全 文 ：Vol. 29 , No. 6
pp. 801～805 　Nov. , 2003
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 29 卷 第 6 期
2003 年 11 月 　801～805 页
An Efficient Protocol for Agrobacterium2mediated Transformation with Microtuber
and the Introduction of an Antisense class Ⅰ patatin Gene into Potato Ξ
SI Huai2Jun1 ,2 　XIE Cong2Hua1 , 3 　LIU Jun3
(1 College of Horticulture and Forestry ; 2 College of Life Science and Technology , Huazhong Agricultural University , Wuhan , Hubei 430070 ; 3 College of Agrono2
my , Gansu Agricultural University , Lanzhou , Gansu 730070 , China)
Abstract 　A simple , rapid and high efficient protocol was established for Agrobacterium2mediated transformation with mi2
crotuber of two potato cultivars , E2potato 3 and Gannongshu 2. Adventitious buds differentiation occurred after 2 - 3 weeks
on selective medium containing 75 mgΠL kanamycin , and transgenic plants could be obtained in 4 - 5 weeks. By combining
the best treatments , especially using 2 mgΠL zeatin riboside ( ZR) in the shoot induction medium , this protocol yielded
transformation efficiency of 45. 5 % and 43. 9 % from E2potato 3 and Gannongshu 2 , respectively. Short duration (4 - 5
weeks) , one2step culture and high efficiency made this system well suited for wider production of transgenic potato plants.
In total 120 positive plants with root on kanamycin2containing medium were obtained using the vector pBSAP carrying an
antisense class Ⅰpatatin gene. Transformants were confirmed by PCR , PCR2Southern blot and Northern hybridization anal2
ysis. Expression of the antisense classⅠpatatin gene resulted in a significant reduction of tubers production in transgenic
plants. The results suggested that the classⅠpatatin gene was possibly involved in regulating tuber formation.
Key words 　Potato ; Agrobacterium tumefaciens ; Transformation ; Microtuber ; Antisense patatin gene
农杆菌介导的马铃薯试管薯遗传转化体系的优化及反义 class Ⅰ patatin
基因的导入
司怀军1 ,2 　谢从华1 , 3 　柳　俊3
(1华中农业大学园艺林学学院 ; 2华中农业大学生命科学技术学院 ,湖北武汉 430070 ; 3甘肃农业大学农学院 ,甘肃兰州 730070)
摘 　要 　用两个马铃薯栽培品种“鄂马铃薯 3 号”和“甘农薯 2 号”的试管薯为供体材料 ,建立了一种农杆菌介导的简单、
快速和高效的遗传转化系统。在含有 75 mgΠL 卡那霉素的选择培养基上 ,2～3 周可产生抗性芽 ,4～5 周获得完整的转基
因植株。筛选出了试管薯遗传转化的优化条件 ,特别是在再生培养基中加入 2 mgΠL 玉米素 ,两个品种的转化频率分别高
达 45. 5 %和 43. 9 %。周期短 (4～5 周) 、一步培养和转化频率高 ,使该转化体系能够广泛用于马铃薯转基因的研究。用
含有反义 class Ⅰ patatin 基因的表达载体 pBSAP转化两个品种 ,共获得 120 株卡那霉素抗性植株。PCR、PCR2Southern 和
Northern 杂交分析证明 ,此反义基因已整合到马铃薯基因组中并在转基因植株中正常转录。反义基因的表达导致部分转
基因植株的试管结薯株率和单株结薯数降低。结果表明 ,该 class Ⅰ patatin 基因可能参与了块茎形成的调控。
关键词 　马铃薯 ;根癌农杆菌 ;转化 ;试管薯 ;反义 patatin 基因
中图分类号 : S532 　　　文献标识码 : A
　　Genetic transformation provides a new approach to im2
provement of potato varieties. Several different transformation
systems have been developed for potato using Agrobacteri2 um [1～9] , direct gene transfer into protoplasts[10] and particlebombardment[11] . Since the potato was shown to be suscepti2ble to Agrobacterium infection[12] , Agrobacterium has beenΞ基金项目 :国家自然科学基金资助项目 (39970464) 。Foundation items : National Natural Science Foundation of China (39970464) .
作者简介 :司怀军 (1971 - ) ,男 ,讲师 ,在职博士研究生 ,主要从事马铃薯生物技术育种的研究。现在甘肃农业大学农学院工作 , E2mail :Si2
huaijun @sina. com. 3 通讯作者 Correspondence author : 谢从华 ,Tel :027287287381 ; E2mail :Xiech @public. wh. hb. cn。
Received(收稿日期) :2002210215 ,Accepted(接受日期) :2003202201.

successfully used in the transformation with the leaf discs , in2
ternodes , tuber , minituber , and microtuber discs of potatoes.
However , most of these transformation systems have some
limitations , such as low transformation frequency[5 ] , geno2
type2dependent [4 ] , and high rate of somaclonal variation[2 ,5 ] .
Development of a simple and widely applicable transformation
system would be very important . Plants regenerated from tu2
ber discs appeared lower somaclonal variation[4 ] than that de2
rived from other somatic tissue sources[13] . Shoots regenerated
from tuber discs have little or no intermediate callus forma2
tion. For these reasons , we adapted the microtuber discs as
donors for the transformation.
Vector pBSAP containing an antisense class Ⅰ patatin
gene was used to establish the system of potato microtuber
transformation. patatin is a trivial name for a family of glyco2
proteins that accounts for up to 40 % of the soluble protein in
potato tubers[14] . patatin is encoded by a multigene family of
approximate 60 members per tetraploid genome[15] . patatin
gene can be divided into class Ⅰand class Ⅱbased on the
absence or presence of a 22 bp insertion in their 5 untranslat2
ed region[16] . Class Ⅰ patatin genes express strongly in tu2
bers. Unlike most other storage proteins , patatin has lipid
acyl hydrolase (LAH) activity[17] . This of course raises the
question about the possible function of such high amounts of
patatin in potato tubers. It was proposed that patatin may be
involved in particular biological processes , especially tuber
formation. Its physiological role and function , especially in
potato tuber formation and regulation , have not been elucidat2
ed. Because no potato mutant or wild potato species devoid of
patatin expression and accumulation in vivo is found. There2
fore , we have isolated a class Ⅰ patatin cDNA clone from a
cDNA library of a diploid species Solanum chacoense[18] and
constructed an antisense patatin gene expression vector. We
introduced it into two potato cultivars for the further study of
the function of class Ⅰ patatin gene and its relationship with
tuber formation and regulation.
1 　Materials and methods
1. 1 　Plant material
　　Two potato tetraploid cultivars , E2potato 3 and Gan2
nongshu 2 , were propagated in vitro by subculturing single2
nodal cuttings on MS medium supplemented with 3 % sucrose
and 0. 8 % agar , pH 5. 8 before autoclaving. Plantlets were
grown in transparent plastic vessels under 16 h light (3000
lx) at 20 ℃.
1. 2 　Construction of antisense patatin gene
A full length cDNA clone SK2421 of a class Ⅰ patatin
gene[18] was chosen for constructing an antisense patatin
gene. In order to introduce Sma Ⅰand Sac Ⅰrestriction en2
donuclease recognition sites , we designed two PCR primers :
L2ATGAGCTCTTGCAAA ATGGCAACTAC and R2TAC2
CCGGGTTACTACTACAACCCGAG. PCR was performed us2
ing plasmid SK2421 as template with Pfu (Promega , USA) in
a thermal cycler (UNO Ⅱ, Biometra) . PCR products were
recovered using UNIQ25 Column DNA Gel Extraction Kit
(Sangon , China) . The recovered 1. 4 kb PCR product was
digested with Sma Ⅰ and Sac Ⅰ, then cloned into the
Sma Ⅰ2Sac Ⅰsite of pBI121. The recombinant plasmid pB2
SAP contained an antisense patatin gene as Fig. 1.
1. 3 　Microtuber transformation
The plasmid pBSAP was transferred into Agrobacterium
tumefaciens strain LBA4404 using freeze2thaw method[19] .
Bacteria was cultured in LB containing 50 mgΠL kanamycin
and 50 mgΠL rifampicin at 28 ℃with shaking at 240 rΠmin
until the optical density of 0. 5 OD600 . Then the bacteria was
collected by centrifuge and resuspended in MS liquid medium
supplemented with 3 % sucrose.
Microtubers grown for 12 - 20 weeks with a diameter of
about 0. 5 cm were cut into 1 - 2 mm discs. For each trans2
formation experiment , 70 - 100 explants per cultivar were
used. Microtuber discs were submerged in 20 mL bacterial
solution for 5 - 10 min in 9 cm diameter petri dishes. The
microtuber discs were then dried on sterile filter papers and
transferred onto petri dishes with different shoot regeneration
medium (SRM) (Table 1) . The petri dishes were sealed with
parafilm and incubated in dark under 26 ℃for 2 days. The
cultures were then transferred onto fresh SRM supplemented
with 75 mgΠL kanamycin and 400 mgΠL carbenicillin and in2
cubated under 16 h light (2000 lx) at 24 ℃until small shoots
were regenerated. When green shoots reached 0. 5 —1 cm in
Fig. 1 Schematic representation of T2DNA of pBSAP
208 　　　作 　　物 　　学 　　报 29 卷 　

Table 1 　Shoot regeneration medium ( SRM) composition
Medium Composition (mgΠL)
SRM1 MS + 1 IAA + 0. 2 GA3 + 0. 5 BA
SRM2 MS + 1 IAA + 0. 2 GA3 + 0. 5 BA + 1ZR
SRM3 MS + 1 IAA + 0. 2 GA3 + 0. 5 BA + 2ZR
SRM4 MS + 1 IAA + 0. 2 GA3 + 0. 5 BA + 3ZR
length , they were excised and transferred onto flasks contain2
ing MS medium supplemented with 50 mgΠL kanamycin and
200 mgΠL carbenicillin. The plantlets with well2developed
roots were propagated for further analysis.
1. 4 　PCR analysis and PCR2Southern blot
Genomic DNA was isolated from 0. 1 g of leaves of posi2
tive and control plants as described by Edwards et al . [20] Pr2
esence of the transferred npt Ⅱgene was demonstrated by us2
ing standard PCR techniques. The npt Ⅱgene was amplified
using the primers L2GCTATGACTGGGCACAACAG and R2
ATACCGTAAAGCACGAGGAA. The PCR product was ex2
pected to be a 676 bp fragment .
PCR products from transformed and untransformed plants
and positive control pBSAP were used for Southern blotting.
Probes were made using PCR product of npt Ⅱgene. Label2
ing , hybridization and detection were carried out using a DIG
High Prime DNA Labeling and Detection Starter Kit Ⅰ
( Roche , Germany) following the manufacturer’s instruc2
tions.
1. 5 　Northern hybridization
Total RNA was isolated from transformed plants using
the guanidine2HCl method[21] . 40μg total RNA was separat2
ed by electrophoresis on 1 % agarose formaldehyde gels and
transferred to nylon membrane. The cDNA clone SK2421 of a
class Ⅰ patatin gene was used to synthesize antisense
probes. The plasmid was linearized with Sma Ⅰand labeled
using DIG RNA Labeling Kit (SP6ΠT7) (Roche , Germany) .
1. 6 　Induction of microtubers in vitro
Microtubers were induced from nodal segments of in
vitro grown plantlets on MS medium supplemented with 8 %
sucrose under 8 h light (2000 lx) at 20 ℃ according to the
method described by Liu et al . [22]
2 　Results
2. 1 　Construction of an antisense patatin gene
　　A 1. 4 kb fragment of patatin gene was obtained by PCR
amplification using a full length cDNA clone SK2421 as tem2
plate and confirmed by sequence analysis. The recombinant
vector pBSAP containing an antisense patatin gene was di2
gested with Sma Ⅰ and Sac Ⅰ and showed that the patatin
gene was cloned in inverted orientation into the plasmid
pBI121. The expression vector pBSAP was transferred into
Agrobacterium tumefaciens strain LBA4404 and confirmed by
PCR analysis (data not shown) .
2. 2 　Optimization of microtuber transformation
Microtuber discs of the two potato cultivars were co2cul2
tured for 2 days with A . tumefaciens LBA4404ΠpBSAP , then
transferred onto the selective SRM medium. After 2—3
weeks , green buds produced directly from the surface of the
microtuber discs ( Fig. 2) . When green shoots reached a
length of 0. 5 —1 cm , they were transferred onto the selective
rooting medium. Roots appeared in about 10 days.
Fig. 2 Shoot formation directly from E2potato 3 transformed microtuber
discs cultured on SRM3 medium for 3 weeks
To test ZR in this transformation system , different ZR
concentrations were used and the results showed that the me2
dium SRM3 containing 2 mgΠL ZR gave the highest transfor2
mation efficiency in the two cultivars , E2potato 3 with 45. 5 %
and for Gannongshu 2 with 43. 9 % (Table 2) . A large num2
ber of buds were obtained 3 weeks later from the cultures sup2
plemented with ZR. In contrast , only a few shoots were re2
generated after 4 ─6 weeks from Gannongshu 2 on the
medium without ZR , indicating that ZR could be helpful for
shoot formation. Additionally , both E2potato 3 and Gannong2
shu 2 produced transgenic plants on the medium with all lev2
els of ZR although transformation efficiency was different .
2. 3 　PCR and PCR2Southern blot analysis
PCR analysis using npt Ⅱgene specific primers was per2
formed on 65 transformed plants which rooted on selective me2
dium containing 50 mgΠL kanamycin. Transformed plants
showed a 676 bp amplification product which did not appear
308　6 期 SI Huai2Jun et al. :An Efficient Protocol for Agrobacterium2mediated Transformation with Microtuber and the . . . 　　　

in the control plant (Fig. 3) . The result of PCR2Southern blot
showed a 676 bp fragment in all of the transformed plants ,
which was absent in the control (Fig. 4) .
Table 2 Transformation efficiency with microtuber discs from two potato cultivars on four kinds of shoot regeneration medium( SRM)
Media
Number of
explants1)
Number of discs with
regenerated shoots
Number of rooted shootsΠexplant
(means ±SD)
Transformation efficiency( %) 2)
(means ±SD)
G2 E3 G2 E3 G2 E3 G2 E3
SRM1 84 72 4 0 0. 8 ±0. 23 0. 0 4. 6 ±2. 75 0. 0
SRM2 100 82 22 10 2. 3 ±0. 29 0. 8 ±0. 13 22. 0 ±0. 34 11. 9 ±5. 67
SRM3 75 90 33 41 3. 2 ±0. 53 3. 6 ±0. 50 43. 9 ±1. 52 45. 5 ±1. 57
SRM4 97 92 28 32 1. 2 ±0. 24 1. 9 ±0. 37 28. 4 ±8. 76 34. 3 ±3. 86
　　Note : 1) G2 and E3 represented respectively potato cultivars Gannongshu 2 and E2potato 3.
2) The percentage was determined as the number of discs producing kanamycin2resistant shoots after 3 weeks from the cultures on SRM medium divided
by the total number of infected discs.
Fig. 3 PCR analysis of transformed potato
M. ФX174 Hae Ⅲdigest DNA marker (TaKaRa) ; P. positive
control ; C. negative control ; 1210. transformed plants
Fig. 4 Detection of transgenic plants by PCR2Southern
P. plasmid pBSAP as positive control ; C. non2transformed plant as
negative control ;1 —8. putative transformed plants
2. 4 　Northern hybridization
Northern hybridization analysis showed that the steady
level of patatin antisense RNA can be detected in the trans2
genic plants while no transcript in the untransformed control
plants ( Fig. 5) . This indicated that the antisense class Ⅰ
patatin gene was integrated in the potato genome and was well
transcribed in the transformed plants.
Fig. 5 Northern hybridization of transformed plants
C. untransformed potato ; 1 —8. transformed potatoes
2. 5 　Influence of antisense class Ⅰ patatin gene on
microtuber formation
The results from tuberization of five antisense plants
grown in vitro of cv. Gannongshu 2 demonstrated that expres2
sion of antisense class Ⅰ patatin gene led to a significant re2
duction in part of the transgenic plants with tubers and the
number of tubers per plantlet while no much influence on tu2
ber weight when compared to the control ( Table 3) . Similar
results were obtained from cv. E2potato 3 (data not shown) ,
suggesting that the class Ⅰ patatin gene was possibly in2
volved in regulating tuber formation.
Table 3 Effect of expression of antisense class Ⅰ patatin gene on
microtuber formation of the transgenic plants in vitro
Plant
Percent of plantlets
formed tubers ( %)
No. of tubers
per plantlet
Mean tuber
weight (mg)
N2 (CK) 41. 1bc1) 0. 41bc 95b
Nb2 49. 2ab 0. 49ab 133a
Nb3 26. 8d 0. 27d 101b
Nb4 41. 0bc 0. 41bc 104b
Nb7 54. 9ab 0. 55ab 103b
Nb9 31. 3d 0. 31d 100b
　　Note : 1) Duncan’s multiple range test ( P < 0. 05) .
3 　Discussion
Most of the published protocols for potato transformation
use a two2step system with two different culture medium , one
for callus induction and another for shoot regeneration[1 ,3 ,8 ] .
The system prolongs the period of tissue culture and shoot re2
generation , and also may lead to a higher level of somaclonal
variation[2 ,5 ] . With our protocol and only one2step shoot re2
generation medium a large number of shoots were regenerated
in a short period (2 —3 weeks) . By using 2 mgΠL ZR in the
shoot regeneration medium , the transformation efficiency
could be improved obviously. Similar results were obtained by
Beaujean[8 ] and Romano[11] in potato internodal transforma2
408 　　　作 　　物 　　学 　　报 29 卷 　

tion systems. This provides a possibility that our microtuber
transformation protocol could be used for wider application of
potato cultivars since nearly all of them are capable to form
tubers in vitro.
Tuber proteins from a number of potato cultivars showed
a wide range of patatin isoforms with LAH activity[17 ,23] .
These isoforms might play different physiological roles in dis2
ease , wounding and development responses in vivo. Since
class Ⅰ patatin gene was expressed in accordance with tuber
initiation , it was speculated that at least one , if not only one ,
of the patatin genes might be involved in control of tuberiza2
tion[24] . The results of the present experiment provided a pre2
liminary evidence that the class Ⅰ patatin gene had strong
influence on tuber formation in some transgenic plants , al2
though some showed no much effect which might be resulted
from a random insertion or unknown expression inhibition. A
possible explanation of how patatin takes part in the process
of tuber formation might be that LAH of patatin could catalyze
the lysis of the membrane lipids to release metabolites such as
linolenic acid , which is the substrate of synthesis of jasmolic
acid that is considered as tuber inducing substance[25] . How2
ever , to confirm the function of patatin in regulation of tuber
formation needs both sense and antisense transformation using
diverse potato genotypes , which are undergoing in our labora2
tory.
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