Two transgenic potato lines csr2-1 and csr4-8, containing two different antisense constructs, csr2 and csr4, respectively, were crossed to investigate the possibility of achieving double transformants with combined effects of the two antisense transgenes on plant phenotypes and cellulose deposition. Molecular analysis revealed an expected segregation ratio of 1:1:1:1 of the four classes. Phenotype characterization revealed that offspring containing either one or both transgenes produced more tubers than the control plants but individual tubers were mostly smaller and had lesser weight than the control tubers

Jacobsen, E.

Olawole, O.

Visser, R.G.F.

English

NARCIS 

Molecular analysis and phenotype characterization of the progeny of two antisense potato plants

Name not available

Wageningen Yield 

African Journal of Plant Science Vol. 2 (6), pp. 038-041, June 2008 
Available online at http://www.academicjournals.org/AJPS 
ISSN 1996-0824 © 2008 Academic Journals 
 
 
Full Length Research Paper 
 
Molecular analysis and phenotype characterization of 
the progeny of two antisense potato plants 
 
Olawole O. Obembe1,2, Evert Jacobsen1 and Richard G. F. Visser1 
 
1Laboratory of Plant Breeding, Wageningen University, Box 386 6700AJ Wageningen, 
The Netherlands 
2Present address: Department of Biological sciences Covenant University, P. M. B 1023 Ota,  
Ogun State, Nigeria. 
 
Accepted 16 June, 2008 
 
Two transgenic potato lines csr2-1 and csr4-8, containing two different antisense constructs, csr2 and 
csr4, respectively, were crossed to investigate the possibility of achieving double transformants with 
combined effects of the two antisense transgenes on plant phenotypes and cellulose deposition. 
Molecular analysis revealed an expected segregation ratio of 1:1:1:1 of the four classes. Phenotype 
characterization revealed that offspring containing either one or both transgenes produced more tubers 
than the control plants but individual tubers were mostly smaller and had lesser weight than the control 
tubers. 
 
Key words: Antisense technology, double transformants, genetic crossing, potato, segregation analysis. 
 
 
INTRODUCTION 
 
Antisense technology has increasingly huge potentials in 
cellular and tissue engineering, in addition to its wide-
spread use in studying gene function as well as control-
ing the expression of unwanted proteins. The basic con-
cept of antisense technology hinges on halting the 
expression of a specific mRNA by the use of a comple-
mentary sequence, which blocks the transfer of genetic 
information from DNA to protein (Jarald et al., 2004).  
There are three ways of producing this specific inhibit-
tion of gene expression. These include the use of single-
stranded oligonucleotides that bind to a specific mRNA, 
forming a DNA-RNA duplex, which in turn inhibits tran-
slation into the corresponding protein. The second credi-
ble antisense strategy is to endogenously express an 
antisense RNA through a recombinant expression vector 
harbouring antisense genes. This antisense RNA is be-
lieved to form a duplex with the complementary mRNA 
sequence and blocks translation by the ribosome. In the 
third strategy, which is RNA interference (Hammond et 
al., 2000), long double-stranded RNA (dsRNA) molecules 
are cleaved by a dsRNA-specific nuclease named Dicer 
(Bernstein et al., 2001) into 21–23  base pairs small inter-
fering RNAs (siRNAs) (Elbashir et al., 2001). The siRNAs 
 
 
 
*Corresponding author. E-mail: 
obembe@covenantuniversity.com. Tel: 234-8060164341. 
are then incorporated into a multi-protein nuclease com-
plex, known as the RNA induced silencing complex 
(RISC). The complex is then guided to the target mRNA 
through conventional base-paring interaction of the anti-
sense strand of the siRNA and eventually degrades the 
target mRNAs homologous to the single stranded siRNA 
(Hammond et al., 2000).  
(Oomen et al., 2004) employed the antisense RNA 
expression vector strategy to generate antisense potato 
plants with varying degrees of down-regulation of cellu-
lose content in tuber cell walls. The transgenic potato 
tuber clones were obtained by transforming the potato 
plant with antisense constructs of the class-specific re-
gions (CSR) of four corresponding potato cellulose 
synthase genes (CSR1, 2, 3 and 4). Two transgenic lines 
csr2-1 and csr4-8, which showed considerable cellulose 
reduction (40 and 60%, respect-ively) in their tuber cell 
walls were identified by the anthrone colorimetric assay 
(Oomen et al., 2004). Hence our interest in exploring the 
possibility of achieving double transformants with com-
bined effects of the two transgenes on organ and/or cel-
lular phenotype, and ultimately on cellulose synthesis and 
deposition.  
In this study, we have crossed the transgenic potato 
lines csr2-1 and csr4-8, whose tuber cell walls exhibit low 
levels of cellulose as compared to the control. We report 
the segregation analysis of the transgenes as well as 
whole plant phenotype characterization, which indicate
Obembe et al.        039 
 
 
 
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gtaatgtatt caacctttag aattgtgcat tcataattag atcttgtttg tcgtaaaaaa 
ttagaaaata tatttacagt aatttggcat acaaagctaa gggggaagta actactaata 
ttctagtgga gggaccagta ccagtaccag tacctagata ttatttttta ttactataat 
aataatttaa ttaacacgag actgatagga atgtcaagtg gtagcggtag gagggagttg 
gtttagtttt ttagatacta ggagacagaa ccggacgggc ccattgcaag gcccaagttg 
aagtccagcc gtgaatcaac aaagagaggg cccataatac tgtcgatgag catttcccta 
taatacagtg tccacagttg ccttccgcta agggatagcc acccgctatt ctcttgacac 
gtgtcactga aacctgctac aaataaggca ggcacctcct cattctcaca ctcaacactc 
aacgagtggt aacttttact catctcctcc aattatttct gatttcatgc atgtttacat 
tctattatgt attatgaatc atagtttcgt gtataaacgt tgtttcatat ctcatctcat 
ctattctgat tttgattctc ttgcctactg aatttgaccc tactgtaatc ggtgataaat 
gtgaatgctt cttcttctca gaaatcaatt tctgttttgt ttttgttcat ctgtagctta 
ttctctggta gattcccctt tttgtagacc acacatcac 
 
 
Figure 1. The 819 base pairs promoter sequence. 
 
 
 
combined effects of the two transgenes on tuber produc-
tion in the progeny.  
 
 
MATERIALS AND METHODS 
 
Plant material and growth conditions  
 
Potato (Solanum tuberosum) plants used for the cross carried, in 
antisense orientation, csr2 sequence of the potato CesA2 gene 
(accession number AY221089) or the csr4 sequence of the CesA4 
gene (accession number AY221088) (Oomen et al., 2004)). 
Expression of the antisense constructs was targeted to the tuber by 
using a granule bound starch synthase (gbss) promoter to drive its 
expression. The 819 base pairs promoter sequence is presented in 
Figure 1.  
Pollen of the plant line csr4-8 was used to fertilize plant line csr2-
1 to produce berries. A total of 488 seeds were removed from the 
berries, dried and prepared for germination. 100 seedlings, repre-
senting the different genotypes, were grown in soil in the green-
house under 3,000 lux and in a light/dark period of 16/8 h. 
 
 
Molecular analysis 
 
Copy number of transgene integration of the parents and 
segregation pattern in the offspring was determined by southern 
analysis. A total of 84 F1 plants were used for segregation analysis. 
Genomic DNA was isolated from young leaves by using GenElute™ 
Plant Genomic DNA Miniprep Kit (Sigma). 8 µg of the prepared 
DNA was digested with restriction enzyme HindIII and electropho-
resed on 0.8% (w/v) agarose gels. Electrophoresed DNA samples 
were transferred onto Nylon N-membrane (Amersham). Hybri-
dization was performed at 65°C with modified Church buffer 
(Church and Gilbert, 1984) containing [32P]UTP-labelled NptII 
probe. 
 
 
Phenotype characterization 
 
Following molecular characterization of the 84 plants and at matu-
rity, the tubers were harvested and grouped accordingly. The tubers 
for each F1 plant were then weighed to generate data on number of 
tubers from a particular offspring plant that correspond to norma-
lized 100 g weight. 
RESULTS AND DISCUSSION 
 
Southern analysis of the parent plants (Figure 2) revealed 
that parent csr2-1 contained 2 copies of the transgene in 
tandem while parent csr4-8 contained 3 copies (1 single 
insertion and 1 tandem repeat). Based on the transgene 
insertion number in the parent, we determined the segre-
gation pattern of the offspring, using Southern analysis. 
This analysis revealed an expected co-segregation of 
both the single copy and the tandem repeat of csr4 
transgenes, which implies that they were integrated in the 
same locus, thus leading to an expected segregation 
ratio of 1:1:1:1 of the four classes (23 = 3.3; P<<0.05). 
The observed segregation pattern of a total of 84 plants 
analyzed was: twenty five plants contained the csr4 
construct, twenty four the csr2 construct, fifteen both the 
csr2 and csr4 construct, and 18 plants contained none of 
the constructs. Although, the number of plants containing 
the double constructs or no construct was lower than that 
of plants containing either of the two transgenes, statis-
tically it is well within the expected segregation ratio. 
There were differences in tuber production whereas 
there was no visible difference in plant morphology, phyl-
lotaxis, growth and development of the offspring. It was 
observed that 100% and 90% of the control and csr4 
plants produced tubers respectively, whereas for the csr2 
and csr2/csr4 plants, only 70% of them produced tubers. 
It was clear that offspring plants containing either one or 
both antisense transgenes produced more tubers than 
the control plants, but individual tubers were mostly 
smaller and had lesser weight than the control tubers. By 
deduction from Figure 2, 88% of the csr2 plants and 64% 
of the csr2/csr4 plants produced tubers with an average 
weight of equal or less than 5 g, whereas 50% of the csr4 
plants and 6% of the control plants produced tubers with 
an average weight of equal or less than 5 g.  
On the whole, our results present molecular evidence 
of Mendelian inheritance of the two antisense transgenes  
040          Afr. J. Plant Sci. 
 
 
 
 
 
Figure 2. Southern blots showing transgene integration in the parents and the observed 
segregation pattern in the offspring. Double insertions of the csr2 construct in the csr2-1 parent 
and triple insertions of the csr4 construct in the csr4-8 parent. 
 
 
 
 
 
Figure 3. Number of tubers of offspring plants corresponding to normalized 100 g weight. Average weight of tuber for a particular 
F1 plant is deduced by dividing the normalized 100 g weight by the corresponding number of tubers. 
  
 
 
genes in the progeny. These plants did not exhibit striking 
additive effects of the transgenes in the whole plant mor-
phology. We are currently analyzing the tubers further for 
cellular phenotypes since the antisense genes were 
targeted to the potato tuber.  
 
 
ACKNOWLEDGEMENT  
 
This work was supported by the Netherlands Foundation 
for the Advancement of tropical Research (WOTRO) The 
Netherlands and by a grant from the Laboratory of Plant 
Breeding, Wageningen University. The authors would like 
to thank Dirkjan Huigen for the maintenance of the plants 
in the greenhouse. 
 
 
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Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001). Role for a 
bidentate ribonuclease in the initiation step of RNA interference. 
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Church GM, Gilbert W (1984). Genomic sequencing. Proc.  Natl. Acad. 
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Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T 
(2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in 
cultured mammalian cells. Nature 411:494-498. 
Hammond SM, Bernstein E, Beach D, Hannon GJ (2000). An 
RNAdirected nuclease mediates post-transcriptional gene silencing in 
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Jarald E, Edwin S, Dubey P, Tiwari AA, Thakre V (2004). Nucleic acid 
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Oomen R, Tzitzikas EN, Bakx EJ, Straatman-Engelen I, Bush MS, 
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An RNAdirected nuclease mediates post-transcriptional gene silencing in Drosophila cells.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

Genomic sequencing.

Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones.

Nucleic acid drugs: a novel approach.

Role for a bidentate ribonuclease in the initiation step of RNA interference.

Molecular analysis and phenotype characterization of the progeny of two antisense potato plants

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