桿菌; 花藥愈傷組織; 遺傳轉(zhuǎn)化; 次生體胚發(fā)

4、生Establishment of Agrobacterium tumefaciens -mediated Anther Calli Transfor-mation System in Hevea brasiliensisHUANG Tian-Dai, LI Zhe, SUN Ai-Hua, ZHOU Quan-Nan, HUA Yu-Wei, and HUANG Hua-Sun*Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences / National Rubber Tree Breedi

5、ng Centre / Key Laboratory of Rub-ber Biology, Ministry of Agriculture / State Key Laboratory Breeding Base of Cultivation Physiology of Tropical Crops, Danzhou 571737, ChinaAbstract: Developping a new Hevea variety by crossing breeding will take 2030 years, but genetic transformation provides an ef

6、fective alternative for shortening breeding cycle. At present, two kinds of recipient materials, anther compact embryogenic calli (ACEC and maintained friable embryogenic calli (MFEC, have been used successfully in Hevea genetic transformation. How-ever, few transformed plants are produced from ACEC

7、-derived embyos, and plants regenerated from MFEC are abnormal, show-ing that they have poorer performance in growth, yield, and disease resistance than the budded control. In order to establish Agro-bacterium tumefaciens-mediated Hevea anther calli transformation system, we surveyed the factors inf

8、luencing transformation frequency, including strains of Agrobacterium , co-culture temperature/time and acetosyringone (AS, and the methods for em-bryos regeneration. GUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture tem-perature/time, which was 5.

9、07 blue spots/callus for EHA105, higher than that for GV3101 and LBA4404. The highest GUS tran-sient expression frequency was 8.43 and 19.10 blue spots/callus under 22 co-cultured temperature and 6 d co-cultured time respectively. Twenty-two thousand ACEC were transformed by the strain EHA105 with p

10、CAMBIA2301, and then transferred to the medium without AS and incubated at 22 for 6 d in the dark. Subsequently, 11 transformants were identified by GUS staining, PCR amplification of target genes, sequencing of PCR products and Southern hybridization. Among them, three planted died di-rectly in the

11、 field; three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field. The re-sults showed that the present transformation system and t

12、he plant propagation by transgenic embryos secondary embryogenesis are efficient for the production of transgenic rubber plants.Keywords: Hevea brasiliensis; Agrobacterium tumefaciens; Anther calli; Genetic transformation; Secondary embryogenesis天然橡膠具有合成橡膠無法比擬的彈性、延展性及導(dǎo)熱性, 是重要的工業(yè)原料1, 主要來源于巴西橡膠樹(Hevea

13、 brasiliensis Mull. Arg.。巴西橡膠樹為多年生異花授粉木本作物, 童期長達(dá)56年,1692作 物 學(xué) 報 第36卷通過常規(guī)雜交育種手段, 培育一個新品種需要20 30年。轉(zhuǎn)基因技術(shù)是縮短橡膠樹育種周期、加快新品種培育十分有效的途徑。農(nóng)桿菌(Agrobacterium tumefaciens 介導(dǎo)的橡 膠樹遺傳轉(zhuǎn)化研究, 始于20世紀(jì)90年代。Arokiaraj 等2-3以花藥致密胚性愈傷組織為受體, 首次成功建立農(nóng)桿菌介導(dǎo)的橡膠樹遺傳轉(zhuǎn)化體系, 獲得3株轉(zhuǎn)Npt II 基因和uidA 基因植株。Montoro 等4-5和Blanc 等6以內(nèi)珠被長期繼代易碎胚性愈傷組織為受

14、體, 通過研究鈣、共培養(yǎng)溫度等因子對農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化效率的影響, 獲得374株轉(zhuǎn)Npt II 基因和uidA 基因橡膠樹。橡膠樹遺傳轉(zhuǎn)化體系的成功建立為功能基因轉(zhuǎn)化橡膠樹奠定了基礎(chǔ)。Arokiaraj 等7和Jayashree 等8均以花藥致密胚性愈傷組織為受體, 分別獲得102株轉(zhuǎn)人血清白蛋白基因(HSA 植株和3株轉(zhuǎn)抗死皮的橡膠樹超氧化物歧化酶基因(HbSOD 植株。Leclercq 等9將另一抗死皮基因銅鋅超氧化物歧化酶基因(CuZnSOD 轉(zhuǎn)入橡膠樹內(nèi)珠被長期繼代易碎胚性愈傷組織, 獲得90個轉(zhuǎn)化愈傷系, 其中13個高表達(dá)CuZnSOD 基因。劉志昕等10和趙輝等11分別以花藥致密胚

15、性愈傷組織和長期繼代的花藥易碎胚性愈傷組織為受體, 均獲得PCR 檢測陽性胚狀體, 但未再生轉(zhuǎn)基因植株, 主要原因在于受體的體胚發(fā)生和轉(zhuǎn)化胚狀體再生能力低。本研究以花藥致密胚性愈傷組織為農(nóng)桿菌侵染受體, 研究影響遺傳轉(zhuǎn)化效率因素的同時, 探討轉(zhuǎn)化胚狀體再生及增殖方式, 以期建立農(nóng)桿菌介導(dǎo)的橡膠樹遺傳轉(zhuǎn)化體系。1 材料與方法1.1 植物材料將橡膠樹大規(guī)模推廣品種熱研7-33-97單核靠邊期花藥接種于胚性愈傷組織誘導(dǎo)培養(yǎng)基上, 培養(yǎng)30 d后用于農(nóng)桿菌侵染。1.2 質(zhì)粒實驗質(zhì)粒為pCAMBIA2301, 其T-DNA 區(qū)域含有CaMV 35S啟動子及其調(diào)控的Npt II 基因和帶一段內(nèi)含子的uid

16、 A 基因(圖1 。1.3 培養(yǎng)基誘導(dǎo)愈傷培養(yǎng)基為MS 培養(yǎng)基添加3.0 mmol L1 CaCl 2、7.0 mol L1 KT、8.1 mol L1 NAA、6.8 mol L 1 2,4-D、204.5 mmol L1蔗糖和2.2 g L1 Phytagel; 分化培養(yǎng)基為MS 培養(yǎng)基添加2.2 mol L1 6-BA、14.0 mol L1 KT、1.4 mol L1GA 3、0.1 mol L1 NAA 、3.8 mol L1 ABA、204.5 mmol L1蔗糖和2.2 g L1 Phytagel; 植株再生培養(yǎng)基為MS 培養(yǎng)基添加0.23 mol L1 KT、0.11 mol

17、L1 IAA、8.7 mol L1 GA 3和4.5 mol L1 2,4-D。1.4 侵染與轉(zhuǎn)化將愈傷組織在3個不同農(nóng)桿菌菌株(EHA105、GV3101和LBA4404 懸浮液中侵染5 min后, 轉(zhuǎn)入添加不同濃度(0、5、10、50、100、500 mol L1 乙酰丁香酮(AS和10 mg L1硝酸銀共培養(yǎng)基(誘導(dǎo)愈傷培養(yǎng)基pH 5.2上培養(yǎng), 2028暗培養(yǎng)19 d。每個處理3次重復(fù)。結(jié)果用SAS9.0進(jìn)行統(tǒng)計分析。將共培養(yǎng)后的愈傷組織轉(zhuǎn)移到添加10 mg L1硝酸銀和500 mg L1特泯丁的愈傷誘導(dǎo)培養(yǎng)基上進(jìn)行恢復(fù)培養(yǎng), 25暗培養(yǎng)710 d。繼而轉(zhuǎn)入添加10 mg L1硝酸銀、

18、50 mg L1卡那霉素和500 mg L1特泯丁的愈傷誘導(dǎo)培養(yǎng)基進(jìn)行抗性愈傷篩選, 每20 d繼代一次, 繼代2次后將抗性愈傷組織轉(zhuǎn)入添加500 mg L1特泯丁和50 mg L1卡那霉素的分化培養(yǎng)基上誘導(dǎo)體細(xì)胞胚胎發(fā)生。最后, 將體細(xì)胞胚轉(zhuǎn)入添加50 mg L 1卡那霉素的植株再生培養(yǎng)基誘導(dǎo)植株再生。 1.5 GUS 的瞬時表達(dá)及穩(wěn)定表達(dá)將共培養(yǎng)愈傷組織及抗性愈傷組織、胚狀體和植株葉片放入X-Gluc 染色液并抽真空510 min后, 置37培養(yǎng)箱中過夜培養(yǎng)。用體視顯微鏡觀察GUS 圖1 質(zhì)粒pCAMBIA2301 T-DNA區(qū)段示意圖 Fig. 1 T-DNA structure of

19、pCAMBIA2301 vectorLB: T-DNA左邊界; RB: T-DNA右邊界; 35S-t : 35S終止子; 35S-p: 35S啟動子; Nos-t: Nos終止子; Npt II: 新霉素磷酸轉(zhuǎn)移酶II 基因;uid A: -葡糖醛酸酶基因。LB: left border; RB: right border; 35S-t: 35S terminator; 35S-p: 35S promoter; Nos-t: nopaline synthase gene terminator; Npt II: neomycinphos-photransferase II gene; uid

20、A: -glucuronidase gene.第10期黃天帶等: 根癌農(nóng)桿菌介導(dǎo)的橡膠樹花藥愈傷組織遺傳轉(zhuǎn)化體系的建立 1693表達(dá)情況, 對每個處理愈傷組織的藍(lán)色斑點進(jìn)行計數(shù)。X-Gluc 染色液的組成為100 mmol L1 NaH2PO 4 2H 2O, 100 mmol L1 Na2HPO 412H2O, 10 mmol L1 Na 2EDTA, 1 mmol L1 K 3Fe(CN6, 1 mmol L1 K 4Fe(CN6, 0.5%(V/V TritonX-100, 20% (V/V 甲醇, 0.5 mg mL1 X-Gluc 。1.6 轉(zhuǎn)化子PCR 檢測及產(chǎn)物序列分析以抗性胚狀

21、體及GUS 染色為陽性植株葉片DNA 為模板, 利用Npt II 和uid A 基因特異引物, 進(jìn)行PCR 檢測。提取DNA 參照Varghese 等12方法。Npt II 特異擴增引物為5-AAGCACGAGGAAGCGG TCAGC-3和5-TGGAGAGGACACGCTGAAATCA CC-3, uid A 特異擴增引物為5-GGTGGGAAAGCG CGTTACAAG-3和5-GTTTACGCGTTGCTTCCGC CA-3。Npt II 、uid A 引物預(yù)期擴增片段長度分別為812和1 203 bp。PCR 程序為94預(yù)變性2 min, 1個循環(huán); 94 40 s, 60 1 mi

22、n, 72 1 min, 35個循環(huán); 72 10 min, 1個循環(huán); 4保存。用1.0%瓊脂糖凝膠電泳分析擴增產(chǎn)物。利用ABI3730X 對PCR 檢測為陽性擬轉(zhuǎn)化子產(chǎn)物進(jìn)行測序后, 通過Blastx 進(jìn)行序列比對分析。1.7 轉(zhuǎn)化子Southern 鑒定按照DIG High Prime DNA Labeling and Detec-tion Starter Kit II (Roche, Germany試劑盒, 以Eco R I過夜消化PCR 檢測陽性植株的總DNA (20 g 為模板, 以隨機引物法標(biāo)記Npt II 基因為探針, 進(jìn)行Southern 雜交鑒定。1.8 轉(zhuǎn)化植株移栽將轉(zhuǎn)基

23、因植株直接移栽或通過兩種方式增殖后移栽, 其一利用次生體胚發(fā)生對獲得的轉(zhuǎn)化體細(xì)胞胚進(jìn)行增殖, 然后誘導(dǎo)體胚植株再生; 其二通過籽苗芽接, 將轉(zhuǎn)基因植株頂芽或芽片嫁接到籽苗, 成活后進(jìn)行移栽。參照Hua 等13的次生體胚發(fā)生方法和黃守鋒14的籽苗芽接法。2 結(jié)果與分析2.1 不同處理對GUS 瞬時表達(dá)的影響農(nóng)桿菌菌株、共培養(yǎng)溫度和共培養(yǎng)時間等對橡膠樹花藥愈傷組織的轉(zhuǎn)化效率具有顯著影響, EHA105菌株侵染力最強, GUS 瞬時表達(dá)率為每個愈傷組織塊5.07個藍(lán)色斑點, 分別是GV3101和LBA4404菌株的11.8倍和72.4倍; 隨共培養(yǎng)溫度的升高和共培養(yǎng)時間的延長, 轉(zhuǎn)化效率先升高再下降

24、, 共培養(yǎng)溫度22, 共培養(yǎng)6 d, 使GUS 瞬時表達(dá)率最高, 每個愈傷組織塊分別達(dá)到8.43個和19.10個藍(lán)色斑點。共培養(yǎng)基中添加AS 無助于橡膠樹花藥愈傷組織轉(zhuǎn)化效率的提高, 隨添加AS 濃度的升高, 轉(zhuǎn)化效率顯著下降, 并且, 當(dāng)添加AS 濃度高于50 mol L1時, 愈傷組織生長開始受到抑制(表1 。2.2 轉(zhuǎn)化植株的獲得及其分子生物學(xué)鑒定利用優(yōu)化后轉(zhuǎn)化體系, 即以EHA105為侵染菌株, 侵染5 min后, 轉(zhuǎn)入未添加AS 的共培養(yǎng)基22暗培養(yǎng)6 d, 轉(zhuǎn)化2.2萬個花藥愈傷組織, 經(jīng)2個周期抗性愈傷組織篩選和3個半月愈傷組織分化后, 共獲得541個抗性胚狀體。隨機取13個抗性

25、胚狀體部分胚塊進(jìn)行PCR 檢測, 其中4個抗性胚狀體檢測到Npt II 基因特異片段, PCR檢測陽性率為30.78%。取其中一個PCR 陽性胚狀體進(jìn)行GUS 染色, 可看到整個胚狀體被染成藍(lán)色(圖2-A 。誘導(dǎo)540個胚狀體再生植株, 共獲得99株抗性植株(圖2-D, 植株再生率為18.33%, 其中11株葉片GUS 染色呈現(xiàn)藍(lán)色(圖2-B 且uid A 基因與Npt II 基因均擴增出與陽性對照相同的PCR 特異擴增片段(圖3, PCR特異擴增片段序列與pCAMBIA2301 (gi:7638149中Npt II 和uid A 基因序列相似率均為97%以上。隨機挑取其中4株擬轉(zhuǎn)化植株進(jìn)行S

26、outhern 雜交, 結(jié)果3株植株具有明顯雜交信號, 2株為單拷貝插入, 1株為三拷貝插入(圖3, 另一單株雜交信號較弱。2.3 轉(zhuǎn)化植株的繁殖及移栽獲得11株轉(zhuǎn)基因植株后, 3株(13未經(jīng)增殖直接移栽到大田, 均未成活; 3株(46通過籽苗芽接進(jìn)行一輪室外嫁接繁殖(圖2-E, 獲得5株轉(zhuǎn)基因單株(圖2-G; 其余5株轉(zhuǎn)基因植株在胚狀體階段通過橡膠樹次生體胚發(fā)生進(jìn)行繁殖研究(圖2-C 。5個轉(zhuǎn)基因胚狀體(711進(jìn)行3次次生體胚發(fā)生增殖后誘導(dǎo)植株再生, 最終獲得676株轉(zhuǎn)基因單株, 增殖系數(shù)為135.20, 移栽成活248株(圖2-F, 成活率為36.69%, 5個轉(zhuǎn)基因胚狀體來源的植株均移栽

27、成活(表2 。3 討論3.1 農(nóng)桿菌介導(dǎo)橡膠樹遺傳轉(zhuǎn)化體系建立農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化在水稻15、玉米16-17、番茄18等作物已經(jīng)成為常規(guī)的轉(zhuǎn)基因手段, 但是對一些重要的木本植物仍然比較困難。自20世紀(jì)90年代以來, 世界各主要植膠國家如馬來西亞、印度及法國, 相繼開展農(nóng)桿菌介導(dǎo)的橡膠樹遺傳轉(zhuǎn)化研1694作 物 學(xué) 報第36卷表1 不同處理對GUS 瞬時表達(dá)的影響Table 1 Effects of different treatments on GUS transient expression 處理 Treatment愈傷數(shù)(塊 No. of calli 斑點數(shù)(個 No. of blue s

28、pots 斑點數(shù)(個/愈傷 Blue spots per callusEHA105 30 152 5.07 a GV3101 30 13 0.43 b 菌株 StrainLBA4404 30 2 0.07 b0 30 142 4.73 a 5 30 96 3.20 ab 10 30 65 2.17 abc 50 30 39 1.30 bc 100 30 12 0.40 bc 乙酰丁香酮AS (mol L1500 30 0 0.00 c20 30 148 4.93 ab 22 30 253 8.43 a 24 30 106 3.53 bc 26 30 160 5.33 ab 共培養(yǎng)溫度Cultu

29、re temp. ( 28 30 9 0.30 c1 30 3 0.10 c 2 30 0 0.00 c 3 30 16 0.53 c 4 30 225 7.50 bc 5 30 211 7.03 bc 6 30 573 19.10 a 7 30 316 10.53 b 8 30 164 5.47 bc 共培養(yǎng)時間Culture time (d9 30 116 3.87 bc數(shù)據(jù)后不同小寫字母表示不同處理間在0.05水平上差異顯著(Duncan法 。The small letters followed the data in the last colume mean significantly

30、 different between the treatments at the 0.05 probability level (Duncans method. 圖2 轉(zhuǎn)基因胚狀體、植株GUS 檢測、植株再生及移栽Fig. 2 GUS staining for transformed embryos and plantlets, plant regeneration and transplantationA: 子葉形胚GUS 染色(右邊為對照; B: 轉(zhuǎn)基因植株葉片GUS 染色(右邊為對照; C: 轉(zhuǎn)基因胚狀體次生體胚發(fā)生; D: 轉(zhuǎn)基因再生植株; E: 籽苗芽接法嫁接轉(zhuǎn)化植株; F: 直接移

31、栽轉(zhuǎn)化植株; G: 嫁接成活轉(zhuǎn)化植株。A: GUS staining for cotyledonary embryo (right is control; B: GUS staining for leaf of transformed plantlet (right is control; C: transformed embryo secondary embryogenesis; D: regenerated Hevea ; E: budded transgenic Hevea by mini-seedling budding; F: transgenic Hevea in soil;G:

32、growing budded transgenic Hevea .第10期黃天帶等: 根癌農(nóng)桿菌介導(dǎo)的橡膠樹花藥愈傷組織遺傳轉(zhuǎn)化體系的建立 1695 圖3 轉(zhuǎn)基因植株分子檢測Fig. 3 Molecular detection of transgenic plantlets左: uid A 基因PCR 特異擴增; 中: Npt II 基因PCR 特異擴增; 右: Npt II 基因Southern 檢測; M: marker; +: 陽性對照, 質(zhì)粒pCAMBIA2301; : 陰性對照, 非轉(zhuǎn)化植株; 0: 空白對照, 水; 111: 轉(zhuǎn)基因單株。Left: PCR analysis of

33、uid A gene; middle: PCR analysis of Npt II gene; right: Southern analysis of Npt II gene; M: marker; +: positive control,pCAMBIA2301; : negative control, untransformed plantlet; 0: blank control, water; 111: transgenic plantlets.表2 轉(zhuǎn)基因植株的增殖及移栽結(jié)果Table 2 Results of multiplication and transplantation f

34、or transgenic Hevea株號 No. 增殖方式Multiplication methods增殖前株數(shù) No. prior to multiplication增殖后株數(shù) No. after multiplication增殖系數(shù) Multiplicationindex移栽成活株數(shù) Survived No.移栽成活率 Survived frequency (%13未增殖Non-multiplication3 3 1 0 04 1 2 2 2 100.00 5 1 1 1 1 100.00 6籽苗芽接Mini-seedling budding 1 2 2 2 100.007 1 272

35、272 27 9.93 8 1 178 178 110 61.809 1 18 18 6 33.33 10 1 199 199 103 51.7611 次生體胚發(fā)生Secondary embryogenesis1 9 9 2 22.22總計Total11 684 45.9 253 36.99究, 先后建立農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化體系, 并利用其轉(zhuǎn)化體系獲得功能基因轉(zhuǎn)化植株2-9,19-21。我國橡膠樹遺傳轉(zhuǎn)化研究起步相對較晚, 劉志昕等10、趙輝等11、王穎等22和洪磊等23對橡膠樹遺傳轉(zhuǎn)化方法及其影響因素進(jìn)行研究, 獲得了PCR 檢測陽性胚狀體, 但未見Southern 檢測、轉(zhuǎn)化再生植株及轉(zhuǎn)化

36、效率報道。本研究獲得11株GUS 表達(dá)、PCR 擴增和PCR 產(chǎn)物測序檢測陽性植株, 隨機選取4株進(jìn)行Southern 檢測, 結(jié)果3株為陽性植株。3.2 轉(zhuǎn)化胚狀體次生體胚發(fā)生是獲得轉(zhuǎn)化植株的有效途徑劉志昕等10、趙輝等11和王穎等22獲得PCR 檢測陽性胚狀體, 但均未獲得轉(zhuǎn)化植株, 特別是趙輝等11以長期繼代的花藥易碎胚性愈傷組織為受體, 胚狀體誘導(dǎo)率達(dá)到30%, 但是絕大部分為畸形胚, 無法再生植株。Arokiaraj 等2以GV2260為侵染菌株獲得65個抗性胚狀體, 但僅再生3株植株, 以LBA4404為侵染菌株獲得38個抗性胚狀體, 未獲再生植株。雖然Blanc 等24利用長期繼

37、代的易碎胚性愈傷組織高效增殖特點, 增殖轉(zhuǎn)化愈傷組織后再誘導(dǎo)體胚發(fā)生, 成功獲得來自6個愈傷系的374株轉(zhuǎn)基因植株, 但是, 長期繼代的胚性愈傷組織再生植株產(chǎn)量低、長勢差、易感病24, 無法實現(xiàn)通過轉(zhuǎn)基因改良橡膠樹目的。轉(zhuǎn)化胚狀體畸形胚比例高、生活力弱, 無法直接再生植株是橡膠樹遺傳轉(zhuǎn)化重要技術(shù)瓶頸。橡膠樹次生體胚發(fā)生是以胚狀體為外植體, 通過循環(huán)體胚發(fā)生, 實現(xiàn)胚到胚的循環(huán)增殖, 最終實現(xiàn)體胚植株增殖的方法, 異常胚狀體和成熟子葉期胚狀體均能誘導(dǎo)次生體胚發(fā)生和產(chǎn)生成熟子葉期胚狀體13。本研究中抗性胚狀體PCR 檢測陽性率高達(dá)30.78%, 但是抗性胚狀體植株再生頻率僅為18.33%, 而且,

38、 利用次生體胚發(fā)生成功獲得5個株系676株轉(zhuǎn)基因單株。因此, 利用次生體胚發(fā)生將抗性胚狀體增殖后, 再誘導(dǎo)抗性胚狀體植株再生和轉(zhuǎn)基因檢測, 不但能降低胚狀體無法再生而造成轉(zhuǎn)化胚狀體丟失的風(fēng)險, 而且可進(jìn)一步提高遺傳轉(zhuǎn)化效率, 在理論上, 本研究建立遺傳轉(zhuǎn)化體系的轉(zhuǎn)化效率, 將由0.05%(11/22000提高到0.75%(54030.78%/22000, 這還需在后續(xù)研究中驗證。1696 作 物 學(xué) 報 第 36 卷 由于轉(zhuǎn)基因植株成活頻率低丟失大量轉(zhuǎn)化子是 橡膠樹遺傳轉(zhuǎn)化另一難題, Arokiaraj 等 移栽 73 株 只成活 16 株; 本試驗直接移栽 3 株, 均未成活。為 此, Ar

39、okiaraj 等 3 直接將轉(zhuǎn)基因植株作為芽條嫁接 于室外實生砧木上, 但是, 這種途徑不能從根本上 解決轉(zhuǎn)化子的丟失問題, 因為無法確保轉(zhuǎn)化胚狀體 全部再生植株; Blanc 等 6增殖長期繼代轉(zhuǎn)化愈傷后 再生植株, 高效獲得 6 個株系 374 株轉(zhuǎn)基因橡膠樹, 但是, 這類受體再生植株農(nóng)藝性狀差 24, 與通過轉(zhuǎn) 基因改良橡膠樹目的相悖。本研究通過次生體胚發(fā) 生成功獲得 5 個株系 676 株轉(zhuǎn)基因單株, 移栽成活 248 株, 每一個轉(zhuǎn)化子均有轉(zhuǎn)化植株移栽成活且室內(nèi)依 然保存轉(zhuǎn)化胚狀體。轉(zhuǎn)化胚狀體次生體胚發(fā)生是從 胚狀體階段進(jìn)行轉(zhuǎn)化胚狀體的增殖, 大大提高了轉(zhuǎn) 化胚狀體進(jìn)入植株再生階

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