生物类英语论文
年欢晚会-
Effect of different carbon source and
nitrogen source on
the cold resistance
of citrus callus induction
Hu Siyuan
(College of Agronomy, Northwest A&F
University, Yangling, Shaanxi 712100,China)
Abstract:
Citrus
callus is ideal material for physical and chemical
mutagenesis
and
genetic
transformation,
the
carbon
source
and
nitrogen
source
is
a
necessary
component
in
the
induction
and
subculture
of
citrus
callus,
so
the
exploration
of
carbon, nitrogen sources
is of great significance to the effect degree of
citrus callus
induction and effect way.
This study choose cold resistance of citrus as
test material,
through
culture
vitro
tissues,
analysis
of
the
effects
of
carbon
source
and
concentration, nitrogen
source and level on the influence of citrus callus
induction, To
provide the basis for the
study of the occurrence and the mechanism of cold
resistance
of Citrus genetic
improvement, somatic embryos.
Key
words:
carbon source;
nitrogen source; citrus; callus
Introduction
Citrus (Citrus
reticulata Blanco) Rutaceae Citrus, small
evergreen tree or shrub,
about
three
meters
high,
weak
branches,
usually
with
thorns.
Leaves
are
alternate,
leathery,
lanceolate
to
ovate-lanceolate,
5.5-8
cm
long,
2.9-4
cm
wide,
apex
acuminate,
base
cuneate,
entire
or
with
fine
blunt
teeth,
petiole
slender,
winged
obvious .
Flowers
small,
yellow-white,
solitary
or
clustered
in
leaf
axils,
sepals
5;
petals
5; stamens 18-24, filaments 3-5 pieces often
connate, ovary 9-15 rooms. Citrus
fruit
flat
spherical,
5-7 cm
in
diameter, orange-yellow
or reddish
yellow, peel
loose
flesh meat easily
separated.
Chenggu citrus cultivation
has a long history dating back more than 2,000
years
of history. Chenggu County is
located in Southern
Shaanxi,
Central Hanzhong Basin,
north
subtropical to temperate transition region, north
of Qinling mountain blocking
the
invasion of cold northwest, south winds Bashan so
warm air can not drive straight.
Inversion
effect
Qinling
belt
and
foothills
sloping
wall,
forming
a
unique
micro-climate
regions,
fewer
winter
cold
and
summer
heat
less.
First,
here's
citrus
high
temperature
hot
wind
does
not
harm
flowering,
fruit
setting
rate;
Second
cool
early
autumn,
temperature,
and
is
conducive
to
fruit
color,
ripe,
citrus
fruit
surface
here is gorgeous, high soluble solids
content, moderately sweet and sour.
Studies
[1-4]
have
shown
that
by
changing
the
carbon
source
to
induce
somatic
embryogenesis.
As
most
of
the
citrus
callus
has
not
regenerate
embryoid
body
phenomena
[5-7]
,
while
the
high-
frequency
induction
of
somatic
embryogenesis
of
citrus cells for genetic
transformation, in vitro mutagenesis, in vitro
screening mutants,
rapid
propagation,
etc.
work
has
special
significance
[8-9]
.
In
this
paper,
sucrose,
glucose, soluble
starch, three kinds of carbon and nitrogen sources
ammonium nitrate
one
kind
of
growth
and
the
ability
of
somatic
embryogenesis
of
citrus
callus
were
studied at different
levels, in order to select the optimal culture
conditions hardy citrus
studies provide
evidence.
1. Purpose and significance
1.1 Purpose
1. Analysis the influence of carbon
types and carbon levels on hardy of citrus in
vitro cultured explants
dedifferentiation;
2.
Explore
the
nitrogen
source
for
citrus
callus
induction
effect
manner
and
extent
of effect;
3.
Screening
suitable
carbon,
nitrogen
conditions
of
citrus
dedifferentiation
in
vitro callus induction.
1.2
Significance
Citrus Calli
is an ideal material for the physical and chemical
mutagenesis and
genetic
transformation,
and
carbon
and
nitrogen
is
a
necessary
ingredient
in
citrus
callus
induction and subculture, therefore it is
important to explore the extent and the
effect of the way on citrus callus
induction of carbon source, nitrogen source.
1.
Explore
the
carbon
and
nitrogen
sources
for
citrus
callus
induction
effect,
especially on the induction rate and
differentiation speed for in-depth study of carbon
and nitrogen in the mechanism of tissue
culture citrus from the body, and to provide
some data to support
2.
Hanzhong
citrus varieties for the study material, try to
filter out the best of its
callus
induction
of
carbon
required,
the
type
and
content
of
nitrogen
for
the
production of virus-free citrus
seedlings through callus ideal way to provide
relatively
basic culture conditions.
2. Materials and methods
2.1
Materials
Seeds of the test material
explants of the same size, healthy full Chenggu
hardy
citrus varieties.
2.2
Preparation
2.2.1 Instrument
HS series horizontal flow bench (Suzhou
Antai Air Technology Ltd.); the dish;
vertical
circular
chamber
pressure
steam
sterilizer
(Shanghai
Medical
Nuclear
Instrument
Factory);
portable
electric
pressure
steam
sterilizer
(Shandong
Xinhua
Medical
Instrument)
electronic
balance
(Shanghai
balance
Instrument
Factory);
electric distilled water (Shanghai
medical nuclear Instrument Factory).
2.2.2 Auxiliary materials
(1) In a planned way of cleaning the
glassware (Erlenmeyer flasks, petri dishes,
etc.), and in natural conditions or
oven drying;
(2) Take a certain amount
of Erlenmeyer flask under seal membrane, with
cotton
rope is secured;
(3)
Take
a
certain
amount
of
the
Erlenmeyer
flask
was
added
to
the
flask
to
approximately 2/3 of pure
water, sealing;
(4) Put the
filter paper into a petri dish, wrap;
(5)
Put
the
sealed
Erlenmeyer
flasks
and
Petri
dishes
into
high-
pressure
steam
sterilizer,
autoclaved at 1.1 kg/cm2 30min;
(6)
The
sterilization
dish,
flask
covered
with
newspaper
spare,
finishing
laboratory.
2.2.3 The basic
culture medium
Table 2.1 the basic
culture medium
Mother liquor
category
A large
number of
element class
KNO
3
N
H
4
NO
3
MgSO
4
·
7H
2
O
KH
2
PO
4
CaCl
2
Trace element class
< br>MnSO
4
·
H
2
O
ZnSO
4
·
7H
2
O
H
3
BO
3
KI
Na
2
MoO
4
·
2H
2
O
CuSO
4
·
5H
2
O
CoCl
2
·
6H
2
O
Lron
class
Organic material
class
Na
2
EDTA·
2H
2
O
FeSO
4
·
7H
2
O
glycine
Thiamine
hydrochloride
Hydrochloric acid
pyrazole
compound
element
niacin
inositol
5
100
250
5000
10
500
Composition
Working liquid
concentration
（
mg/L
）
1900
1650
370
170
332
22.3
8.6
6.2
0.83
0.25
0.025
0.025
37.25
27.85
2.0
0.4
A variety of
practical quantity
（
mg
）
19000
16500
3700
1700
3319
1115
430
310
41.5
12.5
1.25
1.25
1862.5
1392.5
100
20
500
500
500
1000
Constant volume
standard
（
ml
）<
/p>
2.3
Experimental
design
2.3.1
Variable selection
1.
Argument:
(1) Types of
carbon source: sucrose; glucose; soluble starch;
(2) The content of carbon
source:
four levels of
sucrose 15g / L; 30g / L; 45g / L;
60g
/ L;
(3) The content of nitrogen
source: KNO
3
/
NH
4
NO
3
= 1:1 and KNO
3
/
NH
4
NO
3
= 1:2.
2. The dependent
variable:
(1)
Induction
rate:
the
number
of
callus
formation
slices/the
number
of
inoculation slices * 100%;
(2) Expansion rate: the number of in
vitro tissue expansion slices /the number of
inoculation slices * 100%;
(3)
Expansion
speed:
expansion
speed
=
(N
K
-
N
0
)/K
*
number
of
slices.
N
0
represents
the number of
in vitro tissue
expansion
slices
at
first
time, N
K
represents
the number of in
vitro tissue expansion slices at Kth day after the
first time;
(4)
Induction
speed:
induction
speed
=
(N
K
-
N
0
)/K
*
number
of
slices.
N
0
represents the number of in vitro
tissue induction slices at first time,
N
K
represents the
number of in vitro tissue induction
slices at Kth day after the first time.
2.3.2 Design scheme
In this
experiment, three kinds of carbon sources, namely
sucrose, glucose and
soluble starch,
sucrose density gradient set 4, glucose and
soluble starch concentration
gradient
is
not
set;
nitrogen
concentration
gradient
set
2
were
normal
1.65g
MT
medium
/
L
(KNO
3
/NH
4
NO
3
=
1:1)
and
doubling
the
value
of
3.30g
/
L
(KNO
3
/NH
4
NO
< br>3
= 1:2), the specific content is
shown in Table 2.2 table 2.3:
Table 2.2
three kinds of carbon source materials in culture
Composition
Serial number
1
2
3
4
5
6
Sucrose
（
g/L
）
30
30
0
0
0
0
Glucose
（
g/L
）
0
0
30
30
0
0
Starch
（
g/L
）
0
0
0
0
12
12
NH
4
NO
3
（
g/L
）
1.65
3.30
1.65
3.30
1.65
3.30
Table 2.3 four different carbon source
level medium material content
Composition
Serial number
1
2
3
4
Sucrose
（
g/L
）
15
30
45
60
Glucose
（
g/L
）
0
0
0
0
Starch
（
g/L
）
0
0
0
0
NH
4
N
O
3
（
g/L
）
1.65
1.65
1.65
1.65
2.4
Research on
Technology Roadmap
Access
to study material, aseptic inoculation, in vitro
culture observation,
analysis of carbon
and nitrogen source on the effect of citrus in
vitro tissue culture
2.4.1 Aseptic
inoculation
In
the
clean
bench
cut
citrus
seed
cotyledon
about
the
size
of
3mm
×
3mm
random
explants
inoculated
into
each
culture
medium,
the
body
number
of
each
medium was inoculated explant is not
less than 30.
2.4.2 Culture observation
Materials
placed
in
a
temperature
25
±
2
℃
,
relative
humidity
of
50-60%
under conditions of
darkness training, periodic statistical swelling
number of explants
with out a few more,
and so calculate the corresponding secondary
variables.
2.4.3 Statistical analysis
Regular
observation
of
statistics,
data
obtained
entry
Excel
database
and
then
convert the data in SPSS professional
platform for single-factor analysis of variance
and two factor factorial analysis.
3. results
and
analysis
3.1
The
effect of carbon species on citrus callus
induction
In
view
of
the
three
types
of
carbon
in
the
experimental
group
were
provided
with two nitrogen levels, and therefore
can not exclude the impact of nitrogen ratio of
different carbon sources, it should
take two-factor factorial analysis and the results
are
shown in Table 3.1 and Figure 3.1:
Table 3.1 Effect of different carbon
sources on citrus callus
Two-factor
factorial
analysis
Expansion
rate
Induction rate
Expansion speed
Induction
speed
Early expansion speed
Late expansion speed
Early
induction speed
Late induction speed
Sum of squares
0.070
0.047
0.000
2.04E-005
0.003
0.003
2.89E-005
0.001
Degrees of
freedom
2
2
2
2
2
2
2
2
The mean square
0.035
0.023
0.000
1.02E-005
0.002
0.001
1.44E-005
0.000
The F
value
9.422
5.626
2.107
4.711
6.629
4.809
1.284
3.772
Significant
0.008
0.030
0.184
0.044
0.020
0.043
0.328
0.070