Transformation of Drumstick through AgroBacterium Tumefaciens.for the genetic improvement.

2. PRESENTED BY…
KAZIM TANVEER
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Outline of Presentation
Introduction
Aim of the Study
Material and Methods
Results and Discussion
Conclusion
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INTRODUCTION
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 Moringa leaves come from the Moringa oleifera.
 A drought-resistant tree native to the Himalayas
India, Bangladesh, Pakistan and Afghanistan.
 Miracle tree
INTRODUCTION 3

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 Moringa oleifera is a small to medium-sized
tropical tree native to the Indian subcontinent.
 The tree is easily recognizable from its light
colored knotty trunk and highly branched
compound leaves.
PLANT DESCRIPTION 4

7.  The fruit of the Moringa tree is thin,
 12 -24cm. long pods.
 Moringa is commonly known as
drumstick tree. Pods hang from it
during the major part of the year.
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8.  The leaf has been shown to
contain 46 types of antioxidants
and
 92 nutrients, making it one of the
most powerful super foods known.
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PREFERRED SCIENTIFIC NAME DRUMSTICK TREE
KINGDOM PLANTAE
CLASS DICOTYLEDONAE
FAMILY MORINGACEAE
ENGLISH NAME HORSE RADISH TREE
HINDI NAME SHIGRU, SAHAJAN,MUNAGA
URDU NAME SOHANJNA, SAHAJNA OR MUNAGA
GENUS MORINGA
SPECIES OLEIFERA
BOTANICAL CLASSIFICATION OF MORINGA

10. APPLICATIONS
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NUTRITIONAL VALUE

12. TRADITIONAL MEDICINAL USES OF MORINGA 10

13.  1000 ways to eat and cook
 Moringa Juice
 Moringa Salad
 Moringa in Cooked Meals
 Moringa Tea
MORINGA AS FOOD 11
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14.  Moringa leaves Gulay
 Drumstick curry with onions
 Drumstick with rice and coconut
 Drumstick sabzi with gram flour
 Drumstick-aloo sabzi
 Drumstick leaf korma
MORENGA DISHES 12
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15. Aim of the Study
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Aim of the Study
To develop a reliable and stable agrobacterium tumefaciens-mediated
transformation system for the genetic improvement of drumstick
Objective
This transformation protocol provides a basis for the future development of
genetic engineering techniques to improve the performance of drumstick.
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Methodology
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18.  Nodals from the plantlets of three drumstick clones
 M-2, M-5, M-17 were used
 Nodals explants were incubated (Murashige and Skoog)(MS) basal medium
 Regenerated shoots were excised from mother and rooted in rooting medium
 Media pH 5.8-6.0
PLANT MATERIAL AND CULTURE CONDITIONS 16
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19.  Autoclaved 121 °C for 15 min.
Cultures were maintained under 12-h photoperiod
 Using cool light
 Temp 25 °C
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20.  Nodal explants were culture on SIM
 Containing a combination of HYG ( 5-35 mg/L
 Cefalexin (CEF; 150 mg/L for four weeks)
 Explants survival was evaluated at the end
 Regenerated shoots were excised from mother tissue
 Rooted in RM
HYGROMYCIN SENSITIVITY DETECTION 18
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21.  With a combination of HYG(5-25mg/L ) CEF; (150mg/L) for two
weeks
 Exp were performed three times
 Fifteen explants or shoots were used each time
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22.  pCAMBIA1301 (vector)
Containing HYG photophotransferase 2 and β-Glucorinidase
 These genes were driven by CAMV 35S promoter
 It was introduced into A. tumefaciens strain EHA105
 A. tumefaciens EHA105 (1mL) was inoculated into
 100mL yeast extraction broth medium
Binary vector and Agro bacterium preparation 20
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23.  That contain 5 mg chloramphenicol and 5mg kanamycin.
 A. tumefaciens was collected by centrifugation
 O.D AT 600nm of 0.2
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24.  To optimize transformation protocol,
 Pre –culture (0,2,4, or 6 days) and
 Co-cultivation (1,2,3, or 4 days) periods were evaluated
 Explants were infected with A. tumefaciens
 A. tumefaciens-infected plants were blot-dried onto sterile filter paper
 After blot drying explants were transferred to selection medium
Transformation procedure 22
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25.  After approximately 4 weeks
 Putative transgenic shoots (more than 2cm) were excised
 From mother tissues and rooted in RM.
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26.  Gus A expression was analyzed histochemically in tissues
 Samples were soaked in 500 mg/L X-GLUS solution over night
 At 37 °C`
GUS HISTOCHEMICAL ASSAY 24
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27.  DNA was removed from leaves
GusA gene was amplified from genomic DNA
 Using forward (5′- GTCGCGCAAGACTGTAACCA-3′)
 Reverse (5′- CGGCGAAATTCCATACCTG-30) primers
 The products were analyzed by 1.5% agarose gel electrophoresis
Molecular characterization by PCR 25
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28.  The forward primer 5′-GTGAGCGTCGCAGAACA-3′
 And reverse primer 5′-GGCAACAAGCCGAAAGA-3′
 Were used for Gus amplification.
 Actin was amplified as a reference gene
 Using primers ACTf and ACTr.
 The PCRs were stopped after 40 cycles.
Real-time quantitative PCR (RT-qPCR) 26
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29. Results and Discussion
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31.  Different A. tumefaciens strains exhibit
 Different effects on plant transformation frequency
 And transgenic event quality
 In the present study, strain EHA105 played a pivotal role in the
transformation of Drumstick.
The vacuum infiltration technique enhanced the transformation efficiency
A. tumefaciens strain 28
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32.  A suitable vector is also important for genetic transformation.
 The plasmid pCAMBIA 1301 is widely used for plant genetic engineering
 And has been applied to the genetic transformation of various plants,
 Such as rice centipede grass and peanut
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33. Conclusion
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34.  Drumstick has gained interest globally in recent years because of its unique
industrial value.
Therefore, a genetic transformation system which facilitates its
improvement
 By genetic engineering and facilitates functional genomic analyses
 Using molecular tools would be very important.
Conclusion 31
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35.  In the present study, we describe a more efficient
 And reliable genetic transformation protocol for Drumstick,
 Which should open the door for Biotechnological improvements
 Of this important tree species and systematic functional genomic
studies
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36. Agriculture University Faisalabad (AUF)
Nuclear Institute of Agriculture and
Biology (NIAB)

37. 1. Abdulkarim, S. M. and Long, K. and Lai, O. M. and Muhammad, S. K. S. and Ghazali, H. M. 2007. Frying quality and stability of
high-oleic Moringa oleifera seed oil in comparison with other vegetable oils. Food chemistry 4, 1382-1389.
2. Anwar, F. and Latif S.and Ashraf M. and Gilani, A. H. 2007. Moringa oleifera: a food plant with multiple us- dicinal uses.
Phytotherapy research 1, 17-25.
3. Asaolu, M. F. and Omotayo, F. O. 2007. Phytochemical, nutritive and anti-nutritive composition of leaves of Moringa oleifera.
Phytochemistry and pharmacology III, 339-344.
4. Broin, M. and Santaella, C. and Cuine, S. and Kokou, K. and Peltier, G. and Joet, T. 2002. Flocculent activity of a Recombinant
protein from Moringa oleifera Lam. Seeds. Applied Microbiology and Biotechnology 1-2, 114-119.
5. Foidl, N. and Mayorga, L. and Vasquez, W. 1999. Utilization of marango (Moringa oleifera) as fresh forage for cattle. FAO
Animal production and health paper 143, 341-346.
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38. 6. Dahot MU and AR Memon {1987} Properties of Moringa oleifera seed lipase. Pakistan Journal of Scientific and Industrial
Research 30{11}: 832- 835.
7. Dayrit FM, AD Alcantar, and IM Villasenor {1990} Studies on Moringa oleifera seeds, Part I: The antibiotic compound and its
deactivation in aqueous solution. Philippine Journal of Science. 119: 23-32
8. Palaniswamy U {2005}.Purslane—Drumsticks Lok-Vani {e-journal} http://www.lokvani Ramachandran, C,:Peter K.
9. V:Gopalakrishnan,P.K {1980} Drumstick {Moringa oleifera}: a multipurpose Indian vegetables, Economic Botany,34{3}:276-283.
10. Sutherland J.P., G.K. Folkard, M.A. Mtawali and W.D. Grant. 1994. Moringa oleifera as Natural Coagulant. Journal of WEDC
Conference. University of Leicester, UK.
11. Caceres A, A Saravia, S Rizzo, L Zabala, E De Leon, F Nave, 1992. Pharmacologic properties of Moringa oleifera. 2: screening for
antispasmodic, anti-inflammatory and diuretic activity. J Ethnopharmacol, 36: 233–237.
References
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39. A Special Acknowledgment
This presentation was supported by
 Dr. Waqas, Assistant Professor
 Dr. Kazim, Assistant Professor
 Dr. Faisal, Assistant Professor
 Dr. Waseem, Associated Professor
 Dr. Farzana, Assistant Professor
 Dr. Zainab, Assistant Professor
 Madam Faiza, Lecturer
 Madam Erum, Lecturer
 Madam Uzma, Lecturer

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