THE SCOPE OF PLANT TISSUE CULTURE
Introduction:
In this paper we will consider some basic issues that concern plant tissue culture invitro, particularly as applied to plant transformation. We will also look at basic types of cultures involved in plant transformation strategy and also touch a little of some basic technique that can be used to regenerate whole transformed plant from transformed cells or tissues.
Basic Definitions:
Literally, plant tissue culture is define as the act of regeneration of whole plant from isolated plant cells or tissues which have been genetically transformed, this however, take place invitro. Meaning that the regeneration of whole plant from tissue or cell is carried out in a test tube, or any laboratory equipment, example petridish etc. in general invitro simply means the regeneration of whole plant in a sterile controlled environment.
Other definition of plant tissue culture by eminent scholars of repute is as follows;
1- It is said to be the growth of tissues or cells separate from the organism, which is facilitated via the use of liquid, semi liquid or solid growth medium.
2- It is also said to be the collection of techniques used to maintained or grow plant cells, tissues or organs under sterile controlled environment.
3- It is also defined as micro propagation used to produce whole new plant.
Historical usage of plant tissue culture:
In 1885 wilhem Roux removed a section of medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days thus, establishing the basic principle of tissue culture
In 1907, the Zoologist, Ross Granville Harrison, Demonstrated the growth of frog nerve cell process in a medium of clotted lymph. In 1913, E. Steinhardt, C. Israeli and R.A Lambert grew vaccinia virus in fragment of Guinea pig corneal tissue. 1996, the first use of regenerative tissue was used to replace a small distance of a urethra which lead to the understanding that technique of obtaining samples of tissue, growing it outside the body, without a scaffold, and re-applying it can be used for only small distance of less than one centimeter.
Modern usage of plant tissue culture:
In modern usage, tissue culture generally refers to the growth of cells from a tissue of multicellular organism’s in vitro. These cells may be cells isolated from a donor organism, primary cells, or an immortalized cell line.
Tissue Culture in the modern era is therefore an important tool for the study of Biology of cells from multicellular organisms. It provides an invitro model of the tissue in a well defined environment which can be easily manipulated and analyzed.
LITERATURE REVIEW:
As earlier seen in the different Definitions above, tissue culture commonly refers to the culture of animal cells and tissue with the more specific term plant tissue culture been used for plants. Also, known as micro propagation method used to produce new plants under sterile condition. This method uses plants (i.e plant parts) or seeds that have been sterilized before been placed in containers with growing medium (usually gel) with some nutrient added.
The explants or seeds, the containers and medium have all been sterilized and this if successful prevents any cut, or torn tissue or the entire explants or seed itself from becoming infected with micro-organisms of some kind and rotten during the time these plant parts require to become rotted or to multiply.
Many types of seeds germinated in these conditions tend to grow very fast compared to those been sowed in standard growing media outside of sterile, enclosed containers. Using plant tissue culture it is possible to grow the exact copies of the donor plant. This is extremely useful for plant that genetically have desirable traits (i.e. characteristics), because one can create many clones of a particular plant much faster than the traditional propagation methods used by the conventional farmers.
The sterile nutrient media usually contains a nutrient solution (typically salt and vitamins), sucrose (sugar), solidifying or gelling agent such as agar (a product of sea weed).
OBJECTIVES OF PLANT TISSUE CULTURE
The aim of plant tissue culture can simply be summarized as follows:
i- It is aimed at producing as easily and as quickly as possible large number of regenerable cells that are accessable to genetic transfer in a short period of time.
ii- It is also aimed at producing the exact copies of the parent plant that genetically have desirable traits.
In any plant tissue culture experiment, two basic factors have to be considered.
i- Plasticity of the plant
ii- Totipotecy of the Plant
Here, plasticity of the plant simply means the ability of the plant part used in the experiment to alter their metabolism, growth and development ability to best suit their immediate environment. While totipotency of the plant refers to the ability of the plant to express total genetic potential of the parent plant under the correct environmental condition.
THE CULTURE MEDIUM
When plant parts are cultured in vitro, the physical and chemical needs of the plant have to agree with that of the containers, the growth medium and external environment (i.e. light, temperature etc). The growth medium must supply all the essential mineral irons required for growth and development of the plant. The medium must also, supply additional organic supplement such as Amino acids and Vitamins.
We should note that plant cell cultures are not photosynthetic; hence, they require the addition of a fixed Carbon source in the form of sugar (mostly Sucrose). Another vital component that must be supplied is Water which is the principal Biological solvent.
In the culture environment, physical factors like Temperature, pH, gaseous environment, Light and Osmotic pressure must be maintained within acceptable limit.
For practical purposes essential element are further divided into
i- Macro Element (or Macro Nutrient)
ii- Micro Element (or Micro Nutrient)
Macro Element:
As the name implies, these elements are required in large quantities for growth and development. The Macro element includes; Nitrogen, Calcium, Magnesium, Phosphorous and sulphur. All these elements usually comprises of at least 0.1% of total dry weight of plants
Micro Element:
These elements are needed in very small quantities for the plant growth and development, they includes; Manganese, Iodine, Copper, Cobalt, Boron, Molybdenum, Iron and Zinc.
Organic Supplement:
Only two vitamins are considered essential for the culture of plant cells invitro. These are thiamine (Vitamin B1), Myoinositol (considered a B vitamin.). The most frequently used Amino Acid in the organic supplement is Glycine, although Argenine, Asparagines, Aspartic Acid, Alinine, Glutamic Acid, Glutamine and Proline are also used.
Amino Acids usually provide a source of reduced Nitrogen.
Carbon source:
The most commonly used Carbon source in the culture Environment is Sucrose, because it is cheap, easily available, readily assimilated and relatively stable. Other Carbohydrates like Glucose, Maltose, Galactose and sorbitol can also be used
Gelling Agent:
Media for plant cell culture invitro can be used in either liquid, solid or semi solid form; this depends on the type of culture been grown. Agar is most common type of gelling agent and is produce on seaweed.
Plant growth regulators:
These are the critical media component that determines the developmental path ways in any plant cell cultures. Plant growth regulators commonly used are plant hormones or their synthetic analogues.
There are five main classes of these plant regulators, these includes; Auxins, cytokinins, gibberellins, Abscisic Acid, Ethylene
Auxin: these help to promote both cell division and cell growth. The most important naturally occurring Auxin is I.AA i.e Indole-3-acetic acid.
Cytokinin: It promotes cell division in plant. Two naturally occurring cytokinin plays important role in plant tissue cultures, and they are Zeatin, 2ip (2-isopentyle adenine).
Gibberellins: These hormones promote cell elongation. And are agronomically important in determining plant height and fruit set.
Abscisic Acid: This Hormone promotes distinct developmental path ways such as somatic embryo geneses.
Ethylene: This is gaseous in nature and occurs naturally it is associated with fruit ripening.
We should note that Auxins and Cytokinins are the most widely used plant growth regulator in plant tissue culture and are generally used together for the following reasons;
- The ratio of Auxin to cytokinin determine the type of culture that is established or generated
- A high Auxin to Cytokinin ratio generally favors root formation.
- High Cytokinin to Auxin ratio favors shoot formation where as
- An intermediate ratio of both Hormones (i.e 50:50) favors Callus formation
What is Callus?
A callus of a cell is a mass of undifferentiated plant cells. It often starts as lumpy growth on existing tissue and then develop into an odd looking ( hairy) Masses as many tiny plant lets begin to emerge from the Callus.
TYPES OF CULTURE
Cultures are initiated from sterile pieces of a whole plant termed “explants”. These explants may consist of pieces of organs like leaves or roots or may be specific cell type like pollen or endosperms.
Many features of the explants are known to affect the efficiency of culture initiation, but younger, more rapid growing tissue or tissues at an early stage of development are known to be more effective.
The most common features of explants that are commonly used in plant transformations include; Callus, Protoplast, root, shoot tip and meristem, embryo and microspore.
Callus Cultures:
Broadly speaking this type of culture falls into one of two categories
a- Compact
b- Friable
In Compact Callus culture the cells are densely aggregated (i.e the cells are closely packed). While in Friable Callus culture, the cells are always loosely associated with each other and the Callus become soft and breaks apart easily. Friable Callus provides the substance to form cell suspension cultures.
When Friable Callus is placed in a liquid medium and then agitated, single cells and or small clumps of cells are released into the medium. Under the correct condition and manipulation of the Auxin to Cytokinin ratio in the medium can lead to the development of shoots, roots, or somatic embryos from which whole new plant can subsequently be produce.
Proplast Culture:
Protoplasts are plant cell with the cell wall removed. Protoplasts are isolated from either leaf mesophyll cells. There are two general approaches to removing cell wall, these are (i)- mechanical isolation method: when this method is applied it often result in low yield, poor quality and poor performance in culture as a result of substances released from damage cells. (ii)- Enzymatic isolation Method: is usually carried out in a simple salt solution, plus the cell wall degrading enzymes. These cell wall degrading enzymes are; cellulose and pectinase. They are usually mixed with the salt solution during the cell wall isolation process.
Protoplast are fragile and easily damage and therefore must be cultured carefully. Liquid medium here is not agitated and a high osmotic potential is maintained at least in the initial stage. The liquid medium must be shallow enough to allow aeration in the absence of agitation. Protoplast can be plated out on to solid medium and Callus produce. Whole plant can be regenerated by organogenesis or somatic embryogenesis from the Callus.
Root Culture:
Root cultures can be established invitro from the explants of the root tip of either primary or lateral roots and be cultured on fairly simple medium.
Shoot tip and Meristem Cultures:
The tips of shoots (which contain the shoot apical meristem) can be cultured invitro, producing clumps of shoot from either Axilliary or adventitious buds.
Embryo Culture:
Embryos can be used as explants to regenerates Callus cultures, or somatic embryos. Both immature and mature embryos can be used as explants
Stages involved in plant tissue culture:
In any plant tissue culture experiment, the following steps must be strictly observed for a successful plant transformation, thus;
Stage 1- establishment of an asceptic sterile culture.
Stage 2- The multiplication of propagules ( i.e any part of plant used to make or become new plant)
Stage 3- Preparation of propagules for successful transfer to soil (rooting and hardening i.e acclimating) outside of sterile conditions in regular growing media.
Stage 4- Establishment in soil ( or other appropriate growing media).
Plant Tissue Vs Traditional Propagation method.
The major difference between the plant tissue culture and the conventional propagation method is summarized below:
i- The plant grows faster.
ii- Plant can be multiplied rapidly by giving them the right plant Hormones
iii- Using tissue Culture, large number of plant can be created from just one piece of tissue and in relatively short period of time
iv- There is the exact replication of the donor plant with all the desirable characteristics’.
Benefits of plant Tissue Culture:
Plant Tissue has the following benefits to our Conventional farmers;
1- Cultures taken from merismatic tissue are usually virus free (i.e diseases free)
2- It is very fast way to produce a great number of plant
3- The plants are usually very vigorous and healthy since they are grown in a controlled environment.
4- Plant tissue cultures also maintain genes of other plants in the exact form. This is important in hybrid plants because many hybrids can no longer reproduce sexually.
Economic Importance:
Plant tissue culture has for decades played a key role in the improvement of useful crop plant and also the ways it has helped mankind in the field of medicine. It also, forms an integral part of any plant Biotechnology activity, thus; offering an alternative to conventional agricultural practices. Many new pharmaceutical drugs are developed using plant tissue culture techniques.
Conclusion:
This paper has only succeeded in scratching the surface of a sea of knowledge lying to be exploited. However, plant tissue culture has been successfully integrated into plant transformation strategies and the least of plant species that can be routinely transformed continue to grow.
Recommendation:
Plant tissue culture is one area in plant Biotechnology transformation that have been successful, I hereby recommend that BIODEC KATSINA should endeavor to exploit this aspect of modern agricultural Biotechnology strategies.
References:
- Adrian Slater et al : plant Biotechnology “the genetic manipulation of plants”
- Steinhardt et al (1913) : Studies on the cultivation of the virus Vaccnia”.
- Tedtalks (2010): Anthony Atala on growing organs (video file 2011).
