STATISTICAL ANALYSIS

FOR PLANT TISSUE

CULTURE DATA
Introduction
• Statistics provide an objective, non biased way to evaluated
experimental treatments.
• Use probability that an event can occur to determine whether or not
treatment effects are real.
• Measures within and between experimental treatment => allow to
select best treatment.
• Discussed methodologies associated with:
Scientific research
Experimental designs
Methods of comparing treatment means
Methodologies associated with scientific
research
• The hypothesis is tested objectively and either accepted or rejected
according to the result obtained.
• The null hypothesis : all experiment treatments elicit in a similar
response.
• Rejection of null hypothesis -> imposed treatment vary in the
response that they cause.
Methodologies associated with scientific
research
• Steps in scientific research:
1. Define precisely the problem to be solved.
2. Formulate set of experimental objectives.
3. Establish set of treatment.
4. Select experimental materials ( eg. Cultural vessels, culture medium,
growth medium etc.) *selecting treatments and experimental materials
based on knowledge of previous experiments.
5. Choose the experimental design along with the observational units,
number of replicates per treatment and randomization scheme.
Methodologies associated with scientific
research
• Observational unit
• Selection of explant type and source influence the outcome.
• Most culture tissue, observational unit is the culture vessel .
• When there is one explant per culture vessel :
• Vessel : unit of replication
• Explant : subsample
• Number of replication for degree of replication affect precision of statistical
test.
• Precision increase as the number of replicates increase.
• However, plateau when increasing number of replicates no longer improves
precision.
Methodologies associated with scientific
research
Randomization
• Each replication assigned to a treatment.
• Important to make assignment in a manner such that all have an
equal chance of receiving a given treatment.
• Schemes vary according to the experimental design employed.
• After selecting the treatments and experimental design, one should
determine the data to be collected.
• Determine the data to be collected.
• Evaluate and explain treatments effects according to experimental objectives
Methodologies associated with scientific
research
ANOVA(Analysis of variance)
• ANOVA summary tables with all the degree of freedom (DF) and associated
sources of variation (treatment, experimental error, etc.) along with graphs
and tables that show the theoretical results constructed to evaluate the
effectiveness of the proposed experiment.

• Data must be statistically analysed and interpreted as planned before the

experiment, according to the hypothesis , experimental conditions and
previous facts.

• Important to repeat the experiment to confirm results due to some degree

of uncertainty regarding the conclusions from an experiments.
Experimental designs:
completely randomized design
The completely randomized design
• Most popular because cell cultures are generally grown in
environmental chambers that accurately control light, temperature
and humidity.
• The numbers of treatments and replicates per treatment that can be
tested are not limited.
• Applied even there are missing data or unevenly spaced.
• Most precise design because its maximize the degree of freedom (DF)
for estimating error.
• Reduce F-value to detect statistical difference among treatment.
Experimental designs:
completely randomized design
Case study1
• Identify tomato cultivars that displayed high rates of shoot
regeneration from pedicel explants.
• Cultured in test tubes that contained 15ml of shoot regeneration
medium.
• 24 tubes replicates per treatment, with the total of 264 experimental
units.
• Treatments assigned to the experimental units completely at random.
• Experimental data ; number of explants with shoots and number of
shoots per explant.
Experimental designs:
completely randomized design
Case study1
• ANOVA used o analyze the data to determine the genotypes differed
in their ability to produce shoots from pedicel explants.
• Generate degree of freedom (DF), sum of squares (SS) and mean
square values (MS) for treatment and experimental error to perform
statistical test.
• Treatment SS for measures the degree of variation associated with
the treatment.
• Experimental error SS measures variation associated with
experimental units.
Experimental designs:
completely randomized design
Case study1
• In tomato study , the F-value for treatment (cultivitar) calculated by dividing its MS (7.937) by the
mean square error (MSE).
Experimental designs:
completely randomized design
• Case study1
• Significance of the observed F-statistics was determined using 10 and 253 DF.
• A significance of the observed F-statistics indicates there are differences in
shoot regeneration among treatment means, a mean separation procedure
must be performed.
• Mean separation procedure should be chosen prior to ANOVA to avoid
personal bias in the test because each separation test uses a unique formula
when calculating differences among means.
Experimental designs:
completely randomized design
• The CRD is most efficient when there is little variation among
experimental units.
• Efficiency and precision are reduced in situations where there is a
high degree of variation is assigned with treatment effects.
• Non treatment variation is assigned to experimental error.
• In situations, where there is a high degree of outside error and the
source can be identified, designs that employs blocking should be
used.
Experimental designs :
Single factor CRD with subsampling
• Culture several explants in a single culture vessel.
• The response of each explant is measured, resulting in multiple
measurements for each vessel (subsampling).
• More efficient when there is a wide range in response among explants.
• Statistical precision unit when there is a wide range in response among
explants.
• Improved by making several measurement per culture vessel, thus
reducing variation among replicates of the same treatment.
• Subsampling ma be used to study variability among explant, which may be
useful in future experiment.
Experimental designs :
Single factor CRD with subsampling
• Case study 2
• Examined the ability of watermelon cotyledon explant to produce
adventitious shoots illustrates the use of subsampling.
• Objective to identify genotypes with high level adventitious shoot
organogenesis.
• Cotyledons from seedlings of 11 cultivars were cultured in a total of 66 dishes
and 330 explants.
• Explants from each cultivars assigned to petri dish at random , making sure
that only explant from one cultivars cultured together.
• The number of explants with adventitious shoots and the number of shoots
per explants were recorded.
Experimental designs :
Single factor CRD with subsampling
• Case study 2
• ANOVA for CRD experiments with subsampling differs from a CRD with
one observational unit per experimental unit in that SS are generated
for treatment, experimental error and subsampling error.
Experimental designs :
Single factor CRD with subsampling
• Case study 2
• A special error term is used for calculating the treatment F-value and
measures variation among experimental units.
• Subsampling SS represents variation caused by culturing multiple
explants within the same experimental units and not used in
computing treatment differences.
• Improves statistical precision by removing variation among explants
from the experimental error.
• In this example, significance among treatment s detected by dividing
the treatment with MS by the new MSE, resulting in a significance F-
value = 6.83 and indicates that the cultivars displayed varying levels of
shoot regeneration.
• Statistical test be conducted on the treatment.
Experimental designs :
Single factor CRD with subsampling
• Case study 2
• When conducting mean separation tests in subsampling experiments,
the special MSE must be used to calculate values for statistical test.
• Failure specify the correct error term may result in the grouping of
means that should be separated and/or separation of similar means.
• subsampling experiments promote efficient use of space and material
and measures variability among observational units.
• Design not efficient when there is little variation among explants or
when there is a high degree of variability from some other identifiable
source.
• Reduces the DF for experimental error, which means that a higher F-
value is required to detect significant differences among treatments.
• Little variation among explants, a CRD without subsampling should be
used.
• If variation from an identifiable outside source exists, a RCBD with
subsampling should be used.
Experimental designs :
Randomized complete block design (RCBD)
• CRD is only efficient when experimental units are homogenous. This is
because unrecognized variation, regardless of source, is lumped into
the experimental error term.
• Heterogeneity among experimental units results in a high MSE and
reduces statistical precision.
• In experiment with high heterogeneity, it is best to group
experimental units into homogeneous units or block.
• Grouping experimental units into uniform blocks provides a better
estimates of treatment effects ad improves statistical precision.
Experimental designs :
Randomized complete block design (RCBD)
• In RCBDs, treatment are grouped into blocks that contain at least one
replicate from each treatment. Experimental units are randomized
within blocks, each employing a separate randomized scheme. This
minimizes variability within a block while maximizing variability
among blocks.
• Each block should be as uniform as possible.
• The number of treatments should be limited because variability
within each block increases as the number of treatment increase.
Experimental designs :
Randomized complete block design (RCBD)
• Based on Case study 1
• Arrange into 24 blocks , each containing one test tubes with a single pedicel
explant rom each cultivars from each cultivars.
• The ANOVA for a RCBD differs from that of a CRD in that block SS is calculated
in addition to treatment and experimental error SS.
• Block SS identifies variation controlled by blocking.
• Treatment SS represent variability related to the imposed treatment.
• Experimental error SS measures variability among experimental units.
Experimental designs :
Randomized complete block design (RCBD)
• Based on case study 1
• Blocking improves statistical precision by removing variability controlled by
blocking from the experimental error.
• F-value for cultivars (5.99) was obtained by dividing the cultivar MS (7.937) by
the MSE (1.326) and significance determined using 10 and 230 DF.
• A means separation procedure must be performed to determine which
treatment means differ.
Experimental designs :
Randomized complete block design (RCBD)
• Based on case study 1
Experimental designs :
Randomized complete block design (RCBD)
• Based on case study 1
• RCBD useful when some variation is caused by something
there than the imposed treatment.
• If little diffefence among blocks occurs , a RCBD will not
inprove statistical precisio and a CRD should be used.
• If there is much variation among explants , a RCBD with
subsampling should be used
Experimental designs :
Split –plot design
• This design used when two treatment factors are
superimposed on each other.
• One treatment is assigned to main plots that contain all
levels of the second factor.
• Main plot treatments assigned to subplots are randomized
within each main plot.
• Each subplot is randomized differently.
• It identifies experiment error associated with each
treatment factor.
Experimental designs :
Split –plot design
• Treatments with large experimental error rates assigned to
main plots and those with reduced experimental error to
subplots.
• Dividing experimental error into main plots and subplots
improves statistical precision.
• The split plot design is most efficient when treatment
factors have different experimental error between main
plots and subplots.
Factorial experiments
• All possible combinations of two or more factors are examined
simultaneously.
• Allow to examined treatment interdependencies and are more
powerful than multiple single factor experiments.
• Any of the aforementioned treatment randomization schemes can be
used for factorial treatment designs.
Factorial experiments
• Case study 3
• Examining the effects of BA and IBA on tobacco callus growth.
• Treatment ere three levels (0,1,10 μM) of BA and IBA arranged in 3x3
complete factorial.
• Pieces of tobacco callus (1cm3) transferred to test tubes that contained 15ml
of test medium.
• There were six replicates test tubes per treatment that were randomized in a
CRD.
• Callus height , diameter, and dry weight recorded after 1 month after culture
initiation.
Factorial experiments
• Case study 3
Factorial experiments
• Case study 3
• SS are generated for each treatment, treatment interaction and for
experimental error.
• F-value for BA, IBA and their interaction were obtained by dividing the MS for
each by the MSE.
• Significance for the main effects (BA and IBA) was determined using 2 and 98
DF, whereas significance for the BA by IBA interaction was tested at 4 and 98
DF.
• ANOVA indicted that callus growth in the form of dry weight was influenced
by BA by IBA simultaneously.
• Indicate BA affected callus dry weight differently at each level of IBA and IBA
affected callus dry weigh differently at each BA concentration.
Factorial experiments
• Case study 3
Factorial experiments
• Case study 3
• Interactions means for this type data are represented in a line graph with
treatment differences estimates using SE.
• When treatment interactions are significant, the influence of each treatment
separately should not be discussed.
Factorial experiments
• Factorial experiments can become prohibitive with relatively few
factors.
• For example;
• The above 3x3 factorial experiment had 9 treatments.
• If a third factor with three levels was added the number of treatment
combinations would increase to 27.
• Add to that 10 replicates and there would be 270 observational units.
• Interpretation of higher order interactions may be difficult in any factorial
experiment.
Methods for comparing treatment means
• Once a significance F-test with a small P-value is obtained, scientist
must elucidate specific differences among treatments.
• This is not a problem when there are only two treatments because
treatments means are simply presented in a table with the associated
F-test significance level.
• When there are more than two treatment, a post- ANOVA analysis is
necessary.
• The easiest way to compare treatment means is to rank the in
ascending or descending order and pick the best treatments.
Methods for comparing treatment means
• The problem with this method is that natural variation that occurs
within a treatment is not considered.
• There are many mean separation procedures that account for within
treatment variation.
• These are standard error of the mean (SE), multiple comparison and
multiple range tests and regression analysis.
Methods for comparing treatment means :
Standard error of the mean
• SE is obtained by dividing the sample standard deviation by the
square root of the number observations for that treatment.
• The size of SE depends on the magnitude f the data.
• When using SE for mean separation purposes, treatment means are
ranked along with their respective SE and the difference between
paired values calculated.
Methods for comparing treatment means :
Standard error of the mean
• Treatments are declared different if the collective values for the
paired treatments do not overlap.
• Using SE provides an indication of variability within treatments and
allows the reader to make comparisons.
• One disadvantages of using SE for mean separation purposes is that t
is considered conservative and may only detect differences between
means ranked far apart.
Methods for comparing treatment means :
Multiple comparison and multiple range tests
• Only be used when treatments are unrelated e.g different
growth regulators, genotypes etc.

• Multiple comparison test

• use the same critical value to compare adjacent and non adjacent
means.
• Eg: Bonferoni, Fisher’s least significant difference (LSD), Scheffe’s,
Tukey’s Honestly Significant Difference test (Tukey’s HSD) and Waller-
Duncan K-ratio T-test (T-test)

• Multiple range test

• Employ different critical values to compare adjacent and non adjacent.
Protects against commiting type 1 error (instance where treatment
are declared different that truly same).
• Duncan’s New Multiple Range Test (DNMRT), Ryan Eionot Gabriel
Welsh Multple F-test (REGWF) , Ryan Einot Gabriel Welsh Multiple
Range Test (REGWQ) and Student Newman Kuels (SNK).
Methods for comparing treatment means : Multiple
comparison and multiple range tests
• Best used only after obtaining a significant ANOVA.
• Most mean separation procedures may be used in situations when a
significant ANOVA has not been obtained if specific comparisons were
planned prior to data analysis.
• High probability to create type 1 error, especially when comparing
non adjacents means.
• Most recommended mean separation procedures for unrelated
treatments; DNMRT, REGWQ, SNK and Tukey’s HSD and Waller-
Duncan.
Methods for comparing treatment means : Simple linear
regression
• Regression, or trend ,analysis used in experiment with quantitative
treatments where the primary objective is to develop a model that
quantifies the relationship between response variables and treatment
levels.
• Trend analysis is most appropriate when there are three or more
evenly spaced, equally replicated treatments.
• A forward step procedure is most often used to identify the best
model.
• The simplest model (linear) is tested first and more complicated
models (quadratic and cubic) tested after rejection of lower order
models.
Methods for comparing treatment means : Simple linear
regression
• Regression, or trend ,analysis used in experiment with quantitative
treatments where the primary objective is to develop a model that
quantifies the relationship between response variables and treatment
levels.
• Trend analysis is most appropriate when there are three or more
evenly spaced, equally replicated treatments.
• A forward step procedure is most often used to identify the best
model.
• The simplest model (linear) is tested first and more complicated
models (quadratic and cubic) tested after rejection of lower order
models.
Methods for comparing treatment means : Simple linear
regression
• Lack of fit (LOF), T and r-square (r2) values are used to
determine the best model.
• A significant T-value and non-significant LOF signify that the
model accurately describes the relationship between
treatment and response variables.
• A significant LOF vale that the model does not fit the data and
the other models should be tested.
• The r2 value estimates variation described by the model.
• If a low r2 is obtained it may be advantageous to check other
models.
• The most appropriate model generally has significant T-value,
non significant LOF, a high r2.
• Extrapolation must be limited to within treatment boundaries
as the model equation may not accurately describe treatment
effects beyond the tested parameters.
Methods for comparing treatment means : Simple linear
regression
Case study 4
• Regression analysis used to determine the effects of sucrose on the
growth of grape embryogenic cultures.
• Embryogenic cells and somatic embryos (heart and globular stages)
incubated on medium containing 60,90,120,150 or 180 g/l sucrose for
3 months.
• Sbcultured monthly to fresh medium of the sme composition.
• At the end of experiment, embryogenic cultures were dried in oven at
70oC for 72 h and dry weight determined.
Methods for comparing treatment means : Simple linear
regression
Case study 4
• ANOVA determined that the sucrose concentration in the medium
influenced the growth of grape embryogenic cultures.
• Regression and LOF analyses indicated the growth of grape
embryogenic cultures .
• Regression and LOF analyses indicated that the data best fit the cubic
model as indicted by a significant T- value and non significant LOF
value value (-1774.06+57.92-0.4837x2+0.001x3)
Methods for comparing treatment means : Simple linear
regression
Case study 4
Methods for comparing treatment means : Simple linear
regression
Case study 4
• Optimum growth obtained when embryogenic culture incubated on
medium with 9 to 120 g/l sucrose.[Fig.7.2]
• The high 0.8815r2 obtained for this model indicates that much of the
variation in the data was explained by the model.
• The term high or low r2 are relative to the data analyzed.
• High r2 values for biological data may range between 0.50 and 0.90,
whereas a low r2 for non biological data may be 0.90.
Methods for comparing treatment means : Simple linear
regression
Case study 4
Categorical data
• Based on counts of individual that can be placed into groups.
• This occurs when data consists of yes/no values (eg. Yes the explant
responded or no the explant failed to respond) or when explant
response is placed into a category (e.g. explants produced roots,
shoots, somatic embryos or did not respond).
• Categorical data are not continuous and not normally distributed and
therefore, cannot be analyzed with the procedures mentioned above.
• Chi-square or maximum likelihood are used to analyze categorical
data.
Categorical data
Case study 5
• Identifying the best dehydration treatment for maize
somatic embryos.
• Somatic embryo reared on a medium without ABA (M1)
transferred to medium with 0.1M ABA (M2) for 2 weeks ,
or transferred to M2 for 2 weeks before transfer to
medium with 60 g/o sucrose and no ABA (M3) prior to
controlled relative humidity dehydration (CRHD) at 70% or
90% relative humidity for 2 weeks .
• Embryo survival was determined 2 weeks after transfer to
germination medium by the ability of somatic embryo to
produce chlorophyll, roots, coleoptiles or leaves.
• Control embryos obtained from each of the above media
were transferred directly to germination medium without
CRHD .
Categorical data
Case study 5
• surviving embryos were assigned a 1 , whereas those
that failed to survived were assigned 0 (no response).
• According to ANOVA , culture medium and
dehydration treatment simultaneously influenced the
ability of maize somatic embryos to survive
dehydration as determined by the significant
treatment by medium interaction contrast.
• The ability of maize somatic embryos to survive
dehydration depended on the pretreatment medium
and RH level used during CRHD.
Categorical data
Case study 5
• SE was used to compare treatment means .
• Categorical data can be transformed using arc sine prior to
ANOVA and converted back to the original scale for
demonstration in tables or graphs.
INCORPORATION OF TRANSGENES INTO CROPS
• Most of the methods currently used for plant transformation employ a
technique for delivering the DNA into the cell without regard to its ultimate
intracellular location. Once inside the appropriate cellular location by a chance
process, the DNA is integrated into the chromosomal (or organellar) DNA,
usually by a nonspecific recombination process. The exception is
Agrobacterium-mediated transformation, which delivers the DNA specifically
into the nuclear compartment with high efficiency and also provides a
mechanism for its integration.
• In plant transformation, two physical barriers prevent the entry of DNA into a
nucleus, namely the cell wall and the plasma membrane. Most plant
transformation methods overcome these barriers using physical and/or
chemical approaches. In protoplast-mediated transformation, the cell wall is
digested with a mixture of enzymes which attack cell wall components,
yielding individual protoplasts that can be maintained intact using appropriate
osmoticum in the medium. Entry of DNA is then facilitated by the addition of
permeabilizing agents, such as polyethylene glycol, that allow DNA uptake,
presumably by coating the negatively charged DNA inside the cell and various
cellular compartments in a random process.
INCORPORATION OF TRANSGENES INTO CROPS
• A second method, often referred to as biolistic transformation, utilizes fine metal
particles (typically tungsten or gold) coated with DNA that are usually accelerated
with helium gas under pressure. Other methods, such as microinjection,
sonication, and electroporation cause transient microwounds in the cell wall and
the plasma membrane, allowing the DNA in the medium to enter the cytoplasm
before repair or fusion of the damaged cellular structures. However, many of these
methods are tedious and result in variable transformation efficiencies.
• In all transformation techniques, the desired transgene is placed under the control
of a promoter which produces high-level constitutive or inducible expression of
the gene in specific or all tissues. In addition, another gene that allows detection
or selection of the transformed cells is introduced in the same or different vector
DNA. The presence of a screenable or selectable marker gene greatly simplifies the
identification of transformed plants and increases the efficiency of recovery of
transgenic plants. Typical screenable markers are the gus gene, encoding a β-
glucuronidase, and the gfp gene encoding a green fluorescent protein. Selectable
markers that have been most useful are those conferring resistance to antibiotics
such as kanamycin, paromomycin, and hygromycin, or to herbicides such as
bialaphos, glyphosate, or cyanamide.
 METHODOLOGY OF PRODUCING TRANSGENIC PLANTS
• Most of the important crop species have been successfully transformed, at least in the laboratory. Two major
approaches have been widely used to produce transgenic crop plants, both monocots and dicots. One is
biolistic bombardment and the other is Agrobacterium-mediated transformation. In the biolistic protocol, the
primary delivering system is the helium-powered gun. The transgene and the selectable marker are inserted
in the vector between two unique sequences, called the left border and the right border, which are utilized in
insertion of the T-DNA into the host chromosomal DNA.
• Parameters involved in the biolistic gun include pressure (ranging from 900 to 1300 psi), particle size (0.6 to
1.1 µm), and type of material (gold and tungsten), target distance (7.5 to 10 cm), and target material (cell
suspension, callus, meristem, protoplast, immature embryo). These parameters vary somewhat with the crop
involved. Disadvantages of using the biolistic gun are low success frequency, high copy numbers that often
are correlated with gene silencing, patent issues and cost.
• Agrobacterium-mediated transformation may correct some of the weaknesses encountered with the biolistic
approach and has been successful in rice, maize, sorghum, and other crops; its effectiveness with other crops,
especially wheat, remains questionable. Further, some cultivar versus Agrobacterium strain specificity may
limit the range of cultivars that can be successfully transformed. Development of reliable and efficient
protocols are needed to improve the efficiency and range of both approaches in transforming crop plants.
• Transformation techniques not involving tissue culture are desirable for crop species in which many cultivars
do not respond to tissue culture. These techniques include soaking and vacuum infiltration transformation of
Arabidopsis inflorescence with Agrobacterium, transformation via the pollen tube pathway and pollen
transformation via biolistics. The dipping method adopted in Arabidopsis must be modified for cereal crops
by altering the plant stage of infiltration, the concentration of bacterial culture, and the duration of
treatment.
METHODOLOGY OF PRODUCING TRANSGENIC PLANTS
Further, each tiller (for wheat, barley etc) of the same plant must be kept separate. If this technique works,
selection for transformants is made directly from seed-derived seedlings and tissue culture is avoided. Thus,
genotypes that respond negatively to callus induction and plant regeneration will not present a problem;
however, it is likely some genotypes will be more amenable to infiltration transformation than others.
CONFIRMATION OF PUTATIVE TRANSGENIC
PLANTS & TRANSFORMATION EFFICIENCY
Commonly used methods to confirm the putative transgenic plants are
polymerase chain reaction, Southern blotting, Western blotting, Northern
blotting, functional assay (testing the presence of selectable marker and
the target gene), in situ hybridization, and progeny analysis (segregation of
the target gene). Not all transgenic plants produce the same amount of
protein from the target gene and selection based on the Western blot is
necessary. This is because a positive correlation usually exists between the
effectiveness of the gene in the bioassay and the amount of protein it
produces. For example, the level of rice chitinase accumulating in
transgenic sorghum plants with the chi11 gene was positively correlated
with resistance to sorghum stalk rot.
POLYMERASE CHAIN REACTION
PCR, a simple and rapid procedure, is utilized to confirm whether a
putatively transgenic plant that has survived selection is indeed transgenic.
Usually, two primers (one forward and one reverse) specific for the
selectable marker (bar gene, for example) are used in a PCR reaction with
genomic DNA extracted from the transgenic plants. A thermostable DNA
polymerase amplifies the region between the two primers during the
multiple amplification cycles of the PCR, which yields a DNA fragment of
predicted size (the length equal to the number of base pairs between the
two primers in the transgene). This fragment is easily detected on an
agarose gel by staining with ethidium bromide. PCR is a very sensitive and
rapid method for identification of transgenic plants in the seedling stage and
requires only a small amount of plant tissue.
 SOUTHERN BLOTTING/ HYBRIDIZATION ANALYSIS
In Southern blotting, DNA fragments from transgenic plants generated by digestion with restriction enzyme(s)
are first separated according to fragment size by electrophoresis through an agarose gel. The DNA fragments
then are transferred to a solid support, such as a nylon membrane or a nitrocellulose sheet. The transfer is
affected by simple capillary action, sometimes assisted by suction or electric current. The DNA binds to the solid
support, usually because the support has been treated to carry a net positive charge, or some other means of
binding such as inducing covalent binding of the DNA to the support. DNA fragments maintain their original
positions in the gel after transfer to the membrane. Hence, larger fragments will be localized toward the top of
the membrane and smaller fragments toward the bottom. The positions of specific fragments can then be
determined by “probing” the membrane. The probe consists of the DNA fragments of interest, such as a cloned
gene, which has been labeled with a radioactive isotope or some other compound that allows its visual
detection. Under the proper set of conditions, the denatured single-stranded probe will hybridize to its
complementary single strands of genomic DNA affixed to the membrane.
In this way, the size of the fragment on which the probe resides in the genomic DNA can be determined. In
transgenic plant experiments, the Southern blot often is used to determine whether an introduced gene is
indeed present in the plant DNA and whether multiple transgenic plants carry the introduced gene on the same
size of DNA fragment (suggesting independent transformation events). The results of Southern blots also
indicate whether a single copy of the gene has been inserted or if multiple copies are likely to be present.
NORTHERN BLOTTING
Northern blotting – the name was derived as a play on words from the
Southern blot – is very similar to the Southern blot, except that instead of
restriction enzyme-digested DNA, native RNA is separated according to
size by electrophoresis through an agarose gel and then transferred to a
solid support. The rest of the Northern blot procedure is very similar to
that of the Southern blot and it is used to determine whether the
introduced gene has been transcribed into messenger RNA and
accumulates in the transgenic plant.
WESTERN BLOTTING
The Western blotting procedure detects the protein of the transgene in an
extract of proteins prepared from various parts of the transgenic plants
and is, therefore, an assay for a functional transgene. In this technique
the proteins are first electrophoresed in an SDS-polyacrylamide gel and
the proteins are then transferred to nitrocellulose membrane by
electrophoretic transfer. The membrane is then treated with an antibody
specific for the protein encoded by the transgene followed by a second
antibody coupled to an enzyme, which can act on a chromogenic (or
fluorogenic) substrate leading to visualization of the transgene protein
with increased sensitivity. The expression level of the protein can be
quantified using known amounts of the transgenic-encoded protein.
FUNCTIONAL ASSAY
When the selectable markers used are antibiotic-resistant or herbicide-
resistant genes, a functional assay can be made by spraying antibiotics or
smearing herbicide on the leaves of those putative transgenic seedlings or
plants in later segregating populations. Sensitive plants typically will turn
brown and shrivel up whereas resistant (transgenic) plants will stay
healthy and green. Such an assay provides the initial screening of large
number of putative transgenic plants and reduces the work load by
eliminating escapes during selection.
PROGENY TEST
With stable transformed genes, progeny testing should show the presence
and activity of the selectable marker and target genes, such as the gene
gfp encoding green fluorescent protein, or bar and disease resistance.
However, segregation does not always follow the typical Mendelian
fashion. For example, among the progeny in the Agrobacterium-mediated
wheat transformation experiments reported, segregation in the T1
generation had ratios of 32:0, 1:34, 0:40 and 74:0 in addition to the
expected 1:1, 3:1 or 15:1 ratios. This variability indicates aberrant
segregation. However, in other cases, segregation follows the normal
Mendelian pattern. For example, among six sorghum T0 transgenic plants
produced by biolistic bombardment, all showed typical 3:1 segregation
ratios in the T1 generation.
CROP SPECIES AMENABLE TO TRANFORMATION
• Any crop that is able to produce calli from explants and is capable of callus
regeneration into plants with high efficiency is amenable to transformation
using biolistic bombardment and Agrobacterium tumefaciens. However, it is
known that response to tissue culture is highly genotype dependent.
Furthermore, somaclonal variation; spontaneous genetic variations occurring
in cells growing in vitro could occur during tissue culture processes. Thus, to
confirm that the improved phenotype of the transgenic plants is due to the
transgene, two controls should be included – one from seed-derived plants
and the other from non-transformed tissue culture-derived plants.
• For those crops of genotypes that show a negative response to tissue culture,
a transformation procedure independent from tissue culture should be
considered and tested. It will be a great accomplishment to perfect a
procedure bypassing tissue culture, because many cultivars of wheat and rice
and inbred lines of maize and sorghum do not respond to tissue culture
operations.
 Food Prospects
Malthus’s 1798 book: Essay on population: population growth will soon outpace
food production.
Marx Das Kapital: Agric will follow the experience of manufacturing, becoming an
increasingly concentrated sector with many workers per farm with each worker
specializing in small fraction of the tasks involved in farm operation. The USSR and
China tried to implement this vision.

Ecologist Paul Ehrlich’s 1968 book: The Population Bomb predicted that the world
will undergo famines in 1970s, hundreds of millions of people will starve to death in
spite of any crash programs embarked upon now. It is too late!!!

William and Paul Paddock’s 1967 book Famine 1975! America’s Decision: Who will
survive? advocated a triage approach to foreign aid. The “can’t be saved group” that
included India and Philippines should not receive any aid.

Biologist Garrent Hardin became famous for coining the term “the tragedy of the
commons” to describe the problems that can arise from conflicts of interest when
there is open access to exploitation to natural resources. In 1977 he published The
Limits of Altruism in support of “tough-minded” approach recognizing that countries
such as India had exceeded their “carrying” capacity.
Yet over the past century growth in productivity of both land and labor has
enabled world food supplies to outpace the unprecedented increase in food
demand caused by jumps in the growth rate of world income and by
doubling and redoubling of population.

All these theorists were wrong!!

What contributed to this phenomenal increase in ag productivity in last 50
years?

1. Selection of plant varieties: sophisticated genetics based breeding

technique.
2. Crop management.
3. Improvement of animal breeds.
4. New methods of controlling pests and diseases.
 How far from a yield ceiling?
Yield of a crop is a function of biomass x harvest index (HI). Hence yield can be improved by
increasing biomass or HI or both. Since HI of many crops is approaching a ceiling value, so to
increase yield potential we have to increase crop biomass, i.e. there will have to be more
photosynthesis. The theoretical limits of solar energy utilization efficiency in photosynthesis
and the efficiency attained by crop plants provide possibilities and scope for improvement of
photosynthetic productivity.

Tons/hectare
5
4
3
2
1

1866 1936 1956 1996 Year

Despite doubling and redoubling of crop yields seen in some developing countries,
any absolute yield ceiling seems far off.

Scientists have estimated yields that can be generated if a plant is given all the
inputs it needs. For most cereals, potential yields are several multiples of the
present average US yield.
 What role might biotechnology play in sustainable agriculture?
"Sustainable agriculture" is both a term and a concept whose definition has varied a great
deal. As articulated in the 1990 "Farm Bill" Food, Agriculture, Conservation, and Trade Act of
1990, P.L. 101-624, Title XVI, Subtitle A, Section 1603) sustainable agriculture means "an
integrated system of plant and animal production practices having a site-specific application
that will, over the long term: (A) satisfy human food and fiber needs; (B) enhance
environmental quality and the natural resource base upon which the agricultural economy
depends; (C) make the most efficient use of nonrenewable resources and on-farm resources
and integrate, where appropriate, natural biological cycles and controls; (D) sustain the
economic viability of farm operations; and (E) enhance the quality of life for farmers and
society as a whole."

Biotechnology has the potential to assist farmers in reducing on-farm chemical inputs and
produce value-added commodities. Conversely, there are concerns about the use of
biotechnology in agricultural systems including the possibility that it may lead to greater
farmer dependence on the providers of the new technology. Where these two new
developments will lead agriculture is open for debate.