全 文 ：文章编号: 1007-0435( 2006) 02-0196-02 ·博士论文摘要·
利用生物技术提高三倍体狗牙根抗逆性
博士生: 卢少云　导师: 彭新湘,郭振飞
(华南农业大学生命科学学院, 广州　510642)
　　狗牙根是一种重要的暖季型草坪草。三倍体
矮生狗牙根( Cynodon dacty lon×C. t ransvaalen-
sis cv . T ifEagle)已广泛应用。本文研究其抗旱
性、建立组织培养与再生体系、体细胞无性系变异
育种技术及抗逆基因工程等。主要结果和结论如
下:
1.抗旱性及化学调控
测定矮生狗牙根在停止浇水过程中叶片相对
含水量、电导率、脯氨酸含量及抗氧化酶活性的变
化;研究其抗旱性与抗氧化酶活性的关系; 研究
ABA 等生长调节剂提高其抗旱性的效应和机理。
2.建立组织培养再生体系
以亲本匍匐茎节为外植体, 探讨胚性愈伤组
织诱导、继代和再生的条件; 建立快速增殖、高频
组织培养的再生体系; 借助光学组织切片和扫描
电镜, 揭示体细胞胚胎发生是 TifEag le 愈伤组织
的再生方式。
3.体细胞无性系变异体的筛选及鉴定
从连续悬浮继代愈伤组织再生的植株中, 筛
选出夏季和春季匍匐生长速度较快的株系; 在不
同年份越冬中多次获得评分较高的株系;研究愈
伤组织进行长时间连续继代悬浮培养能有效地诱
导体细胞变异,从再生植株中筛选有益的性状。
4.矮化变异体
从再生植株中选出一株矮化变异体( T V4)。
测定该变异体及其亲本的形态和抗逆性特征; 矮
化变异体具有矮化、密度大、颜色浓绿和根系发达
等特点,其耐冷性略有提高,是一个很有应用潜力
的新株系。
5. 耐盐变异体的筛选及生理鉴定
以 0. 3 mo l/ L 的 NaCl 作为选择压力, 采用
间歇筛选法从悬浮继代 18 个月以上的愈伤组织
中筛选耐盐的细胞系, 并获得再生植株;耐盐性测
定结果表明, 50%变异体株系的耐盐性高于亲本;
测定变异体和亲本在正常和盐胁迫条件下的
Na
+ / K
+比、脯氨酸含量、可溶性糖含量和部分抗
氧化酶活性; 探讨耐盐变异体的耐盐机理。结果表
明,耐盐株系具有不同的耐盐机理,但在正常生长
条件下的脯氨酸含量均高于亲本, 而 Na+ / K + 比
则低于亲本。
6. 遗传转化、转基因检测及抗逆性鉴定
构建 BA DH 和CMO的表达载体。采用基因
枪转化狗牙根愈伤组织,获得 PCR检测呈阳性的
植株;对转 BA DH 植株进行 Souther n杂交分析,
出现多条杂交信号,证明基因以多拷贝方式整合
到狗牙根基因组中;对部分转基因植株进行耐盐
性和耐旱性分析, 结果表明,一些转基因株系耐盐
性和耐旱性明显提高。
关键词: 狗牙根; 生物技术; 抗逆性; 组织培养; 体细胞变异; 转基因
中图分类号: S812; Q943　　　　文献标识码: A
完成时间: 2005年 10月
收稿日期: 2006-04-11; 修回日期: 2006-05-14
基金项目: 国家转基因植物研究专项( JY03-B-32-02)、863项目 ( 2001AA244082)、国家自然科学基金( 30371014)、教育部优秀青年
教师基金、广东省自然科学基金课题( 032263、010281)、广州市科技攻关项目( 2002Z3-E0281)等资助
作者简介: 卢少云( 1967-) ,云南广南县,理学博士,研究方向为草业生物技术, E-mail: turf lab@ scau. edu. cn
导师简介: 彭新湘( 1962-) ,男,研究员,研究方向为植物逆境生理与分子生物学;郭振飞( 1964-) ,男,研究员,研究方向为草业生物
技术, E-mail: zhfguo@ scau . edu. cn
第 14卷　第 2期
　Vo l. 14　 No . 2
草　地　学　报
ACT A AGRESTIA SIN ICA
　　　2006年　 6 月
　June　　 2006
Biotechnological Approaches to Improvement of Triploid Bermudagrass
for Abiotic Stress Tolerance
Candidate: LU Shao-yun　A dviso r: PENG Xin-x iang , GUO Zhen-fei
( Col leg e of Life Science, South Chin a Agricu lture Un ivers ity, Guan gzhou 510642, Ch ina)
Object ives of our research w ere to invest ig ate the improvement o f t riploid bermudagrass ( Cynodon dacty lon
×C. transv aalensis cv. T ifEag le) for st ress tolerance by biotechnolog ical methods. The resul ts w ere summarized
in the follow ing :
1. Drought tolerance and its chemical control in turfgrass species
The drought tolerance in 3 turfgrass species w ere comparatively studied by measuring relative water
content ( RWC) , ion leakage, antio xidants enzyme act ivity during w ithholding irrig at ion. T he results indicated
t riplo id bermudag rass cult iv ar , T ifdw arf , had highest drought to lerance, follow ed by manilagrass and
carpetg rass. ABA and S3307 tr eatments increased the drought tolerance of turfg rasses, w hich w ere associated
w ith their induced ant iox idants enzyme act iv ity .
2. Establishment of tissue culture and regeneration in triploid bermudagrass
Sterilized node segments w ith 5 mm wer e used as explants and callus induct ion, suspension subculture,
and regenerat ion w ere studied. Basal medium composition, pro line, phy tohormones were compared in the
callus induct ion, suspension cul ture and r egeneration. High effect ive tissue cul ture sy stem w as established in
t riplo id bermudag rass. Somatic embryogenesis dur ing regenerat ion in t riploid bermudagrass was demonst rated
by using scanning elect ron m icroscope and light microscope observat ions.
3. Somaclonal variations and selections for cold resistance in triploid bermudagrass
Morphological var iat ions in leaf co lor, leaf length and w idth, stolons internodes leng th w ere obser ved
among the regenerated plants f rom the init ial 138 embryogenic calluses, w hich had been cont inuously
subcultured fo r over 18 months by suspension culture. Sever al lines grew fast in the field dur ing spring and
summer . Part ial lines maintained gr een color and cont inuous g row th in the w inter. T he results indicated that
somaclonal variat ions w er e induced during the long-term subculture o f callus and among them useful variants
could be selected. It is suggested that somaclonal variation may be used for tr iploid bermudagrass breeding.
4. A dwarf somaclonal variant in triploid bermudagrass
A dwarf somaclonal variant , T V4, and its par ent T ifEagle w ere comparat ively studies in morpholog ical
characters and r esponse to chilling str ess. In comparison to T ifEag le, TV 4 show ed signif icant morpholog ical
variat ion w ith decrease in stolons internodes length and leaf length, increase in leaf w idth. TV 4 had ex tensiv e
roo t sy stem which w ould allow it higher dr ought tolerance, showed higher chilling resistance than TifEag le.
5. Selection of salt-tolerant lines in triploid bermudagrass and their physiological analysis
The salt-tolerant callus l ines selected on the medium contained 0. 3 mol/ L NaCl w ere regenerated on the
medium contained 0. 1 mol / L NaCl. Half o f the regener ated plants l ines show ed tolerance to 0. 25 mo l/ L NaCl
st ress, compar ed to it s par ent T ifeagle. Na
+ , K
+ , Na
+ / K
+ , proline, soluble sugar s and ant iox idant enzyme
act ivity w ere measured af ter the plants w ere subjected to salt st ress. The results show ed that the tolerant lines
show ed diversity in physiolog ical index in r esponse to salt st ress. All the tolerant lines had much higher pro line
content and low er Na+ / K + rat io under non-st ress condit ion.
6. Transgenic plants production, molecular analysis and their stress tolerance in triploid bermudagrass
Tr ansgenic plants fo r betaine aldehyde dehydrogenase gene ( BA DH ) under the contr ol by Ubi promoter
w er e produced by part icle bombardment . PCR ampl if icat ion and PCR-Southern blot analy sis show ed part ial
regenerated plants positive. Southern blo t of genomic DNA filter demonst rated that BADH was integr ated into
the genomics in t ransgenic plants in a manner of mult iple copies. T hree t ransgenic lines show ed greatly
increased to ler ance to sal t and drought str ess.
Key words : T riploid bermudagrass; Bio technolo gy; St ress to ler ance; T issue cultur e; Somaclonal
variat ion; T ransgenic plants
(责任编辑　孟昭仪)
197第 2期 卢少云等:利用生物技术提高三倍体狗牙根抗逆性