Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
1
Chapter 1
INTRODUCTION
1.1 Sunflower
The cultivated sunflower (Helianthus annuus L.) is an annual plant with high nutrit...
2
1.2 Agrobacterium tumefaciens
Agrobacterium tumefacians (updated scientific name: Rhizobium radiobacter) is the
causal a...
3
AIM, OBJECTIVES AND SCOPE
AIM
The Aim for this study is to standardize a simple protocol for inplanta transformation of
...
4
CHAPTER 2
REVIEW OF LITERATURE
Agrobacterium mediated transformation is one of the most widely used mode of
transformati...
5
2.2 Genetic transformations in Helianthus annuus L.
Lappara et al (1995) evaluated three methods of transformation in su...
6
CHAPTER 3
METHODOLOGY
3. a Requirements for the project
 Collection of samples:
The Sunflower seeds of variety CO4 were...
7
3.1 TRANSFORMATION OF pCAMBIA 1305.2 VECTOR INTO Agrobacterium
tumefaciens STRAIN LBA4404
3.1.1 Preparation of Competant...
8
 Aliquots of 200µl were transferred to prechilled 1.5 ml tubes and was then used for
transformation.
3.1.2 Transformati...
9
plates were then incubated at 28ºC for 2 days. Then the transformed colonies were confirmed by
plasmid isolation.
3.1.4 ...
10
Solution II (Lysis Buffer)
Solution III (DNA neutralization buffer)
Sterile Microtips
Sterile microfuge tubes
Isopropan...
11
3.2 STANDARDIZATION OF GENETIC TRANSFORMATION PROTOCOL FOR
Helianthus annuus L.
3.2.1 Sterilization of seeds
Materials:...
12
Protocol:
 A loopful of Agrobacterium strain LBA4404 containing pCAMBIA1305.2 culture was
inoculated in 20 ml YEP medi...
13
3.3.1.1 Expression of β-glucuronidase
Materials:
0.1% HgCl2
GUS staining solution.
Sterile distilled water
75% ethanol
...
14
CHAPTER 4
RESULT AND DISCUSSION
4.1 TRANSFORMATION OF pCAMBIA 1305.2 VECTOR INTO Agrobacterium
tumefaciens STRAIN LBA44...
15
roots of the plants put in the soilrite started decaying and hence the plants did not grew and
resulted in no transform...
16
No.
of
Seeds
(a)
No. of Seeds
Contaminated
(b)
No. of
Seeds
Germinated
(c)
Percentage of
Contamination
(b/a*100)
Percen...
17
Fig. 4.1a Fig. 4.1b
Kanamycin resistant blue colonies of Negative control plate without any growth of
Agrobacteriumtume...
18
Figure 4.2. Sunflower seedlings after inplanta transformation
Seedlings to the left are control plants and the one to t...
19
REFERENCES
 Bottinger P., Steinmetz, Schieder O. and Pickardt T. 2001. Agrobacterium-mediated
transformation of Vicia ...
20
 Jones.H.D, Doherty.A and Wu.H 2005. Review of methodologies and a protocol for the
Agrobacterium-mediated transformat...
21
 Ping, L.X., M.Nogawa, M. Nozue, M. Makita, M.Takeda, L. Bao and M. Kojima,
2003. In planta transformation of mulberry...
22
 Theriappan.P and Gupta.A.K 2014. Development of a protocol for Agrobacterium
mediated transformation of Brassica oler...
23
APPENDIX
1. SOLUTIONS FOR BACTERIAL TRANSFORMATION
100mM CaCl2
Dissolve 1.4702 g of CaCl2 in 20 ml of sterile water. Fi...
24
1% SDS
Solution III
Dissolve 29.45g of Potassium acetate in 80 ml of distilled water. Add glacial acetic acid
till the ...
25
Dissolve 50mg of Rifampicin in 500μl of methanol and make up the volume to 1ml using
sterile distilled water.
5. MS MED...
26
Stock solution of Meso-inositol (x500)
Dissolve 1gm meso-inositol in 20 ml of Water. Take 1mlfor 1 liter media
Stock so...
Upcoming SlideShare
Loading in …5
×

Final Report

371 views

Published on

  • Login to see the comments

  • Be the first to like this

Final Report

  1. 1. 1 Chapter 1 INTRODUCTION 1.1 Sunflower The cultivated sunflower (Helianthus annuus L.) is an annual plant with high nutrition value. Sunflower (Helianthus annuus L.) as an economical important crop, is relatively young, having been selected and cultivated on a large scale since the latter part of the nineteenth century. Presently, it is the fourth most important oilseed crop in the world. It was grown worldwide on over 21 million ha in 2000, in intermediate, temperate, subtropical and parts of tropical climates. Sunflower species are allelopathic in nature and this crop appears to have a bright future, especially if the scientists can translate the cutting-edge research into technologies that will reduce the reliance on synthetic herbicides, pesticides, and crop protection chemicals. Sunflower oil world production now ranks 4th after soybean. It is a major target for the food and feed industry. It is not only known for its richness in polyunsaturated fatty acids but also for its relative high content of miner constituents such as tocopherols (vitamin E)and phytosterols, known to lower plasma cholesterol levels (Hewezi et al 2004). Sunflower seeds are the achenes fruits of sunflower plants (Helianthus annus L.), They are grayish –green with an outer shell (pericarp) that appears black, white or striated, depending on the variety of plant. Sunflower seeds contain an important source of key nutrients in a healthy diet: vitamin E, vegetable protein, potassium, phosphorous, calcium, iron, magnesium, thiamin, riboflavin, niacin, with very low intake of saturated fatty acids. In recent years, there has been a very interesting assessment of the content of sunflower seeds and their medicinal properties (Liu et al., 2011).Tissue culture technology of sunflower has also been under investigation since the 1980s, but regeneration of this crop is still limited to date (Liu et al., 2011).
  2. 2. 2 1.2 Agrobacterium tumefaciens Agrobacterium tumefacians (updated scientific name: Rhizobium radiobacter) is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram - negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for ‘transfer the DNA’), from plasmid, into the plant cell, which is incorporated at semi-random location into the plant genome. Some Agrobacterium cells carry only one vector but some of them have two, each carrying two different combination of gene. There are several significant advantages to transferring DNA via Agrobacterium, including a reduction in transgene copy number, the stable integration with fewer rearrangements of long molecules of DNA with defined ends and the ability to generate lines free from selectable marker genes (Jones et al 2005). 1.3 Plant transformation methods Agrobacterium mediated transformation. Biolistic (or) particle bombardment. 1.3.1 Agrobacterium mediated transformation Agrobacterium mediated transformation method are thought to induce less rearrangement of the transgene. This method produces lower transgene copy number than direct DNA delivery method. It is capable of transferring large fragments of DNA very efficiently without substantial rearrangement. The stabily of gene transferred is excellent. 1.3.2 Biolistic (or) particle bombardment. This method can be use to transform all plant species. No binary vector is required. Transformation protocol is relatively simple. High velocity micro projectile were utilize to deliver nucleic acids into loving cells. This method uses the instrument called as biolistic gun or gene gun.
  3. 3. 3 AIM, OBJECTIVES AND SCOPE AIM The Aim for this study is to standardize a simple protocol for inplanta transformation of sunflower (Helianthus annuus L.) OBJECTIVES  Transformation of pCAMBIA 1305.2 vector into Agrobacterium tumefaciens Strain LBA4404.  Standardization of genetic transformation protocol for Helianthus annuus L.  Screening of transgenic Helianthus annuus L. SCOPE Once a simple protocol for inplanta transformation is standardized, this method can be utilized to do genetic engineering in sunflower easily with any given gene of interest.
  4. 4. 4 CHAPTER 2 REVIEW OF LITERATURE Agrobacterium mediated transformation is one of the most widely used mode of transformation in plants. Several Researchers throughout the world are involved in this research working with several plants. This is more preferred method of transformation because it is economical, does not require any high end instrumentation, and also produces stable lines of transformants. The choice of tissue or organ in the plant of study has been found varying. Feldmann and Marks (1987) developed a non-tissue culture approach of Agrobacterium- mediated transformation in germinating seeds of Arabidopsis thaliana. Stable transformed lines were obtained from apical shoots of sunflower (Burrus et al 1996) and callus in Vicia faba (Bottinger et al., 2001). Keshamma et al., (2008) successfully developed an inplata method of Agrobacterium mediated transformation in Cotton (Gossypium hirsutum L.). This tissue culture independent method was found to be an effective method of obtaining stable transformants in recalcitrant plant species like cotton. 2.1 Genetic transformation in plants Protocols were developed by Mukopadhyay et al (1992) for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. ‘Pusa Kalyani’. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and β-glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Fursova et al (2012) transiently expressed three hydrolase genes in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.
  5. 5. 5 2.2 Genetic transformations in Helianthus annuus L. Lappara et al (1995) evaluated three methods of transformation in sunflower viz., direct gene transfer into protoplasts, particle bombardment and Agrobacterium co-culture. All techniques allowed efficient short-term or transient expression of the introduced gene(s) in the respective tissues, stable transformation was only observed after transformation with Agrobacterium. Burrus et al (1996) developed stable lines of Helianthus annuus L. through agrobacterium mediated transformation in the apical shoots. In 1999, Rao and Rohini developed a very simple protocol of transformation in sunflower using Agrobacterium. In this method, two days old seedlings with one cotyledon detached were infected with Agrobacterium, this resulted in stable transformation. Weber et al (2003) assessed the macerating enzymes and sonication methods of treatment in improving Agrobacterium -mediated transformation of sunflower (Helianthus annuus L.). Liu et al (2011) optimized Agrobacterium mediated transformation in Helianthus annuus L. using immature embryos.
  6. 6. 6 CHAPTER 3 METHODOLOGY 3. a Requirements for the project  Collection of samples: The Sunflower seeds of variety CO4 were obtained from Tamil Nadu Agricultural University, Coimbatore. Few of these seeds were sown in our college garden for further studies.  Bacterial strain: Agrobacterium tumefaciens strain LBA4404 Agrobacterium tumefaciens (Rhizobium radiobactor) is capable of T-DNA transfer to plant cells. The T-DNA (transfer DNA) is located in the Ti plasmid and is capable of integration into the host plant chromosomal DNA. Integrated genes derived from T-DNA are expressed and the transformed plant cells typically become Crown gall tumor cells.The strain LBA4404 has rifampicin resistance gene present in its chromosome and streptomycin resistance gene on the Ti plasmid.  Plasmid Vector: pCAMBIA 1305.2 These vectors contain minimal heterologous sequences for plant transformation and selection of transformants; they allow insertion of desired genes for transformation into plants but require all promoter and terminator sequences for plant expression of newly cloned genes.  Vector contains kanamycin resistance gene for bacterial selection and hygromycin B resistance gene for plant selection.  It also incorporates the GusPlus reporter gene.  The reporter gene of pCAMBIA 1305.2 lacks the bacterial ribosome binding site and shows no expression in Agrobacterium but good expression in plant cells.
  7. 7. 7 3.1 TRANSFORMATION OF pCAMBIA 1305.2 VECTOR INTO Agrobacterium tumefaciens STRAIN LBA4404 3.1.1 Preparation of Competant cells of Agrobacterium tumefaciens strain LBA4404 Most species of bacteria take up only limited amounts of DNA under normal circumstances. For efficient uptake, the bacteria have to undergo some form of physical and /or chemical treatment that enhances their ability to take up DNA. Cells that have undergone this treatment are said to be competent. The fact that Agrobacterium cells that are soaked in an ice- cold salt solution are more efficient at DNA uptake than unsocked cells. Traditionally, a solution of CaCl2 is used is used to make competent Agrobacterium cells. Materials: YEP broth 100mM CaCl2 solution 250 ml conical flask 1.5 ml centrifuge tube Microtips and 1.5ml microfuge tubes Protocol:  Agrobacterium strain LBA4404 was grown over night at 28˚c in YEP medium containing 50 μgmlˉ¹ rifampicin.  The overnight culture was then chilled on ice for 30 mins.  It was then transferred into prechilled 15ml falcon tubes and was then centrifuged at 4000 rpm at 4ºC for 10 mins.  The supernatant was then discarded and the pellet was dissolved in 10ml 100mM ice cold CaCl2.  The tubes were then incubated on ice for 20 mins.  Later it was centrifuged at 4000 rpm at 4ºC for 10 mins.  The supernatant was then discarded and the pellet was gently resuspended in 2 ml 100mM ice cold CaCl2.
  8. 8. 8  Aliquots of 200µl were transferred to prechilled 1.5 ml tubes and was then used for transformation. 3.1.2 Transformation of pCAMBIA 1305.2 vector into Agrobacterium tumefaciens strain LBA4404 by free-thaw method Transformation broadly means uptake of any DNA molecule especially plasmid by living cell like bacteria. Agrobacterium cells that are soaked in CaCl2 solution affects only DNA binding, and not the actual uptake into cell. The actual movement of DNA into competent cells is stimulated by briefly raising the temperature to 37˚c by heat shock treatment. Materials: Competent cells (200µl) Plant transformation vector – pCAMBIA 1305.2 YEP broth YEP agar plates with Kanamycin and Rifampicin each 50μg/ml. Sterile Microtips IPTG X-Gal Protocol:  Plasmid DNA, 500ng (3μl) was added to the tube of competent cells of Agrobacterium.  This was then mixed well and incubated in -20 ºC for 30 mins.  Heat shock treatment was then given by immediately transferring into 37ºc water bath for 5 mins and was soon placed on ice for 5 mins.  YEP broth, 800 μl was added and then was incubated at 28ºC for 3-4 hrs.  After incubation the culture was spread plated YEP plate with Rif + Kan containing IPTG and X-Gal  These plates were then incubated at 28ºC for 2 days. 3.1.3 Selection of transformed colonies Selection of transformed colonies was done by picking the blue colored colonies. The selected colonies were then again grown on YEP agar plates with Rif + Kan by streaking. These
  9. 9. 9 plates were then incubated at 28ºC for 2 days. Then the transformed colonies were confirmed by plasmid isolation. 3.1.4 Isolation of plasmid from transformed colonies containing the pCAMBIA 1305.2 vector Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs. The term 'plasmid' was introduced by an American molecular biologist Joshua Lederberg. Plasmids are considered as transferrable genetic elements or 'replicons'. They are actually naked DNA. Plasmids are important tools in genetic and biotechnology labs where they are commonly used to multiply or express particular genes. Plasmids are also used to make large amounts of proteins. Plasmids encoding Zinc Finger Nucleases are used to deliver therapeutic genes to a preselected chromosomal site with a frequency higher than that of random integration. Mainly there are two types of plasmids: conjugative and non conjugative. Conjugative plasmids have tra- genes (tra-transfer) and can perform conjugation. Non conjugative plasmids cannot perform conjugation. There is an intermediate class of plasmid called mobilizable plasmid. Mobilizable plasmid can carry only a subset of genes required for transfer. They can parasitize a conjugative plasmid transferring at high frequency only in its presence. 3.1.4.1 Plasmid Isolation by Mini-Prep Method Mini-Prep method is commonly used protocol for plasmid isolation. Materials: Bacterial culture YEP broth with kanamycin Solution I (Suspension Buffer)
  10. 10. 10 Solution II (Lysis Buffer) Solution III (DNA neutralization buffer) Sterile Microtips Sterile microfuge tubes Isopropanol 70% ethanol Protocol: 1. Five ml of sterile medium was incubated with a single bacterial colony and kept overnight at 280C. 2. Next day, 2 ml of bacterial culture was taken in microfuge tubes and centrifuged for 10 min at 10,000 rpm. 3. The supernatant was decanted till the last drop. 4. The cells were in 100μl of ice cold DNA suspension buffer (Solution I). The bacterial cells were completely suspended by vortexing until no cells clumps remain. 5. DNA Lysis buffer (Solution II) 200μlwas added and mixed gently by inverting 3-4 times. 6. Immediately 150μl of prechilled DNA neutralization buffer (Solution III) was added. It was then mixed immediately by gently inverting the vial 3-4 times and incubated in ice for 10 min. 7. The tubes were then centrifuged at 10,000 rpm for 12 min at 40C. The clear supernatant containing plasmid was collected into a fresh centrifuge tube. 8. If the supernatant was turbid, it was re-centrifuged at 10,000 rpm for 10 min at 40C. The cleared supernatant containing plasmid was collected into a fresh centrifuge tube. 9. Equal volume of isopropanol was added to the supernatant and was mixed properly. 10. It was then centrifuged at 10,000 rpm for 15 min at room temperature. Supernatant was discarded and the pellet was washed with 70% ethanol. 11. The pellet was dried at 370C for 10 min and was then suspended in 50-100μl of glass- distilled water (or) nuclease free water.
  11. 11. 11 3.2 STANDARDIZATION OF GENETIC TRANSFORMATION PROTOCOL FOR Helianthus annuus L. 3.2.1 Sterilization of seeds Materials: 1% Bavistin 0.1% HgCl2 Steriile petri dishes Germination sheets (Sterile) Sterile forceps and conical flasks Sterile distilled water Protocol:  The seeds of sunflower were soaked overnight in distilled water and were surface sterilized first with 1% Bavastin for 10 mins.  It was followed by distilled water wash thrice.  Later it was treated with 0.1% HgCl2 for 30 seconds and washed thoroughly with distilled water.  The seeds were then transferred onto sterile petri plates with moist germination sheets and was allowed to germinate on petriplates 30˚C in dark.  Two-day old seedlings were taken as explants for Agrobacterium infection. 3.2.2 Transformation of sunflower seeds Materials: Agrobacterium strain LBA4404 + pCAMBIA1305.2 culture YEP broth with Kanamycin ½ MS media Soilrite Sterile Tissue culture bottles Sterile forceps and conical flasks Sterile distilled water
  12. 12. 12 Protocol:  A loopful of Agrobacterium strain LBA4404 containing pCAMBIA1305.2 culture was inoculated in 20 ml YEP media supplemented with kanamycin for overnight  Overnight culture, 5ml was resuspended into 100 ml of YEP with kanamycin media and incubated for 2 days.  The culture was centrifuged and the pellet was resuspended into 100 ml ½ strength MS media and acetosyringone to final concentration of 50µM was added and incubation was continued at 28˚c for 5 hours.  The seeds with emerging plumule were infected by separating the cotyledons without damaging the meristem with a sterile sewing needle.  Subsequently the seeds were dunked into Agrobacterium culture in ½ MS media and was incubated at 28˚c for 60 mins.  Then the seedlings were washed with Sterile water and few of them were transferred into autoclaved soilrite and remaining few in ½ MS agar for germination under aseptic condition (5 seedling per Jar).  The growth chamber was maintained at 26-28˚c under photoperiod of 14hours with florescent light of intensity of approximately 2500lux. 3.3 SCREENING OF TRANSGENIC Helianthus annuus L. 3.3.1 GUS assay Plants contain endogenous β-galactosidase activity, so lacZ is not generally a useful reporter gene for plants. A widely used reporter gene in plants is the uidA, or gusA, gene that encodes the enzyme β-glucuronidase (GUS). This enzyme can cleave the chromogenic (colorgenerating) substrate X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronic acid; Fig. 2), resulting in the production of an insoluble blue color in those plant cells displaying GUS activity. Plant cells themselves do not contain any GUS activity, so the production of a blue color when stained with X-gluc in particular cells indicates the activity of the promoter that drives the transcription of the gusA-chimeric gene in that particular cell. The GUS assay is easy to perform, sensitive, relatively inexpensive, highly reliable, safe, requires no specialized equipment, and is highly visual (Jefferson 1987; Jefferson et al. 1987; Jefferson and Wilson 1991)
  13. 13. 13 3.3.1.1 Expression of β-glucuronidase Materials: 0.1% HgCl2 GUS staining solution. Sterile distilled water 75% ethanol Protocol:  Five days old transformants were screened for expression of β-glucuronidase.  For analysis of the transformants, tissue that were tested and found free of residual Agrobacterium were used.  The persistence of Agrobacterium in the putative transformants was largely controlled by a brief agitation of the co-cultivated seedlings with 0.1% HgCl2 for 30s followed by through washes with distilled water.  The method of Jefferson (1987) was used to assess histochemical assay of uid A gene expression in the tissues of primary transformants,  The complete seedlings as such were incubated overnight at 37˚C in GUS staining solution.  The next day they were washed in water and later soaked with 75% ethanol to clear chlorophyll.
  14. 14. 14 CHAPTER 4 RESULT AND DISCUSSION 4.1 TRANSFORMATION OF pCAMBIA 1305.2 VECTOR INTO Agrobacterium tumefaciens STRAIN LBA4404 The current study was initiated by transforming pCAMBIA 1305.2 vector into Agrobacterium tumefaciencs strain LBA4404. For this standard method of competent preparation was followed and competent cells for Agrobacterium tumefaciencs strain LBA4404 was prepared. The prepared competent cells were used for transformation of the plasmid vector. The transformation of plasmid into Agrobacteium was performed by freeze thaw method which is also called as heat shock method. Successfully, 330 transformed colonies were obtained (Fig. 4.1). The transformation efficiency was calculated as 2.75x103 transformants / μg DNA. Further all the point colonies obtained on the selection plate were blue in colour since IPTG and X-Gal were added onto the plate for screening of transformants. The presence of blue colored colonies meant that the plasmid did not have any gene inserted within its Multiple Cloning Site (MCS) region and thus the LacZ gene was functional and was able to utilize the X-Gal in presence of IPTG, thus releasing the chromogenic blue substrate resulting in blue colour. 4.2 STANDARDIZATION OF INPLANTA GENETIC TRANSFORMATION PROTOCOL FOR Helianthus annuus L. The method of seed sterilization published by Keshamma et al., (2008) was modified and used for this study. The sterilization procedure was repeated thrice with different number of seeds and resulted in overall percentage of germination as 84%. Also it was found the overall percentage of contamination was 1.33%. Thus this protocol for sterilization of seeds proved to be efficient as it resulted in lesser contamination with greater percentage of germination. The efficiency of this protocol, after considering the rate of germination as well as contamination resulted as 82.16%. Data shown in Table 4.1. The overall transformation efficiency for the inplanta transformation method that we adopted was 11.32%. But the transformation efficiency for the plants cultured in ½ MS media grew better and gave better results with transformation efficiency of 19.35%. The lower overall percentage of transformation was due to the transformants grown in soilrite. The growth in all the plants after transformation which were grown in soilrite was stagnant. This was because the
  15. 15. 15 roots of the plants put in the soilrite started decaying and hence the plants did not grew and resulted in no transformation. So ½ MS proved to be a better medium of potting soon after transformation in invitro studies. Data shown in Table 4.2 and Fig. 4.2. 4.3 Screening of transgenic Helianthus annuus L. Though the transformation efficiency was less, still the GUS staining showed that there was uniform expression of GUS in the transformed plants above the hypocotyls (Fig. 4.3). In the present study, a tissue culture-independent in planta transformation protocol was used to develop transformants (Rohini and Sankara Rao, 2000a; Rohini and Sankara Rao, 2000b; Rohini and Sankara Rao, 2001). Such in planta transformation techniques have also been standardized in other crops like, buckwheat (Kojima et al., 2000), mulberry (Ping et al., 2003), kenaf (Kojima et al., 2004), soybean (Chee et al., 1989) and rice (Supartana et al., 2005) etc. In this method, Agrobacterium is targeted to the wounded apical meristem of the differentiated seed embryo. Therefore, Agrobacterium tumefaciens transfers the gene into the genome of diverse cells which are already destined develop into specific organs and the meristematic cells still to be differentiated. This results in the primary transformants being chimeric in nature. The method therefore is advantageous because it avoids the need for tissue culture. Nevertheless, transformability depends on the susceptibility of the variety to Agrobacterium. A number of factors affect transformability by inplanta transformation. Finally, all these factors including stability of the transgene can only be assessed after getting the T1 and T2 generations.
  16. 16. 16 No. of Seeds (a) No. of Seeds Contaminated (b) No. of Seeds Germinated (c) Percentage of Contamination (b/a*100) Percentage of Germination (c/a*100) Efficiency [(c- b)/a*100] I 18 1 18 5.5% 100% 94.44% II 30 0 22 0.0% 73.33% 73.33% III 27 0 23 0.0% 85.16% 85.16% T 75 1 63 1.33% 84% 82.76% Table 4.1 Efficiency of the sterilization protocol after considering the rate of germination and rate of contamination Media No of seedling used for transformation (a) No. of transformed seedlings (b) No. of seedlings not transformed (c) Transformation percentage (b/a*100) Soilrite 35 0 35 0.00% ½ MS 18 6 12 33.33% Total 53 6 47 11.32% Table 4.2 Total inplanta transformation percentage.
  17. 17. 17 Fig. 4.1a Fig. 4.1b Kanamycin resistant blue colonies of Negative control plate without any growth of Agrobacteriumtumefaciens transformed competent cells due to the presence of with pCAMBIA 1305.2 antibiotic Kanamycin Figure 4.1: Transformation of pCAMBIA 1305.2 vector into Agrobacterium tumefaciens Strain LBA4404
  18. 18. 18 Figure 4.2. Sunflower seedlings after inplanta transformation Seedlings to the left are control plants and the one to the right are putative transformants (a) (b) Figure 4.3. Expression of GUS enzyme (a) Control showing no blue colour stain after GUS staining (b) Transformats stained blue above hypocotyls after GUS staining
  19. 19. 19 REFERENCES  Bottinger P., Steinmetz, Schieder O. and Pickardt T. 2001. Agrobacterium-mediated transformation of Vicia faba. Molecular Breeding Volume 8, Issue 3, pp 243-254  Burrus M., Molinier J., Himber C ., Hunold R., Bronner R., Rousselin P. and Hahne G. 1996. Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns. Molecular Breeding, Volume 2, Issue 4, pp 329-338  Chee, P.P., A.K. Fober, and L.J. Slightom, 1989. Transformation of soybean (Glycine max (L.) Merrill) by infecting germinating seeds with Agrobacterium tumefaciens, Plant Physiol. 91: 1212-1218.  Feldmann K. A and Marks M.D. 1987,Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach. Molecular and General Genetics Volume 208, Issue 1-2, pp 1-9  Fursova.O, Pogorelko.G and Zabotina.O.A 2012. An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall, Annals of Botany, 1-10.  Heweli.T, Alibert.G and Kallerhoff.J 2004. Genetic transformation of sunflower (Helianthus annuus L.) transgenic Crops of the World p435-451.  Jefferson, R. A., T. A. Kavanagh, and M.W. Bevan. 1987. GUS fusions: β- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6: 3901- 3907.  Jefferson, R.A. 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molecular Biology Reporter 5: 387-405.  Jefferson, R.A., and K.J. Wilson. 1991. The GUS gene fusion system. In Gelvin, S.B. and R.A. Schilperoort (eds.) Plant Molecular Biology Manual, Kluwer Academic Publishers (Dordrecht). B14: 1-33.
  20. 20. 20  Jones.H.D, Doherty.A and Wu.H 2005. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat, Plant method 2005, 1:5 doi:10.1186/1746-4811-1-5.  Keshamma.E, Rohini.S, Rao.K.S, Madhusudhan.B, and kumar.M 2008.Tissue culture-independent inplanta transformation strategy: An agrobacterium tumefaciens- mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.) The Journal of Cotton Science 12:264-272.  Khan M. M. robin A. B. M. A. H. K, nazim-ud-dowla.M.A.N, Talukder. S. K. and Hassan.L 2009. Agrobacterium-mediated genetic transformation of two varieties of brassica: Bangladesh J. Agril. Res. 34(2) : 287-301.  Kojima, M., H. Shioiri, M. Nogawa, M. Nozue, D. Matsumoto, A. Wada, Y. Saiki, K. Kiguchi, 2004. In planta transformation of kenaf plants (Hibiscus cannabinus var. aokawa no.3) by Agrobacterium tumefaciens, J. Biosci. Bioeng. 98: 136-139.  Kojima, M., Y. Arai, N. Iwase, K. Shiratori, H. Shioiri, and M. Nozue, 2000. Development of a simple and efficient method for transformation of buckwheat plants (Fugopyrum esculentum) using Agrobacterium tumefaciens, Biosci. Biotechnol. Biochem. 64: 845-847.  Kumar.M, shukla.A.K, singh.H, verma.P.C and Singh.P.K 2013. A Genotype- independent agrobacterium mediated transformation of germinated embryo of cotton (Gossypium hirsutum L.), International Journal of Bio-technology and Research (IJBTR) vol.3,issue1,81-90.  Lappara H., Burris M., Hunold R., Damm B., Bravo-Angel A.M., Bronner R., and Hahne G. 1995, Expression of foreign genes in sunflower (Helianthus annuus L.) — Evaluation of three gene transfer methods. Euphytica Volume 85, Issue 1-3, pp 63-74  Liu.H, Xie.X, Sun.S, Zhu.W, Ji.J, Wang.G 2011. Optimization of agrobacterium- mediated transformation of sunflower (Heliathus annuus L.) AJCS(12):1616-1621.  Mukhopadhyay A., Arumugam N., Nandakumar P.B.A., Pradhan A.K., Gupta V and Pental D. 1992. Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration. Plant Cell Reports, Volume 11, Issue 10, pp 506-513
  21. 21. 21  Ping, L.X., M.Nogawa, M. Nozue, M. Makita, M.Takeda, L. Bao and M. Kojima, 2003. In planta transformation of mulberry trees (Morus alba L.) by Agrobactetium tumefaciens, J. Insect Biotechnol. Sericol. 72: 177-184.  Piqueras.A, Alburquerque.N and Folta K.M 2010.Explants used for the Generation of transgenic plants. DOI 10.1007/978-3-642-04809-8_2.  Radonic.L.M, Zimmermann.J.M, Lopez.D.Z.N, Bilbao.M.L 2008. Introduction of antifungal genes in sunflower via Agrobacterium. Electronic Journal of Biotechnology ISSN:0717-3458.  Rao.K and Rohini.V.K 1999. Agrobacterium-mediated transformation of Sunflower (Helianthus annuus L.): Annals of Botany 83: 347-354.  Rohini, V.K. and K. Sankara Rao, 2000a. Transformation of peanut (Arachis hypogeae L.): a non-tissue culture based approach for generating transgenic plants, Plant Sci. 150:41-49.  Rohini, V.K., and K. Sankara Rao, 2000b. Embryo transformation, a practical approach for realizing transgenic plants of safflower (Carthamus tinctorius L.), Annals of Botany 86: 1043-1049.  Rohini, V.K., and K. Sankara Rao, 2001. Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease, Plant Sci. 160 (5): 883-892.  Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989. Molecular cloning, Plain view, New York, Cold Spring Harbor Laboratory Press.  Schrammeijer.B, Sijmons.P.C, Van.P.J.M Elzen and Hoekema.A 1990. Meristem transformation of sunflower via Agrobacterium,volume 9,Issue2 ,pp 55-60.  Sujatha.M, Vijay.S, Vasav.S, Veera.P Reddy, Rao.S 2012. Agrobacterium-mediated transformation of cotyledons of the mature seeds of multiple genotypes of sunflower (Helianthus annus L.) Plant Cell Tiss Organ Cult 110:275-287.  Supartana, P., T.Shimizu, Shioiri.H, M. Nogawa, M. Nozue, and M. Kojima. 2005. Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens, Journal of Bioscience and Bioengineering 100(4): 391-397.
  22. 22. 22  Theriappan.P and Gupta.A.K 2014. Development of a protocol for Agrobacterium mediated transformation of Brassica oleraceae Lvar botrytis cv Early Kunwari, European Journal of Biotecnology and Bioscience 1(3):34-38.  Weber S., Friedt W., Landes N., Molinier J., Himber C., Rousselin P., Hahne G. and Horn R. 2003. Improved Agrobacterium -mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication. Plant Cell Reports Volume 21, Issue 5, pp 475-482  Weber.S, Friedt.W, Landes.N, Molinier.J, Himber.C, Rousselin.P, Hahne.G, Horn.R 2003. Improved Agrobacterium-mediated transformation of sunflower (Hellianthus annuus L.): assessment of macerating enzymes and sonication, Plant cell Rep 21:475-482.  Zombori.Z, Szecsenyi.M, Gyorgyey.J 2011. Different approaches for Agrobacterium- mediated genetic transformation of Brachypodium distachyon, a new model plant for temperate grasses,volume 55(1):193-195.
  23. 23. 23 APPENDIX 1. SOLUTIONS FOR BACTERIAL TRANSFORMATION 100mM CaCl2 Dissolve 1.4702 g of CaCl2 in 20 ml of sterile water. Filter sterilize it and add it it 80 ml of sterile water. YEP Broth Bacto Peptone 10.0 g Yeast Extract 10.0 g NaCl 5.0g YEP Agar Bacto Peptone 10.0 g Yeast Extract 10.0 g NaCl 5.0g Bacto Agar 15.0 g H2O 1000 ml IPTG Stock 1M Dissolve 0.238g of IPTG in 1ml of sterile distilled water. Store it in -20ºC X-Gal (20%) Dissolve 200mg X-Gal in 1 ml of Dimethyl sulphoxide. Store it in -20ºC 2. SOLUTIONS FOR PLASMID ISOLATION Solution I (Suspension Buffer) 50mM Glucose 10mM EDTA 25mM Tris cl pH 8.0 100µg/ml RNase A Solution II (Lysis Buffer) 0.2N NaOH
  24. 24. 24 1% SDS Solution III Dissolve 29.45g of Potassium acetate in 80 ml of distilled water. Add glacial acetic acid till the pH reaches 5.5. Make up the volume to 100ml and sterilize by autoclaving. Store in 4 ºC TE Buffer 10mM Tris cl pH 8.0 1mM EDTA 3. SOLUTIONS FOR INPLANTA TRANSFORMATION 1% Bavastin Bavastin 1g Distilled Water 100 ml 0.1% HgCl2 HgCl2 100mg Distilled Water 100 ml GUS staining solution 0.1M phosphate buffer-pH 7.0 2mM X-Gluc 5mM Potassium Ferricyanie 5mM potassium Ferrocyanide 0.1% Triton X 75% Ethanol Measure 75 ml of ethanol using a measuring cylinder. Make the volume to 100ml by adding sterile distilled water. 4. ANTIBIOTIC STOCK SOLUTIONS Kanamycin stock (50mg/ml) Dissolve 50 mg of Kanamycin in 1ml of Sterile water. Rifampicin stock (50 mg/ml)
  25. 25. 25 Dissolve 50mg of Rifampicin in 500μl of methanol and make up the volume to 1ml using sterile distilled water. 5. MS MEDIA COMPOSITION Constituents Mg/l g/1 20x Volumes to be taken 500ml 1 lit MACRONUTRIENTS MgSO4.7H2O CaCl2.2H2O KNO3 NH4NO3 KH2PO4 370 440 1900 1650 170 3.7 4.4 19 16.5 1.7 (500ml) 3.7 4.4 19 16.5 1.7 25ml 50ml MICRONUTRIENTS MnSO4.4H2O(or) MnSO4.H2O ZnSO4.7H2O CuSO4.5H2O COCl2.6H2O H3BO3 Na2MOO4.2H2O (1000x) 22.3 16.9 8.6 0.025 0.25 6.2 0.25 0.223 0.169 0.086 0.00025 0.00025 0.062 0.0025 100(100ml) 2.23 1.69 0.86 0.0025 0.0025 0.62 0.025 0.5ml 1ml IRON SOURCE FeSO4.7H2O Na2 EDTA 27.85 37.25 278.5 372.5 140x(100ml) 0.2785 0.3725 0.5ml 1ml Carbohydrate Source Sucrose 30-50(g/l) 30g/l Constituents Mg/l Stock (1000x) Volume to be taken 500ml 1000ml VITAMINS Nicotinic acid Pyridoxine HCl Thiamine HCl 0.5 0.5 0.5 50ml 0.025g 0.025g 0.025g 0.5ml 1ml KI Stock Solution Dissolve 83mg of KI in 100 ml Double distilled water. Take 1 ml KI solution for 1 liter media
  26. 26. 26 Stock solution of Meso-inositol (x500) Dissolve 1gm meso-inositol in 20 ml of Water. Take 1mlfor 1 liter media Stock solution of Glycine (x1000) Dissolve 40 mg glycine in 20 ml of double distilled water. Take 1ml for 1 liter madia 1N HCl solution Take 8.3 ml of HCl and make up the volume to 100ml using distilled water. 1N NaOH solution Dissolve 4g NaOH in 100 ml water.

×