Introduction what is isotope ? Types of Isotopes Isotopic Labelling ADVANTAGES AND DISADVANTAGES OF ISOTOPIC STUDY APPLICATIONS OF ISOTOPES IN AGRICULTURE Principle isotopes used in plant-soil studies Case studies FUTURE THRUSTS OF ISOTOPIC STUDY CONCLUSIONS REFERENCES

2. CREDIT SEMINAR
ON
USE OF STABLE AND RADIOISOTOPES TO UNDERSTAND THE PHYSIOLOGICAL PROCESS
SUBMITTED BY
RAHUL GOAPLE
(PG19PHY-1937)
ADVISORY COMMITTEE
CHAIRMAN - DR. R. D. DEOTALE SIR
MEMBER – DR. S. B. BAVISKAR MAM
MEMBER – DR. S. R. KAMDI SIR
MEMBER – MR. D. J. JIOTODE SIR
SUBMITTED TO
SECTION OF AGRICULTURAL BOTANY,
COLLEGE OF AGRICULTURE, NAGPUR

3.  INTRODUCTION
A. WHAT IS ISOTOPE ?
B. TYPES OF ISOTOPES
C. ISOTOPIC LABELLING
 ADVANTAGES AND DISADVANTAGES OF ISOTOPIC STUDY
 APPLICATIONS OF ISOTOPES IN AGRICULTURE
 PRINCIPLE ISOTOPES USED IN PLANT-SOIL STUDIES
 CASE STUDIES
 FUTURE THRUSTS OF ISOTOPIC STUDY
 CONCLUSIONS
 REFERENCES
CONTENTS

4. USE OF STABLE AND RADIOISOTOPES TO UNDERSTAND THE PHYSIOLOGICAL PROCESS
INTRODUCTION –
(A) WHAT IS ISOTOPE ?
Atoms of element with different numbers of neutrons are
called "isotopes" of that element. The term isotope is
formed from the Greek word “isos” and “topos”, meaning
"the same place"; thus, the meaning behind the name is that
different isotopes of a single element occupy the same
position on the periodic table. It was coined by Scottish
doctor and writer Margaret Todd in 1913 in a suggestion to
chemist Frederick Soddy.
For example, 12C, 13C, and 14C are three isotopes of the
element carbon with mass numbers 12, 13, and 14,
respectively. The atomic number of carbon is 6, which
means that every carbon atom has 6 protons, so that the
neutron numbers of these isotopes are 6, 7, and 8
Fig. – Isotope
Symbols
Fig. Isotopes of
Carbon

5. (B) TYPES OF ISOTOPES
 STABLE ISOTOPES - Stable isotopes have a stable
combination of protons and neutrons, so they have stable
nuclei and do not undergo decay.
 RADIOACTIVE ISOTOPES - Radioactive isotopes have an
unstable combination of protons and neutrons, so they
have unstable nuclei. Because these isotopes are
unstable, they undergo decay, and in this process can
emit alpha, beta and gamma rays.
(C) ISOTOPIC LABELLING
Technique used to track the passage of an isotope
through a reaction of metabolic pathway is called isotopic
labelling. A radioactive compound is introduced into a
living organism and the radio-isotope provides a means
to construct an image showing the way in which that
compound and its reaction products are distributed
Fig. Stable Isotopes of
Oxygen
Fig. – Radioactive Isotope
of Hydrogen (Tritium)

7. ADVANTAGES DISADVANTAGES
 With the help of radio-isotope we can easily locate the
presence of single atom and molecule and their movement.
 Very small quantities of labelled nutrients can be accurately
measured in presence of large quantities of other nutrients.
 Tracer technique enables one in tracing those elements taken
by the plants accurately and precisely.
 It also helps to study accurately the interaction among the
mineral nutrients.
 You can label specific atoms (say carbon-1 in glucose) to
follow where each one goes.
 A radioactive molecule is chemically exactly like the
unlabelled form. Thus, it will behave just like the unlabelled
form so you don’t have to worry about effects due to the
labelling itself.
 Since carbon, hydrogen and phosphorus can be easily
purchased in radioactive forms, you can make any
biomolecule in a radioactive form.
 During the stable isotope application no radiation hazard
takes place and also it is non-toxic.
 Wide spectrum of stable isotopes available which provide
broad area of application
 In the stable isotope labelling multiple labels possible and it
has smaller isotope effect
 Radioisotopes are rather expensive.
 Radioisotopes are hazardous and must be handled with
extreme care. By the same token, they present a disposal
hazard.
 Some radioisotopes (like 32P and 125I) have short half-lives, so
have to be used quickly.
 Stable isotopes are expensive high cost and sophistication of
instrumentation
 Isotopic studies are subjected to the labour intensive analysis.
 The radiations emitted by these isotopes will induce damage
in the developing embryo and other cells. Internally deposited
isotopes can cause more harm to the particular organ and
tissue in which they are localized
 Experimental evidence shows that for the same administered
activity, the organically bound tritium (triturated thymidine) is
much more harmful than tritiated water (UNSCEAR 1986).
ADVANTAGES AND DISADVANTAGES OF ISOTOPIC
STUDY

8. Radioisotopes are used as a research tool to develop new strains of agricultural crops that
are drought and disease resistant, are of higher quality, have shorter growing time and
produce a higher yield. Radioactive elements emit a variety of radiations and energy particles
during decay which are used in health care, agriculture and physical sciences for basic
research and in wide range of applications (Sahoo & Sahoo, 2006).
There are many applications of isotopes in various agricultural prospects which are –
 PLANT PHYSIOLOGY AND ECOLOGY
 PLANT NUTRITION STUDIES
 CROP IMPROVEMENT
 INSECT PEST MANAGEMENT
 FOOD PROCESSING AND PRESERVATION
 INCREASING ANIMAL PRODUCTION AND HEALTH
 WATER RESOURCES MANAGEMENT
APPLICATIONS OF ISOTOPES IN
AGRICULTURE

9. PRINCIPLE ISOTOPES USED IN PLANT-SOIL STUDIES

11. CASE STUDY - 1
Materials and Methods
Experimental design – RBD
Replication – Five
Isotopic labelling method – PETIS
Measurement of transpiration rate -Portable leaf carbon dioxide analyzer
(LCA-4)
Parkinson chamber (PLC-4)

12. RICE VARIETIES
Pot Solution Spacin
g
Growth Environment
Acid-tolerant varieties Reference
intolerant varieties
1. GP15,
2. Luk-daeng (L-
daeng)
3. KDML105
4. Suphanburi90(S-
buri90)
5. IR53650
6. Norin30 (N30)
7. Hatsusakumochi(
H-mochi)
1. Kinangdang
Puti (KP)
2. IR36
3. Nipponbare
(N-bare)
Yoshida's
nutrient
solution
1×1
cm2
 Germinated seeds were sown on
a sand nursery bed, and 21-
days old seedlings were
transplanted.
 The plants were cultured in a
plant growth cabinet.
 air temperature was controlled
at 25ºC/ 20ºC (day / night), the
PAR intensity was 1,000 µmol m-
2 s-1 at the level of the plants
provided by mixed cool-white
fluorescent and halogen lamps,
the day length was 14 h day / 10
h night, and the CO2
Plant materials and growth conditions -

13. RESULTS AND DISCUSSION
Fig.2. Water uptake curve at collar part of the uppermost fully expanded
leaves, 4th leaf, 48 h after low pH treatment, for 10 rice varieties. : Control
plants (pH 5.3), Low pH-treated plants (pH 3.8)
 The rate of water uptake was determined
continuously by measuring radioactivity
in the collar part (target area) of rice
leaves exposed to 18F-labeled water (18F-
water) using a Positron Emitting Tracer
Imaging System (PETIS).
 The PETIS measurement showed that the
radioactivity in the target position of
leaves of acid-tolerant varieties increased
faster than that of the intolerant varieties
after the 18F-water was applied into the
vial.
 In the plants subjected to low pH
conditions, the transpiration rate (Tr) and
leaf water potential (Ψ) decreased in both
acid-tolerant and intolerant varieties.
 the extent of reduction was less in the
acid-tolerant varieties than in the
intolerant ones. Moreover, the dry matter
production rate of the acid-tolerant

16. CASE STUDY - 2
Materials and Methods –
 Soybean seedlings - Samples aged from 18 to 20 days old were used for
this experiment.
 Measurement of the vessel area - To measure the vessel area, a 0.05%
safranine solution was applied to the plant for 30 min with 50% humidity at
280C, with light. Stem sections of 150 mm thickness were cut with a
Vibratome (VT1000S)
 Generation of H2
15O - the gas 15O2 was generated by the reaction of 14N (d,
n)15O; the H2
15O vapor was then synthesized by the reaction of the oxygen
15

17. The quantitative and real-time measurement of H2
15O in the stem internode - A
soybean plant, from which the cotyledons and the root 8 cm below the cotyledons
had been removed, was fixed between lead blocks to shield radiation, and placed in
the Biotron about 1 h before the measurement. A pair of gamma ray detectors was
placed immediately adjacent to either side of the internode of the plant, 2 cm above
the cotyledons and the counting efficiency of the gamma ray detector was calculated
to be 0.120%, by comparing the count of the gamma ray detector with that of a
gamma counter (Auto-gamma scintillation spectrometer) for the plant sample
supplied with H2
15O (200 M bq ml–1) The measurement of H2
15O volume was
performed on plants with two treatment :-
a. A plant with vaseline spread on the surface of the internode to be measured
between the cotyledon and the first leaf
b. A plant with a 5 mm portion of the tissue outside of the cambium removed by a
razor blade, from 3 cm above to 1 cm below the measuring position.
 Quantitative measurement of water volume by two pairs of gamma ray detectors
 The measurement of the effect of the exchange of the isotope 15O for 16O – By

18. RESULT -
The lateral water movement in the intact stem of a transpiring soybean
plant was analyzed quantitatively by a real-time measurement system
utilizing labeled water, H2
15O and gamma ray detectors.
A large volume of water escaping from xylem vessels during its transport
was detected. The escape of water was not influenced by evaporation from
the stem surface or mass flow in the sieve tubes.
It was assumed that the total amount of water transported through xylem
vessels was kept almost completely constant along the internode. As a
result, most of the escaped water was found to re-enter the xylem vessels,
i.e. Water exchange occurred.
An analysis based on a mathematical model showed that the net volume of
water which escaped from xylem vessels was not dependent on the

22. CASE STUDY - 3
Mycorrhizas (fungal roots) play vital roles in plant nutrient
acquisition, performance and productivity in terrestrial
ecosystems.
15N external labeling or enrichment (usually expressed as atom%)
and 15N naturally occurring abundance (§15N) techniques have
been employed to trace the direction and magnitude of N
transfer between plants, with their own advantages and

23. METHODS FOR INVESTIGATING NITROGEN TRANSFER BETWEEN PLANTS –
15N stable isotope - In general, 15N isotopic composition can
provide information on
a) N inputs through N2 fixation by free-living and symbiotic
organisms
b) Inputs of fertilizer N
c) The extent of N cycling
d) Sources of N available for plant growth
15N labelling or enrichment method – In the 15N labelling or
enrichment method, an external N source (up to 99.90% at 15N
enrichment) is used to trace N transfer or N metabolism over
short-term periods (days to months). Measurement of isotopic
effects is straight-forward because there is a large enrichment of

24. Continued….
Several sources of error have been
identified. Nitrogen transfer has been
quantified in three ways: percentage of
N transfer (% N transfer, equations 1–3),
amount of N transferred (mg/plant,
equation 4) and percentage of NDFT (%
NDFT, N in the N receiver derived from
transfer, equations 4 and 5) . Equations
(5) and (6) are equal if the 0.3663% 15N
of atmospheric N2 is subtracted from
the measured atom% 15N in equation
(5).
Two-way(bidirectional) or net N
transfer can be calculated as the
difference between N transfer from
plant A to plant B, and the reverse.
Then, net N transfer could benefit one

25. PERSPECTIVES –
 Does N transfer occur between any mycorrhizal plants, irrespective of their N2
fixation characteristics? What determines the magnitude and direction of such N
transfers in monocultural and/or mixed-species plant systems?
 Do AM and EM networks behave similarly on N transfer, considering that AM and
EM networks are structurally different and that EM fungi can acquire n from organic
sources?
 Are there interactions between AM and EM networks on N transfer since some
plants do have dual AM/EM associations?
 Can the 15N natural abundance method be employed to detect N transfer, similar to
the 15N enrichment technique?
 Is there reciprocal C and N transfer? If yes, using 13C/15N to study C/N transfers
between plants could provide clues on C translocation or cycling that plays crucial
role on global C/N balance and environmental change.
 Is it possible to employ network theory and computer modelling to simulate the

26. FUTURE THRUSTS OF ISOTOPIC STUDY
Stable isotope analysis is a powerful tool for assessing plant carbon and
water relations and their impact on biogeochemical processes at different
scales.
Our process based understanding of stable isotope signals, as well as
technological developments, has progressed significantly, opening new
frontiers in ecological and interdisciplinary research.
At the advent of development of new techniques for nuclear magnetic
resonance spectroscopy (NMR), new options arise for studies of, e.g.
Starch-sugar partitioning and complementary information on photo-
respiration by analyses of non-homogeneous distribution of 13C within
carbohydrate molecules.
Radioisotopes are used in the nutritional studies of major and minor
elements, mechanism of photosynthesis studies, and uptake of nutrients
and ions mobility in soil. Assessment of the available soil nutrient is of
considerable importance in planning optimum fertilizer use.

27. CONCLUSIONS
 Atoms of element with different numbers of neutrons are called isotopes. Most elements
have both stable and radioactive isotopes.
 Applications of Radioisotopes and radiations are helping us to find the solution of problems
in much shorter time.
 Isotopic labelling is a technique used to track the passage of an isotope through
a reaction, metabolic pathway, or cell.
 Every botanist is aware of the great progress achieved in the fields of biochemistry and
plant physiology using radioactive isotopes with their easily and sensitively detectable
radiation. The most striking example is the exploration of the carbon pathway in
photosynthesis.
 The rate of water uptake was determined continuously by measuring radioactivity in the
collar part (target area) of rice leaves exposed to 18F-labeled water (18F-water) using a
Positron Emitting Tracer Imaging System (PETIS).
 The lateral water movement in the intact stem of a transpiring soybean plant was analysed
quantitatively by a real-time measurement system utilizing labelled water, H2
15O and
gamma ray detectors. A large volume of water escaping from xylem vessels during its
transport was detected.
 Both 15N external labelling or enrichment (usually expressed as atom%) and 15N naturally

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