This document discusses genetic engineering techniques used to modify ornamental plants. It describes using Agrobacterium-mediated transformation to insert genes like ipt and barnase-barstar into pelargonium plants. The ipt gene driven by the male specific promoter pSAG12 delayed leaf senescence when expressed. Barnase-barstar induced male sterility. Transgenic plants were selected using antibiotic resistance and validated by PCR and phenotypic analysis. GFP was also used as a visual marker for transformation efficiency. Together these techniques allow genetic engineering of important traits in ornamental crops.

4. Ornamental floriculture is becoming an
important industry .
Ornamentals include a large variety of
crop plants
Cut flowers,
Bulbs and corms,
Foliage and Flowering pot plants.
All the present day ornamental varieties
and novelties are as a result of extensive
hybridization, induced mutation and
selection .

6. Genetic engineering: The technology of
preparing recombinant DNA in vitro by
cutting up DNA molecules and splicing
together fragments from more than one
organism.
Genetic engineering is a laboratory
technique for gene manipulation.
Genetic engineering brings about novel
combination of genes by using
recombinent DNA technology which is not

7. Genetic engineering of plants is much
easier than animals.
there is natural transformation system for
plants(Agrobacterium).
plant tissue can redifferentiate.
plant transformation and regeneration are
relatively easy for a variety of plants.
Agrobacterium tumefaciens can infect
wounded plant tissue, transferring a large
plasmid, the Ti plasmid, to the plant cell.

8. Important methods in
recombinant DNA technology are
Isolation of desired gene Insertion
of isolated gene into a suitable
vector Introduction of recombinant
vector in to host Selection of
transformed host cells
(A.C.Dutta 2005)

9. Digestion of the cell wall by enzymatic
action, dissolution of the biological
membranes by detergent losses,
centrifugation to isolate pure DNA.
DNA cut into no. of fragments by
restriction endonulcleases “molecular
scissors” with sticky ends.

10. Isolating
Genomic
DNA
Fragmenti
ng DNA
Screening
DNA
fragments
Insertion
of DNA in
vector
Introducin
g DNA in
host
Culturing
the cells
Transformatio
n of host cell

11.  Most widely used
 More economical
 More efficient
Agrobacterium mediated
gene transfer
 Particle bombardment
or
micro projectile .
 Direct DNA delivery by
PEG .
Electroporation .
 Microinjection .
Chandler and Brugliera, 2011

13. 1-2 µm of tungsten or gold particles
(microprojectiles)coated with DNA to be used for
transformation are accelerated to velocities using
pressurized Helium gas

14. DNA solution is injected directly inside the
cell using capillary glass micropipetts .

15. 2
3
1T - DNA
Ti - Plasmid

16. 2 The same
restriction
enzymes cut the
same base
sequences in
plasmid DNA. 5 Recombinant DNA
inserted into host cells
is copied each time the
host cells divide.
1 Restriction
enzymes cut
specific base
sequences.
4 The result is recombinant DNA
molecules with both Target and
plasmid DNA.
3 The plasmid
DNA and the target
DNA fragments are
mixed in a solution
with enzymes that
link them together.

17. First discovered in plants
(R. Jorgensen, 1990)
•When Jorgensen introduced a re-
engineered gene into petunia that had a
lot of homology with an endogenous
petunia gene, both genes became
suppressed!
First discovered in plants
(R. Jorgensen, 1990)

18. Wild-type petunia
producing purple
anthocyanin
pigments
Chalcone synthase
(CHS) is the enzyme at
the start of the
biosynthetic pathway for
anthocyanins
Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25.
Anthocyanins
Chalcone synthase
(CHS)

19. Attempted to overexpress chalone synthase (anthrocyanin
pigment gene) in petunia.
(trying to darken flower color)
Caused the loss of pigment.

20. Small RNAs are a pool of 21 to 24 nt
RNAs that generally function in
gene silencing .
Small RNAs contribute to
post-transcriptional gene silencing by
affecting mRNA stability or translation
AAAAA
RNA Pol
Histone modification, DNA methylation

21. Sense RNA
Antisense
RNA
Sense construct:
PRO CHS
Endogenous gene
mRNA
Transgene
PRO CHS
mRNA
Protein translated
mRNA
mRNA
Extra protein translated
Antisense construct:
PRO
CHS
Transgene Sense-antisense duplex
forms and prohibits
translation

22. Surprisingly, both antisense and sense gene constructs
can inhibit pigment production
Photo credit Richard Jorgensen
Plants carrying CHS transgene
CaMV 35S pro : CHS CaMV 35S pro :
CHS
Sense Antisense
OR

23. No blue rose - naturally – incapable of
synthesizing delphinidin
• Molecular geneticists with
Florigene and Suntory achieved by
combining something old,
something new,
Something borrowed,
and something blue.

24. 'something
blue'
the delphinidin
gene cloned from
a pansy.
'something
borrowed
an iris gene for
an enzyme, DFR,
required to
complete the
delphinidin-
synthesis reaction
'something
new'
man-made gene
designed by
geneticists exploited a
powerful new
developed technology
- to switch off a rose
gene .
'something
old '
Roses are very old
garden subjects

25. Use of RNAi technology to switch off
DFR gene in a red rose to block
cyanidin pathway,
and then install the delphinidin gene –
plus a new DFR gene to complete
delphinidin synthesis

26. The three-gene package (pansy
delphinidin, iris DFR, anti - rose DFR
)package worked:
Suntory's transgenic rose produced
very high levels of delphinidin in its
petals,
and a small residue of cyanidin.
The new rose is an attractive
shade of mauve - lilac roses like
'Blue Moon' and 'Vol de Nuit'.

28. Mercuri et. al (2001)
 GFP detection in Eustoma
(Lisianthus) flower petals. (A)
 GFP detection in
Osteospermum ligules
flower petals. (A)
GFP = Green Fluorescent Protein

29. García-Sogo et al. BMC
Plant Biology 2012, 12:156
http://www.biomedcentral.c
om/1471-2229/12/156

30. Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Rosids
Order: Geraniales
Family: Geraniaceae
Genus: Pelargonium

34. Plant
material
Surface
sterilization
Morphogen
esis
Induction
Medium
(MIM)
Elongation
Medium
Rooting
Acclimatization

35. Surface
sterilization
Morphogen
esis
Induction
Medium
(MIM)
Calculate
activity cost
drivers’
rates
Rooting
Acclimatization
Plant
material
Pelargonium zonale
cv. 370
Explants: Young
leaf explants
from 30–40 days old
plantlets
Pelargonium peltatum
cv. Aranjuez

36. Plant
material
Morphogen
esis
Induction
Medium
(MIM)
Elongation
Medium
Rooting
Acclimatization
Rinsed three
times with sterile
distilled water.
Iimmersion in a 2.5%
solution of sodium
hypochlorite for 20 min.
Surface
sterilization

37. Plant
material
Surface
sterilization
Calculate
activity cost
drivers’
rates
Rooting
Acclimatization
leaves was cut into 1
cm2 pieces and
cultured on MIM
MS basal medium
and Shahin [46]
vitamins
Morphogen
esis
Induction
Medium
(MIM)
supplemented with
50 mg l-1 kanamycin
Regeneration in
Pelargonium zonale was
carried out via direct
organogenesis and
in Pelargonium peltatum
via somatic
embryogenesis.

38. Plant
material
Surface
sterilization
Morphogen
esis
Induction
Medium
(MIM)
Rooting
Acclimatization
•After 2.5 - 3 months
in culture, calli
showing well
developed
morphogenetic
structures (shoots in
the case of P. zonale
and somatic embryos
in P. peltatum) were
transferred to a
selective Elongation
Medium .
• Elongation
Medium (EM: MS
basal medium and
Shahin vitamins,
supplemented with
50 mg l-1
kanamycin)
• All explants
were subculture
every 2 weeks
onto the same
fresh medium until
shoots were long
enough to be
separated ..
Elongation
Medium

39. Plant
material
Surface
sterilization
Assign
costs to
activity cost
pools
Elongation
Medium Acclimatization
• After 1 – 1.5 months
in EM, the shoots were
cut and cultivated in
Rooting Medium (RM).
Rooting

40. Plant
material
Surface
sterilization
Morphogen
esis
Induction
Medium
(MIM)
Elongation
Medium
Rooting
•and acclimatized
in growth
chambers under
(16-h light/8-h
dark photoperiod)
and then
transferred to a
greenhouse until
they flowered..
• Regenerated
plantlets with
welldeveloped
roots were
transferred to
plastic pots
containing peat
moss and perlite
(3:1).
Acclimatization
Transformation efficiency was
estimated
as the number of independent
transformation
events (one transgenic plant per
explant) in relation to the total
number of inoculated explants.

41. Cytokinins have been implicated in several aspects of
plant development, including plant senescence [15-20],
and are thought to be synthesized mainly in the roots
and transported to the shoots via the xylem.
Overexpression of the ipt gene in transgenic plants led to
elevated foliar cytokinin concentrations and delayed leaf
senescence, but high cytokinin levels have been reported
to be detrimental to growth and fertility [26 30].
To circumvent these effects :
Specificgene promoter (pSAG12 )

42. Promoter which
induces transcription
in male reproductive
specifically
Gene which disrupts
normal function of cell
Agrobacterium-
mediated
transformation
regeneration
male-sterile
plant

43. (A portmanteau of "BActerial" "RiboNucleASE")
is a bacterial protein that consists of 110 amino acids
and has ribonuclease activity.
It is synthesized and secreted by the bacterium
Bacillus amyloliquefaciens, but is lethal to the cell
when expressed without its inhibitor barstar .
The inhibitor binds to and occludes the ribonuclease
active site, preventing barnase from damaging the
cell's RNA

45. • LBA4404 cells were electroporated
to carry different plasmids a pBIN19 binary vector .

46. nptII nos
•GFP
•report
er
gene
CaMV
•Barnase
•barstar
TA29ipt pSAG
12GUS 35SCa
MV
•npt
•marker
gene
nos
T- DNA region
Bacterial DNA
Virulence region
Oregion of replication

47. Bacteria were grown at 28°C on solid LB
plates supplemented with 40 mg l-1
rifampicin and 100 mg l-1 kanamycin
Single colony was used to inoculate 25 ml of LB
liquid medium with the same antibiotics ,
maintained at 28°C and 200 rpm for 24 h
Inoculate a liquid MS medium supplemented
0.2 mM acetosyringone dissolved in 70%
ethanol (sterilized by filtration), which was
cultured at 28°C for 12 h.
Inoculation of explants was conducted in
bacterial culture

54. Identification of the ipt transgene (460 bp fragment) by PCR in different P. zonale pSAG12::ipt
transgenic plants. C + (positive control: pVDH393-pSAG12::ipt) and TI (negative control).

55. Identification of the barnase-barstar transgene (544 bp fragment) by PCR in different P.zonale male
sterile plants. C + (positive control: pBI101-PsEND1::barnase-barstar) and TI (negative control).

60. (a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34 (h) days of incubation in darkness.

61. (i) Mean concentration (±SE) of chlorophyll a + b (mg/g fresh weight) from detached
leaves of control (WT) and pSAG12::ipt (TRG) plants at 0, 6 and 8 days of incubation
in darkness .

62. (j) Senescence delay of detached leaves from pSAG12::ipt plants. Fresh weight changes in detached
leaves of WT P. zonale and a transgenic line carrying the pSAG12::ipt chimaeric gene over the
time course analyzed. Data are the means of sixteen leaves ± SE. Bars: 1 cm.

64. 50. Rogers SO, Bendich AJ: Extraction of total cellular DNA from plants, algae and fungi. Plant Mol Biol
Manual 1994, D1:1–8.
51. He J, Gray J, Leisner S: A Pelargonium ARGONAUTE4gene shows organspecific express
53. Lichtenthaler HK: Chlorophylls and carotenoids: Pigments of photosynthetic biomenbranes. Met Enzymol
54. Elliot AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL: Green fluorescent protein facilitates rapid in
vivo detection of genetically transformed plant cells. Plant Cell Rep 1999, 18:707–714.
55. Escobar MA, Park JI, Polito VS, Leslie CA, Uratsu SL, Mc Granahan GH, Dandekar AM: Using GFP as a
scorable marker in walnut somatic embryo transformation. Ann Bot 2000, 85(6):831–835.
56. Ghorbel R, Juárez J, Navarro L, Peña L: Green fluorescent protein as a screenable marker to increase the
efficiency of generating transgenic woody fruit plants. Theor Appl Genet 1999, 99:350–358.
57. Pérez-Clemente RM, Pérez A, García L, Beltrán JP, Cañas LA: Transformation and regeneration of peach
plants (Prunus persica L.) from embryo sections using the green fluorescent protein (GFP) as a vital marker. Mol
58. Rakosy-Tican E, Aurori CM, Dijkstra C, Thieme R, Aurori A, Davey MR: The usefulness of the gfp reporter
gene for monitoring Agrobacteriummediated transformation of potato dihaploid and tetraploid genotypes.
59. Yancheva SD, Shlizerman LA, Golubowicz S, Yabloviz Z, Perl A, Hanania U, Flaishman MA: The use of
green fluorescent protein (GFP) improves Agrobacterium-mediated transformation of ‘Spadona’ pear (Pyrus
60. Baranski B, Klocke E, Schumann G: Green fluorescent protein as an efficient selection marker for
Agrobacterium rhizogenes mediated carrot transformation. Plant Cell Rep 2006, 25:190