Regeneration of Plant in plant biotechnology

Contents

• Introduction
• History
• Regeneration Of Plants
• Types of regeneration in plant
• Plant tissue culture
• Types of tissue culture
• Application of tissue culture
• Advantages & Disadvantages of tissue culture
• Conclusion
• References 

Introduction

An entire plant can be regenerated from an adult tissue or organ, a mass of unorganized calli, or even a single cell in a process referred to as plant regeneration. Plant regeneration refers to the physiological renewal, repair, or replacement of tissue in plants. The totipotency or pluripotency of plant cells underlies the ability of plants to regenerate, reflecting the high plasticity of cell fate. Totipotency refers to the ability of a cell to differentiate into a complete individual, whereas pluripotency involves the differentiation of a specific group of tissues or organs from a cell. The concept of tissue culture was proposed as early as a century ago and envisaged the regeneration of whole plants from somatic cells in vitro. The tissue culture system has matured since the historical discovery that different concentration ratios of auxin and cytokinin (CK) are critical to regenerating adventitious roots and shoots. Steward et al. (1958) successfully regenerated new somatic embryos and subsequently developed roots and shoots by using isolated phloem cells from carrot roots, which confirmed the totipotency of plant cells. Since then, tissue culture technology based on regenerative ability has been extensively used in various fields, including basic research, micropropagation, and transgenic breeding. The ability of plant regeneration is affected by multiple factors, including use of a plant growth regulator, the composition of basic medium and explant type. Importantly, plant tissue culture presents strong species dependence and genotype specificity. Some plants, such as tobacco (Nicotiana tabacum), Arabidopsis thaliana, and rice (Oryza sativa), can be easily regenerated in vitro, whereas other plants, such as soybean (Glycine Max), wheat (Triticum aestivum), and maize (Zea mays), are more difficult to regenerate. Moreover, Japonica varieties show a higher capacity for callus formation than Indica varieties in rice. The tissue culture capacities of hybrid lines are higher than those of inbred lines in maize (Duncan et al., 1985). Clarifying the regulatory network and genetic control of plant-regeneration ability in tissue culture is helpful to improving plant-regeneration rates and genetic transformation efficiency.

• Regeneration: - Regeneration is the natural process of replacing or restoring damaged or missing cells, tissues, organs, and even entire body parts to full function in plants and animals.
• The process of growing an entire plant from a single cell or group of cells.
• Regeneration is possible because plant cells can be made totipotent using hormones.
• Differentiated tissue: stems, leaves, roots, etc.
• Undifferentiated (embryonic) cells are totipotent can become a whole new plant by differentiating into a whole new.

History

•  The theoretical basis for plant tissue culture was proposed by Gottlieb Haberlandt in his address to the German Academy of Science in 1902 on his experiments on the culture of single cells
• Historically, Henri-Louis Duhamel du Monceau (1756) pioneered the experiments on wound healing in plants through spontaneous callus (unorganized mass of cells) formation on decorticated region of elm plants.
• Vochting (1878) suggested the presence of polarity as a key feature that guide the development of plant fragments.
•  In 1902, a German Botanist Gottlieb Haberlandt developed the concept of culture of isolated cells of Tradescantia in artificial condition. Though his experiment failed to induce the cells to divide.
• Then the possibility for cultivation of plant tissues for unlimited period was announced simultaneously by P.R. White (1939) and R.J. Gautheret (1939).
•  In 1959, discovery of kinetin promoted by F. Skoog along with C.O. Miller and co-workers and demonstration of induction of regeneration of shoots in tobacco callus paved the way for multiplication of plant by tissue culture.

 

THE PLANTS CAN BE REGENERATED BY: -

 

Organogenesis in plants

The process of development of plant organs such as shoot, flower, and root system from either an ex-plant or from the callus of culture is known as organogenesis in plants.

A completely developed plant consists of organs specialized in a particular function such as roots are responsible for absorbing nutrients and water from the soil, leaves are necessary for photosynthesis, and flowers for reproduction. Tissues such as meristem, cortex, phloem, and epidermis are organized together to form these organs. Developing and initiating these organs is called organogenesis.

 Meristematic cells are responsible for the development of plant organs like the root system, flowers, and shoot system. Shoot apical meristem or shortly known as SAM is responsible for generating or developing organs above the root, later organ. Shoot apical meristem (SAM) regenerated organs such as leaves, stems, buds, flowers, etc. hold organogenesis capability on their edges. When these cells are induced in-vitro a whole new plant grows from it. This whole process is called organogenesis.

Dedifferentiation and redifferentiation are the two steps that are involved in organogenesis.

Dedifferentiation is a process that helps in the formation of callus from the tissues of explant with acceleration in cell division. Cells multiply and divide very quickly to grow their number to form undifferentiated cells, i.e. callus, this process of dedifferentiation.

Redifferentiation is the process of developing a permanent organ by converting the cells that were formed during dedifferentiation. Cells lose their capability to multiply and divide in this process so that they can be converted into permanent tissue.

The process of organogenesis can take place in three ways:

• From an explant
• From the callus culture
• From the axillary buds

Types of Organogenesis in plants

There are two types of organogenesis in plants that are:

• Direct organogenesis
• Indirect organogenesis

Direct organogenesis in plants tissue culture

When buds and shoots are directly developed from tissue and there is no need for the callus stage then this process is known as direct organogenesis in plant tissue culture.

Direct organogenesis results in the development of planting material with no genetic variation therefore cloning. Uniformity in the planting material is ensured. This process is also useful in propagating plants with a better multiplication rate (the number of plants per explant is higher).

Direct organogenesis is more of an industrial process as it provides plants with better multiplication rates and cloning propagation where the genetic variation is zero.

Indirect organogenesis in plant tissue culture

In this process of indirect organogenesis, a plant’s organ is developed from the callus of an explant (tissue that developed at the site of a cut or wound). The process of indirect organogenesis is more useful in the development of a transgenic plant. There are two ways that can be used to develop a transgenic plant in the indirect organogenesis method:

• Transformed callus is used to regenerate a new plant that is transgenic
• A modified explant is used to develop callus in the shoot, transform explant is initially used.

Factors that affect organogenesis:

We can divide the factors into two major groups that is:

• External factors
• Internal factors

External factors that affect organogenesis are

• The medium

 The medium has a great impact on organogenesis

•  The chemicals and medicines

Curtain chemicals such as auxin and cytokinin show a great impact on the growth of plant organs such as roots and shoots. Experiments and studies have shown that auxin simulates information of the root system and stops the formation of the shoot. Whereas, cytokinin promotes the development of shoot.

• The environmental conditions

The environment and surroundings play a key role in organogenesis. A good and rich nutrient environment promotes organogenesis whereas a harsh and tough environment does the vice versa.

Internal factors that effects organogenesis is

•  Gibberellin

Gibberellins is a hormone that restricts the formation of shoot and root both. Not only this but also gibberellins lower the content of starch and prohibit bud formation.

•  Carbohydrates

Carbohydrates work as osmotic agents and as a respiratory energy source. Callus development and growth are affected by osmotic stress. Sucrose which is a form of carbohydrates is essential for this process.

•  Ethylene

Ethylene is a hormone that enhances the development process during the primordial process but the process of organogenesis is blocked by ethylene hormones.

SOMATIC EMBRYOGENESIS

Somatic embryogenesis is the process wherein somatic cells differentiate into somatic embryos. It is not a naturally occurring process, an artificial one wherein an embryo or plant is obtained from one somatic cell. Somatic embryos take form from the cells of the plants, which usually do not take part in embryo development. Neither a seed coat nor endosperm is formed around the somatic embryo.

In the process, one cell or a cluster of cells initiates the developmental route, which results in reproducible regeneration of non-zygotic embryos, which can germinate for the formation of an entire plant.

The cells which are derived from potential source tissues are subject to a culture medium for the formation of an undifferentiated cluster of cells referred to as the callus. In the tissue culture medium, the plant growth regulators can be formed for the induction of the formation of calluses and hence modified to induce the embryos for the formation of calluses.

Process of Somatic Embryogenesis

The somatic embryogenesis procedure is a three-step procedure, which causes the induction of embryogenesis, development of the embryo and its maturation.

The principle of somatic embryogenesis finds its basis on the topic of totipotency of the plant cells; it illustrates two facets of plant embryogenesis:

• The process of fertilization can be replaced by an endogenous mechanism.
• The other types of cells of the plant, apart from the fertilized egg cells, can retrieve the capacity to form an embryo.

Since the process of somatic embryogenesis does not entail the procedure of fertilization, it promotes the large-scale propagation of plants at a faster rate. In addition, it also assists in the genetic transformation of plants, serving as a promising resource for the cryo-storage of the embryo and germplasm.

Somatic embryogenesis – Induction

Cells are reactivated to differentiate and develop embryos, which occur through two processes: direct somatic embryogenesis and indirect somatic embryogenesis.

Direct somatic embryogenesis

It involves the development of the embryos in a direct way from the cells of the explants, such as the cells of the immature embryos. Here, there is no intermediary stage (like the formation of the callus). The explants of the somatic embryogenesis are seen to entail PEDCs (pre-embryogenic determined cells).

Indirect somatic embryogenesis

It includes the formation of somatic embryos by reiterating numerous cycles of cell divisions. It includes intermediary steps of growth of the callus, and hence the process includes multiple steps.

The cells which do not carry the pre-embryogenic determined cells are caused to differentiate for the formation of the embryo by revealing different treatments. The cells modify into IEDs (induced embryogenic pre-determined cells).

Types of Somatic Embryogenesis

Somatic embryogenesis is of two types:

• Direct somatic embryogenesis

Here, the embryos start directly from the explants when callus formation does not take place. Embryos, in this case, are formed as a result of Pre-induced Embryogenic Determined Cells (PEDCs).

• Indirect somatic embryogenesis

The callus from the explants occurs from where the embryo develops. Here, the embryos are formed as a result of Induced Embryogenic Determined Cells (IEDCs).

Advantages of Somatic Embryogenesis

In comparison with zygotic embryogenesis, somatic embryogenesis has these benefits:

• A huge number of embryos are obtained
• The development and environmental stage of somatic embryos can be regulated
• This process of embryogenesis can be monitored easily

The significance of somatic embryogenesis is as follows:

• Production of artificial seeds
• Higher rate of propagation
• Apt in suspension culture
• Labour savings

Factors Affecting Somatic Embryogenesis

The aspects which affect the process of somatic embryogenesis are as follows:

Traits of explant

Despite the fact that variations of explants can be used, the apt stage of development of explants is vital too to initiate the embryogenic callus; whereas juvenile explants tend to give rise to more somatic embryos compared to older explants. Also, different explant explants tissues from the same mother plant generated embryogenic callus at varying frequencies.

The desired species of plants to be induced for embryogenesis decides the choice of explants. For the majority of plant species, explants of immature zygotic embryos are apt for somatic embryogenesis.

Growth regulators

Cytokinins: -These have been in use in the primary medium consistently at the time of embryogenesis of the crop plants. They are vital in speeding up the process of maturation of somatic embryos, the cotyledon development, precisely.

Auxins: - These alone or in combination with cytokinin seemingly are vital for the start of growth and the induction of the embryogenesis of all the auxins. Auxins find immense importance in the first step of this process – the step of induction. High levels of auxins can lead to the inhibition of embryogenesis in the explants of the citrus plants.

Abscisic acid: -These are supplied at the inhibitory levels. It facilitates the development and maturation of the somatic embryos, while also inhibiting the unusual proliferation and the initiation of the accessory embryos.

Genotype

The process of embryogenesis is also affected by the genotypic variation seen in different plants; as per research, it can also be as a result of the endogenous levels of the hormones.

Sources of nitrogen

Nitrogen forms that are utilized in the media have an influence on the process of embryogenesis in plants. Forms of nitrogen have a marked influence on somatic embryogenesis. Somatic embryo development takes place on a medium that contains       NO^–₃ as the only source of nitrogen.

Polyamines

The concentration of polyamines in media or explants is said to have an effect on the process. Experts observe the concentration of polyamines to be seen in higher concentrations in the polyembryonates compared to monoembryonates.

Electrical stimulation

Electrical stimuli apparently facilitate the differentiation of the structured embryo by influencing the cell polarity via modifications in the structure of the microtubules and the induction of first asymmetric division.

 

REGENERATION OF PLANTLETS

1. Preparation of Suitable Nutrient Medium: Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers.
2. Selection of Explants: Section of explants such as shoot tip should be done.
3. Sterilization of Explants: Surface sterilization of the explants by disinfectants and then washing the explants with sterile distilled water is essential.
4. Inoculation: Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilized by filter-sterilized to avoid microbial contamination) in culture vessels under sterile conditions is done
5. Incubation: Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e, artificial light; 16 hours of photoperiod), temperature (-26°C) and relative humidity (50-60%) is required.
6. Regeneration: Regeneration of plants from cultured plant tissues is carried out.
7. Hardening: Hardening is gradual exposure of plantlets to an environmental condition.
8. Plantlet Transfer: After hardening plantlets transferred to the green house or field conditions following acclimatization (hardening) of regenerated plants.
   

                                 Fig: - The schematic diagram of plant tissue culture       

Plant tissue culture

• Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition.
• It is widely used to produce clones of a plant in a method known as micropropagation.
• Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (Cellular totipotency)
• single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.
•  Preparation of plant tissue for tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet.

•      Following are the main categories of Cultures: -

Primary Culture: - These model the natural function of the Tissue and are generally mortal. They consist of natural Tissues excised from the living organisms by biopsy.                                                         

Culture of Established Cell Lines: - These are derived from tumor biopsies, or from the primary cells that had undergone mutation and continued to replicate.

Some Types of Tissue Culture techniques

Seed Culture: In seed Culture, explants are obtained from an in- vitro derived plant and hence are introduced into a laboratory where they proliferate. To prevent the plants from Tissue damage it should be sterilized.

Embryo Culture: Embryo Culture involves the in-vitro development of an embryo. For this process, an embryo is isolated from and living organism, both a mature and an immature embryo can be used. Mature embryos can be obtained from ripe seeds whereas immature embryos are obtained from the seeds that failed to germinate. The ovule, seed, or fruit has already been sterilized, hence there is no need to sterilize them.

Callus Culture: - A callus can be defined as an unorganized, dividing mass of cells. A callus is the explants are Cultured in a proper medium good. The growth of callus is followed by organ differentiation. This Culture is grown on a gel-like medium composed of agar and specific nutrients which are required for the growth of the cells.

Organ Culture: - In organ Culture, any organ of the plant such as a shoot, the leaf can be used as an explant. Many methods can be used for the organ Culture such as the plasma clot method, raft method, the grid method, and Agar gel method. This method can be used to preserve the structure and functions of an organism

Meristem Culture: - meristems have the main function of the production of new cells and the synthesis of protoplasm. Shoot meristem consists of a group of certain actively dividing cells that are being protected by the developing leaves.

Protoplast Culture: - It can be defined as a cell without a cell wall. The hanging-drop method or micro-Culture chambers can be used to Culture a protoplast. A number of phases can be observed in protoplast Culture, development of cell walls, cell division, regeneration of a whole plant.

Suspension Culture: - suspension Culture can be defined as a form of Culture in which single cells or small aggregates of cells multiply while suspended in an agitated liquid medium. It can also be called cell Culture or cell suspension Culture.

Steps of Tissue Culture

• Following are the steps of Tissue Culture

Initiation Phase: -

• This is a stage when the Tissue is initiated into the Culture. To prevent the process from any contamination the Tissue of interest is obtained, introduced, and sterilized.

Multiplication Phase: -

• In the multiplication stage, the sterilized ex-plant is introduced into the medium which consists of growth regulators and appropriate nutrients, they are responsible for the multiplication of cells. Hence this undifferentiated mass of cells is known as a callus.

Root Formation: -

• This is the stage when the root starts forming. To initiate the formation of root plant growth hormones are added. Consequently, complete plantlets are obtained.

Shoot Formation: -

•  For the formation of the shoot, plant growth hormones are added and growth is observed for a week.

Acclimatization: -

• When the plant starts to develop, the plant is transferred to a greenhouse for it to develop under controlled environmental conditions. Thereafter it is finally transferred to the nurseries for its growth under natural environmental conditions.

HORMONES USED IN PLANT TISSUE CULTURE: -

Auxins: - Any of a group of plant hormones that regulate growth, particularly by stimulating cell elongation in stems.

Cytokinin's: - Cytokinin’s are a group of plant growth regulators which are primarily involved in performing cell division in plant roots, shoot system. 

Gibberellins: - One of the plant hormones that regulate a wide range of processes involved in plant growth, organ development, and environmental responses.

Abscisic Acid: -A plant growth regulator known for its functions, especially in seed maturation, seed dormancy, adaptive responses to biotic and abiotic stresses, and leaf and bud abscission.

Polyamines: - Polyamines can increase the activity of various antioxidant enzymes in plants, so that it can effectively regulate oxidative stress in plants caused by various environmental factors. 

Application of plant tissue culture

• Plant tissue culture technology has been used in almost all the field of biosciences Its applications include
• Production of phytopharmaceuticals and secondary metabolites.

1.  Biotransformation (Biochemical Conversion)
2.  Plant cell immobilization
3.  Genetic transformation (Transgenic plant)
4.  Elicitors

• Micropropagation (Clonal Propagation)
• Synthetic seed Protoplast culture and somatic hybridization
• The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum.
• A Differential production of tropane alkaloids in hairy roots and in vitro culture
• A Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.
• Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don.
• Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii
• Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system.
• Extracts from black carrot tissue culture as potent anticancer agents.
• Enhanced production of tropane alkaloids in transgenic Scopolia parviflora hairy root cultures over-expressing putrescine N-methyl transferase (PMT) and hyoscyamine-6B-hydroxylase (H6H).
• Taxus globosa S. cell lines: initiation, selection and characterization in terms of growth, and of baccatin III and paclitaxel production.
• Production of camptothecin in cultures of Chonemorpha grandiflora.
• Regeneration, in vitro glycoalkaloids production and evaluation of bioactivity of callus methanolic extract of Solanum tuberosum L.

 

Conclusion: -

Plant regeneration is the major outcome of plant tissue culture, which is based on the principle of totipotency. Plant regeneration can be achieved by organogenesis and somatic embryogenesis. Organogenesis means formation of organs from the cultured explants. The shoot buds or monopolar structures are formed by manipulating the ratio of cytokinin to auxin in the cultures. In somatic embryogenesis, the totipotent cells may undergo embryogenic pathway to form somatic embryos, which are grown to regenerate whole plants. It was first established in carrots (Daucus carota), where bipolar embryos developed from single cells. The somatic embryogenesis is influenced by herbal extracts, phytohormones, and the physiological state of calli.

Plant regeneration involves the in vitro culture of cells, tissues, and organs under defined physical and chemical conditions. Critical for in vitro plant propagation and biotechnology, this phenomenon is also applicable to studies of plant developmental regulatory mechanisms.

 

References: -

• D.Dumet, A. Adeyemi, O.B.Ojuederie (2008). Yam invitro genebanking. Genebank manual. http://www.iita.org/genebank/manual
• D.Dumet, A.Adeyemi, O.B.Ojuederie (2008). Cassava in vitro processing and the genebanking. Genebank manual. http://www.iita.org/genebank/manual
• HORT689/AGRO689 Biotechniques in Plant Breeding
• H.S Chawla.2002 Introduction to Plant Biotechnology 2nd edition. Oxford & IBH Publishing C./ Pvt. Ltd New Delhi India
• Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassay with Tobacco tissue culture. Physiologia plantarum 15: 473-497.