Final PhD Synopsis

2. Genetic Transformation and Expression
Analysis of Cold Tolerant gene in Tomato
(Lycopersicon esculentum Mill)
Synopsis
By
Sabir Hussain Shah
Ph.D Student
Department of Plant Genomics and Biotechnology
PARC Institute of Advanced Studies in Agriculture
KPK Agricultural University, Peshawar
Pakistan

3. SUPERVISORY COMMITTEE
 Supervisor: Dr. Shaukat Ali
 Member: Dr. Ghulam M. Ali
 Member: Dr. Jalal-ud-Din

4. Tomato (Lycopersicon esculentum)
 Area: 0.0534 million hectares
 Production: 0.562 million tons
 Average yield: 10.5 tons/ha.
(Agricultural Statistics of Pakistan. 2008-09)

5. Importance of Tomato
• Tomato is rich in vitamins A, C, K and fibre (USDA, 2009)
• It contains approx. 20-50 mg lycopene/100 g of fruit weight.
(Kalloo, 1991)
• Lycopene protects humans from cancer (Rao, 2000)

6. Factors affecting Yield (Problems)
 Low yield due to:
 Abiotic and Biotic stresses
 Abiotic stresses:
Cold, drought and salinity etc - serious threat to
crops (Maqbool et al., 2002)
Cold may cause up to 70% losses in tomato
(Lysak, 2010)

7. Mechanism of Cold Stress
• In cold, ice formation occurs in apoplast space.
• Ice formation in apoplast establishes a vapour
pressure gradient.
• Unfrozen water migrates down from cell cytosol to
apoplast.
• It causes enlargement of ice crystals and cause
mechanical strain on cell wall.
• Due to it, plasma membrane leading to cell ruptures.
• Freezing induces cell dehydration also.

8. Cold ??
 the sensation produced by low temperatures.
 Effect of Cold on tomato Physiology:
 Limits the plant growth
 Effect on seed germination
 Transport rate of assimilates is affected
 Closure of stomata
 Photosynthesis stop

9. Cold Tolerance?
 Cold tolerance refers to the degree to which a plant
is adapted to low temperature conditions.
 Plant's ability to stay alive, grow, and ultimately
produce fruit, with part of its life cycle under cold
stress.

10. Possible ways to solve cold problem
 Genetic Improvement of tomato for cold stress:
 Through conventional breeding approach
 Through genetic engineering approach

11. Genetic Improvement of tomato for
cold stress:
 Conventional Breeding Approach
 Take greater time
 Present in same species or closely related
species
 Genetic Engineering Approach
 Take Less time
 Provide wider gene pool and not restrict in same
or closely related species

12. Cold tolerance gene
 DREB1A
 It is a transcription factor
i.e. regulate the expression of several genes related
to cold stress.

13. Aim and Objectives of Project
 Aim:
 To improve tomato for cold tolerance
 Objectives:
 To establish tissue culture protocol for different
genotypes of tomato.
 To develop protocol for transformation.
 To incorporate the DREB1A gene in various
genotypes of tomato.
 To check the expression by different parameters

14. Materials and Methods
This research project will be performed in National
Institute of Genomics and Advanced
Biotechnology (NIGAB), NARC, Islamabad.
 Experiment No. 1
(Standardization of In Vitro Culture System)
• Plant Material: Riograndae, Money maker and
Roma.
• Callus Induction Protocol Optimization:
MS media (Murashige and Skoog, 1962) with
different levels of growth regulators (IAA, BAP and Kinetin).

15. • Regeneration Protocol Optimization:
MS media + different levels of growth regulators (IAA, BAP and
Kinetin)
Experiment No. 2
(Agrobacterium-mediated Transformation)
 Explants pre-culturing
 Agrobacterium culturing
 Subculturing of Bacteria
 Co-cultivation

16. Agrobacterium mediated transformation
 Pre-selection
 Selection
 Regeneration
 Green house/field

17. Transformation---
 Co-Cultivation
 Explants + Agrobacterium
 Co-cultivation media
 MS media
 Minimum 24 hours
 Pre-Selection
 MS media + Cefotaxime
 Time: 4-5 days

18. Transformation---
 Selection:
 Regeneration medium + Cefotaxime +
Hygromycin
 Time required 3-4 weeks
 Regeneration:
 Optimized regeneration medium+ Hygromycin
+ Cefotaxime
 Shoots development
 Roots development

19.  Acclimatization
 Shifting into green house
 Transgenic analysis
 PCR---detection of gene
 RT-PCR---for gene expression
 Statistical Analysis
Each experiment will be repeated thrice and their
means will be compared by using ANOVA and
their significance level will be analyzed by using
Duncan’s Multiple Range Test (DMRT) .

20. Physiological Parameters for
Evaluating Cold Tolerance in tomato
Cold tolerance will be evaluated by following physiological parameters:
 Membrane Leakage:
• Leakage of ions from the leaves will be measured according to a
method devised by Sairam et al., 1997.
 Proline Accumulation:
• Proline will be isolated by a method given by Bates et al., 1973
• Quantitative assay of proline will be done on the basis of Optical
density by comparing with a standard curve with known amounts of
Proline.

21.  Total Soluble Sugars Accumulation:
• Total soluble sugars will be extracted from lyophilized leaf material in
80% ethanol.
• Sugar contents will be analyzed by a method devised by Dubois et al.,
1956.
 Chlorophyll Concentration:
• Chlorophyll contents will be extracted by the protocol devised by
Wettestein, 1959
• Quantitative assay of chlorophyll concentration will be done on the basis
of optical density by spectrophotometer.

22. References
 Agricultural Statistics of Pakistan. 2008-2009. Government of Pakistan. Ministry of
Food and Agriculture (Economic Wing). Islamabad.
 Bates, L-S., R. P. Waldren and I. D. Teare. 1973. Rapid determination of free
proline for water stress studies. Plant and Soil, 39: 205-208.
 Chaudhry, Z., and H. Rashid. 2010. An improved Agrobacterium mediated
transformation in tomato using hygromycin as a selective agent. African J. of
Biotech., 9(13): 1882-1891.
 Chaudhry, Z., S. Abbas, A. Yasmin, H. Rashid, H. Ahmed and M. A. Anjum. 2010.
Tissue culture studies in tomato (Lycopersicon esculentum) var. Money maker.
Pak. J. Bot., 42(1): 155-163.
 Dubois M, K. A. Guilles, J. K. Hamilton, P. A. Rebers, F. Smith. 1956. Colorimetric
method for determination of sugars and related substances. Anal Chem. 28: 350-356.
 Hu, H., J. You, Y. Fang, X. Zhu, Z. Qi and L. Xiong. 2006. Characterization of
transcription factor SNAC2 conferring cold and salt tolerance in
rice. Plant Mol. Biol. 67: 169-181.

23.  Kalloo, G. 1991. Introduction. In: Kalloo G (eds). Monographs on Theoretical and
Applied Genetics 14, Genetic Improvement of Tomato (pp. 1-9).
Springer-Verlag, Berlin, Heidelberg, New York.
 Khoudi, H., A. Nouri-Khemakhem, S. Gouiaa and K. Masmoudi. 2009. Optimization
of regeneration and transformation parameters in tomato and improvement of
its salinity and drought tolerance. African J. Biotech., 8(22): 6068-6076.
 Lysak, A. 2010. Florida tomato crop declines by 70% due to freezing weather
conditions. Agriculture Featured TNM Florida United States.
 Mamidala, P. and R. S. Nanna. 2009. Efficient in vitro plant regeneration, flowering
and fruiting of dwarf tomato cv. Micro-Msk. Plant Omics J. 2(3): 98-102.
 Maqbool, S., H. Zhong, Y. El-Maghraby, A. Ahmad, B. Chai, W. Wang, R.
Sabzikar and M. Sticklen. 2002. Competence of oat (Avena sativa L.) shoots
apical meristems for integrative transformation, inherited expression and
osmotic tolerance of transgenic lines containing hva1. Theor. Appl. Genet. 105:
201-208.
 Murashige, T. and F. Sakoog. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue culture. Physiol Plant. 15: 473-497.

24.  Paramesh, H., Fakrudin, B. and Kuruvinashetti, M.S. 2010. Genetic transformation of a local
variety of tomato using gus gene: an efficient genetic transformation
protocol for tomato. J. Agri. Technology. 6(1): 87-97.
 Rao A & Agarwal S. 2000. Role of antioxidant lycopene in cancer and heart disease.
J.Am.College Nutr. 19: 563-569.
 Rhodes D. 2002. Tomatoes – Notes (Purdue University).
 Sairam, R. K., P. S. Deshmukh and D. S. Shukla. 1997. Tolerance to drought and temperature
stress in relation to increased antioxidant enzyme activity in wheat. J. Agron. Crop. Sci., 178:
171-177.
 USDA National Nutrient Database for Standard Reference. May 5,
2009: http://www.nal.usda.gov/fnic/foodcomp/search/
 Wettestein, D. V. 1959. Chlorophyll- letate and Der supmkroskopisch fromwecksee Der
plastiden Exp. Cell Res., 17: 427.

25. THANKS