Chapter 2 Basic Principles of Plant Tissue Culture

Totipotent theory of plant cell is the core theory of plant tissue culture. Under suitable nutrition and environmental conditions, cells cultured in vitro, undergo dedifferentiation and redifferentiation, and can form the regenerated plants. Dedifferentiation Redifferentiation

Section 1 Plant cell totipotent and cell differentiation

1、Plant cell totipotent 1902，Haberlandt 1970s. Totipotent ：Each cell has the full genetic information of the plant, which has the ability to develop into a complete plant.

1980s. Totipotent ：differentiate different types of cells, forming different types of organs and even somatic embryos, until the formation of complete regeneration plants.

Now. Totipotent ：Formation of secondary metabolites in plant cell culture and generation of whole plant from single cells.

2、Plant cell differentiation Cell differentiation: the process that causes the cell to form different structures, causing functional changes or potential changes of developmental pathway.

Cell differentiation is the basis of tissue differentiation and organ differentiation, which is the basis of differentiation of cell cultured in vitro and plantlet regeneration.

Some rules and mechanisms of differentiation of cell cultured in vitro: ①Cell differentiation divide into two categories: morphological structure differentiation and physiological and biochemical differentiation；

②The permanent closure of part genome does not exist in the plant ②The permanent closure of part genome does not exist in the plant. If the condition is appropriate, it can express the totipotent；

③In the whole plant, once the cell development pathway is determined, it is not easy to change, but it can be lost the determination in culture in vitro；

④Polarity is closely related with differentiation；

For example: Regenerated shoots of stem segments only formed at the upper end of the morphology, while regenerated shoots of root segments often occur at the lower end of the morphology.

⑤Promoting effect of physiological isolation or mechanical isolation in cell differentiation；

⑥Cell division plays an important role in cell differentiation；

⑦Plant growth regulators (or plant hormones) have a significant regulatory effect on cell differentiation, which is closely related to some processes of cell differentiation, such as cell growth and division；

For example: The root or shoot differentiation was determined by the ratio of auxin to cytokinin (CTK).

The high ratio promotes rooting, and the low ratio promotes bud differentiation. When the ratio is 1, there is no formation of morphological structure.

⑧Role of change of cell nuclear chromosome and DNA in cell differentiation；

The most common change of chromosome is endopolyploid (核内多倍性) and polyteny (多线染色体), which has been reproduced many times and the cells do not divide.

Differential replication (差异复制) of DNA plays an important role in the development and differentiation of some organisms.

Section 2 Occurrence of plant organs in vitro

1、Dedifferentiation and redifferentiation The loss of the original structure and function; The recovery of the meristem state; The formation of the cell clusters (callus) without organization structure.

Cell differentiation doesn’t only exist in cell cultured in vitro, also exists in nature. For example： When leaves fall in autumn, abscission layer is formed at the base of petiole.

（2） Redifferentiation Calli redifferentiate and form cells, tissues, organs and plants. ①Redifferentiation in the cell level Form various types of cells.

②Redifferentiation in the tissue level The most common is vascular tissue (维管组织) differentiation. Friable callus: Containing meristemoid or tumor-like structure.

Compact callus: Few differentiation, most of whose cells are rich in vacuoles.

③Redifferentiation in the organ level Also known as organogenesis.

Redifferentiated tissues can form various organs, such as roots, buds, leaves, flowers and abnormal organs such as bulbs, tubers, etc.

According to the different origin, organogenesis can be divided into： ——Organ type： The cells in explant directly form organ primordia and then develop into the organ.

——Organogenesis type： Calli are firstly formed in the explant, and then calli produce different organ primordia, which then directly developed into the organ.

④Plant regeneration Plant reconstruction at the level of organ.

There are three ways to form regenerated plants through roots or buds cultured in vitro. ——Firstly budding then rooting (common)；

——Firstly rooting then budding;

——Bud and root are formed respectively in different parts of the calli, and then vascular tissue is also formed to connect bud and root in order to form a plant.

Main regeneration pathways of plants in culture in vitro ——Organogenesis Protocorm is a specific plant regeneration pathway of Orchidaceae plant.

——embryogenesis Forming a structure similar to seed embryo——somatic embryo(体细胞胚) or embryoid (胚状体), which directly rooting and budding at the same time.

2、Factors affecting cell dedifferentiation and redifferentiation ①Damage: promoting cell proliferation

②Growth regulators: The role of auxin (2,4-D)

③Light: dark or weak light

④Cell location: The sensitivity that different plant responding to stimulation is different.

⑤Physiological state of explants: Different physiological state, different responses.

⑥Plant species differences：Dicotyledons dedifferentiate more easily than Monocotyledons and Gymnosperm.

（2）Factors affecting cell redifferentiation Plant species are different, their redifferentiation ability is also very different.

Plant regeneration conditions for some plants are not fully mastered.

Light: normal photoperiod.

Growth regulators: The role of auxin (NAA) and cytokinin (CTK, such as KT and 6-BA). In general, CTK is higher than auxin.

3、Callus culture （1）Callus formation, growth and maintaining The cell structure of calli is often heterogeneous, and has no obvious polarity.

The formation process of callus can be divided into： ——Induction period：also known as starting period. It is starting point of callus formation and the period that cells prepare to divide.

——Division period：Cell division is very fast, the structure is loose and has no organization and the color is light and transparent.

If calli are continued to culture in the original culture medium, the cells will be differentiated into new structures.

But if calli are transferred to fresh culture medium, they can carry sustained cell division without restriction and maintain callus state with no differentiation.

——Differentiation period: the formation of different forms and functions of cells. Note: Division of above three periods is artificial.

②Callus growth Major changes in appearance: Rapid increase in cell number

Major physiological and biochemical changes: ——RNA content decrease (increase in induction period)

——The changes in isozymogram （isoenzyme spectrum）: First decreased and then recovered to normal.

——Continuous subculture can change the accumulation level or ability of secondary metabolites.

——Prolonged culuture period may make the need of callus to auxins change, resulting in auxin self-sufficiency or autotrophy, which is known as habituation phenomenon.

Loose The quality of callus can be transformed into each other. Dense Reducing or removing auxin or adding high concentration of cytokinin (CTK) Loose Dense Adding high concentration of auxin

②Callus maintaining With the increase of subculture times and the extension of culture time, The callus will have following problems.

Callus growth potential falls； Callus browning； Callus differentiation and morphogenetic capacity gradually reduced or even died ； Even callus morphogenetic capacity loses completely.

But in some cases, callus can be maintained for a long time. One is habituation, the other is alternating culture in two kinds of different media.

（2）Callus morphogenesis ①Somatic embryogenesis ②Organgenesis ——Firstly budding then rooting; ——Firstly rooting then budding; ——Bud and root are formed respectively in different parts of the calli, and then vascular tissue is also formed to connect bud and root in order to form a plant; ——Only rooting or buding.

Section 3 Plant somatic embryogenesis

Embryogenesis：a series of continuous processes, i. e Embryogenesis：a series of continuous processes, i.e., regular changes of occurrence and development of mature embryo from the fertilized egg. In this case, the embryo is called zygotic embryo.

Under the condition of culture in vitro, cells, tissues and organs of plant can also produce the structure similar to embryo, and its formation is also experience an occurrence and development process similar to embryo.

This kind of structure similar to embryo is called the embryoid This kind of structure similar to embryo is called the embryoid. Mature embryoid can directly grow roots, buds and regenerate plants like zygotic embryo.

Establishment of embryoid generated from plant cells cultured in vitro is called somatic embryogenesis.

Roots, stems, leaves and other organs can induce diploid embryoids, pollen can induce haploid embryoids and endosperm can induce triploid embryoids.

（1）Somatic embryogenesis process 1、Plant somatic embryogenesis process （1）Somatic embryogenesis process In general, it can be divided into 5 stages: embryonic callus induction, somatic embryo induction, early differentiation and development of somatic embryos, somatic embryo maturation, somatic embryo germination and plant regeneration.

In Dicotyledons, somatic embryogenesis experienced 5 stages: proembryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo and mature embryo.

（2）Differences in physiological state of embryogenic or non-embryogenic cells or callus Physiological states are significantly different.

Embryogenic cells: unique, similar to meristem, small, nearly round, nuclear and nucleolus are large and can highly staining, cytoplasm is dense. Embryogenic callus: Incompact graininess.

Compared with non-embryogenic callus, embryogenic callus showed： DNA, RNA and protein synthesis is active； starch and soluble sugar content increases；

The content of endogenous polyamines increase; The level of reactive oxygen species (ROS) increases; Isozyme patterns and endogenous hormone synthesis has obvious changes.

（3）Gene expression mechanism of somatic embryogenesis Somatic embryogenesis involves more than 4000 genes.

During the developmental late stage of somatic embryos, the expression of the gene can reach more than 10000.

The first key link in the study of somatic embryogenesis is obtainment of plant embryonic cell , with which cdc2 （cell division cycle 2）gene and other cell cycle regulatory genes have a certain relationship.

In Arabidopsis thaliana (model plant): LEC1, LEC2 and BBM genes can launch the transformation of plant cells from vegetative growth to embryonic growth.

PKL gene regulates expression of LEC1. Normally, LEC1 is closed by PKL, and the plant is normally developed. However, if the PKL gene is lost, LEC1 will cause the cell arise embryonic development, and the plant can not grow normally.

Early embryonic development status has a great influence on the subsequent embryo development. That is, in a large extent, the late embryonic development has been decided in the early embryonic development.

In the late embryonic development, there are a large number of late embryogenesis abundant protein (LEA) expression.

In addition, the occurrence of somatic embryogenesis in plants is closely related to the methylation of DNA. Methylation: Methyl selectively added to cytosine in order to form 5- cytosine.

The DNA methylation degree of non-embryogenic cells was the best, and the expression of the related genes was inhibited.

With the decrease of the degree of DNA methylation, embryonic related genes begin to express, embryogenic cells begin to form.

With formation and further development of somatic embryos, DNA methylation degree gradually increases and inhibits the expression of embryonic related genes, which made somatic embryos develop and mature.

2、Plant somatic embryogenesis pathway （1）The way of somatic embryogenesis Direct pathway：The embryonic cells in some parts of explant are directly induced and differentiated into somatic embryos. This embryonic cell has been determined before the occurrence of the embryo.

For example, nucellus（珠心组织）, cotyledon（子叶） and inflorescence（花序） can directly induce somatic embryo.

Indirect pathway：The explants were first differentiated to form callus, and then some cells, which were redetermined to be embryonic cell from callus, differentiate into somatic embryos. Most somatic embryos were produced by indirect pathway.

（2）Origin of somatic embryos Most somatic embryos originated from single cells；

The essence of somatic embryogenesis is cell differentiation. Under the condition of cell culture in vitro, partial genes are induced to open and genetic information are expressed.

（3）Polarity and physiological isolation of plant somatic embryogenesis Somatic embryos have two distinct characteristics： Double polarity and physiological isolation

①Double polarity Single embryonic cell has obvious polarity and in most cases the first division is unequal.

Inducing factor of somatic embryogenesis is plant hormones and external stimuli, such as 6-BA, which can change the polarity and the division plane of the cell.

Polarity is important for cell differentiation and somatic embryogenesis.

② physiological isolation Somatic embryos have no direct relationship with vascular bundle system of parent tissue or explant, which is in a relatively isolated state.

In the early stage, there is still plasmodesma (胞间连丝 ) in the embryonic cell and the surrounding cells; but with the development of the embryonic cells, cell wall was thickened and plasmodesma disappears or is blocked.

Physiological isolation is a prerequisite for somatic embryogenesis, which is conducive to the expression of embryogenesis potential.

Physiological isolation is relative, and does not mean that it is completely isolated from the surrounding tissues.

For somatic embryos that are indirectly induced from the explants, the material, energy and information obtained from the surrounding cells may be necessary.

（4）Physiology and biochemistry of plant somatic embryogenesis ①Protein and nucleic acid Protein content and synthesis rate increase Large-Scale ribosome RNA was synthesized, and the amount of DNA also increases.

②Polyamine metabolism Polyamines (PAs) are related to cell division, which participate in morphogenesis. High content of polyamines and polyamine synthase activity was closely related to somatic embryogenesis.

③Sugar, metal ions and microelements Sugar is an important component of somatic embryogenesis, which provide carbon source, maintain osmotic pressure, and also play a role in signal molecules.

Metal ions can control the synthesis of ethylene and increase the frequency of somatic embryogenesis. Such as Ag+、Co2+ and Ni2+.

Some rare earth elements can also increase the frequency of somatic embryogenesis, and promote the normal differentiation and development of somatic embryos.

④Endogenous hormone Auxin: used for induction of cell division and embryogenic potential and promotion of early development of somatic embryos.

Cytokinin (CTK): High level of CTK is important for cell division and growth, but not involved in the process of embryogenesis.

Gibberellin (GA): has inhibitory effect on longan (龙眼) and citrus (柑橘) somatic embryos and has promotion effect on pomelo (柚) somatic embryo.

Abscisic acid (ABA): It is related to the initiation or expression of embryogenic ability.

ABA regulates the expression of specific genes in somatic embryogenesis, which can activate the expression of related genes.

ABA synthesizes a large number of storage protein, late embryogenesis abundant (LEA) protein and a small amount of embryogenesis specific protein.

⑤Reactive oxygen species (ROS) Oxidative stress is related to cell differentiation, and the process of somatic embryogenesis certainly has the effect of oxidative stress.

Section 4 Factors affecting plant morphogenesis in vitro

1、Plant species and genotypes In different species and different genetic types in the same species, their morphogenesis ability has a huge difference.

The more similar the genetic relationship of culture material is, the more similar the conditions of morphogenesis are.

2、Physiological state of culture material （1）Plant developmental age Immature tissue has stronger morphogenesis ability than mature tissue.

（2）Culture organ or tissue type Seeds, immature embryos and hypocotyls (下胚轴) were easy to form embryoid, but stem segment and leaf is more difficult to form embryoid.

The size of explant and the contact location with culture medium also has effect on organ regeneration.

（3）Culture time and cell ploidy (倍性) The longer the culture time is, the more delayed or reduced the morphogenesis ability is.

Cell ploidy also affected morphogenesis ability. For example: In the anther culture, in the condition of high osmotic pressure, haploid pollen is easy to form haploid embryoid, and the growth and differentiation of diploid cells derived from anther wall were significantly inhibited.

3、Culture medium （1）Plant nutrition Ammonium nitrogen and K+ are beneficial to the formation of the embryoid.

NH4NO3 in MS culture medium had a certain effect on the occurrence of somatic embryos.

No sugar or low sugar can not form embryoid.

（2）Plant growth regulator 2,4-D: It is widely used in the induction of somatic embryos. Cytokinin (CTK): 6-BA, TDZ. GA: GA3. Ethylene: promotion or inhibition, Related to plant type and processing time. Abscisic acid (ABA): It is very important for somatic embryogenesis and maturation.

（3）Culture medium physical properties Such as solid or liquid, osmotic pressure and pH, etc., also have effect on the formation and development of organs or embryos.

4、Cultivation condition （1）Light Light intensity: 1000-5000lx Light length: expressed as a response to photoperiod. long day and short day. generally 10-16h/d. Light quality: Plantlet regeneration: blue; rooting: red.

（2）Temperature The optimum temperature for different plant is also different. Most of culture room was 25±2℃.

Temperature has a close relationship with organ differentiation and formation of embryoid.

Some plants also require changing culture temperature.

Plants that are required by the seasonal temperature difference, in particular the bulbs (鳞茎) or corms (球茎), need to be treated at low temperature before transplanting. 荸荠 bí qi

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