1) The document summarizes antisense RNA technology, which involves producing RNA sequences that are complementary to target mRNAs to inhibit gene expression. 2) Two case studies are described where antisense RNA was used to suppress ethylene biosynthesis genes (ACC oxidase) in orchids and carnations, extending their vase life. 3) The technology has potential applications in crop improvement by extending shelf life and improving traits like fruit ripening and flower longevity.

1. DAYANANDA SAGAR COLLEGE OF ENGINEERING
DEPARTMENT OF BIOTECHNOLOGY
Seminar Presentation
Antisense RNA Technology
S. Kiruthika
(14163PGDPGT0006)
1st Semester, BTFS, DSI, Bengaluru-560 078
Under the Guidance of
Ms. Supreetha. K
Department of Biotechnology
Dayananda Sagar College of Engineering
S. M. Hills, Kumaraswamy Layout, Bengaluru -560 078

2. INTRODUCTION
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When expression of a transgene is desired, the arrangement of
promoter and sequence to be expressed mimics the usual
arrangement of the gene, so that the promoter directs
expression of sense RNA that is in the correct orientation to
be translated, The simplest RNAi—related technology is to
produce so called antisense RNA.
In vectors, designed to produce antisense RNA, the sequence
to be expressed(which can be all or part of the coding
sequence of an endogenous gene) is placed in the reverse
orientation with respect to the promoter. The promoter
therefore directs the production of antisense RNA, which is
complementary to the endogenous mRNA.

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The complementarity between antisense RNA and mRNA results in the
production of dsRNA, RNAi can be deliberately induced to silence the
expression of specific genes in transgenic plants.
BASIS OF THE TECHNOLOGY:
A sense strand is a 5’ to 3’ mRNA molecule or DNA molecule. The
complementary strand to this strand is called an antisense. The antisense
technology works by binding the antisense strand with the targeted sense
strand through hydrogen bond. The double stranded molecule will be
recognized as the foreign molecule and is susceptible to degradation.
Though, as the DNA is double helix, it can be used in antisense
technology; resulting in the formation of triplex.
Antisense strand can be:
1. DNA: A DNA antisense strand is of approximately 17 bases long.
2. RNA (either catalytic or non-catalytic): 13 bases long RNA antisense
strand functions properly.

4. ANTISENSE RNA TECHNOLOGY FOR IMPROVING VASE LIFE OF
CUT FLOWERS
ETHYLENE BIOSYNTHESIS AND ITS REGULATION
Methionine
SAM Synthetase ATP
PPi + Pi
S-Adenosylmethionine (SAM)
ACC Synthase
1-Amino-cyclopropane-1-carboxylic acid (ACC)
ACC Oxidase O2
Ethylene
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5.  The vase life of cut flowers can be extended by adding silver salts to the
water, which blocks the response to ethylene.
 Antisense ethylene technology is applicable to other systems triggered
by ethylene like senescence of picked flowers.
 The phytohormone ethylene is essential for the senescence of fruits and
flowers. In the biosynthesis of ethylene in plants, the conversion of
SAM into ACC and the conversion of ACC into ethylene is catalysed
by ACC synthase and ACC oxidase respectively.
 The introduction of antisense ACC synthase or ACC oxidase would
have a similar effect of extending vase-life of cut flowers by
suppressing ethylene synthesis.
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6. CASE STUDY-1
Agrobacterium-Mediated Transformation of Dendrobium secundum (Bl.) LindI with
Antisense ACC oxidase
Prontip et al., (2007), Asian Journal of Plant Sciences 6 (7): 1065-1071.
Objectives:
The objectives of this study were to improve the efficiency of regeneration and
transformation of Dendrobium secundum with antisense ACC oxidase gene using
Agrobacterium tumefaciens for prolonging the longevity and vase life of orchid flowers.
Design of study:
1. Agrobacterium mediated transformation: Agrobacterium tumefaciens strain LBA4404
(pCAMBIA 1305.1) containing Antisense ACC oxidase, β-glucuronidase (GUS), and
hygromycin resistance (hptII) gene with CaMV 35S promoter. Protocorms were used
as the explant for transformation.
2. Assay for β-glucuronidase (GUS) activity.
3. PCR analysis.
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7. Results:
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11. RESULTS ON EFFECT OF ANTIBIOTICS
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12. Conclusion:
 The protocorms with high proliferation efficiency capacity were cultured on
the modified VW medium with 0.5 mg/L NAA.
 The efficiency of transformation was 40 min of co-cultivation of protocorms
with Agrobacterium tumefaciens that has been induced with 200 µM
acetosyringone.
 Effect of antibiotics on Protocorms:
1. Hygromycin at 25 mg/L was effective for the transformant selection
2. Maximum concentration of cefotaxime that protocorms could tolerate
was 500 mg/L
 Hygromycin resistant protocorms showed histochemical blue staining due to
GUS activity was 60%.
 PCR analysis of fragment sizes 195, 180 and 843 bp for the 35S, NOS and
Antisense ACC oxidase respectively confirmed the successful transformation.
 This methodology can be proposed as one of the efficient method used to
transform novel genes into the other orchid species.
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CASE STUDY-2
Antisense ACC oxidase RNA delays Carnation petals Senescence.
Keith et al., (1995), Hortscience, Vol 30(5): 970-972.
Objective:
The transgenic carnation plants producing antisense ACC oxidase RNA were generated
to create a carnation line with low ethylene synthesis and delayed petal senescence, and to
more clearly define the role of ethylene in the senescence of carnation petals.
Design of study:
1. Agrobacterium mediated transformation for the production of transgenic carnation
plant: T-DNA based expression vector (pCGP407) has carnation ACO cDNA fragment
contained within pCGP363 was inserted in reverse orientation into a binary vector
pCGP293 between MAC promoter and mas 3’ end (Agrobacterium mannopine
synthase gene). MAC is a strong constitutive promoter and is able to direct the
expression of reporter gene (uidA).
2. Southern Analysis.

14. 3. Northern Analysis
4. Gas chromatography
Results:
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18. Conclusion:
• The antisense ACO gene has succeeded in suppressing the level of ACO
mRNA and hence all but eliminated ethylene production and extended
carnation flower vase life.
• Expression of ACS and ACO genes in petals must be dependent on the
presence of ethylene.
• The generation of transgenic carnation plants in which the ability to
synthesis ethylene has been reduced or eliminated by use of antisense
technique reflects similar findings in tomato and reinforces the concept
of improving properties, such as postharvest qualities, by genetic
engineering rather than by classical long term breeding or by
application of chemicals.
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19. Conclusion
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 Antisense technology was successfully used to reduce or eliminate the
softening and ripening process in fruits and vegetables which results in the
improvement of the shelf-life.
 Examples:
o Calgene’s Flavrsavr fresh tomato with antisense polygalacturonase,
antisense phytoene synthase, and antisense ACC oxidase gene for delayed
softening, red colouration and ripening respectively.
o Down regulation of Pectin Methyl Esterase by Antisense PME delayed the
softening process in apple (MacIntosh).
 Similarly, the antisense RNA technology can be used for the down
regulation of the ethylene synthesis which would enhance the vase-life of
the cut flowers after post harvesting. This technology could be an effective
means rather than using chemicals for improving the vase-life of the cut
flowers.

20. Future Perspective
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1. Application in Crop Improvement.
2. Artificial Antisense RNA Regulation of Gene expression.
3. Virus resistance by Antisense RNA.
4. Antisense Technology in the Treatment of Genetic
disorder.
5. Development of New methods of Drug Design.

21. References
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1. Prontip Atichart, Sumontip Bunnag, and Piyada Theerakulpisut (2007).
Agrobacterium-Mediated Transformation of Dendrobium secundum (Bl.) LindI
with Antisense ACC oxidase. Asian Journal of Plant Sciences 6 (7): 1065-1071.
2. Keith W. Savin, Stanley C. Baudinette, Michael W. Graham, Michael Z. Michael,
Greg G. Nugent, Chin – Yi Lu, Stephen F. Chandler, and Edwina C. Cornish (1995).
Antisense ACC oxidase RNA delays Carnation petals Senescence. Hortscience, Vol
30(5): 970-972.
3. Adrian slater, Nigel W. Scott, and Mark R. Fowler : Plant Biotechnology, the
genetic manipulation of plants (Second Edition), Oxford University Press, ISBN
9780199560875.

22. Acknowledgement
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1. Dayananda Sagar Institutions, S. M. Hills, Kumarasamy Layout,
Bangalore – 560078.
2. Dr. G. A. Ravi Shankar, Vice President, Research and Development
Life Sciences, Dayananda Sagar Institutions, S. M. Hills,
Kumarasamy Layout, Bangalore – 560078.
3. Dr. Kiran, BTFS Co-ordinator and Head of Department, Department
of Biotechnology, Dayananda Sagar College of Engineering, S. M.
Hills, Kumarasamy Layout, Bangalore – 560078.
4. Ms. Supreetha. K, Assistant Professor, Department of Biotechnology,
Dayananda Sagar College of Engineering, S. M. Hills, Kumarasamy
Layout, Bangalore – 560078.

23. Thank You
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