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1、三色堇花药培养技术研究初探刘辉基金项目：杭州市科技局科技计划项目（20101032B10）第一作者简介：刘辉（1979-），男，山东泰安人，在职博士，农艺师，现从事观赏植物栽培技术与分子育种研究.通信地址：E-mail:liuhui518lh;Tel:0571-873132441，傅巧娟1，陈一1（1杭州市农业科学研究院 园艺研究所，浙江 杭州 310024）摘要：研究了三色堇花蕾消毒方法、花蕾低温预处理和不同培养基组分对花药愈伤组织诱导率及其分化的影响。结果表明，采用0.1% HgCl2灭菌20 min是三色堇花蕾较为理想的表面灭菌时间。基因型不同花药愈伤组织的诱导率也不尽相同，而且愈伤组织
2、的诱导率总体不高。选择合适的培养基组分、激素种类及其配比以及合适的培养条件可能是影响三色堇花药培养成功的关键因子。关键词：三色堇；花药；组织培养Elementary Study on Anther Culture of Pansy (Violawittrockiana)LIU Hui*1, FU Qiao-juan1,CHEN Yi1,(Department of Horticulture,Hangzhou Academy of Agricultural Sciences,Hangzhou 310024,China)Abstract: Pansy (Violawittrockiana),a b
3、edding plant is used widely in landscape in China.The effects of different ways of bud sterilization,bud cold pretreatment, different combinations of plant growth regulators on callus induction rate of anther and plant regeneration of Pansy were investigated.The results showed that 20 min with 0.1%
4、HgCl2 was a ideal bud surface sterilization time,and obvious difference was detected between two genotypes in their effect on callus initiation of anther,but the overall rate of callus induction was not high.It may be suggested that the kind and concentration of hormones and culture conditions shoul
5、d be key factors for successful anther culture of pansy.Key words:Pansy;Anther;Tissue Culture;三色堇又名蝴蝶花、猫儿脸，属堇菜科、堇菜属多年生草本植物，因其色彩丰富艳丽、品种繁多、花色多变、花期长、适应性强，是当前我国优良的重要花坛绿化草花。另外，三色堇较耐低温，可耐零下15 低温，在昼温15-25、夜温3-5 条件下植株发育良好，因此也是我国冬、春季节不可多得的重要花坛绿化草花。由于三色堇具有明显的杂种优势，观赏性强，目前市面上销售的三色堇种子多为F1代。利用杂交优势进行杂交育种的关键是获得优良性状
6、的纯系父母本（自交系）。为了获得自交系，按常规的自交方法，需要耗费大量的田间土地、时间和劳力,一般需要5-6年的时间，而且手续十分繁琐，最终的纯度也不够理想。而采用花药培养获得单倍体再加倍的方法，只需1年时间就可以获得和多代自交效果相同的纯系，可以极大地缩短杂交育种年限，提高育种效率。本试验研究了三色堇花药培养的技术和条件，初步探讨了影响三色堇花药组织培养的因素，以期最终获得单倍体植株,在较短的时间内培育出三色堇育种亲本。1 材料与方法1.1 材料选用生产上常用的两个三色堇品种：清新绯红和想象力紫色，种子由浙江虹越花卉有限公司提供。三色堇于2009年9月中旬种植在杭州农科院园艺所实验基地,于2
7、010年3月上旬开始采集三色堇花蕾进行研究。根据杨宏光1等对三色堇小孢子发育时期与花器形态相关性分析的研究，选取纵径长度1415mm与横径在35mm的花蕾，此时期的花蕾多处于单核靠边期。1.2 花蕾消毒方法研究：将选好的花蕾放在自来水下冲洗5 min，再用蒸馏水冲洗3次后用吸水纸吸干水分。在超净工作台上，用75%的酒精消毒1 min，再用无菌蒸溜水冲洗3次后用0.1% HgCl2进行消毒，消毒时间设为10、15、20、25和30 min。消毒完后用无菌蒸溜水冲洗4次，然后接种在Nitsch1培养基上，培养室温度251，暗培养。每一处理10瓶，每瓶接种6个花蕾，接种7 d后统计一次污染率。Nit
8、sch1基本培养基，附加7g/L琼脂和20g/L 麦芽糖，pH值为5.8。污染率=（污染的花蕾数/接种的花蕾总数)100%1.3 花蕾低温预处理对选定好的花蕾以5冷藏1、2、3和4 d；采用0.1% HgCl2消毒20 min的消毒方法对处理好的花蕾消毒，然后用镊子剥去花萼、花瓣，取出花药、去净花丝后接种在Nitsch2培养基上，每瓶接种10个花药，各接5瓶。每隔3 d观察一次各瓶花药的启动情况，接种后30 d统计各花药愈伤组织的启动率情况。培养室温度251，培养条件暗培养。Nitsch2基本培养基，附加1.0mg/L 6-BA、0.5 mg/L NAA、0.5 mg/L IBA、7g/L琼脂
9、和20g/L 麦芽糖，pH值为5.8。启动率=（膨大的花药数/接种的花药总数）1.4 花药愈伤组织的诱导对选定好的花蕾以5 冷藏2 d，按1.3所述花蕾消毒方法和花药剥取方法，将花药接种在含不同激素的培养基上（表3），每瓶接种10个花药，各接5瓶。培养基种类分为Nitsch基本培养基和MS基本培养基，各培养基附加7g/L琼脂和20 g/L 麦芽糖，pH值为5.8。培养室温度251 ，培养条件暗培养。愈伤组织诱导前期每20 d换一次新鲜的原培养基，待愈伤组织形成后及时将初代愈伤组织转接到新鲜的原培养基上进行继代培养。每隔3 d观察一次各瓶花药的诱导情况，接种后40 d统计各处理花药愈伤组织诱导率
10、。诱导率（形成愈伤组织的花药数/接种的花药总数）100%1.5 花药愈伤组织的分化将继代一次后获得的愈伤织织转接到分化培养基上进行芽的分化。各处理共接10瓶，每瓶接种6块愈伤组织。接种后每30 d统计一次分化率。分化培养基：Nitsch基本培养基，7g/L琼脂，20 g/L 麦芽糖和0.5 g/L 脯氨酸，pH值为5.8，分别附加1 mg/L 6-BA和1 mg/L KT，或1 mg/L TDZ和0.1 mg/L IBA。培养室温度251，光照培养（光照强度为200 molm-2s-1），光照周期14h（5：00-19：00）。2 结果与分析2.1不同灭菌时间对三色堇花药污染率的影响由表1可见
11、，短时间的0.1% HgCl2灭菌，三色堇花蕾污染率较高，而随着灭菌时间的延长，接种的花蕾污染率逐渐下降，当灭菌时间为20 min时，污染率下降到10%，25 min以上时基本没有污染。由于HgCl2是一种杀伤力很强的消毒剂，消毒时间过长，会对细胞产生毒害作用，抑制细胞生长。表1表明两个三色堇品种的花蕾在HgCl2灭菌时间为20 min时污染率较低，因此为了减少HgCl2对花蕾内花药的损伤，进而影响愈伤组织的诱导，采用0.1% HgCl2 灭菌20 min是三色堇花蕾较为理想的表面灭菌时间。表1不同灭菌时间对三色堇花药污染率的影响品种消毒时间（min）污染数（瓶）污染率（%）清新绯红10860
12、155502011025003000想象力紫色10760154402011025003000注：每一处理10瓶，每瓶接种6个花蕾，接种7 d后统计一次污染率。2.2不同低温预处理时间对三色堇花药愈伤组织启动率的影响一些研究表明，在花药培养前对花蕾预处理可提高花药的出愈率，花蕾预处理包括低温、高温等处理，其中低温处理方法比较常用2。本实验以花药初期膨大即表明花药有可能诱导出愈伤组织为判断依据，从表2可见，不同的低温预处理时间对两个品种的花药愈伤组织启动率有明显差异，5 处理2 d的两个品种花药均有明显膨大现象，清新绯红和想象力紫色的启动率分别为25.6%和26.3%，表明低温处理2 d能明显促进
13、花药愈伤组织的形成。表2不同低温预处理时间对三色堇花药愈伤组织诱导率的影响品种低温处理时间（d）启动率（%）清新绯红113.5225.6321.2418.3想象力紫色110.5226.3324.3415.6注：每瓶接种10个花药，各接5瓶。2.3不同培养基组分对三色堇花药愈伤组织诱导率的影响培养基的种类、激素水平和配比对诱发细胞的分裂、生长和分化起着决定性作用。采用不同的培养基种类和激素配比对三色堇花药愈伤组织诱导率的研究表明（表3A），在相同培养基成份的条件下，Nitsch培养基比MS培养基有利于三三色堇花药愈伤组织的形成，而MS培养基对三色堇花药愈伤组织诱导率差异不明显；结果还表明在同为N
14、itsch基本培养基条件下，6BA对诱导三色堇花药愈伤组织起着重要作用，两个品种间差异明显，清新绯红在添加有1.0 mg/L 6BA培养基上有最高的花药愈伤组织诱导率，达到21.4%，而想象力紫色则在1.5 mg/L 6BA培养基上有最高的花药愈伤组织诱导率，达到25.6%。为了获得更为理想的诱导三色堇花药愈伤组织的激素水平和配比，本试验还研究了6BA、IBA和NAA组合对三色堇花药愈伤组织诱导率的影响。表3B表明，不同激素种类及浓度对两个品种的花药愈伤组织诱导率有明显差异，清新绯红在添加有1.0 mg/L 6BA、0.2 mg/L IBA和0.5 mg/L NAA培养基上有最高的愈伤组织诱导
15、率，达到27.6%，而想象力紫色则在1.0 mg/L 6BA、0.5 mg/L IBA和0.5 mg/L NAA培养基上有最高的花药愈伤组织诱导率，达到26.9%。表3不同培养基水平和配比对三色堇花药愈伤组织诱导率的影响A培养基种类生长调节剂诱导率（%）6-BA（mg/L）IBA（mg/L）清新绯红想象力紫色Nitsch00.5000.517.412.31.021.420.81.520.325.62.014.518.6MS05.610.50.512.914.51.015.614.61.517.312.62.014.513.7注：每瓶接种10个花药，各接5瓶。B培养基种类生长调节剂诱导率（%）6
16、-BA（mg/L）IBANAA（mg/L）清新绯红想象力紫色Nitsch0.50.20.521.319.81.00.20.527.622.50.50.50.515.617.31.00.50.512.326.90.50.51.011.312.7注：每瓶接种10个花药，各接5瓶。2.4 不同培养基组分对花药愈伤组织分化的影响本研究将继代一次后获得的各花药愈伤组织选取较好的转接到不同的分化培养基上进行不定芽的诱导，30 d后的统计结果表明两个品种的所有愈伤组织在2种培养上均不能分化，最后褐化死亡。可见，三色堇花药虽能诱导形成愈伤组织，但形成的愈伤组织进一步分化难度很大，不得获得再生植株。3 讨论与结
17、论目前，三色堇是我国重要的花坛和盆栽花卉，市场需求量大，然而其育种研究远远落后于其广泛的园林应用现状3。国内有关三色堇花药培养的研究特别少，而通过三色堇花药培养成功获得再生植株的研究更是鲜有报道。本文对三色堇花药组织培养进行了初步研究，结果表明三色堇花蕾通过低温预处理2 d后能提高花药愈伤组织诱导率，但进一步的研究显示继代后的愈伤组织分化出不定芽的能力很差，不能获得再生植株。有关园艺植物花药培养的研究认为，基因型、小孢子发育时期、培养基成分（基本培养基种类、激素、碳源、附加成分）及培养条件是影响花药培养成功的主要因素4。本研究采用2个不同基因型的三色堇品种进行花药培养研究，尽管基因型不同花药愈
18、伤组织的诱导率也不尽相同，但愈伤组织的诱导率总体不高，最高不到30%，而且最终均没有成功获得再生植株。因此，选择合适的培养基组分、激素种类及其配比以及合适的培养条件可能是影响三色堇花药培养成功的关键因子。在下一步的试验中，我们将在此基础上对三色堇花药培养做更为深入的研究。参考文献1 杨宏光,孙晓梅,王孝鹏,等.三色堇小孢子发育时期与花器形态相关性分析J,沈阳农业大学学报,2008,39(5):621-6242 Yurong chen,Edward O,Kenaschuk,J Doug Proeunier.Plant regeneration from anther culture in Can
19、adian cultivars of flax(Linum usitatissimum L.)J,Euphytica,1998,102:183-1893 张其生,包满珠,卢兴霞,等.大花三色堇育种研究进展J,植物学报,2010,45(1):128-1334 张绿萍，陈红.园艺植物花药培养研究进展J,安徽农业科学,2007，35(17)：5140-5142Editors note: Judson Jones is a meteorologist, journalist and photographer. He has freelanced with CNN for four years, cov
20、ering severe weather from tornadoes to typhoons. Follow him on Twitter: jnjonesjr (CNN) - I will always wonder what it was like to huddle around a shortwave radio and through the crackling static from space hear the faint beeps of the worlds first satellite - Sputnik. I also missed watching Neil Arm
21、strong step foot on the moon and the first space shuttle take off for the stars. Those events were way before my time.As a kid, I was fascinated with what goes on in the sky, and when NASA pulled the plug on the shuttle program I was heartbroken. Yet the privatized space race has renewed my childhoo
22、d dreams to reach for the stars.As a meteorologist, Ive still seen many important weather and space events, but right now, if you were sitting next to me, youd hear my foot tapping rapidly under my desk. Im anxious for the next one: a space capsule hanging from a crane in the New Mexico desert.Its l
23、ike the set for a George Lucas movie floating to the edge of space.You and I will have the chance to watch a man take a leap into an unimaginable free fall from the edge of space - live.The (lack of) air up there Watch man jump from 96,000 feet Tuesday, I sat at work glued to the live stream of the
24、Red Bull Stratos Mission. I watched the balloons positioned at different altitudes in the sky to test the winds, knowing that if they would just line up in a vertical straight line we would be go for launch.I feel this mission was created for me because I am also a journalist and a photographer, but
25、 above all I live for taking a leap of faith - the feeling of pushing the envelope into uncharted territory.The guy who is going to do this, Felix Baumgartner, must have that same feeling, at a level I will never reach. However, it did not stop me from feeling his pain when a gust of swirling wind k
26、icked up and twisted the partially filled balloon that would take him to the upper end of our atmosphere. As soon as the 40-acre balloon, with skin no thicker than a dry cleaning bag, scraped the ground I knew it was over.How claustrophobia almost grounded supersonic skydiverWith each twist, you cou
27、ld see the wrinkles of disappointment on the face of the current record holder and capcom (capsule communications), Col. Joe Kittinger. He hung his head low in mission control as he told Baumgartner the disappointing news: Mission aborted.The supersonic descent could happen as early as Sunday.The we
28、ather plays an important role in this mission. Starting at the ground, conditions have to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The balloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) where o
29、ur day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 miles/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. As he crosses the boundary layer (called the tropopause), he can
30、 expect a lot of turbulence.The balloon will slowly drift to the edge of space at 120,000 feet (22.7 miles/36.53 kilometers). Here, Fearless Felix will unclip. He will roll back the door.Then, I would assume, he will slowly step out onto something resembling an Olympic diving platform.Below, the Ear
31、th becomes the concrete bottom of a swimming pool that he wants to land on, but not too hard. Still, hell be traveling fast, so despite the distance, it will not be like diving into the deep end of a pool. It will be like he is diving into the shallow end.Skydiver preps for the big jumpWhen he jumps
32、, he is expected to reach the speed of sound - 690 mph (1,110 kph) - in less than 40 seconds. Like hitting the top of the water, he will begin to slow as he approaches the more dense air closer to Earth. But this will not be enough to stop him completely.If he goes too fast or spins out of control,
33、he has a stabilization parachute that can be deployed to slow him down. His team hopes its not needed. Instead, he plans to deploy his 270-square-foot (25-square-meter) main chute at an altitude of around 5,000 feet (1,524 meters).In order to deploy this chute successfully, he will have to slow to 1
34、72 mph (277 kph). He will have a reserve parachute that will open automatically if he loses consciousness at mach speeds.Even if everything goes as planned, it wont. Baumgartner still will free fall at a speed that would cause you and me to pass out, and no parachute is guaranteed to work higher tha
35、n 25,000 feet (7,620 meters).It might not be the moon, but Kittinger free fell from 102,800 feet in 1960 - at the dawn of an infamous space race that captured the hearts of many. Baumgartner will attempt to break that record, a feat that boggles the mind. This is one of those monumental moments I will always remember, because there is no way Id miss this.
