NTHEMIND OF GREATCOMPANIESBy Scott BlanchardThe.docx

NTHE
MIND OF GREAT
COMPANIES?
By Scott Blanchard
T
he old saying, "money isn't
everything," rings hollow in
today's business world.
where rninute-by-minute
stock quotes scroll across
our computer monitors, and
careers are won or lost based
on Wall Street's analysis of a
company's perforniance. Throw in giob-
al competition, outdated products and
services, increased costs, corporate silos
and other business challenges, and it's
no wonder that tnatiy of today's compa-
nies focus solely on their bottom line,
ofteti at the expense of customer service
and employee satisfaction.
It need not be this way. Great compa
nies focus on more than one bottom
line when gauging their perforniance.
Ttiey choose to be not only the invest-
ment of choice, but also the provider of
choice for their products or services, as
well as the employer of choice for work-

ers in their industry. By looking beyond
immediate, short term results and focus-
ing on strategies to make their compa-
nies successful for the long-term, they
recognize challenges sooner, identify
solutions more quickly and deliver re-
sults ahead of their competitors. In short,
they learn to lead at a higher level.
A clear warning sign that your busi-
ness is trapped in a short-term mindset
is the presence of an "either/or" philoso-
phy. Managers either believe they can
achieve profitability or they can develop
a great workplace, but not both. These
leaders don't always take morale and job
satisfaction into consideration. Their
focus is only their financial bottom line.
From there, it's a short leap to the false
notion tlrat making money is the sole
reason to be in business.
A NEW APPROACH
Contrary to the either/or philosophy,
leading at a higher level requires man-
agers to embrace a "both/and" approach.
In great companies, the development of
people is of equal importance to finan-
cial performance. As a result, the focus
is on long-term results and human satis-
faction. Accordingly, great companies
begin by both creating and nurturing a
vision of the future, and then measuring
progress against that vision.

There are three questions to ask,
which represent the main components
of a corporate vision. By focusing on
these questions, companies are more
likely to ensure they don't lose sight of
their path to success. They are:
• What business are you in? This will
help you identify your company's signif-
icant purpose.
• What will the future look like if you
are successful?
• What guides your behavior and deci-
sions on a daily basis? This will help
you identify clear values.
Great companies keep al! three of
these ideas clearly in mind and make
necessary course corrections when they
realize they are off track.
The next step is to create a corporate
culture that both reflects and reinforces
the corporate vision. The culture con-
sists of the values, attitudes, beliefs,
behaviors and practices of the organiza-
tion's members. Culture is an organiza-
tion's personality, and it can help or hin-
der an organization looking to achieve
greatness.
Many companies develop a corporate
culture over time, but if it wasn't active-

ly sculpted wilh the company's long-
term vision in mind, it may not reflect
tlie company's ultimate mission. When
organizations seek greatness, they often
find that aspects of their organizational
culture need to be changed.
Certainly, values are an important part
of the company s corporate culture, but it
is not enough to simply identify what
those values are. Great companies zero in
on their top three or four values and rank
them to help botli management and the
work force make the right decisions.
Ranking the list is essential, because val-
ues are sometimes in conflict. Wben con-
flicts arise, people need to know which
value should take priority. For example, if
you value financial growth, but integrity
is your core value, any activities that
could lead to financial gain must first be
checked against your integrity value.
THE ROLE OF TRAINING
Once great companies establish a long-
term corporate vision and have identi-
fied and ranked the three or four values
that help make up corporate culture, the
next step is to develop and implement a
comprehensive, integrated training pro-
gram. Businesses that consistently invest
in training outperform their competitors
in both good and bad times.
Without committed and empowered
employees, companies can never achieve

greatness. Employees learn by example.
Managers can't treat their employees
poorly and expect them to treat cus-
tomers well. Part of treating employees
well means providing them with high-
impact training programs. These need to
be aligned with specific, measurable
goals that are fully supported and in
alignment with organizational objectives.
Without this continuous learning, em-
ployees cannot develop tbe skills and atti-
tudes needed to succeed.
A successful training program has the
following components:
• Top management buy-in - Nothing
derails a performance-improvement ini-
tiative faster than a lack of support from
the leaders in an organization. However,
getting this support means convincing
executives of the return on investment
of the training.
• Demonstrated tangible value - Senior
leaders (and training parti( ipants) want
evidence that any new training initiative is
going to result in new skills tbat positive-
ly impact the organization's bottom line.
• Follow-up/reinforcement - The compa-
nies tbat are the most successful spend
lo times tbe amount of effort reinforcing
the training they deliver as opposed to
moving on to the next initiative.
LEAP TO GREATNESS

A segment of RR Donnelley's business
that was, until recently, known as Banta
provides an excellent case study ot a
company that made the leap to great-
ness by following the steps outlined
above. The Minneapolis-based business
provides catalog production services
including prepress, printing, binding, list-
processing services and distribution.
In 2004, tbe organization found itself
challenged witb issues of overcapacity
in an uncertain market and an industry
that was shifting from traditional offset
printing to digital and Web-based solu-
tions. In addition, margin pressures,
increased costs, competition and a silo-
structure mentality amplified the organi-
zation's problems and prevented team-
ing, process improvement, innovation
and customer focus.
The company assembled a team ot
consultants and trainers to provide solu-
tions for its situation. An analysis soon
revealed Banta's employees couldn't d e
scribe the company's core business beyond
"printing catalogs" or "making a profit."
Furthermore, tbey were out of touch witb
their role in the company and that of oth-
ers. The solution was to create a corporate
culture in which every employee's action
was guided by a set of shared values.
Management crafted a new corporate
vision: "To deliver quality, on-time mer-

chandising solutions that drive our cus-
tomers' success." It incorporated new val-
ues into the employee-evaluation sys-
tem, and developed slide shows and a
brochure to show examples of behaviors
that supported Banta's stated values.
"These values became our compass,"
former Banta President Mark Deterding
said. "They needed to guide each and
every decision we made and every
action we took. No matter how tough it
was some days, we knew it was essential
that senior leadership set the tone and
walked the talk. And we asked every
individual to hold us to these values."
The company also conducted training
that supported and reinforced the appli-
cation of its values in the day-to-day
business of the company, It asked each
department to relate bow its work sup-
ported the overall mission of the compa-
ny and contributed to its success. Com-
pany leaders participated in special
training to help them get the most from
themselves and their employees.
THE PAYOFF
The results were impressive. Soon after
completing its organizational assessment
and training, profitability had increased
36 percent and employee retention had
improved 17 percent. Recruiting and
training costs for new employees de-

creased. Surveys also showed employee
engagement improved 20 percent in the
first six months, and employees actively
looked for ways to cut costs and improve
the work environment.
It's hard to argue with numbers like this.
Banta truly made a commitment to a both/
and proposition - that it could combine
profitability with a long term corporate
vision and an investment in people. Busi-
ness leaders should take a close look at
their own companies to see whether their
visions need correcting, or if they're tnily
on the path to long-term greatness,
Scott Blanchard is executive vice presi-
dent of client solutions with The Ken
Blanchard Cos. He is also a co-author of
Leading at a Higher Level: Blanchard on
Leadership and Creating High Performing
Organizations. For more information, visit
vmw.kenblanchard.com or call 800-728-6000.
Thi Lan Phuong Nguyen 42777982
1

Contents
I. Abstract
...............................................................................................
................................................. 3
II. Introduction
...............................................................................................
......................................... 3
2.1. Plant – microbial interaction
...............................................................................................
......... 4
2.2 Lectin protein
...............................................................................................
................................. 5
2.3. Arabidopsis plant
...................................................................................... .........
.......................... 8
2.4. Metagenomics
...............................................................................................
.............................. 9
III. Research objective
...............................................................................................
............................ 10
IV. Material and methods
...............................................................................................
...................... 11
4.1. Plant growth conditions, chemical treatment and
rhizosphere soil sampling .......................... 11

4.1.1. Germinating Arabidopsis thaliana seeds
................................................................................ 11
4.1.1.1. Germinating Arabidopsis thaliana seeds on soil
.................................................................. 12
4.1.1.2. Germinating Arabidopsis thaliana seeds in sterilize
conditions .......................................... 13
4.1.1.3. Checking homozygous seeds
...............................................................................................
15
4.1.2. Preparation of E.coli for inoculation around plant roots
........................................................ 16
4.1.2.1. Growth of E.coli on LB (Luria-Bertani) agar plate
................................................................ 16
4.1.2.2. Growth of E.coli on LB Broth.
...............................................................................................
16
4.1.2.3. Wash LB Broth by PBS (Phosphate Saline Buffer) to
get the pellet of E.coli ....................... 16
4.2. Sample DNA, RNA extraction, PCR amplification, Real
time PCR and data processing. ............ 17
4.2.1. Plant genomic DNA and RNA extraction.
................................................................................ 17
4.2.1.1. Plant genomic DNA extraction by CTAB (Cetyl
Trimethyl Ammonium Bromide) ................ 17
4.2.1.2. Plant RNA extraction.

...............................................................................................
............ 19
4.2.2. Bacterial genomic DNA extraction.
......................................................................................... 20
4.2.4. Measure DNA concentration and checking the quality of
DNA. ............................................ 23
4.2.4.1. Quantification of DNA concentration
.................................................................................. 23
4.2.4.2. DNA quality confirmation
...............................................................................................
..... 24
4.2.5. DNA amplification by PCR (Polymerase Chain Reaction).
....................................................... 25
4.2.6. Clean up PCR products
...............................................................................................
............. 25
4.2.8. Quantitative RT – PCR (qRT-PCR)
............................................................................................
27
V. Results
...............................................................................................
................................................ 28
5.1. Screening and selecting homozygous lectin-1-
overexpressing Arabidopsis plants ................... 29
5.1.1. Response of lectin-1-overexpressing Arabidopsis plants
to Basta herbicide. ........................ 29

5.1.2. Measurements of RNA concentrations from Arabidopsis
plants ........................................... 31
2
5.1.3. Quantification of Lectin 1 gene expression by qRT-PCR
......................................................... 32
5.2. Evaluation of survival bacteria around Lectin-1–
overexpressing Arabidopsis thaliana. ........... 33
5.2.1. Quantify the concentration of DNA extracted from root
and rhizosphere ............................ 34
5.2.2. Quantification of E.coli 16S copies using ER-F2 and ER-
R2 primers. ...................................... 35
5.2.3. Quantification of E.coli 16S copies using Univesal E.coli
16S primers (906F and 1062R) ....... 36
VI. Discussion
...............................................................................................
......................................... 36
VII. Conclusion
...............................................................................................
........................................ 38

3

I. Abstract
The rhizosphere soil is an environment where different plant-
microbe interactions occur.
Beside beneficial interactions that result in plant growth
promotion or disease resistance,
plants also usually face a variety of pathogen and diseases. The
protein Lectin belongs to a
group of carbohydrate-binding proteins and can be synthesized
in various organs,
particularly in roots, tubers and seeds [1]. This protein plays a
role in the recognition of
rhizobia by legume plant species and is related to different
pathogen defence activities. This
research using lectin-1-overexpressing Arabidopsis thaliana was
undertaken to investigate
whether lectins had ability to increase or decrease populations
of rhizoshere bacteria. An
experiment for checking if the lectin-1-overexpressing
transgenic line is homozygous was
also initially performed.
II. Introduction
Activities of microbial communities are key elements to

determine biogeochemical
transformations in nature. They, therefore, can play an
important role in managing and
engineering ecosystems [2]. The soil environment influenced by
root is called rhizosphere. It
harbours the microbial diversity that affects plant health and
nutrient [3]. However, the
mechanisms underlying these plant-microbe interactions are
currently not well understood.
Improving methods to perform the whole community level
characterisation of microbe
genome as well as the gene expression is an essential task which
facilitates a comprehensive
profiling of rhizosphere communities [4].
4
2.1. Plant – microbial interaction
Plants and microorganisms become involved in a close
interaction in soil environments. In
such relationships, it can be seen that plants play a role as a part

of the microbial residence
environment and provide nutrient released from exudates as a
substrate for microbe
growth, and microorganisms probably interact directly with
plants via altering their
environment. For instance, saprophytic fungi play a role in
decomposing complex organic
compounds and consequently plants can acquire readily
available nutrients [5] [6]. On the
other hand, production of organic acids and/or proton extrusion
can lead to a drop in pH in
soils. This change results in solubilisation of phosphate from
precipitate form into the soil
solution and subsequently phosphate becomes available for
plant uptake [6]. In addition,
locating with a large number in soil environment, once microbe
die, the carcasses become
also a source of nutrient for plants. Bacterial rhizosphere
microflora is also related to plant
health as it plays an important role in suppression soil borne
plant diseases relied [7]. The
processes associated with such suppression that are involved in
systemic acquired
resistance (SAR) or induced systemic resistance (ISR) could be

antibiosis, lytic activity,
competition for substrates, or competition for iron caused by
siderophores [7, 8]. In regard
to self-defence, due to the sessile lifestyle that consequently
leads to the invasion of more
pathogen than other mobile eukaryotes, plants tend to secrete a
wide range of chemical
defences against biological attacks. They can be antibiotics, or
antibiotic precursors that
collectively called phytoanticipins [9]. Some of the best known
group of phytoanticipins are
the saponins, steroid and terpenoid glycosides [10]. When the
antibiotics are absent or
present with a very low concentration, phytoanticipins can be
performed secondary
metabolites with antimicrobial activity [11]. Hence, it can be
said that the characteristics of
plants as well as exudates released by roots definitely play the
role in shaping the
composition of rhizosphere microbial community [12, 13]. It is
also obvious that
maintenance of plant defences is costly. As a result, the
mechanisms utilised by plants that
determine allocation of resources to defence microbial threats

or growth have been
attracting scientists’ interest.
The major endogenous low molecular weight signal molecules
involving in regulating the
plant defence signaling are the plant hormones salicylic acid
(SA), jasmonic acid (JA),
ethylene (ET), and abscisic acid (ABA) [14, 15]. These
hormones activate specific pathways
and can act individually, antagonistically or synergistically
depending on the pathogen
5
involved [15]. In addition to local resistance, many of these
phytohormones can also induce
defence responses in systemic tissues. An example is the ISR,
which is triggered upon root
colonization by some non-pathogenic rhizobacteria. Meanwhile,
SAR is induced in distal
tissues upon pathogen infection and generates a long-lasting
resistance to secondary

infections caused by a broad spectrum of pathogens [15].
Furthermore, ISR is triggered by
application of methyl jasmonate while salicylic acid is an
important phenolic compound for
establishment of SAR. Using Arabidopsis thaliana as a model
plant, a complex interplay of
signal molecules in various defence-signaling pathways could
be determined. Of them, JA is
a key member in the jasmonate family that plays a role in
regulating plant defence to both
biotic and abiotic stresses. As other signalling pathways, the
JA pathway includes the
perception of stress stimulus leading to local and systemic
signal transduction, perception of
specific signal, followed by synthesis of jasmonic acid, and
subsequently, responsiveness to
JA involving induction of subsequent downstream effects [15].
A study involving JA-
biosynthesis mutants showed that the triple mutant fad3 fad7
fad8 is deficient in the JA-
precursor leading to an inability in accumulating JA and
consequently higher susceptibility to
infection by insect larvae [16]. Further experiments showed that
the fad3 fad7 fad8 mutant

line is hypersusceptible to root rot caused by Pythium
mastophorum. Alternatively, an
exogenous application of methyl jasmonic acid confers less
susceptibility to soil-borne
pathogens [17]. Thus, both production of JA in wounded tissues
as well as perception of JA
in distal tissues are vital for activation of systemic responses. In
other words, JA molecules
function as a signal of ISR [18].
It can be seen that plant and microbe are involved in a
consistent interaction that play a
vital role in natural balance in an ecosystem. In which, a
numerous beneficial
microorganisms even have been described for plants. However,
negative impacts of
soilborne bacteria is also considerable [19, 20]. Hence, studies
of increasing the ability of
defending against plant pathogens need to be attentions for
further researches.
2.2 Lectin protein
Lectins are carbohydrate-binding proteins that reversibly bind
to specific mono or
oligosaccharides with high affinity [21]. Such proteins have
been found in plants, animals

and microorganisms and are widely implicated in immune
responses as pharmaceuticals
[20]. Lectins from about 80 species have been characterized to
identify structures and
6
specific biological functions. Lectin-like proteins work as plant
agglutinins and are involved
in plant’s defence against variety of plant-eating organisms
[21]. They can be found in
different parts of various plants such as legume, cereals (eg.
rice, wheat), solanaceae (eg.
tomatoes, potatoes) and particularly in tissues or organs that
need extra protection such as
seeds or storage organs. A possible reason for extra protection
is that they are susceptible
to attack by foreign organisms including conventional parasites
and predators [21]. Based
on the structure, lectins are classified into three major types,
including merolectins,

hololectins, and chimerolectins [21]. Merolectins are single
polypeptide and contain
exclusively one single carbohydrate-binding domain, for
example Hevein from rubber tree
[22] or monomeric Man-binding proteins of orchids [21].
Meanwhile, hololectins include
two or more carbonhydrate – binding domains and such domains
are either identical or very
homologous [21]. However, the majority of well-known lectins
are chimerolectins which
contain one carbonhydrate-binding domain arrayed tandemly
with other unrelated domain
which acts independently on such carbonhydrate-binding
domain. The pathogen defence
activity of lectin varies depending on its familiarity with the
environment. For instance,
lectins that work as ribosome inactivating protein type 2 (type 2
RIPs) belong to the lectin
major type of chimerolectins and are extremely toxic to all
eukaryotes when they reach the
cytoplasm [21]. There isevidence that type 2 RIPs which
critically affect on higher animals
involving humans have been found since ancient times [21].
One kind of type 2 RIPs called

Ricin exhibits toxicity to the coleoptera Callosobruchus
maculates and Anthonomus grandis
[23]. Another lectin which comes from winter aconite (Eranthis
hyemalis) is highly toxic to
the larvae of the insect Diabrotica undecimpunctata, which is
known to attack maize) [24].
Though type 2 RIPs are also toxic to fungi, deleterious effects
due to invasion are normally
prevented since the presence of a rigid and thick cell wall. As a
result, type 2 RIPs cannot
penetrate the cytoplast [21]. Although the evidence of antiviral
activity of plant lectin is not
obvious, some plants expose indirectly antiviral action. For
example the existence of
insecticidal lectin results in preventing or decreasing the spread
of insect-transmitted viral
diseases. Meanwhile, the understanding about antibacterial
activity of plant lectin seems to
be more convincing. In a research in 1977, Sequeira and
Graham showed that potato lectins
that exist as cell wall proteins have the ability to immobilize
avirulent strains of
Pseudomonas solanacearum when they attack the cell wall [25].
Because of the presence of

7
cytoplast, the mechanism of plant’s defence against bacteria
must be indirect through
interactions of the protein lectin with extracellular glycans or
carbonhydrates exposed on
the cell wall. Another example of such indirect defence is the
interaction of thorn apple
(Datura stramonium) seed lectin with normal motile bacterial
community around the air-
water interface resulting in blocking the movement of such
bacteria [21]. The lectin-
mediated block of bacterial motility in the experiment expressed
correlatively with the
highly specific release of lectin from the seed coat and the seed
epidermis during
imbibitions [21]. Thus, by suppressing the chemotactic motility
of soil bacteria toward
germinating seed, the protein lectin can protect seedling roots
from harmful bacteria [21].
Brieftly, it can be concluded that most plant lectins are reported

to be involved in plant
defences. From this viewpoint, the preferential accumulation of
lectins in storage organs
has been the focus of attention for further research.
Furthermore, lectins in plants are
typically present in large amounts and therefore also behave as
storage proteins, as plants
can accumulate them as a nitrogen reserve [21]. Recently, by
using microarray screening
and a subtractive cDNA library from Alternaria brassicicola-
inoculated Arabidopsis thaliana
plants, Prof Schenk’s research team discovered a gene
(At3g15356) that encoded a lectin-
like protein [20]. The gene was named Protectin 1 and
particularly responds to methyl
jasmonate signal and is up-regulated by all common plant
defence pathways as well as by
the attack of pathogens and nematodes [20, 26]. Protectin 1
gene expresses one of the most
abundant transcripts during defence response against pathogen.
Quantitative real-time PCR
(qRT-PCR) revealed an increase of 10% in gene expression of
Protectin 1 when Arabidopsis
plants were exogenously treated with methyl jasmonate.

Furthermore, the increase in
Lectin expression was also observed in treatments that included
other defencedefence
signaling hormones, such as ethylene (ET) and salicylic acid
(SA) (up to 13.1-fold and 11.1-
fold, respectively) [20]. However, such gene was suppressed by
the compound abscisic acid
(ABA), which is a stress signaling compound. A clear
repression of 5.6-fold of Protectin in
Arabidopsis was revealed after a 24-hour treatment with ABA
[14]. Utilizing SDS-PAGE and
mass spectrometry, P. Schenk et al. envisaged two isoforms of
Protectin-1, an
unglycosylated (29.989 kDa) and a heavily glycosylated (31.175
kDa) protein. The fully-
formed glycosylate harbours six or seven sugar residues binding
to protein while the hypo-
glycosylated form consisted of just one sugar residue attached
[20]. Plant expression studies
8

with fusion proteins that accumulate a green fluorescent protein
(GFP) indicated that the
protein Protectin 1 is expressed in the root cell wall and may be
able to act as a defence
barrier to the plant [20]. Lectin-overexpressing plants showed
higher resistance against
bacteria possibly by immobilising them at the root surface. The
present study aimed to
investigate whether the same mechanism of immobilising
bacteria may also enable plants to
obtain nutrients by direct uptake of bacteria.
2.3. Arabidopsis plant
Since the research time is short, we used Arabidopsis thaliana
which has a short life cycle. In
addition, Arabidopsis thaliana is an excellent model for
investigating plant biotechnology
[27]. Although it has no major agronomic significance,
Arabidopsis facilitates basic research
in genetics and molecular biology because these plants harbour
a simple genome, short life
time, and a large number of mutant lines and genomic resources

[28]. As rhizosphere and
plant roots are colonised by soil bacteria that are attracted by
rhizodeposits, roots possibly
manipulate the microbial flora when they need to allocate
resources for plant defence [29,
30]. Results shown by Hein et al. (2008) indicated that the
diversity of rhizosphere microbes
is different between Arabidopsis thaliana salicylic acid-
mediated systemic resistance mutant
and the wild-type. This insight opened the door towards a
thorough understanding as well
as application of inducible plant as a control force in shaping
soil bacterial assemblages [31].
As a first step to gain a further understanding about how
Arabidopsis thaliana can
manipulate their soil environment via inducible defence
mechanism, we attempted to
quantify Escherichia coli cells in the rhizosphere and roots of a
lectin over-expressing line
and wild-type Arabidopsis thaliana. In nature, the Lectin gene is
expressed in some plant
species including the wild-type Arabidopsis. A Lectin over-
expressing line was generated by
transforming Arabidopsis plants with the 35S overexpressing

promoter upstream the Lectin-
encoding gene. [26]. In order to facilitate the convenient
selection of successful transgenic
plants, an anti-herbicide gene was inserted to the synthetic
vector simultaneously.
Therefore, before conducting a germinating experiment, a step
of checking homozygous
lectin-overexpressing Arabidopsis plants was performed by
spraying basta, an herbicide that
9
should select for transgenic plants co-expressing the basta-
resistance (BAR) gene. A 100%
survival rate indicated that mother plants are homozygous [32].
The 35S lectin
overexpression line at the 2
nd
day and the 5
th
day of post infection showed a lower amount

of bacteria Pseudomonas expression compared to the T-
DNA/knock out line and the wild
type. Furthermore, less nematode eggs were also found in the
rhizosphere of lectin
overexpressing plants in comparison to wild type. Surprisingly,
none of these independent
overexpressing transgenic lines showed any discernible
morphological phenotype [20].
2.4. Metagenomics
Metagenomics is the culture-independent genomic analysis to
study potential functions of
microbial communities directly from their natural environments
[33]. This term is combined
between the statistical concept of meta-analysis (a process of
statistically combining
separate analyses) with genomics (the comprehensive
acknowledge of an organism’s
genetic material) [34].
The soil microbial community is considered to have a highest
level of microbial diversity
compared with other environments [35, 36]. The number of
bacterial species per gram of
soil is estimated to vary between 2000 and 8.3 million [36].
This soil species pool confers a

gold-mine for genes servicing in applications in industry, such
as pharmaceutical products as
well as in biodegradation of human-made pollutants [37, 38].
However, it is estimated that
only less than 1% of this diversity can be cultured by traditional
techniques [39]. Thus,
culture-independent approaches including a variety of methods
to extract DNA from soils
have been developed [40, 41] and if coupled to next generation
sequencing, this approach
can significantly improve our access to these communities [35].
Metagenomics has recently
been advanced in microbial genomics, in polymerase chain
reaction (PCR) amplification and
in cloning of genes that share a sequence which is similar to the
16S rRNA directly from
environmental samples [39]. In bacteria, archaea, chloroplasts,
and mitochondria, a small
ribosomal subunit possesses the 16S rRNA (letter S in “16S”
stands for Svedberg unit) while
the large one contains two rRNA species which are the 5S and
23S rRNAs. All of the bacterial
16S, 23S, and 5S rRNA genes are typically organized as a co-
transcribed operon [42] and

generally, the rRNA genes are the most conserved (least
variable) in all cells. So, portions of
the rDNA sequences from distantly-related organisms are very
much alike and sequences
from such organisms can be precisely aligned. They generate
the true differences for an
10
easy measurement. Consequently, the rRNA-coding genes are
typically used to determine
taxonomy, evolutionary relationship, and the rate of species
divergence among bacteria
[42]. Currently new deep sequencing methods confer a
convenient platform to characterise
efficiently the composition of microbial communities [43, 44].
16S rRNA pyrosequencing
that is used for quantification of bacteria presence recently has
been become one of the
most striking means to tackle that issue. The 16S rRNA gene
consists of highly conserved

regions which are interspersed with variable regions. Therefore,
the PCR primers were
designed to be complementary to universally conserved regions
and to flank variable
regions [45]. The results that are acquired from amplification
and sequencing then are
compared to databases to allow the generation of bacterial
lineages and proportions in their
community [46, 47]. Un-cultured rhizosphere bacteria have been
also studied extensively
using 454/Roche pyrosequencing to identify the 16S rRNA gene
sequence, and multiple
studies then have been conducted to optimise the method [45].
In briefly, this new and
potential technique can also become a powerful mean to
evaluate the effect of plants to the
diversity of rhizophere bacteria.
III. Research objective
In this study we used the lectin-overexpressing Arabidopsis
plants for controlled microbial
inoculation experiments. We aimed to quantify inoculated
bacterial cells in the rhizosphere
and roots and to explore whether inoculated bacteria may
survive differently around lectin-

overexpressing plants. The following two objectives help to
achieve the above-mentioned
aim.
Objective 1: Screening and isolating homozygous lectin-
overexpressing Arabidopsis plants.
Objective 2: Using controlled E. coli plant root inoculation
experiments coupled with
quantitative PCR (qPCR) in order to evaluate the survival of
these bacteria around lectin-
overexpressing plants. This may provide clues whether these
plants can increase direct
nutrient uptake from bacteria.
11
IV. Material and methods
Firstly, plants of Lectin-1-overexpressing Arabidopsis line were
cultivated in soil. After 3
weeks when plants achieve 10-leaf-stage, Basta herbicide 1%
was sprayed over plants. Since

the BAR gene for Basta resistance was co-transformed with the
35S promoter, survival rates
of plants after spraying were used to evaluate whether plants
were offspring of homozygous
or heterozygous lines. The homozygous seeds that showed high
percentages of germinating
and low rates of plants depicting any yellowing symptoms were
chosen to be utilised in the
next screening step to isolate the best Lectin-1-overexpressing
Arabidopsis plant. To
perform this screening, the best Lectin-1 seeds that were
isolated from the Basta-spraying
experiment were germinated again and the plant tissues were
collected for RNA extraction.
After cDNA synthesis, a qRT-PCR was performed utilising
primers (provided by Shenk lab) to
amplify the Lec-1 gene to confirm which over-expressing line
contained highest levels of Lec-
1 transcripts.
Other experiments later were carried out to determine
differences of bacterial densities in
the rhizosphere of Arabidopsis between the wild type (WT) and
the Lec-1 over-expressing

line. One was performed with surface sterilized seeds
germinated on sterile solid medium of
Murashige and Skoog (MS) mineral salts. Seedlings were grown
under axenic conditions.
After 2 weeks when these plants reached 6-8 leaf-stage, they
were transferred to sterilised
vessels containing autoclaved soils and Escherichia coli was
inoculated on the soil around
plants. E. coli was chosen since it does not form any kind of
association with Arabidopsis
thaliana and is not ordinarily found in soils.
DNA then was extracted from roots and from the rhizosphere
soil. qRT-PCR was performed
again using two sets of primers to amplify 16S rRNA gene.
4.1. Plant growth conditions, chemical treatment and
rhizosphere soil
sampling
4.1.1. Germinating Arabidopsis thaliana seeds
Arabidopsis thaliana can be grown in various environment
conditions, for instance, growth
chambers, growth rooms, window ledges, outdoors, or
greenhouses [48]. Peat moss-based
mixes, defined agar media, relatively inert media watered with

nutrient solutions and
commercial greenhouse mixes and can all be used as plant
substrates [49]. However, this
12
study only focused on growth of plants on soil and on agar
plates which are placed in
growth rooms. Arabidopsis seeds are typically stored at 4
o
C for three days after sowing.
4.1.1.1. Germinating Arabidopsis thaliana seeds on soil
In this study we used the product Real Premium Potting Mix
manufactured by J.C. & A.T.
Searle Pty. Ltd, Queensland, Australia. The recipe contains
Flourish soluble plant food,
Penetraide, Robust Plus, complete plant food plus trace
elements, water crystals & zeolite,
and fully organic compost & peat.
Different containers or pots can be used for the growth of
Arabidopsis plants on soil [49].

The preparation of pots and planting can be conducted as
follows:
1. Potting soil was autoclaved first to give plants the best
growing environment by killing
disease pathogens and weed seeds that might be lingered in soil.
Typically, most
commercial products had been already done this step but it
should be repeated again.
2. Several pots were placed in a tray or in another similar
container which was covered by a
plastic wrap. Additionally, each pot was also covered by a piece
of mesh fabric to keep the
soil inside as well as maintain enough humidity.
3. Humidified soil with tap water then place loosely soil in pots
or flat chambers. The soil
was not compressed to give a soft and uniform bed. At this
stage, pots were ready for
germinating.
4. Sowed Arabidopsis seed to the surface of soil pots. Try not to
cover plant seeds by soil
since they needed light for germination.
5. Covered the trays by a clear plastics lid to maintain humidity
for germination and avoid

seed desiccation.
6. The whole tray was covered more by a plastic bag and placed
in the dark and cold room
at the refrigerator temperature (3-4°C) for 3 days to break
dormancy and improve
germination rate and its synchrony. This treatment stage was
especially important to freshly
harvested seeds that had more pronounced dormancy [49].
7. After the cold treatment stage, they were moved to the
growth room and watered every
one or two days to maintain approximately 2 cm of water
around seed during germination
phase.
13
Arabidopsis seedlings were grown in a growth chamber at 25°C
with a photoperiod of 16
hour light and 8 hour dark. Under optimal conditions of water
supply and good nutrition,
seeds started to germinate within 3-5 days [49].

After germination, plants were needed to avoid water stress. So,
sub-irrigation was only
applied when the soil begin to dry. When plants had got true
leaves, watering frequency
was decreased [49].
4.1.1.2. Germinating Arabidopsis thaliana seeds in sterilize
conditions
It is necessary to grow Arabidopsis thaliana axenically for this
specific experiment of
determining the survival of bacteria around lectin-
overexpressing plants compared to the
wild type. Firstly, we used petri dishes to germinate surface-
sterilised seeds and then plants
were transferred to vessels containing soils.
The media that was used for Arabidopsis culture was Murashige
and Skoog (MS) mineral
salts added Bacto Agar TM 1.5% with optional 1.5% sucrose
[49].
The recipe for 0.5L MS agar media:
- Sugar Sucrose 7.5g
- Agar 7.5g

- MS salt 1.1g
- Distilled water 500ml
Preparation of 500ml media was conducted as follows:
- Added 7.5g of MS salts and 7.5g sugar to 450ml of distilled
water, stired to dissolve;
- Checked and adjusted to pH 5.7. Adjustment was supported by
1M KOH;
- Added 7.5g agar and diluted with distilled water to final
volume of 500mL;
- Autoclaved for 20 minutes at 121
o
C, 15 psi.
The solution was then divided into petri dishes and waited until
the agar surface was hard
enough.
Seed sterilization was also required before using as follows:
1. Add ed1ml Ethanol 70% into tube of seed;
2. Vortexed or shaked for 2 minutes;
3. Poured away;
4. Added 1ml Bleach (Hypoclorit) 50%, , repeated every 1
minute in 10 minutes;

14
5. Washed at least 4 times with distilled water;
6. Added 1ml distilled water for using.
The stage of placing seeds on media plate was conducted in
flow cabinet condition;
sterilized tips and pasteur pipet were also required. Otherwise,
contamination can possibly
occur. Exhausted air from the pipet, soaked its tips into the seed
tube and used slow release
pressure on bulb to take a single seed into tip. The seed then
was dropped at the expected
location on agar surface. Try to design a fair density with about
64 seed per plate. These
seed plates were then covered and sealed with parafilm to
prevent desiccation and
contamination and placed in the growth room under condition of
16 hour light and 8 hour
dark photoperiod. This photoperiodic lighting program
stimulated the quick growth of

plants.
The agar plate is a nice environment for Arabidopsis growth,
however there is not enough
space for plant maturation. Thus, after 2 weeks when these
plants got 6-8-leaf stage, they
were transferred to sterilize soil environment with E.coli
inoculated in simultaneously as
shown in the figure 1 below. We used 7.5 cm-diameter clear
transparent tissue culture jars.
Each one contains:
- University of California mix 25g
- Commercial compost soil 25g
These jars of soil blend then were undergone double sterilize
treatment on the same day.
This soil mixture facilitated optimized water drainage for
growth of Arabidopsis in tissue
culture jars. For treatment we applied to each jar: Plants taking
out from agar plates were
grown into soil. The roots were buried well into medium soils
before adding 1.2ml solution
of bacteria inoculation around each plant. Each of jars
harboured 3 plants, so, 3.6ml

inoculation solution was added totally per container. Finally,
4.8ml of distilled water was
provided to ensure enough humidity for Arabidopsis growth.
Closed tightly cap then placed
jars in growth environment. The environment inside jars
currently liked a closed system.
15
Figure 1: Arabidopsis thaliana in the process of transferring
form agar plate to soil
environment.
(A) Arabidopsis thaliana after two weeks of germination on the
MS media
(B) Arabidopsis thaliana were grown in jar of soil
(C) Arabidopsis thaliana in jar of soil after two weeks
4.1.1.3. Checking homozygous seeds
In this research, the transgenic BAR gene against the herbicide
BASTA was investigated as a

physiological marker. Each of grown plant was progeny of an
independently-derived lectin-
transformed line. Such lectin-transformed plants typically
carried one T-DNA insertion
hemizygously at a single locus, since plants harbouring 2
independent in sertions were not
common. As a result, lectin transformants needed to be selected
for homozygosity via Basta
resistance, self-pollinated, and harvested individually. Among
lectin-transformed lines, we
found the homozygous ones by checking the resistance of them
to 1% Basta herbicide.
Prepare 1% Basta solution:
Basta herbicide: 150µL
Distilled water: 15mL
16
4.1.2. Preparation of E.coli for inoculation around plant roots
4.1.2.1. Growth of E.coli on LB (Luria-Bertani) agar plate

* Recipe for 1L LB medium without antibiotic:
- Bacto Tryptone 10 g
- Yeast extract 5 g
- NaCl 10 g
- Agar 15g
This medium was autoclaved on liquid cycle at 15 psi for 20
minutes; cooled to
approximately 55°C and poured into petri dishes. Let harden,
and then the plates were
inverted and stored at +4°C in the dark room. These plates were
used for 16 streaking with
E.coli then inoculated overnight at 37
o
C.
4.1.2.2. Growth of E.coli on LB Broth.
On the following day, these isolated bacteria continued to be
inoculated in LB broth within 3
hours before being added into soil jars.
- Bacto Tryptone 10 g
- Yeast extract 5 g

- NaCl 10 g
For this inoculated treatment, we used 2 flasks of 250ml with
100ml of LB Broth inside. One
very full loop of E.coli was added into the media. After 3 hours
of inoculation at 37
o
C in
shaking machine, the number of bacteria was possibly generated
enough for the next
inoculation in rhizosphere Arabidopsis environment.
4.1.2.3. Wash LB Broth by PBS (Phosphate Saline Buffer) to
get the pellet of
E.coli
PBS recipe (1L)
1. Dissolved the following in 800ml distilled water;
- 8g of NaCl
- 0.2g of KCl
- 1.44g of Na2HPO4
- 0.24g of KH2PO4
2. Adjusted pH to 7.4;

17
3. Added distilled H2O to the final volume 1L;
4. Autoclaved
The procedure was conducted as follows:
1. Divided 200ml LB inoculum into 5 tubes of 50ml;
2. Centrifuged;
3. Discarded suspension;
4. Added 30ml PBS into each tube;
5. Vortexed well;
6. Centrifuged again;
7. Discarded suspension;
8. Added 40ml PBS into each tube;
9. Vortexed well before using.
4.2. Sample DNA, RNA extraction, PCR amplification, Real
time PCR and data
processing.
4.2.1. Plant genomic DNA and RNA extraction.

4.2.1.1. Plant genomic DNA extraction by CTAB (Cetyl
Trimethyl Ammonium
Bromide)
Essentially, the extraction requires any mechanical means that
can break down cell wall and
cell membranes to allow access to nuclear material without
damaging DNA. This method
which uses CTAB can give intact genomic DNA from plant
tissues.
After harvesting plant leaf, liquid nitrogen was employed in
initial grinding stage for
breaking down cell wall material while harmful cellular
enzymes and chemical remained
inactivated. The tissues were ground sufficiently then
resuspended in CTAB buffer. Soluble
proteins and other material were separated by mixing with
chloroform and centrifugation
while insoluble particulates were removed through
centrifugation to purify DNA. Such
nucleic acid were then precipitated from aqueous phase and
washed thoroughly to remove
contaminating salts.
Material and methods

- CTAB buffer;
- Mortar and Pestle;
- Microfuge tubes;
http://en.wikipedia.org/wiki/Cetyl_trimethylammonium_bromid
e
http://en.wikipedia.org/wiki/Cetyl_trimethylammonium_bromid
e
18
- Microfuge;
- Liquid Nitrogen;
- 70 % Ethanol (ice cold);
- Absolute Ethanol (ice cold);
- 7.5 M Ammonium Acetate;
- 55
o
C water bath;
- Distilled water;
- Chloroform: Iso Amyl Alcohol (24:1);

- RNase (10mg/mL).
CTAB buffer 100ml
- 2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)
- 10.0 ml 1 M Tris pH 8.0
- 28.0 ml 5 M NaCl
- 4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic
acid Di-
sodium salt)
- 1 g PVP 40 (polyvinyl pyrrolidone (vinylpyrrolidine
homopolymer)
Molecular weight 40,000)
- 40.0 ml H2O
Adjusted the solution to pH 5.0 with HCL and made up to 100
mL with H2O.
1 M Tris pH 8.0
Dissolved 121.1g of Tris base in 800 ml of H2O. pH was
adjusted to 8.0 (by adding HCL). The
solution was allowed to cool down to room temperature before
making the final
adjustments to the pH of 8.0. Added more distilled water to the
final volume 1L then

sterilized by autoclaving.
Procedure
1. Ground 200 mg of tissue sample to a fine paste with
approximately 500 μL CTAB
buffer; 1μL of GFP plasmid was also added at the same time to
calculate the PCR efficiency
later.
2. Transfered all extract mixture to a microcentrifuge tube;
3. Incubated for about 15 min in a recirculating water bath at 55
o
C;
19
4. The CTAB/plant extract mixture was spin at 12000 g for 5
min to spin down cell
debris then the supernatant was transferred to fresh microfuge
tubes;
5. To each tube 250 μL of Chloroform: Iso Amyl Alcohol (24:1)
is added. The solution

was mixed by inversion then spun at 13000 rpm for 1 min. The
upper aqueous phase was
transferred to a clean microfuge tube. This stage was repeated
twice;
6. 50 μL of 7.5 M Ammonium Acetate was added to each tube
before adding 500 μL of
ice cold absolute ethanol. These tubes were then incubated over
night at -20
o
C;
7. After incubation, spined to form pellet at 13.2 x 1000g for 30
minutes. Discarded the
supernatant and the DNA pellet was washed by adding two
changes of ice cold 70 %
ethanol;
8. The DNA pellet was then formed again by centrifugation at
12000 for 5 minutes;
9. The DNA was let to be dried at room temperature for 15
minutes and then
resuspended in 50 μL ultrapure water followed by adding 1 μL
RNase (10 ng/mL) and
incubating at 37
o
C for 1 hour to remove RNA in the preparation;
10. The resuspended DNA was then incubated at 65

o
C for 20 minutes to destroy any
contaminated DNase and stored at 4
o
C until using.
4.2.1.2. Plant RNA extraction.
Total RNA from leaves were extracted by SV Total RNA
isolation Kit (Promega). All the work
places and pipettes were decontaminated with the solution RNA
AWAY (Invitrogen).
The RNA concentration was measured by spectrophotometer
(NanoDrop® ND-1000). To
check the quality of the RNA, an agarose gel was run with
ethidium bromide. The procedure
was conducted as follows:
1. Samples (leaves) harvesting was placed in centrifuge tube
then put in liquid nitrogen
that employs in initial grinding stage. Abrasive sticks were used
to break down cell wall and
cell membrane in about 30 seconds before the sample can be
defrosted.
2. Added 175 μL SV RNA Lysis Buffer (already added BME) to
tubes. Mix through by

inversion.
3. Added 350 μL SV RNA Dilution Buffer. Mix briefly by
inverting 3 – 4 times.
4. Centrifuged for 10 minutes then transfered the clear lysate to
a clear
microcentrifuge tube.
5. Applied 200 μL Ethanol 100% to clear lysate, mixed well by
repeat pipetting.
20
6. Transfered the mixture to Spin Basket Assembly then
centrifuged for 1 minute.
Eluate was discarded.
7. Added 600 μL SV RNA Wash
Solution
(already added ethanol) then centrifuged for 1

minute and discard eluate.
- Prepared DNase incubation mix as follows:

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