作物杂志, 2018, 34(6): 162-167 doi: 10.16035/j.issn.1001-7283.2018.06.026

种子科技

消毒方法对红花种子发芽率的影响和种子环境细菌研究

马梦雪, 赵玲玲, 唐思, 陈贤军, 覃瑞

中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北重点实验室,430074,湖北武汉

The Effects of Different Disinfection Methods on Seed Germination and Study on the Environmental Bacteria in Safflower (Carthamus tinctorius L.)

Ma Mengxue, Zhao Lingling, Tang Si, Chen Xianjun, Qin Rui

College of Life Sciences, South-Central University for Nationalities/Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China, Wuhan 430074, Hubei, China

通讯作者: 覃瑞,教授,研究方向为植物细胞遗传

收稿日期: 2018-05-9   修回日期: 2018-10-11   网络出版日期: 2018-12-15

基金资助: 湖北省科技条件平台建设专项.  2017BE014
湖北省技术创新专项重大项目.  2018ABA093

Received: 2018-05-9   Revised: 2018-10-11   Online: 2018-12-15

作者简介 About authors

马梦雪,硕士研究生,研究方向为植物细胞遗传 。

摘要

红花(Carthamus tinotorius L.)种子在发芽和组培中染菌情况严重,发芽率较低,针对这一现象,对供试红花种子进行消毒以降低染菌率并提高发芽率。选择种子预浸泡时间、消毒剂种类、消毒时间3个因素,采用L9(3 4)正交试验,以种子的发芽率、发芽势和染菌率为考察指标,综合分析种子消毒效果。结果表明,消毒剂种类是影响红花种子消毒效果的最重要因素。红花种子最佳消毒方法为种子消毒前用无菌水预浸泡12h,再经2% NaClO消毒10min,染菌率仅为8%,发芽率高达90.67%。另外,对分离纯化消毒后种子所带的细菌16S rDNA(GenBank登录号:MH190221)进行了PCR扩增,序列Blast结果显示,该菌为阿耶波多芽孢杆菌(Bacillus aryabhatti),初步判断该菌可能为红花种子内生菌。该研究结果为快速获得生长良好的无菌苗、后续的组织培养以及遗传转化奠定了基础。

关键词: 红花 ; 种子消毒 ; 发芽率 ; 正交试验 ; 细菌

Abstract

Contamination was a serious problem in seed germination and tissue culture of safflower (Carthamus tinctorius L.). In our study, seeds were disinfected in order to reduce the contamination rate.Three factors including soaking time, disinfecting agents and disinfecting time were used for L9(3 4) orthogonal experiment. Results were comprehensively evaluated by using the weighted scoring method based on indexes such as germination rate, germination vigor and contamination rate. Orthogonal test showed that the disinfecting agent was the most important factor affecting the seeds germination. Among various treatments, seeds soaked in sterile water for 12h followed by the 2% sodium hypochlorite (NaClO) for 10min was the best disinfecting method in safflower seeds which yield a contamination rate of 8% and a germination rate of 90.67%. In addition, 16S rDNA (GenBank Accession No. MH190221) of a bacterium was isolated from seeds and purified after disinfection which was amplified by PCR. BLAST results showed that it belonged to Bacillus aryabhatti which was most likely to be entophytes in safflower seeds. The results of this study provided an important basis for obtaining well-grown aseptic seedlings, tissue culture as well as genetic transformation.

Keywords: Carthamus tinctorius L. ; Seed disinfection ; Seed germination rate ; Orthogonal experiment ; Bacteria

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本文引用格式

马梦雪, 赵玲玲, 唐思, 陈贤军, 覃瑞. 消毒方法对红花种子发芽率的影响和种子环境细菌研究[J]. 作物杂志, 2018, 34(6): 162-167 doi:10.16035/j.issn.1001-7283.2018.06.026

Ma Mengxue, Zhao Lingling, Tang Si, Chen Xianjun, Qin Rui. The Effects of Different Disinfection Methods on Seed Germination and Study on the Environmental Bacteria in Safflower (Carthamus tinctorius L.)[J]. Crops, 2018, 34(6): 162-167 doi:10.16035/j.issn.1001-7283.2018.06.026

红花(Carthamus tinotorius L.)属菊科(Compositae)红花属(Carthamus)的一年生草本植物,又名刺红花、菊红花和红花菜等,是一种药食同源的经济作物[1]。红花的适应性较强,能抗寒、耐旱、耐盐碱[2],在世界各地理区域均有分布,哈萨克斯坦和印度是其主产国[3]。在我国,红花的种植遍布25个省市自治区,河南、四川、浙江和新疆为其主要产区[4]。红花花丝含有活血通经、去淤疗伤、解郁安神等药用成分,是我国传统的中草药;此外,花丝中还含有天然色素,可用于食品、化妆品等领域。红花种子含油率较高,亚油酸的含量高达73%~80%,有“亚油酸之王”之称[5]。亚油酸为不饱和脂肪酸,具有降低血清中的胆固醇、软化血管、防治动脉粥样硬化、调节血脂等作用[6,7]。红花还被开发用来生产生物燃料和工业用油[8,9]。近年来,转基因红花还被用于生产人类感兴趣的重要药物,如胰岛素和载脂蛋白[10,11,12]

目前,红花已经得到了广泛的种植,但是在传统的遗传育种过程中,红花易受病虫害的影响,尤其是球腐病、叶斑病、根腐病、锈病等病害严重影响着红花的生产和繁殖[13]。此外,传统育种还存在周期长、见效慢的缺点。红花育种计划旨在获得对病虫害抵抗力强、优质、高产并且抗逆性强的油药兼用品种。随着生物技术的不断发展,基因工程和组织培养技术可用来进一步改善农艺性状。用红花种子培育无菌苗,再获取外植体用于组织培养,构建红花再生体系不仅为快速扩繁红花奠定基础,也为其基因改造提供平台,有利于红花功能基因的验证和红花资源的保护[14]。然而,红花种子在栽培和组织培养过程中易染菌,种子消毒剂的选择和最佳消毒时间的确定能在很大程度上减少种子染菌现象,提高种子发芽率。红花种子的灭菌主要是用70%乙醇、0.1%升汞、2% NaClO或10% H2O2等溶液漂洗[15,16,17,18,19]。李威等[20]研究表明在5℃~25℃温度条件下红花种子发芽率较高。

本研究以种子发芽率、发芽势和染菌率为考察指标,通过不同的种子预处理时间、消毒剂种类和消毒时间对红花种子消毒效果影响的分析,探索红花种子最佳消毒方法,旨在提高种子发芽率、缩短种子萌发时间,为快速获得生长良好的无菌苗和后续的组织培养、遗传转化奠定基础。

1 材料与方法

1.1 材料

试验于2017年6-7月进行。供试红花种子为中国农业科学院油料作物研究所严兴初研究员提供的安徽红花品种,保存于4℃条件下。选用无菌水于25℃条件下浸种;选用75%乙醇作为种子表面消毒剂;种子消毒剂有0.1% HgCl2、2% NaClO、10% H2O2;选用MS培养基[21](购自PhytoTech公司)作为无菌苗生长培养基。

1.2 方法

1.2.1 最佳预处理时间、消毒剂及消毒时间的选择挑选饱满无破损的红花种子,用无菌水预浸泡;将预浸泡后的种子放入已提前灭菌的小瓶中,倒入适量的75%乙醇表面消毒30s,无菌水润洗3次,每次2min;消毒剂处理后,用无菌水润洗5次,每次3min。用镊子将处理过的种子取出置于滤纸上晾干,然后接种于MS培养基。根据种子消毒和萌发相关文献[22,23,24],对影响种子萌发的种子预浸泡时间、消毒剂种类、消毒时间3个主要因素进行3水平正交试验(表1),共9种处理组合,每处理3次重复,每重复50粒种子。

表1   正交试验因素水平

Table 1  The list of different factors for orthogonal experiment

水平
Level
因素Factor
A预浸泡时间(h)
Soaking time
B消毒剂
Disinfecting agent
C消毒时间(min)
Disinfecting time
D对照
Control
100.1% HgCl210
262% NaClO15
31210% H2O220

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1.2.2 种子发芽率、发芽势、发芽指数和种子染菌率 种子萌发以突破种皮的胚轴长度达到种子自身长度一半为标准[25]。记录种子起始萌发的天数,以7d为标准统计种子发芽率和发芽指数等各项指标。各指标计算公式[26,27]如下:

发芽率(GR)=(7d内萌发的种子数/供试种子总数)×100%

发芽势(GV)=(前3d发芽种子数/种子总数)×100%

发芽指数(GI)=∑(Gt/Dt),式中:Gt为不同发芽时间t的发芽数;Dt为不同的发芽试验天数(d)。

种子染菌率(CR)=(污染种子粒数/种子总数)×100%。

1.2.3 数据分析 利用SPSS 17.0对上述数据进行处理,用Excel 2003绘图。

1.2.4 红花种子染菌的初步分析 挑选种子染菌区域菌落,转接至牛肉膏蛋白胨固体培养基(牛肉膏3g/L,蛋白胨10g/L,氯化钠5g/L,琼脂粉15g/L,pH 7.5)中进行划线分离,于37℃倒置培养24h后观察菌落特点并记录,平板放于4℃冰箱保存备用。用20μL枪头挑取单克隆,于加入200μL ddH2O的离心管中来回旋转吸打,以此菌悬液作为菌落PCR的模板。根据16S rDNA通用引物(27F:AGAGTTTGATCCTGGCTCAG,1429R:GGTTACCTTGTTACGACTT)进行PCR扩增,试验采用50μL体系:模板2μL,两种通用引物各2μL,PCR Buffer 5μL,dNTP 3μL,Taq酶2μL,MgCl2 4μL,ddH2O 30μL;扩增程序为:94℃预变性5min;94℃变性30s,56℃退火45s,72℃延伸2min,35个循环;72℃再延伸10min;4℃保温。PCR产物由武汉华大基因科技有限公司测序,获得的序列与GenBank中的序列进行BLAST比对,初步判定菌株种类和染菌原因。

2 结果分析

2.1 不同种子预浸泡时间对红花种子消毒效果的影响

采用9种不同的处理组合,对红花种子进行消毒处理,7d后对种子的染菌率进行测定,以此评价其消毒效果,结果如表2所示。可以看出,10% H2O2消毒处理的种子染菌率相对较高,最高可达27.33%(T6),0.1% HgCl2消毒处理的种子染菌率低,最低仅为2.00%(T7),说明0.1% HgCl2对红花种子的消毒效果良好,2% NaClO次之,10% H2O2消毒效果较差。此外,随着无菌水浸种处理时间的增加,种子染菌率均有一定程度的下降,表明浸种处理可以在一定程度上减少种子的染菌率。

表2   正交试验设计L9(34)对种子发芽率的影响

Table 2  The effects of orthogonal test of L9(34) to seeds germination rate

处理
Treatment
A
预浸泡时间(h)
Soaking time
B
消毒剂种类
Disinfecting agent
C
消毒时间(min)
Disinfecting time
D
对照
Control
染菌率(%)
Contamination
rate
发芽势(%)
Germination vigor
发芽指数(%)
Germination index
发芽率(%)
Germination
rate
综合评分
Overall score
T101(0.1% HgCl21019.3334.6733.6564.670.27
T202(2% NaClO)15219.3334.0038.6871.330.15
T303(10% H2O220324.0024.0028.9548.67-0.06
T461(0.1% HgCl21533.3350.0045.3775.330.45
T562(2% NaClO)20110.6755.3357.2280.670.39
T663(10% H2O210227.3336.0040.0261.33-0.01
T7121(0.1% HgCl22022.0053.3351.1085.330.53
T8122(2% NaClO)1038.0059.3363.1790.670.48
T9123(10% H2O215118.6744.6746.0867.330.17
K10.361.240.740.83
K20.831.020.770.67
K31.180.110.860.88
k10.120.410.250.28
k20.280.340.260.22
k30.390.040.290.29
R0.280.380.040.07

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2.2 不同种子预浸泡时间对红花种子发芽率的影响

不同处理组合对红花种子发芽率的影响见表2。T1~T9处理的发芽率分别为64.67%、71.33%、48.67%、75.33%、80.67%、61.33%、85.33%、90.67%、67.33%。就种子发芽率而言,无菌水预浸泡12h、2% NaClO消毒10min的种子发芽率(90.67%)最高;未进行预浸泡的种子发芽率则相对较低。由此可得,预浸泡处理的种子发芽率高于未浸泡处理,且随着浸种时间的增加种子发芽率也有一定程度的增加;NaClO和HgCl2对种子的消毒效果较好,对种子的萌发抑制作用相对较小,种子发芽率相对较高。

2.3 不同种子预浸泡时间对红花种子发芽势和发芽指数的影响

表2可知,未预浸泡时HgCl2消毒处理与NaClO消毒处理的种子的发芽势无明显差异,但两者消毒处理的种子发芽势均高于用H2O2消毒处理的种子。当对种子进行预浸泡时,随着预浸泡时间的增加,种子的发芽势也随之增大。发芽指数不仅包括发芽的种子数,而且强调发芽速度和整齐度,与种子活力呈正相关关系,其值越大,表明种子活力越高[20]。由图1可知,随着种子预浸泡时间的增加,各组试验红花种子发芽指数变化的趋势与发芽率和发芽势的趋势基本一致。说明浸种处理能够促进种子的萌发,从而缩短种子萌发所需的时间,为后续红花离体再生体系的建立带来便利。用2% NaClO处理12h的种子发芽指数最高为63.17%,0.1% HgCl2处理12h的种子发芽指数较高的为51.10%,10% H2O2处理的种子发芽势相对较低。因此,预浸泡12h、用2% NaClO消毒处理的种子的发芽势较高,种子活力也更高。

图1

图1   不同处理对种子发芽率、发芽势和发芽指数的影响

Fig.1   The effects of different treatments on seed germination rate, germination vigor and germination index


2.4 红花种子消毒方法的优化及验证试验

优选红花种子消毒技术,要满足种子染菌率低,在提高种子发芽率的同时尽量缩短种子发芽时间。以种子发芽率、发芽势和染菌率为指标,采用加权综合评分法考察红花种子消毒的方法,其中以发芽率和染菌率为主要考察指标,均按40%的系数计分,发芽势按20%的系数计分。评分标准:0.4GR/GRmax+0.2GV/GVmax-0.4CR/CRmax,结果见表2。方差分析结果(表3)表明,因素A和B对试验结果具有显著性影响,各因素对红花种子消毒效果的影响顺序为B>A>C(消毒剂种类﹥预浸泡时间﹥消毒时间),由综合得分可知,A3B1C3(0.53)消毒效果最好,A3B2C1(0.48)次之。但是从发芽率和消毒剂对种子活性的影响角度出发,可得红花种子最佳消毒方法为A3B2C1,即种子无菌水预浸泡12h,再用2% NaClO消毒10min。

表3   各因素间方差分析

Table 3  Variance analysis of different factors

因素Factor偏差平方和
Square of deviance
自由度
Degrees of freedom
F
A预浸泡时间(h)
Soaking time
0.113237.667*
B消毒剂种类
Disinfecting agent
0.247282.333*
C消毒时间(min)
Disinfecting time
0.00321.000
D对照Control0.0002

Note: F0.05(2,2)=19.00, F0.01(2,2)=99.00

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按上述红花种子消毒最佳方法进行3次平行试验,分别按上述方法计算各项指标,结果(表4)表明,各项指标稳定,能达到预期结果,说明此方法稳定可行。

表4   验证试验结果

Table 4  Results of verification tests %

编号
Number
发芽率
Germination rate
发芽势
Germination vigor
染菌率
Contamination rate
191.3360.007.33
290.6760.677.33
390.0058.008.67

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2.5 红花种子染菌初步分析结果

菌株16S rDNA序列如下:

TGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACG
TGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGA
TGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAG
GCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA
GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCT
GTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGC
CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTC
TGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCC
ACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGG
CGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTT
TCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTG
ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAC
AACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG
GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAA
CCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAA
GGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGA
AGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC
ACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCTATAAG

该序列GenBank登录号为MH190221,与NCBI中阿耶波多芽孢杆菌(Bacillus aryabhatti)的序列相似度达100%。

3 结论与讨论

种子在无菌条件下萌发并成为健壮无菌苗的过程受多种因素的影响,如种皮的限制、消毒剂类型、灭菌时间、培养温度、润洗方式等都会影响种子的萌发[28,29],因此选择高效快速的灭菌方法对后续试验具有良好的促进作用。本研究探讨了不同种子预浸泡时间、消毒剂及消毒时间对红花种子消毒效果及种子萌发的影响,以种子发芽率、发芽势和染菌率为考察指标,采用多指标综合加权法[27],可以在一定程度上避免单一指标评价的片面性,使评价结果更加科学合理。

种子经浸泡后,其种皮结构变松软[30],消毒剂更易渗入,种子也更易萌发。本研究中,随着预浸泡处理时间的增加,种子的发芽率、发芽势和发芽指数都随之增加,表明浸种处理能够促进种子的萌发,缩短种子萌发所需的时间,同时在一定程度上降低种子的染菌率。各因素对红花种子消毒效果的影响程度从大到小依次为消毒剂种类、种子预浸泡时间、消毒时间。种子消毒中常用的消毒剂有HgCl2、H2O2、NaClO、Ca(ClO)2、抗生素等[22-24,31],其中75%乙醇和0.1% HgCl2被广泛使用。张付远等[32]研究表明,0.1% HgCl2对野生凤仙花消毒时污染率仅为10%;朱淑颖等[24]研究表明,用0.1% HgCl2消毒3min草珊瑚种子,其发芽率可达68.89%。HgCl2的消毒效果好,但残留的Hg2+渗入种子不仅难洗净而且废液还会给环境造成污染。陈芳等[33]、鲁黎明等[34]研究表明,在亚麻种子和烟草种子中NaClO的消毒效果最好。NaClO可以利用自身分解产生的氯气进行杀菌,且对生物组织毒害作用较小,残留液易挥发,对环境的污染也相对较小。本研究结果表明,用10% H2O2对红花种子消毒处理时,消毒时间短,培养基污染比较严重,消毒时间长,种子的发芽率和发芽势较低,所以H2O2不适宜作为红花种子的消毒剂。当种子经预浸泡处理后用0.1% HgCl2消毒20min时效果最好,种子的发芽率较高,为85.33%;用2% NaClO消毒10min的效果次之,但是种子发芽率最高,为90.67%。这表明HgCl2和NaClO对种子的处理效果明显优于H2O2的处理效果,对种子的萌发抑制作用相对较小。就种子染菌率而言,HgCl2的消毒效果最好,但是其降低了种子的发芽率,考虑到HgCl2对后续愈伤生长的抑制作用及对环境的污染,选定NaClO作为红花种子最理想的消毒剂。本研究解决了红花种子在发芽过程中污染严重和发芽率低的问题,为今后红花高效再生体系的建立和基因工程研究打下了基础。

本研究还对红花种子的染菌原因进行了初步分析。本试验中消毒后的种子仍然带菌,菌落PCR和BLAST比对结果表明本试验中染菌菌株为阿耶波多芽孢杆菌。阿耶波多芽孢杆菌为芽孢杆菌属,是一种好氧产孢、革兰氏阳性细菌,广泛存在于水、土壤和空气中[35]。它们通过空气传播附着在种子表面,也可借助操作人员皮肤、培养器皿、接种工具传播,特别容易沾染在超净工作台和接种工具的一些灭菌死角上。因此,选择合适的种子消毒技术和完善无菌操作技术对于减少组培过程中染菌情况是至关重要的。但是,采用多种消毒方式和完善无菌操作技术后,该菌仍然存在。芽孢杆菌属本身是一类常见的植物内生菌属,所以我们推测阿耶波多芽孢杆菌很可能是红花的内生菌,这为进一步研究和应用红花内生菌提供了参考。

The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。

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红花籽油被广泛用作优质食用油,且已产业化发展.研究显示,红花籽油不仅含有多种对人体有益的成分,如亚油酸、亚麻酸、维生素E、油酸、磷脂等,而且具有抗炎、降血脂、减肥、抗衰老等多种药理活性.针对目前红花籽油应用前景广阔、基础研究较少、产品优势缺乏等现状,本文对红花籽油主要成分、药理作用、应用范围等内容作一综述,为进一步开发和利用红花籽油提供参考.

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Safflower ( Carthamus tinctorius L.) seeds have a tocopherol fraction dominated by α -tocopherol, which accounts for more than 95% of the tocopherols. α -Tocopherol exerts a high vitamin E activity, but a low in vitro antioxidant action. For non-food applications, replacement of high α -tocopherol by γ -tocopherol is preferred. Because of the limited variability found in germplasm of cultivated safflower, the objective of the present research was to search for variability for tocopherol profile in germplasm of wild safflower species. Bulk seed samples of 77 accessions from six species were analysed for total tocopherol content and profile. One accession of C. oxyacanthus showed increased γ -tocopherol content (36%). Single-seed analysis showed the existence of a bimodal distribution, which included high α -tocopherol (>90%) and high γ -tocopherol (HGT) seeds (>85%). Plants from the HGT seeds bred true for the trait and showed introgression of cultivated safflower, which allowed a rapid selection for cultivated safflower traits. Genetic analysis revealed that the HGT trait was controlled by partially recessive alleles at a single locus Tph1, which will facilitate the transference of the novel trait to diverse safflower germplasms.

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Safflower has been transformed for field scale molecular farming of high-value proteins including several pharmaceuticals. Viable safflower seed remaining in the soil seed bank after harvest could facilitate seed and pollen-mediated gene flow. Seeds may germinate in subsequent years and volunteer plants may flower and potentially outcross with commodity safflower and/or produce seed. Seeds from volunteers could become admixed with conventional crops at harvest, and/or replenish the seed bank. Seed in following crops could be transported locally and internationally and facilitate gene flow in locations where regulatory thresholds and public acceptance differ from Canada. Seed-mediated gene flow was examined in three studies. Safflower seed loss and viability following harvest of commercial fields of a non-transgenic cultivar were determined. We assessed seed longevity of transgenic and non-transgenic safflower, on the soil surface and buried at two depths. Finally, we surveyed commercial safflower fields at different sites and measured density and growth stage of safflower volunteers, in other crops the following year and documented volunteer survival and viable seed production. Total seed loss at harvest in commercial fields, ranged from 231 to 1,06902seeds02m 612 and the number of viable seeds ranged from 81 to 51802seeds02m 612 . Safflower has a relatively short longevity in the seed bank and no viable seeds were found after 202years. Based on the seed burial studies it is predicted that winter conditions would reduce safflower seed viability on the soil surface by >50%, leaving between 40 and 26002viable02seeds02m 612 . The density of safflower volunteers emerging in the early spring of the following year ranged from 3 to 1102seedlings02m 612 . Safflower volunteers did not survive in fields under chemical fallow, but in some cereal fields small numbers of volunteers did survive and generate viable seed. Results will be used to make recommendations for best management practices to reduce seed-mediated gene flow from commercial production of plant molecular farming with safflower.

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Plants producing industrial oils of high quality and low price will facilitate a switch to vegetable oil-based raw materials from the current petroleum-based in the chemical industry. Oils have qualities designed to meet the stringent requirements of the industry, thus it is necessary that they are kept separate from farming of food staples. It is therefore a sensible strategy to develop such production in oil crops dedicated for industrial production. Genes for the special features of the non-food industry oil are missing and must be brought to plants by genetic engineering to improve or develop new crops tailoring the industry's needs. This requires that the crop platforms designated for the industrial oil production will need to be transformable. This review provides an overview of potential non-food oil crops that could become production platforms for oil of industrial quality. It also gives a short state of the art regarding the transformation of these plants producing quality non-food oil.

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红花(Carthami Flos)为菊科植物红花(Carthmus tinctorius L.)的干燥管状花,具有抗炎镇痛、扩张冠状动脉、降血压等多种药理作用,临床应用广泛。其中黄酮类成分羟基红花黄色素A (HSYA)是红花的主要活性成分,由此羟基红花黄色素A含量的高低,直接影响红花品质的优劣。因此,从基因水平研究与HSYA生物合成相关的基因,最终从分子水平上定向调控红花品质,为红花的育种提供极大的便利。另外,利用组织培养技术,培育红花的再生再生植株,为快速扩繁红花和验证基因的功能奠定基础。 本研究获得的主要结果如下: 1.筛选出红花种子的最佳0.1%HgCl2消毒时间为:30分钟;通过对不同部位的外植体(子叶、根、茎、真叶)的诱导比较获知再生体系建立的最佳诱导材料是:子叶;通过试验比较细胞生长素NAA和细胞分裂素6-BA、KT、TDZ不同配比的培养基,筛选获得了最有利于诱导愈伤和诱导形成再生芽的培养基:MS+0.1mg/L NAA+2.0mg/L6-BA+15mg/ LKT和MS+0.1mg/L NAA+2.0mg/L6-BA+20mg/LKT。 2.利用高效液相技术完成了对09年红花中HSYA的含量的测定,含量分为0%-2.6%不等. 3.采用cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)技术,首次从红花植物中克隆得到黄烷酮3-羟化酶(F3H)基因。并对该基因进行了蛋白结构的预测和初步的生物信息学分析,F3H基因其全cDNA为1415bp(GenBank登录号:JF737995)包含一个1086bp的开放阅读框(ORF),编码361个氨基酸残基。 4.利用实时定量PCR检测了F3H基因在HSYA含量不同的红花样本中的表达差异。发现在F3H在白花(不含HSYA)中的表达远远低于在黄花(含有HSYA)的表达。

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红花是重要的药油兼用作物,通过基因工程手段提高红花的产量与质量具有重要意义.红花的组织培养为其基因 改造提供了平台,是实现活性化合物工业化生物合成的关键步骤.本文对近年来国内外红花组织培养的研究进展进行了综述,包括植物激素、温度、湿度、光照、酸 碱度等因素对红花组织培养的影响,并对该领域的发展前景进行了展望.

丁亦男, 魏健, 张丽娇 .

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浙江大学学报(农业与生命科学版), 2000,26(6):643-646.

DOI:10.3321/j.issn:1008-9209.2000.06.016      URL     Magsci     [本文引用: 1]

<p>植物有机营养试验中供试无茵苗通常由种子获得,因此,种子灭菌非常重要.采用多种消毒剂(70%酒精,10%H<sub>2</sub>O<sub>2</sub>,2%次氯酸钠和0.1%HgCl<sub>2</sub>等)对几种作物种子进行组合灭菌.结果表明,灭菌效果与作物种类、种子贮存时间及不同消毒剂组合等有很大关系.采用70%酒精浸1min&rarr;无菌水冲3次&rarr;2%次氯酸钠浸5min&rarr;无茵水冲5次&rarr;0.1%HgCl<sub>2</sub>浸5min&rarr;无茵水冲6次的组合灭菌程序,可取得较好的灭茵效果;若种子对2%次氯酸钠敏感,则可用10%H<sub>2</sub>O<sub>2</sub>代替.</p>

刘西莉, 牡丽丹, 王红梅 , .

红花种子带菌检测及药剂消毒处理

植物保护, 2003,29(6):49-51.

DOI:10.3969/j.issn.0529-1542.2003.06.017      URL     [本文引用: 1]

采用平皿法检测了红花种子的带菌情况,并研究了5种杀菌剂对红花种子的消毒效果。结果表明,红花种子携带的主要真菌类群为链格孢属(Alternaria spp.)、黄曲霉属(Aspergillus spp.)、镰孢霉属(Fusarium spp.)、黑根霉属(Rhizopus spp.)和青霉菌属(Penicillium spp.)。福美双与多菌灵混剂、噁霉灵、甲基立枯磷、种衣剂1号、种衣剂2号分别对不同批次红花种子所带真菌均具有一定的消毒效果。

李威, 谭勇, 陈文 , .

温度对不同品种红花种子萌发的影响

安徽农业科学, 2013,41(10):4299-4301.

DOI:10.3969/j.issn.0517-6611.2013.10.023      URL     [本文引用: 2]

[目的]探讨温度对不同品种红花(Carthamus tinctorius L.)种子萌发的影响。[方法]选取裕民无刺、新红4号和吉红1号3个红花品种种子作试验材料,在不同温度下进行发芽试验。[结果]温度对3个品种红花种子萌发的影响很大。红花种子在温度5~35℃下均有萌发,随着温度升高,始发芽时间提前,发芽周期缩短,发芽率降低。在温度5~25℃下都可以保证很高的发芽率,最高发芽率在10℃,为87.89%。但考虑到地温与大气温度的差异性,所以播种红花的适宜温度范围为10~15℃。[结论]栽培红花时宜根据当地气候条件选择合适品种。

Murashige T, Skoog F .

A revised medium for rapid growth and bio assays with tobacco tissue cultures

Physiologia Plantarum, 1962,15(3):473-497.

DOI:10.1111/ppl.1962.15.issue-3      URL     [本文引用: 1]

尚丹, 文昭竹, 龚梨霞 , .

Ca(ClO)2消毒剂对高羊茅种子的消毒效果

作物研究, 2016,30(5):563-568.

DOI:10.16848/j.cnki.issn.1001-5280.2016.05.19      URL     [本文引用: 2]

为研究Ca(ClO)2对高羊茅种子作为外植体诱导培养愈伤组织时的消毒效果,以3种不同的前处理方式配合Ca(ClO)2溶液不同浓度(3%、20%)和不同消毒时间(15、30、45、60min)进行高羊茅种子消毒处理,以升汞消毒剂处理的高羊茅种子为对照。愈伤组织诱导培养结果表明:仅用Ca(ClO)2搅拌的前处理的感菌率显著高于其余两种前处理;Ca(ClO)2高浓度处理比低浓度处理的出愈率低;Ca(ClO)2处理的出愈率和愈伤总数均优于升汞处理;最佳的消毒组合是无菌环境下用Ca(ClO)2搅拌+3%Ca(ClO)2浸泡60min,该消毒处理方式可降低种子感菌率、缩短出愈时间、提高出愈系数,为建立高羊茅高效再生体系奠定实验基础。

江涛, 李晨曦, 陈磊 , .

毛竹组织培养中成熟种子消毒方法

江苏农业科学, 2017,45(4):103-106.

DOI:10.15889/j.issn.1002-1302.2017.04.032      URL     [本文引用: 1]

成熟毛竹种子污染严重和萌发率低是制约毛竹组织培养发展的关键因素。研究发现氯气消毒优于常规的次氯酸钠和高锰酸钾消毒法。试验结果表明,氯气消毒毛竹种子未污染率、萌发率分别可达45.60%±2.06%、33.30%±5.51%,极显著高于次氯酸钠和高锰酸钾消毒法;并且进一步优化试验条件,经过1 d浸种,在0.01 mol/L氯气浓度下处理7 h,毛竹种子的未污染率、萌发率分别可达96.23%±0.19%、74.00%±1.89%;且以氯气消毒的种子能发育成实生苗和进行愈伤组织的诱导。成熟毛竹种子的氯气消毒方法,可为今后更好地利用毛竹种子进行组织培养研究奠定基础。

朱淑颖, 杨帆, 姜叶琴 , .

草珊瑚的无菌播种和丛生芽诱导研究

种子, 2016,35(8):32-36.

DOI:10.16590/j.cnki.1001-4705.2016.08.032      URL     [本文引用: 3]

以去除果肉的草珊瑚种子为试验材料,采用无菌播种方式培育出种子苗,并以此为组织培养的外植体来源,诱导出丛生芽,为离体再生体系的建立奠定基础。结果表明:种子的最佳消毒方法为先用75%酒精消毒30s,无菌水冲洗3~4次,再用0.1%升汞消毒3min,无菌水冲洗3~4次;最佳播种培养基为1/4MS+NAA 0.05mg/L,温度25℃,每天光照12h,种子发芽率可达68.89%;切取种子苗的顶芽接种于MS+6-BA 4.0mg/L培养基上可诱导出丛生芽,且丛生芽易于增殖和生根。

李威, 张曦, 谭勇 , .

干旱胁迫对3个品种红花种子萌发的影响

中国农学通报, 2013,29(34):207-211.

[本文引用: 1]

朱艳蕾, 安登第, 曾献春 .

银沙槐种子表面消毒技术及无菌萌发条件研究

新疆师范大学学报(自然科学版), 2014,33(3):17-20.

DOI:10.3969/j.issn.1008-9659.2014.03.004      URL     [本文引用: 1]

为获得银沙槐种子表面消毒的最佳方法及无菌萌发的最适条件,采用不同消毒剂和不同培养基对种子消毒方法和消毒后种子无菌萌发条件进行了研究。结果表明:种子消毒的最佳方法为自来水冲洗2-3次(振荡5min/次),蒸馏水冲洗3次(振荡5min/次),75%酒精预处理12min,无菌水冲洗1次,5%NaClO溶液消毒15min,最后用无菌水冲洗6-8次。发芽率可达64%,并彻底无污染;在MS、1/2MS、1/4MS、PDA及不同成分PDA的固体和液体培养基中,除MS外,种子在液体培养基中的萌发率要高于固体培养基,最适萌发培养基为MS固体培养基,其次是PDA(含葡萄糖)液体培养基,提示培养基中水分和营养成分可能共同影响银沙槐种子的萌发率。

李宏, 程平, 郑朝晖 , .

盐旱胁迫对3种新疆造林树木种子萌发的影响

西北植物学报, 2011,31(7):1466-1473.

URL     [本文引用: 2]

以3种新疆常见造林树木白榆、大叶白蜡、梭梭种子为材料,通过测 定不同土壤含盐量(0.1%~0.5%)和含水量(9%~23%)复合胁迫作用下种子的发芽率、发芽指数,以探讨3种种子萌发对盐分和干旱胁迫的响应特 征.结果显示:(1)梭梭种子对盐和干旱胁迫的适应范围最广,大叶白蜡次之,然后是白榆.(2)在土壤含盐量不超过0.2%情况下,土壤含水量达到17% 以后,3种树木种子发芽率都在50%以上;而当土壤含盐量为0.3%时,3树种种子仅在23%含水量下有较高发芽率;0.4%土壤含盐量条件下,只在含水 量为23%时,梭梭、大叶白蜡萌发率能达到50%以上;0.5%土壤含盐量时,仅梭梭种子萌发率达到50%.(3)白榆、大叶白蜡、梭梭的耐旱临界值分别 为15.1%、10.7%、9.6%,耐盐临界值分别为0.34%、0.41%、1.03%.研究表明,盐旱胁迫存在明显互作效应,盐旱复合胁迫对种子萌 发的危害性明显强于单一的盐胁迫或旱胁迫.

谢翔, 顿子云, 刘瑞 , .

不同消毒方法及浓度对冰菜种子萌发与幼苗生长的影响

天津农业科学, 2017,23(6):92-95.

DOI:10.3969/j.issn.1006-6500.2017.06.023      URL     [本文引用: 1]

试验研究升汞和Na Cl O等2种消毒剂对冰菜种子萌发与幼苗生长的影响。初步预试验结果表明,Na Cl O处理组萌发率优于升汞处理组。设置不同浓度Na Cl O处理组进行研究,结果表明,不同消毒剂处理组,通过对种子表皮的侵蚀,影响种子萌发过程中负责分解内含物的α-淀粉酶、β-淀粉酶及蛋白酶活性,进而影响种子的萌发与幼苗的生长,其中,6%Na Cl O处理组对冰菜种子的萌发率及幼苗生长效果最佳。

张晶华, 马亚丽, 兰海燕 .

不同灭菌方法对烟草和拟南芥种子萌发及无菌苗生长的影响

新疆农业科学, 2016,53(9):1683-1691.

[本文引用: 1]

彭璐, 张志杰, 龚千锋 , .

多指标加权法优选醋五倍子炮制工艺

药物分析杂志, 2017(9):1733-1738.

URL     [本文引用: 1]

目的:研究醋五倍子的炮制工艺,确定其最佳工艺参数。方法:选用L_9(3~4)正交表,化学指标选用没食子酸、鞣花酸的含量,药理指标选用牛津杯法测定抑菌圈大小,数据分析方法选用多指标综合加权评分法。炮制工艺选择三因素三水平,考察不同的因素和水平对其成分含量及活性的影响,影响因素及水平依次为用醋量(因素A:10%、15%、20%),浸润时间(因素B:12、14、16 h),蒸制时间(因素C:1、1.5、3 h)。没食子酸和鞣花酸的色谱条件:色谱柱为Kromasil C_(18)(4.6 mm×150 mm,5μm),流动相为乙腈(A)-0.1%三氟乙酸水(B),梯度洗脱,流速0.8 mL·min~(-1),柱温25℃,检测波长280 nm。结果:正交试验结果显示,醋五倍子的最佳工艺条件为A_3B_2C_3,即因素A用醋量20%,因素B浸润时间14 h,因素C蒸制时间3 h。结论:该炮制工艺可操作性强,方法稳定可行,可为醋五倍子的炮制工艺规范化研究提供实验数据。

胡重怡, 郑少清, 陈兴江 , .

烟草无菌苗培养前的种子消毒技术研究

中国烟草科学, 2007,28(2):45-46.

DOI:10.3969/j.issn.1007-5119.2007.02.012      URL     [本文引用: 1]

不同消毒处理对烟草种子消毒效果的影响研究表明,在种子消毒之前,经无菌水浸泡24h(预处理)的比不浸泡的消毒效果好;在10%H2O2、2%有效氯次氯酸钠溶液、1‰升汞3种杀菌剂中,10%H202消毒效果最好,种子带菌率为0%,用75%乙醇预处理可降低种子带菌率;3种消毒剂中以10%H2O2处理烟草种子发芽率最高。

张付远, 李丹丹, 廖忠明 , .

不同消毒剂及激素对江西省野生白花凤仙花组培诱导的影响

现代农业科技, 2015(15):164-165.

DOI:10.3969/j.issn.1007-5739.2015.15.098      URL     [本文引用: 1]

【目的】研究不同消毒剂和激素对江西野生白花凤仙花外植体诱导效果的影响。【方法】把江西省铜鼓县大沩山野生白花风仙花移植温室内,待其长出新枝后,剪去带有腋芽的嫩梢为材料,研究0.1%升汞、2%次氯酸钠、10%次氯酸钙和饱和漂白粉溶液等不同消毒剂在不同处理时间对外植体的消毒效果,以及不同浓度的6-BA和NAA组合对外植体诱导率的影响。【结果】以酒精消毒30s后再用0.1%4t汞消毒,白花凤仙花外植体诱导的培养基为MS+2.0mg/L6-BA+0.4meCLNAA,诱导率高达94%。[结论】消毒剂和激素对江西野生白花凤仙花外植体的诱导均有一定程度的影响。

陈芳, 党占海, 张建平 , .

不同基因型亚麻下胚轴不定芽诱导的研究

作物杂志, 2014(3):39-43.

URL     [本文引用: 1]

以5个亚麻品种陇亚10号、张亚2号、CDC-scroll、低亚麻酸及不育系1S为试验材料,对不同基因型亚麻下胚轴的不定芽诱导进行研究。确定了5个亚麻品种最适的种子消毒处理为75%乙醇消毒0.5min、1%次氯酸钠消毒10min。筛选出亚麻下胚轴不定芽诱导分化的最适培养基为MS培养基附加0.5mg/L 6-BA和0.02mg/L NAA。讨论了不同基因型的亚麻下胚轴不定芽诱导分化及其不定芽生根之间的差异,最终得到陇亚10号的下胚轴不定芽诱导分化率及不定芽的生根率最高,宜用来做后续的研究工作。

鲁黎明, 安影 .

次氯酸钠对烟草无菌苗生理指标的影响

作物杂志, 2012(3):141-143.

[本文引用: 1]

倪鑫鑫, 龚海燕, 马顶虹 , .

芽孢杆菌生理生化特性分析

安徽农学通报, 2015(7):47-48.

URL     [本文引用: 1]

芽孢杆菌是自然界中常见的细菌类群,被广泛应用于工农业生产。该文从芽孢杆菌结构性状、生理特征、营养和生化特征等方面对10种芽孢杆菌进行研究,补充、完善了芽孢杆菌的生理生化特性,为芽孢杆菌的分类鉴定提供思路。

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