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Chapter 11
Conservation In Vitro of Plant
Germplasm Resources
• Maintaining biodiversity is an international
issue.
• Due to the destruction of the ecological
balance, a large number of species are
gradually lost or destroyed.
• Artificial selection and extension推广of improved
varieties良种, species composition is gradually
simplified, resulting in many potentially useful
precious germplasm resources are lost;
• Germplasm resources are the basis of plant
breeding育种work.
• Therefore, the collection and conservation of
germplasm resources have been valued by
countries around the world.
• The conservation of plant germplasm
resources
• It refers to germplasm resources being
conserved using natural or artificial creating
suitable environment in order to maintain
the genetic integrity完整性and vitality
contained in the individual's genetic
material遗传物质 and pass on their genetic
traits by breeding繁殖.
• There are two types of germplasm
resources conservation, that is, in situ
conservation (原生境保存) and ex situ
conservation (非原生境保存).
• In situ conservation
• The genetic material of plants is conserved
in their natural environment.
• The most place of in situ conservation is
plant natural reserves (植物自然保护区),
and an alternative method is field planting
conservation (农田种植保存).
Wolong
national
nature
reserve
Wuyishan
national
nature
reserve
Jiangxi Lushan national nature reserve
Field planting conservation
Field planting conservation
• Ex situ conservation
• The genetic material of plants is not conserved in
the place of their natural habitats.
• Methods of ex situ conservation are as follows:
• Field gene bank (Germplasm collection, 种质圃);
• Botanical garden (Arboretum, 植物园);
• Seed bank;
• Conservation in vitro;
• Etc..
[cōn
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• Although in situ conservation, botanical
garden, and seed bank are very important,
but they requires a lot of manpower,
material resources and land, and they are
vulnerable to natural disasters and the
invasion of pests and diseases.
• Therefore, these ways are not conducive to
the exchange and conservation of plant
germplasm resources.
• Limiting factors of seed conservation:
• ①Seed life decreased with time;
• ②Asexual propagated plants have no
seeds;
• ③Plants that maintains excellent traits by
asexual reproduction may mutate by seed
reproduction;
• ④The plants producing recalcitrant-type
seeds are sensitive to dehydration;
• ⑤Seeds are easy to lose when they are
attacked by natural disasters.
• In vitro conservation
technique was born.
• In 1960s,
conservation in vitro:
• Plant tissue culture
technique is used to
limit, delay or stop the
growth of plant
materials.
• Advantages of in vitro conservation:
• The occupied space is small, saving
manpower, material resources and land;
• Convenient for the exchange and utilization
of germplasm ;
• When needed, materials can be taken out
for mass reproduction;
• To avoid the germplasm loss caused by
natural disasters.
• Shortcomings of in vitro conservation:
• Need continuous subculture;
• Contamination or human error is easy to
occur;
• Subculture for many times is easy to result
in variation.
• Section 1
• Minimal growth conservation
• 限制生长保存
• Minimal growth conservation is a
common strategy for germplasm
conservation in vitro by limiting the growth
rate to preserve germplasm.
• For example:
• ①Low temperature;
• ②Increase the osmotic pressure渗透压;
• ③Add plant growth inhibitor or plant growth
retardant延缓剂;
• ④Dry;
• ⑤Reduce oxygen partial pressure分压;
• ⑥Mineral oil矿物油cover;
• etc..
• ☆ ☆ ☆But pay attention to two points:
• ①In order to reduce the evaporation蒸发
rate of the culture medium, the type and
the sealing密闭method of the storage
container should be paid attention to
• The death of the long-term conserved
materials is often caused by the water
deficit亏缺 of the medium.
• ②Minimal growth conservation has a
greater chance of variation whose genetic
stability must be periodically定期identified
(cytology细胞学, genetics遗传学, and
production traits生产性状).
• 1、 Coneservation method by low
temperature
• The most widely used method;
• 1-9 ℃ (tropical 热带and subtropical 亚热带
plants 10-20 ℃).
•
• 2、Conservation method by high osmotic
pressure
• Increase the osmotic pressure of the medium,
inhibit the growth rate;
• Generally the sucrose concentration was 3%. If it
increased to 10%, the growth rate is inhibited;
• But the effect of mannitol甘露醇and sorbitol山梨
醇 is better, because they are inert matters惰性物
质 and are not easily absorbed by plantlets
• 3、 Conservation method by plant growth
inhibitor or plant growth retardant延缓剂
• Commonly used is ABA;
• Also including:
• Maleichydrazide (MH)青鲜素;
• chlormequat (CCC)矮壮素;
• N- two methylamino succinamic acid (B9)丁酰肼,
比久;
• Paclobutrazol (PP333)多效唑 .
• 4、Conservation method by reducing oxygen
partial pressure
• To reduce the oxygen partial pressure in the
culture vessel容器, and change the gas condition
in the culture environment;
• Similar to the method of storage and keeping
fresh of fruits and vegetables;
• However, if the oxygen partial pressure of the
culture vessel is too low, it will produce toxic
effect毒害作用 because of anaerobic respiration
无氧呼吸.
• 5、Conservation method by drying
• Reduce the moisture水分 content of the
culture;
• Similar to seed drying storage.
• 6、Advantages of minimal growth
conservation
• ①Simple and easy to operate, rapid
recovery, suitable for the management of
modern germplasm genebank;
• ②But different materials, the methods used
are different;
• ③Two or more than two methods are often
combined and used.
• 7、Disadvantages of minimal growth
conservation
• Still need subculture, which is easy to
cause contamination污染;
• Continuous subculture will occur genetic
variation, so it can only be suitable for short
and long term conservation.
• Section 2
• Cryopreservation of germplasm
resources
• In 1970s, Nay and Street successfully
conserved carrot suspension culture cells
in liquid nitrogen (LN, 液氮) for the first time.
• Cryopreservation is the method of plant's
cells or tissues being antifreezing treated
and conserved below 80 ℃.
• Generally liquid nitrogen is used as a cold
source of cryopreservation, so that the
temperature can be maintained at -196 ℃.
•Question:
•Why not use ultra-low
temperature freezer?
• Under the ultra-low temperature, the
metabolism and growth activity of living
cells was almost completely stopped.
• Therefore, the change of genetic traits of
the cell, tissue and organ will not cause in
the cryopreservation process, which will
not lose the potential of morphogenesis形
态发生.
• At the same time, under the condition of
super low temperature, the metabolism and
the aging衰老 process of organism are
greatly slowed down, or even completely
stopped, so the plant materials can be
conserved for a long time.
• Significance of cryopreservation
• ①To prevent the extinction灭绝 of
resources, saving manpower and material
resources, easy to exchange.
• ②It can conserve a lot of resources in a
limited space.
• ③Compared with seed storage, it can not
be restricted限制by time and high water
content.
• ④Compared with the plantlets, it can avoid
the genetic variation caused by frequent
subculture.
• 1、The basic principle of cryopreservation
• ①Theory of cell protective dehydration
• At room temperature, the cell and its solution
are in osmotic equilibrium state.
• When the temperature drops below the
freezing point, the water in the extracellular
solution firstly freezes into ice, extracellular
solution concentration begins to increase, the
balance of intracellular and extracellular
solution is destroyed, the water in the cell
permeates outwards through cell membrane,
cell begins to contract, intracellular
concentration also increased.
• When the temperature continues to decrease,
freezing and penetration process
continuously, the process is called cell
protective dehydration.
• When the temperature rises, the frozen ice
continues to melt, the water is permeated
into cells, which causes contracted cells
begin to swell膨胀, which may be restored
恢复 to the original state原状.
• Control precisely the above process,
which can make the cells be not damaged
and die in the process of cooling,
rewarming, permeation.
• ② Theory of solution vitrification
• When the solution is cooled, the
supercooled过冷 solution can be formed at
first, if there is not the formation of
homogeneous均一 nucleation晶核 or there
is not enough time for the nucleation晶核
growth.
• If continuing to cool, homogeneous
nucleation begins to form.
• If the cooling rate is not fast enough, the
sharp尖锐ice crystals冰晶 begin to form.
• The cooling rate is fast enough, there is
little or almost no formation of
homogeneous crystal nucleus均一晶核, or
the homogeneous nucleation growth lack
sufficient time, the solution goes into a
amorphous无定型的 state (vitrification
state).
• It is a transparent透明的 solid, compared
with the liquid, the molecule does not occur
rearrangement, so it is different from the
crystal晶态, which Is called vitrification
state, the temperature is called
vitrification formation temperature.
•
• In the process of vitrification, there is no
solution effect on cell damage, and no
mechanical damage caused by the
formation of ice crystals.
• ③Measures to improve the effect of
cryopreservation
• Because plant cells contain more water
than animal cells, cryopreservation of plant
cells is very difficult.
• If plant cells are directly plunged into投入
liquid nitrogen, cells and tissues will die
because of the freezing结冰 of their
intracellular water.
• Therefore, plant material can only be
successfully cryopreserved by the aid of借
助于 cryoprotectant冷冻保护剂.
• ④ Protection mechanism of
cryoprotectant
• In aqueous solution水溶液, it can strongly
combine with water molecules, which can
increase the solution viscosity黏度.
• When the temperature descends下降, the
freezing point of the solution is decreased,
that is, the growth rate of ice crystal center
is decreased, so that the solidification
degree of water decreased.
• Thus, cryoprotectant plays an important
role in reducing the freezing point of culture
medium and the freezing point of the plant
tissues and cells.
• The use of cryoprotectant increases the
osmotic pressure of the medium, which
leads to slight plasmolysis质壁分离 of the
cell and relatively improves the cold
resistance of tissues and cells. .
•
•
•
•
⑤Commonly used cryoprotectant
※ ※Permeable cryoprotectant:
Most are neutral materials of small molecule;
In the solution, they are easy to combine the
water molecules and occur hydration水合作用, so
that the solution viscosity粘性 is increased, the
crystallization结晶process of the water is
weakened, and the purpose of protection is
achieved.
• Permeable cryoprotectant mainly includes
the following substances:
• ★ ★ DMSO (二甲亚砜);
• Note: DMSO is very easy to penetrate into
the cell and prevents the cell from
excessive dehydration and damage in the
freezing and thawing化冻.
•
•
•
•
•
★ ★ Glycerol甘油;
★ ★ Mannitol甘露醇;
★ ★ Proline脯氨酸;
★ ★ Glycol乙二醇;
★ ★ Propylene glycol丙二醇.
• ※ ※ Non permeable cryoprotectant:
• Non permeable cryoprotectant is
polymeric聚合物 molecules, which can
dissolve in water, but can not enter the cell;
• Non permeable cryoprotectant can
cause the solution to become supercool to
play a protective role.
• This kind of cryoprotectant has protective
effect on fast and slow cooling.
• Non permeable cryoprotectant mainly
includes the following substances:
• ★ ★ Polyvingyl pyrollidone (聚乙烯吡咯烷
酮, PVP);
• ★ ★ Dextrane (葡聚糖);
• ★ ★ Polyethylene glycol (聚乙二醇, PEG);
• ★ ★ Hydroxyethyl starch (羟乙基淀粉,
HES).
•
•
•
•
•
•
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2、Basic procedures of cryopreservation
Generally include the following steps:
①Selection of plant materials;
②Material pretreatment;
③Freezing treatment;
④Storage in LN (liquid nitrogen);
⑤Thawing；
⑥Re-culture.
These steps are described below.
•
•
•
•
•
①Selection of plant materials
★There are three main categories:
★ Callus, suspension cell, protoplast;
★ Pollen and pollen embryo;
★ Shoot-tip, axillary bud primordium原基,
embryo, young plant.
• Generally choose the materials with a good
genetic stability, easy regeneration, and
strong anti-freeze;
• Shoot-tip, axillary bud primordium原基,
embryo, and young plant are ideal
materials.
• ②Material pretreatment
• In order to ensure the stable and high
survival rate of shoot-tip after conserved in
liquid nitrogen, it is necessary to carry out a
certain pretreatment:
• Or be precultured in the presence of a
cryoprotectant;
• Or be pretreated directly at low
temperature (0-10℃).
• ③Freezing treatment
• The method of freezing treatment has fast
freezing method, slow freezing method,
pre-freezing method and drying freeze
method.
• ★ ★ Fast freezing method
• Refers to the method of plant material
directly plunging into投入 liquid nitrogen
from 0 ℃ or other pretreatment
temperature, whose cooling rate is 1000
℃/min.
• The materials require small volume, dense
cytoplasm, low water content and low
degree of vacuolization液泡化.
• ★ ★ Slow freezing method
• 0℃ or other pretreatment temperature of
the materials drops to -100 ℃ from the
initial temperature.
• After stabilized for 1h, the materials are
plunged into投入 liquid nitrogen and
conserved.
• Or after the temperature of the materials
continues to drop to -196 ℃ at cooling rate
of 1-2℃/min, they are plunged into投入
liquid nitrogen and conserved.
• The method is suitable for mature cells
containing large vacuoles液泡 and high
water content.
• Especially suitable for the the suspension
culture cells.
• ★ ★ Pre-freezing method
• Refers to the method of the plant material
being trained at low temperature for a
short period of time before being plunged
into liquid nitrogen.
• Pre-freezing method can be divided into
two step两步freezing method and step by
step逐级freezing method.
• Pre-freezing method can make cells of
conserved materials fully dehydrate, thus
can avoid the emergence of irreversible不
可逆 damage due to the freezing of the
cells.
• ★ ★ Drying freeze method
• Refers to the method of plant material
being dehydrated by being dried in 27 - 29
℃ oven and then plunging into投入 liquid
nitrogen.
• Drying freeze method can prevent the
materials from freezing and death.
• ④Storage in LN (liquid nitrogen)
• Materials are conserved in liquid nitrogen
for long periods, which need a suitable
containers to hold liquid nitrogen.
• Generally liquid nitrogen tanks are used.
Liquid nitrogen
need be
continuously
added during
cryopreservation.
• ⑤Thawing
• The freezing injury of the plant material
occurred in the process of cryopreservation
is produced in the freezing and thawing
process.
• The freezing injury occurred in the process
of thawing is caused by intracellular
secondary freezing次生结冰.
• The cell membrane system is destroyed by
the osmotic shock渗透冲击of water in the
process of thawing.
• Note: Thawing method has two kinds: Fast
thawing method and slow thawing method.
• ★ ★ Fast thawing method
• Refers to the method of material stored in liquid
nitrogen being directly put into the 37-40℃
constant temperature water bath for thawing.
• The thawing speed is 500-750℃/min, and most
of the plant materials can be used in this way.
• ★ ★ Slow thawing method
• Refers to the method of the material stored
in liquid nitrogen being firstly thawed at 0 ℃,
whose thawing temperature being
gradually increased to the room
temperature.
• This method is suitable for the materials
whose cells have low water content.
• For example: Winter buds of woody plants.
• ⑥Re-culture
• Process of thawed materials being reinoculated in the culture medium to restore
growth.
• Re-culture is the most basic method to test
the effect of cryopreservation or to
determine whether the method is suitable.
• Section 3
• Genetic stability of germplasm
resources conserved in vitro
• Objective of conservation in vitro:
maintaining genetic stability.
• In the process of conservation in vitro,
there will be a different degree of genetic
variation.
• 1、Changes of genetic integrity in the
process of conservation in vitro of
germplasm resources
• Genetic stability is to maintain the original
state原始状态of germplasm.
• The important sign of the success of
germplasm conservation is whether it can
maintain its genetic stability.
• Namely, in the course of germplasm
conservation, the lowest degree of genetic
variation can be maintained, and the
maximum genetic similarity can be also
maintained after re-propagation.
• In the process of conservation in vitro,
due to the frequent subculture in the longterm conservation, the variability of the
materials conserved in vitro usually
increases, which is manifested by
chromosome aberration畸变 and gene
mutation.
• Therefore, in order to detect the genetic
stability, it is necessary to carry out the
identification of cytology细胞学, genetics遗
传学(Biochemistry, molecular marker
detection, etc.) and production traits生产性
状of the conserved materials.
• 2、The main factors affecting the
genetic integrity of germplasm
resources conserved in vitro
• There are many factors that affect the
genetic stability of germplasm resources
conserved in vitro, such as the type of
explant, the composition of culture medium,
the conservation in vitro method, the
conservation time and so on.
• In order to improve the genetic stability of
germplasm resources conserved in vitro,
How to reduce the genetic variation in the
process of conservation in vitro of
germplasm resources as much as possible
should be considered.
• The explants should be able to represent代
表 the genetic characteristics of the plant
material.
• The dose of substances which are easy to
cause chromosomal aberration畸变 and
gene mutation should be reduced as much
as possible.
• For example: 2,4-D and other growth
regulators.
• Because of high concentration of 2,4-D will
increase the frequency of chromosome
aberration畸变 of tissue and cell.
• In minimal growth conservation method,
regardless of the culture medium
formulation配方, with the extension of
conservation time and the increase of
subculture times, the possibility of variation
will increase.
• Therefore, we should try to reduce the
subculture times as much as possible in
the course of the conservation in vitro of
germplasm resources.
• In the process of detecting genetic stability,
attention is paid to distinguish between
genetic and non genetic variation.
• This is the end of the semester!
• I wish you all success in the future!