1. Generation of transgenic plant
LECTURE 9
Generation of transgenic plant
Dr. Aparna Islam

2. Generation of Transgenic Plants
Plant regeneration technology
Designing vector with the transgenes
» Promoter
» Manipulation of gene expression integration vs. transient expression
» Targeting gene (genome/ chloroplast)
Gene Transfer to Plant Cell
» Direct DNA transfer
» Agrobacterium-mediated gene-transfer
Regeneration and selection of transformants

3. Plant Regeneration Protocol
Explant
Organogenesis
Direct
Indirect
Shoot
Root
Plantlet
Callus
Embryogenesis
Embryo Induction
Embryo development
Maturation
Germination

4. Organogenesis

5. Embryogenesis

6. Induction, development, maturation
How does plant tissue culture fits with plant transformation technology?
Embryogenesis
Transformed explant
Vector construct
Transgenic Agrobacterium production
Direct
Indirect
Organogenesis
Putative transgenic plants
Selection
Transformation
So. Plant tissue culture provide the basis for plant transformation. Exception is in planta transformation
Explant
Indirect
Organogenesis
Direct
Shoot
Root
Plantlet
Embryogenesis
Embryo
Induction, development, maturation

7. In Planta Transformation
In planta is a indirect transformation technique which is mediated by Agrobacterium tumefaciens and tissue culture independent.
It is necessary for the plants which are in vitro recalcitrant plants and have no strong regeneration and tissue culture protocol.
Approaches:
Target the meristem or tissues giving rise to gametes for transformation

8. Advantage: Disadvantages: it avoids the tissue culture method
special skill and special aseptic conditions required if tissue culture technique fully avoided
rapid screening of transformant
avoid somaclonal variation
thousands of transformants were produced in a few year.
Disadvantages:
Difficult to produce genotype consistently
Low expression of transgenes
Low transformation frequency
Plant Physiol 124: (2000)

9. A. Seed treatment: (K. Feldman and D. Marks 1987)
Method: Incubated Arabidopsis seeds with Agrobacterium, grew the plants to maturity without any selection, collected progeny seeds and germinated them on antibiotic media to identify transformed plants.
Advantages:
1. Avoided somaclonal variation, but difficult to reproduce genotype consistently
2. Thousands of transformants were produced in a few years
3. Helped speed gene cloning by Arabidopsis researchers.

10. B. “Clip ‘n Squirt” methods (Chang et al., 1994; Katavic et al., 1994)
Method: Clipped off the reproductive inflorescences, applied Agrobacterium to the center of the rosette. New inflorescences formed a few days later, was removed again and Agrobacterium re-applied. Next set of inflorescences maintained. Plants were allowed to develop and set seed.
Advantages & Disadvantages:
More reliable than the seed treatment method, but transformation efficiency was only marginally better than traditional tissue culture based methods.

11. C. Vacuum infiltration method (Bechtold et al., 1993)
Method: Arabidopsis plants at the reproductive stage were uprooted and placed into a bell jar in a solution of Agrobacterium. Vacuum was applied and then released – causing air trapped within the plant to bubble off and be replaced with the Agrobacterium solution. Plants were transferred back to soil and allowed to set seed. Seeds were screened on antibiotic media.
Transformation rate: 0.4-1%

12. D. Arabidopsis Floral Dip Transformation (Clough and Bent, 1998)
Method: Simplified medium {with sucrose and a surfactant (Silwet L-77)} used for infiltration of floral tissues at an early stage of flowering. Penetration of Agrobacteria in plant tissues can be achieved without vacuum. Grow the plants to seed and screen seeds under antibiotic selection.
Advantages:
Extremely simple, widely used by Arabidopsis researchers, avoids somaclonal variation, no tissue culture skills required, no need for plant regeneration.

13. Arabidopsis Floral Dip Transformation

14. E. Whole Embryo infection
Method: Mature embryo is pricked and dipped in Agrobacterium solution. Without co-culture the embryos are transferred to vermiculite to geminate into a whole plant. Seeds are collected which are screen under antibiotic selection.
Advantages: No need for regeneration protocol, extensively used for recalcitrant species.

15. Generation of Transgenic Plants
Plant regeneration technology
Designing vector with the transgenes
» Promoter
» Manipulation of gene expression integration vs. transient expression
» Targeting gene (genome/ chloroplast)
Gene Transfer to Plant Cell
» Direct DNA transfer
» Agrobacterium-mediated gene-transfer
Regeneration and selection of transformants

16. Designing the Vector LB RB
transgene consists of the following parts: a promoter (a DNA on/off switch), followed by a gene of interest, terminator, then another promoter, followed finally by a selectable marker gene, terminator.
Genes of interest: The trait we want to introduce
Selectable marker genes: two types of marker genes are used. Screenable and selectable marker gene.

17. Promoters
All working genes (genes that are "expressed") naturally have their own promoters that serve as on/off switches. To make the gene of interest work, to have it expressed, there are several kinds of promoters from which to choose:
1. Constitutive promoters: will be "on" at all times, in all tissues of the plant body, and in all kinds of plants (both monocots and dicots); will result in high levels of gene expression; the most common is the “CaMV35s" promoter derived from the Cauliflower Mosaic Virus (a virus that targets plants). In monocot sometime this CaMV35s doesn’t work efficiently. In that case other constitutive promoters, like, actin 1 (in rice), ubiquitin 1 (in maize) promoters are used.

18. Sometime constitutive promoter cause deleterious effect on transgenic plants due to continuous expression of transgenes. In such cases more regulated and controlled expression is needed. For this reason many more types of promoters have been identified and in use.
2. Tissue specific promoters: will express the gene only in one specific tissue, such as fruits, seeds, leaves etc.
3. Time specific promoters: will express the gene only at a specific time in the plant's life cycle, such as, germination or flowering or fruit senescence (ripening).
4. Regulated promoters: will express the gene only under specific conditions--such as heat, light, or lack of moisture (draught)--or only in the presence of specific biochemical signals, such as, those that naturally occur when the plant is injured or damaged.

19. Expression of Transgenes
Expression of the transgene can be of two kinds
Transient expression
Lots of DNA taken up, BUT no incorporation of exogenous DNA into the genome, most degraded before integration, transgene express at extra-chromosomal state.
» Molecular farming – transient
2. Stable expression
Incorporation of exogenous DNA into a plant genome, incorporated in host nuclear DNA, nuclear transformation, plant regeneration and expression of the transgene(s) through the life cycle of the plant.
» Development of resistance in plant - stable

20. Generation of Transgenic Plants
Plant regeneration technology
Designing vector with the transgenes
» Promoter
» Manipulation of gene expression integration vs. transient expression
» Targeting gene (genome/ chloroplast)
Gene Transfer to Plant Cell
» Agrobacterium-mediated gene-transfer
» Direct DNA transfer
Regeneration and selection of transformants

21. Plant Transformation Method
i. Agrobacterium-mediated
ii. Direct method

22. Generation of Transgenic Plants
Plant regeneration technology
Designing vector with the transgenes
» Promoter
» Manipulation of gene expression integration vs. transient expression
» Targeting gene (genome/ chloroplast)
Gene Transfer to Plant Cell
» Agrobacterium-mediated gene-transfer
» Direct DNA transfer
Regeneration and selection of transformants

23. Selection for Transformed Plants
Those plant cells that have successfully received the T-DNA from the Agrobacterium strain or a complete transgene from the bombardment of gold pellets need to be identified out of the many that did not.
There are two basic strategies:
1. Display of a reporter gene. (screenable marker or reporter gene)
2. Survival of transformed cells (or young plants) on a selection medium. (selection marker genes)

25. Screenable Marker: GUS gene
During the mid 1980's E. coli ß-glucuronidase gene (uidA) was recognized as an alternative to several other marker (reporter) genes.
ß-glucuronidase (ß – D - glucuroniside glucuronosohydrolase) is an acid hydrolase that catalyses the cleavage of a wide variety of ß-glucuronides.
Substrates for ß-glucuronidases are generally water-soluble, and many substrates are commercially available, including substrates for spectrophotometric, fluorometric, and histochemical analyses.

26. GUS Assay
The expression of the ß-glucuronidase gene will be determined by the use of a synthetic substrate for the enzyme, X-Gluc (5-bromo-4-chloro-3-indolyl-glucuronide). ß-glucuronidase catalyses the oxidative dimerization of X-Gluc, and the product of this reaction is an insoluble blue ClBr-indigo which precipitates in the cytoplasm. Transformants can thus be identified by their blue tissue.
In most plant tissues no background endogenous enzyme activity is found.
It is still wise to include negative control tissue in every experiment.

27. gus-staining of a transgenic plantlet (chimeric) (Phaseolus acutifolius)
gus-staining in epicotyls (pea):

28. GFP-assay (GFP= green fluorescent protein from jelly fish)
Cloned from the jellyfish Aequoria victoria.
27 kDa monomer that fluorescences green under UV (365nm) or blue (490) light
Must be highly expressed

29. GFP-assay (GFP= green fluorescent protein from jelly fish)

30. Learn yourself
What is the reaction of GUS and GFP?
Comparison between GUS and GFP marker genes?

34. Kanamycin Selection in Eggplants

35. What else do we have to consider?
1. Molecular evidence for the transgenic nature of the transformed plants
DNA level: PCR, Southern blot hybridization
RNA level: Northern blot hybridization, RT-PCR
2. Expression of transgenes
Protein level: Western blot analysis, ELISA, Bioassay of the transgene (to see the effectiveness of the transgene)
3. Transmittance of transgenes to subsequent generations (chimaeras??)
Germination, molecular and expression analysis through data analysis (Biostatistics).

36. Transformation of peanut (Arachis hypogaea L
Transformation of peanut (Arachis hypogaea L.): a non-tissue culture based approach for generating transgenic plants
V.K. Rohini, K. Sankara Rao
Plant Science 150 (2000) 41–49

37. Bacterial strains and vectors
Plant material
Seeds of peanut cultivar TMV-2 were soaked overnight
The next day surface sterilized with 0.1% mercuric chloride for 5– 7 min
followed by thorough rinses with sterile water
Embryos with one of the cotyledons cut off at the site of attachment to the primary axis
incubated on semisolid MS basal medium for two days prior to infection
Bacterial strains and vectors
Agrobacterium strain LBA 4404
Binary vector pKIWI105 having β glucuronidase (uid A) and neomycin phosphotransferase (npt II)
driven by CaMV 35S and nopaline synthase promoters, respectively.

38. Infection with Agrobacterium and recovery of transformants
The Agrobacterium strain LBA 4404:pKIWI105 was grown overnight at 29– 30°C in LB medium (pH 7.0) containing 50 mg/ ml kanamycin.
The bacterial cells ere later re-suspended in Winans’ AB medium (pH 5.2) and grown for 18 h.
Wounded tobacco leaf extract was later added to this suspension.
The embryo axes were pricked randomly with a sterile sewing needle and dunked in the suspension of Agrobacterium in Winans’ AB medium
The infection was carried out by gentle agitation at 28–30°C.
The embryos were blot-dried, washed thoroughly with 500 mg/ml of cefotaxime for 18 h and placed on autoclaved gelrite for germination under aseptic conditions in capped bottles.

39. After 5–6 days, the germlings were transferred to soilrite in pots and the seedlings were followed to grow under growth room conditions for at least 10 days before they were transferred to the greenhouse.
The pots were initially covered with polythene bags to maintain humidity.
The growth chamber was maintained at 26–28°C under a 14 h photoperiod with fluorescent light of intensity 35 mmol m2 s1.
For each experiment 50 embryos were infected and the experiments were repeated thrice.
Various transformation conditions viz., infection time, effect of acetosyringone and addition of wounded tobacco leaf extracts on transformation efficiency were evaluated.
Wounded tobacco leaf extracts preparation: Winans’ medium (100 ml) added with wounded tobacco leaf extract (2 g in 2 ml sterile water) and a 16 h infection time were used for all the transformation experiments.

40. Optimization of culture conditions and development of transformation protocol
The feasibility of the transformation strategy adopted in the study was initially evaluated by the number of peanut embryo axes germinating into normal seedlings following wounding by excision of one cotyledon and by pricking with a needle.
infection with Agrobacterium and decontamination treatment
Preliminary experiments to develop transformation protocol involved optimization of infection time and vir gene induction treatments to enhance the transformation efficiency.

41. Effect of infection time on the transient GUS expression in peanut cultivar TMV-2.
Maximum number of embryos expressing GUS when infection was carried out for 16 and 24 h. Only two to four embryos per ten sampled have expressed GUS when infected for two and four hours. GUS expression increased with increased time of infection. It attained a plateau when the
infection as for 16 h. Though the blue sectors appeared were more with 24h infection, a 16 h period was chosen for further transformation experiments because of a reduction in the germination rate observed on infection for 24 h or longer.
As the infection treatment given was for an extended period, and that any further exposure of explants to bacteria appeared deleterious, a discrete co-cultivation step that generally follows infection was not included in the protocol.

42. The results of different vir gene induction treatments
Infection of peanut with Agrobacterium previously treated with acetosyringone improve transformation efficiency (Fig. up).
The wounded tobacco leaf extract added to the AB induction medium enhanced the transformation efficiency in terms of the number of embryos expressing GUS.
Winans’ medium (100 ml) added with wounded tobacco leaf extract (2 G in 2 ml sterile water) and a 16 H infection time were used for all the transformation experiments.
Embryos of peanut cv. TMV-2 when infected in the absence of acetosyringone or tobacco leaf extract did not show GUS expression.
Effect of varying concentrations of acetosyringone on the transformation efficiency in peanut cultivar TMV-2.
Effect of varying quantities of tobacco leaf extract on transformation efficiency in peanut cultivar TMV-2.

43. Kanamycin susceptibility of the non-transformed embryos:
In the experiments performed initially to determine the tolerance to kanamycin, it was observed that uninfected embryos (control) did not germinate beyond 150 mg/ml of kanamycin. Further, there was a reduction in germination rate when wounded uninfected as well as infected embryos were germinated in the presence of kanamycin. Therefore, selection on kanamycin was eliminated.
Thirty percent of the seeds survived wounding, infection and germinated into healthy plants with 16 h of infection.
The seedlings transferred to pots were initially covered with polythene bags while they were grown in the culture incubation room and before they were shifted to the greenhouse.
GUS expression in the tissues of peanut cultivar TMV-2 explanted at various stages of recovery of transgenic plants. (a) Embryo axis and the plumule, five days after infection (bar0.5 mm); (b) embryo axis of peanut cultivar JL-24 (bar4.5 cm); (c) leaflet from a month old plant (bar3.5 cm); (d) embryos with one cotyledon removed (T0, primary transformant; T1, progeny) (bar4.0 cm).

44. Six primary transformants resulted from three independent transformation events involving a total of 150 embryos.
Five plants, which survived hardening and transfer to greenhouse were analyzed for the presence and transmission of the transgenes. These plants have put forth healthy, green and expanded leaves.
The rate of growth of these infected seedlings was however slow when compared to uninfected seedlings, which might be due to wounding and prolonged infection with Agrobacterium.
Greenhouse established peanut transformants in peanut cultivar TMV-2. (a) A fertile plant of T0 generation; (b) plants of T1 generation.

45. Expression of marker genes
GUS enzyme activity was assessed in the tissues sampled from different phases of development of putative transformants.
Expression of GUS was also ascertained by Western blotting. Total proteins were extracted from about 1g of leaf issue of two months old transformants and resolved on a 10% polyacrylamide gel.
Immunostaining of the immobilized proteins was performed using the GUS antibody.
The expression of the npt II gene in the genome of the transformed peanut plants was checked by the NPT II SDS-PAGE assay.

46. Molecular analysis
Plant genomic DNA was isolated.
Plasmid DNA from A. tumefaciens strain LBA 4404:pKIWI105 and E. coli strain L-1 Blue:pUCGUS121 was prepared
For PCR analysis of the uid A gene in the genome of transformants, a 21 mer primer that amplifies a 514 bp fragment was employed.
The PCR was initiated by a hot start at 94°C for 4 min.
For Southern analysis, DNA (10 mg) was digested with the appropriate endonuclease, electrophoresed on a 0.8% agarose gel, and blotted on a nylon membrane (Hybond, Amersham). Dot blot was performed with uncut DNA (5 mg). The uid A gene probe was prepared from pUCGUS121 from E. coli strain XL-1 Blue, by releasing a 2.1-kb uid A fragment using Bam H1 and Eco R1. This fragment was labeled using the random primingkit supplied by Amersham. Hybridization signals were detected following exposure of X-ray film to the membrane for 16 h at 70°C.

47. Expression of the npt II gene
The NPT II assay performed, resulted in a signal at the expected position of 14 kDa with the total protein extracts of the primary transformant and the T1 progeny indicating co-integration, expression and transmission of npt II gene. There was no signal seen in case of uninfected peanut plants.
Fig. 6. Expression of neomycin phosphotransferase II gene.
(a) Expression in T0 plants. Lane 1: total protein extract from uninfected plant (negative control). Lane 2: total protein extract from a transformant. (b) Expression in T1 plants. Lane 1: total protein extract from uninfected plant. Lanes 2 and 3: total protein extract from the progeny of transformants.

48. Expression and inheritance of the uid A gene
The expression of uid A gene assayed in the greenhouse-established plants by Western blotting has shown a protein band at the expected position of 74 kDa in the total protein profile.
This band was not detected by the antibody in the total proteins of uninfected plants.
Western blot analysis of b–glucuronidase in T0 plants. Lane 1: purified GUS protein (20 mg) (Clonetech) positive control. Lane 2: total protein extract (50 mg) from a transformant. Lane 3: total protein extract (50 mg) from an uninfected plant.
Molecular analysis of the T0 plants
In PCR analysis expected size of 514 bp in length were amplified from the total DNA of the putative transgenic plants. These DNA fragments were not detected in the DNA of uninfected plants.
PCR of peanut cultivar TMV-2, T0 plants using a 21 mer primer, which amplifies a 514-bp uid A gene fragment. Lane 1: pKIWI105 DNA (positive control). Lane 2: DNA from the leaves of uninfected plant. Lanes 3–7: DNA from the leaves of T0 plants.

49. Dot blot analysis of DNA samples of the five PCR positive plants further confirmed the presence of the uid A gene in the primary transformants .
DNA (5 mg) from the five PCR positive T0 plants was loaded on a dot blot apparatus and probed with a 2.1-kb uid A gene fragment. Lanes 1–5: DNA from the five PCR positive T0 plants. Lane 6: positive control (pKIWI105 DNA). Lane 7: uninfected plant DNA (negative control).
Southern blot of uncut DNA of one of these plants gave a hybridization signal with a 2.1 kb uid A gene probe. This DNA when digested with Sma I gave a signal at the 10 kb position (lanes 1 and 7). A hybridization signal was not obtained with the DNA from the non-transformed plant (lanes 6 and 10). The protocol facilitated recovery of transformants in as short a time as 4 weeks.
Lanes 1 and 7: uncut and Sma I digested DNA of a primary transformant (T0) respectively.
Lanes 2–5: uncut DNA of T1 plants. Lanes 6 and 10: uncut and digested DNA of uninfected plant (negative control).
Lanes 8, 9, 11 and 12: DNA of T1 plants digested with Sma I.

50. Molecular analysis of the T1 and T2 plants
Seeds of T0 plants (Table) were germinated in the greenhouse (Fig. 5b) and DNA was prepared from leaves of all the plants that germinated. PCR analysis of 52 DNA samples showed the presence of the uid A gene in 36 samples. These 36 DNA samples gave a hybridization signal in the Dot blots too.

51. Southern hybridization of four PCR positive T1 plants was carried out in order to further confirm uid A gene inheritance and integration. Undigested DNA from all the four T1 plants gave a signal at around 23 kb indicating the integration of the uid A gene.
Variation in hybridization pattern was obtained in the case of Sma I digested DNA confirming integration of the gene.
Five T1 plants monitored, produced fertile seeds. These seeds were germinated and assayed for GUS expression.
A 3:1 segregation ratio for GUS expression was observed in most of the T1 and T2 plants. Abnormal ratio was observed for the T01 plant, which shows its chimeric nature. These data confirmed the inheritance and integration of the uid A gene in both T1 and T2 generations.