This document summarizes an experiment studying the effects of genetically engineering tobacco plants to overproduce citric acid. The study found that transgenic tobacco lines that expressed the citrate synthase gene produced more citric acid and had enhanced growth and productivity compared to control plants when grown in low-phosphorus soil conditions. Specifically, the citrate-overproducing plants were taller, had larger leaves, and produced more shoot and capsule biomass than controls in soils with limited phosphorus availability. Biochemical analysis also showed the transgenic lines accumulated more phosphorus in their leaves. These results suggest citric acid overexpression enables plants to better utilize insoluble sources of phosphorus in the soil.

1. Kifayat Ullah
(SP17-R02-003)
MS 2 BTY
12/04/17 1

2. Contents
Introduction
 Problem
Methodology
Experimental Protocol
Results and discussion
Conclusion
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3. Introduction
Phosphorus (P) is an essential nutrient
 Nucleic acids, phospholipids, ATP, and other
biologically active compounds
P is the second most important nutrient after Nitrogen,
limiting agricultural production.
P reactive available for plant uptake at a
narrow range of neutral soil pH values.
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4. In acidic soils,
 P forms low-solubility molecules with aluminum (Al)
and iron (Fe),
Alkaline soils,
 P + (Ca) and (Mg) phosphate compounds
Therefore, although the total amount of phosphorus in the
soil may be high, in most cases it is unavailable for plant
uptake.
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5. To reduce P deficiencies and ensure plant productivity,
 Nearly 30 million tons of P fertilizer are applied
worldwide every year
 Up to 80% is lost because it becomes immobile and
unavailable for plant uptake because of adsorption,
precipitation or conversion to organic forms
Excessive use of fertilizers, particularly P, environmental
pollution
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6. Plants Adaptations
In response to P-limiting conditions, some plants undergo
physiological and developmental adaptations :
 Changes in root architecture,
 Induction of genes encoding high-affinity P transporters,
 Rhizosphere acidification, and
 Exudation of organic acids (Citrate)
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7. An increase in the excretion of citrate (Brassica napus,
Lupinus albus, and the Proteaceae family of plants)
 as a potential mechanism to enhance P uptake.
Citrate high affinity for divalent and trivalent cations
 Citrate can displace P from insoluble complexes,
 Making it more available for plant uptake.
Two to threefold increase in the concentration of soluble P
in the rhizosphere. 7

8. Here we report that plant‘s ability to use insoluble P
compounds can be significantly enhanced by engineering
plants to produce more organic acids
And that citrate overproducing plants yield more leaf and
fruit biomass when grown in soils with P- limiting
conditions.
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9. Methodology
Generated transgenic tobacco plants (CSb lines )
 Express the citrate synthase coding sequence from
Pseudomonas aeruginosa.
 Under control of the 35s CaMV promoter by
Agrobacterium mediated transformation.
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10. 10

11. Experimental protocol
Biochemical analysis.
P-Soluble Experiments
Use of hydroxyapatite
P-Determination
Mycorrhizal colonization experiments.
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12. Results and discussion
Trangenic lines - CSb 4 , CSb 18
Transgenic control plant (line 1522, transformed with the
same vector but lacking the bacterial CS coding sequence
3 treatments of water soluble P was applied (NaH2P04 at
22,44, and 108 mg kg-' soil).
Analysis was made after 30 days and at flowering
Significant differences between CSb and control
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13. Growth and productivity of CSb tobacco plants
subjected to different P treatments.
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14. Figure 1. Transgenic (CSb-4 and CSb-18) and control (1522) plants
were grown in a sterile low-P, alkaline soil with or without
application of P. After four months in the greenhouse, plants were
photographed.
(A) Plants grown without added P or nutrients.
(B, C) Plants grown in soil adjusted to 22 or 108 mg of P per kg of soil
with NaH2P04, respectively, and watered with a nutrient solution
lacking P.
(D) Fruits from plants grown at 22 p.p.m. of added NaH2P04.
(E) Top view of a pot containing a CSb-18 plant, in which calcium
citrate precipitate is clearly visible. (
F-H) Trypan blue staining of transgenic CSb-18 roots. F) Non
inoculated plant.
(G, H) Plant Inoculated with G. fasciculatum (x20 and x40
magnification, respectively).
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15. Height and total foliar area of transgenic
and control plants grown in alkaline soil.
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Figure 3. Transgenic (CSb-4 and CSb-18) and control (1522) plants were
grown in soil adjusted to 22 mg per kg of soil with NaH2P04 and watered
with a nutrient solution lacking P. Height was measured at flowering. Total
foliar area was measured at harvest time using a leaf area meter (CI-202;
CID Inc., Vancouver, WA). Data are representative of at least two
independent experiments conducted in different seasons (n = 8 plants).

16. Accumulation of biomass in CSb lines grown
under limiting P conditions.
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Figure 2. Transgenic CSb and control tobacco lines were grown at two levels
(22 and 44 p.p.m.) of added NaH2P04. (A) Shoot and (B) capsule dry weight.
Plants were harvested at the following times: V, 30 days after transplanting; F,
at the initiation of anthesis; R, at the completion of fruit set. The data represent
average and standard error (n = 4 plants).

17. 17
Biochemical analysis

18. P level in leaves of CSb and control
lines.
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Figure 4. P level in leaves of CSb and control lines.
Total content of phosphorus in shoots of control and CSb-4 and -18 lines grown
at two levels (22 and 44 p.p.m.1 of NaHzP04 was determined. Measures
were made at the following times: V, 30 days after transplanting; F, at the
initiation of anthesis. Bars show mean and standard errors (n = 4 plants).

19. Mycorrhizal colonization experiments.
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20. Insoluble source of phosphate
(hydroxyapatite)
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Figure 5. Fruit dry weight of CSb-4, CSb-18, and control lines grown at
two levels (22 and 44 p.p.m.) of hydroxyapatite in an alkaline
soil.
Fruits were harvested at the completion of fruit set. The data represent
the average and standard error of 10 plants per treatment.

21. 21
Figure 6. Effect of citrate on the biomass accumulation of tobacco
plants grown in media containing an insoluble source of phosphate.
Transgenic (CSb-4 and CSb-18) and control (1522) plants were grown in MS
nutrient media (pH 8.0) with or without application of 1 mM of citric acid.
Soluble and insoluble sources of phosphate were adiusted to 1 mM. Seedlinas
were harvested at 15 - days of age and their dry weight was determined. Data
show the average and standard error of at least three independent experiments
(n = 20 seedlings).

22. Conclusion
Results demonstrate that transgenic plants with increased
levels of citrate exudation :
 Have a higher capacity to use insoluble forms of P,
 This characteristic is enhanced, when the plant
associates with mycorrhizae.
Since citrate overproduction has been achieved in plant
species other than tobacco, it is quite likely that the ability
to use P insoluble compounds can be enhanced in other
plant species as well. 22

23. Two advantages are evident in plants that have a high
capacity to extract P from soils:
 Better exploitation of natural soil P reserves, and
 Ability to meet P requirements with a smaller
application of P fertilizer.
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24. Thank You…..
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