Plants improvement via plant cell culture

Plants improvement via plant cell culture

Plant tissue culture

Introduction:

Tissue culture:

A method of biological research in which fragments of tissue from an animal or plant are grown in vitro in artificial medium under aseptic condition and continue to survive and function. (Omen et al)

Plant tissue culture:

It can be defined as method which we can use to grow whole plant from a single cell by growing it artificially prepared nutrient medium under aseptic conditions.

Requirements of tissue culture

Explant
Suitable growth medium (some of the common media are MS, LS, Gamboges, Heller

For haploids etc.)

Aseptic(sterile) condition
Plant cell culture depends on:
Totipotency

The ability by which plant cell can regenerate into a whole plant. (Haberlandt G 1902).

Plasticity:

It is the ability of plant to alter their metabolism, growth, and development to best suit their environment.

We can say it is the ability to regenerate lost organs or undergo developmental pathway when it is exposed to particular stimuli.

HISTORY:

Science of tissue started from the discovery of cells when in 1838 Schwann and schleiden discovered that cell is the basic unit of life. They visualized that cell is capable of autonomy and because of this property it should be able to develop into a whole plant.

Father of plant tissue culture:

In 1902, the experiment of tissue culturing by culturing isolated single palisade leaves from cells in a salt solution enriched with sucrose was done by a German physiologist Gottlieb Haberlandt, although cells failed to divide but they remained alive for one month and increased in size. Though he did not succeed in regenerating cell but he was the one who gave the idea of regenerating whole plant from single cell.

Some landmark discoveries

1902-concept of in vitro cell culture was given by Haberlandt

1904-culturing of embryos from several cruciferous species was done by Henning.

1922-kolte cultured root, robin successfully cultured stem tips.

1926-indole acetic acid-growth hormone was discovered by went.

1934-white gave the idea of introducing vitamin c as growth supplement in culture media for tomato tip.

1939-Establishment of endless proliferation of culture by Gautheret, white, and Nobecourt.

1941-overbreek added coconut milk for cell division in Datura.

1957-Skoog and Miller gave the idea of hormonal control of organ formation

1959-Reinet and Steward regenerated embryos from callus clumps.

1960-Cocking isolated protoplast by enzymatic degradation of cell wall.

Nutrient Media

It is basically a composition of essential minerals and vitamins which are required for the growth and development of plants. Minerals consist of macronutrients as well as micronutrients. It contains iron, vitamins, sugars-all are in sterile or axenic condition. Ph of nutrient media adjusted to 5.2-5.8(Owen et al, 1991). Acidic. may delay the growth of potential contaminants. However, certain cultures require a higher ph.ph influences the uptake of various components and also regulates the various biochemical reactions occurring in plant tissue cultures. Vitamins required include pyridoxine, nicotinic acid and thiamine. Sometimes other vitamins such as folic acid, biotin, ascorbic acid and vitamin E are also added to media formulations. We can use solid as well as liquid medium. However mostly we use solid media because it provides support system to the explant and is easier to handle. Agar is used for the solidification of media. agar is derived from seaweed but sometime we also use agar substitutes such as phytagel and gel rite which is much clearer than agar.The concentration of hormones which is required for plants growth depends on specie of plant. (Ting IP 1982)

Most commonly used nutrient media include:

MS (Morishige and skoog 1962)
WPM- McCowns woody plant medium (McCowns and Lloyd 1981)
Knudson’s medium (knudson 1946) was developed for Orchid tissue culture.

Choice of Medium:

we choose media dependingon response shown by explant to that particular media. All the media have different composition of nutrients. For example, macronutrient concentration is higher in MS media as compared to WPN media which suits plant species while woody plants grow better in WPN media. It means nature of explant decides which medium should be used

Plant growth regulators:

Auxin, gibberellin and cytokinin are the most commonly used regulators. High concentration of auxin promotes root formation while high concentration of cytokinin promote shoot formation so if we add balanced concentration auxin and cytokinin then an undifferentiated mass of cells -callus will be developed. Auxins which are most commonly used include IAA (indole acetic acid), NAA (naphthalene acetic acid), IBA (indole butyric acid) and 2, 4-D (2, 4-Dichlorophenoxyacetic acid). While most commonly used cytokinin include BAP (benzyl aminopurine), zeatin and kinetin. In addition, some other hormones have also been used which include abscisic acid (Augustine et al.1997), jasmonic acid (Blazquez et al.2004) etc.

Stevia rebaudiana ( Rafiq et al.2007)showed maximum root induction when it was supplemented with 0.5mg/l NAA.While phalaenopsis showed maximum shoot elongation when it was provided with 0.5mg/l GA.

Plant improvement via plant tissue culture

With increase in population, the demand for food is also increasing day by day. in order to meet food demand we should develop methods through which we can produce more food using less land. Tissue culture technique is helping mankind in this concern. This technique also helps to bring soma colonel variations. Quality of food can also be improved using gene transformation technique. as we provide nutrient media by ourselves in which plant cells into whole plant cell grows into whole plant so we can improve nutritional quality of food crops. During tissue culture we can introduce genes of our interest in the plant so that it may become genetically modified plant. For example, we can introduce genes which are resistant to viruses, diseases etc. Genetic variability which may be introduced through tissue culture can help us bring variations in the plants so that we can get new and stable genotypes. Gene transformation techniques can help us produce varieties which have more potential and resistance against pests.

Improvements of crops using tissue culture technique:

Improved nutritional quality
Insect resistance
Disease resistance
Salt tolerance
Delayed fruit ripening
Biopharmaceuticals and vaccine
Secondary metabolites production
Heat or stress resistance varieties.
Plant for commercial purpose such as potting, florist varieties and landscape may be produced.
Rare or endangered species of plant may be conserved
We can screen cells instead of plants for advantageous characters i.e. tolerance/resistance etc.
Plants can be grown at large scale using single cell

Following methods may help us in plant improvement

There are many methods of tissue culture which can be used to produce improved crop varieties which have better nutritional quality.

These methods include:

Germplasm conservation
Protoplast fusion
Embryo culture
Haploid production
Genetic transformation
Soma colonel variations

Soma colonel variations

During invitro growth some cytological and genetic variations may occur in plants. such variations are known as somaclonal variations. Somaclonalvariation may be heritable or non-heritable. Somaclonal variations may be due to:

Explant may have some preexisting aberrant cell
Culture environment may cause developmental or physiological changes

Epigenetic changes may produce somaclonal variations which are not transmitted to progeny while somaclonal variations may also be due to some specific changes which are transmitted to progeny. Although somaclonal variations may restrict colonel propagation (a technique which we can use to conserve best characteristics of plant) but on other hand somaclonal variation may produce such desirable characters which may be used to develop novel cell lines. In addition heavy-metal resistant (Chakravarty and Srivastava 1997), herbicide resistant (smith and chaleff 1990), disease resistant (Jones 1990), salt-tolerant (ochatt et al.1990) have been selected by callus tissue in whichsomaclonal v variations took place.so somaclonal variations may help us in crop improvement, to create genetic variations and to produce toxin resistant plants.

Protoplast fusion:

“Components of cell remained after removing cell wall are collectively called protoplast”

cell – remove cell wall – protoplast

If we grow a whole plant from protoplast then it will be called as protoplast culture.

Here protoplast is explant .we will provide nutrition medium to explant .Protoplast will start cell division and a mass of cells will be formed which will eventually grow into a plant .we can extract protoplast from leaf, tissue, callus, roots etc. . . .But our most priority is leaf because we can extract protoplast from leaf conveniently. As cell wall is removed which is hard covering and it protects cell from drastic conditions so protoplast is fragile and difficult to handle. It helps to get desired characters in plants by transferring genes through protoplast. (Evans et al.1988)

Two general approaches to remove cell wall:

Mechanical method
Enzymatic method

Mechanical method:

Removal of cell wall by cutting it with any equipment

Now mechanical methods are not applied for removal of cell wall because it has following disadvantages:

Low yield
Poor quality

Enzymatic method:

As name indicates this method involves the use enzymes. These enzymes include cellulose and pectinase because cell wall is made up of cellulose and pectin (Dodds and Roberts 1995) so for their breakdown we need cellulase and pectinase respectively.

Procedure:

Take shoot and separate a leaf from it and cut the leaf and then incubate it in flask having enzymes i.e. cellulase and pectinase .Enzymes will digest cell wall and we will have protoplast in the flask. Purify protoplast from cellular debris by centrifugation .we will culture protoplast in nutrient medium.Mostly we use solid medium because in solid medium we have better regeneration rate and it will start growing into a plant by a process known as organogenesis.

Organogenesis; formation of organs from cells

Somatic embryogenesis; Formation of whole plant body from single somatic cell or a group of somatic cells

A portion of leaf is made up of mesophyll tissue and these mesophylls are made up of mesophyll cells which are also called somatic cells.

.Genetic Transformation:

This technique ensures transfer of genes having desirable characters between two or more plants (Hinchee et al 2011). Using this technique, we can improve crops because we can introduce characteristics of our interest as we can increase yield, produce better quality crops and make plants more resistant to diseases and pests. (Sinclair et al 2004)

There are two ways by which we can transform genes:

Vector-mediated: This is an indirect method of gene transfer

Vector-less: This is direct method of gene transfer. (Sasson A 1993)

An example of vector mediated method is genetic transformation through agrobacterium. Here agrobacterium is vector and it can be used to introduce foreign traits in plant. Jatropha plant has been successfully transformed genetically by DNA delivery through direct bombardment at shoot apices which were derived from seed (Pukaryashta et al 2010). Using this technique, we can reduce toxic substances in seed (Misra et al 1993). It lessens the obstacles in seed utilization in industries. Production of potato transgenic plant is a big success in crop improvement because it produces potato plant resistant to potato virus Y which destroyed a major portion of potato crop every year (Bukovinszki et al.2003). Using multi auto transformation system petunia hybrid has been produced which is resistant to Botrytis cinerea (an agent responsible for causing grey mold). (khan et al.2011)

Micropropagation:

“In vitro production of clones at large scale using explant is known as micropropagation”

Using this technique, we can produce plants of desirable characters at large scale. Hence crop yield can be increased using this method.

Haploid production:

“Homozygous plants can be produced via tissue culturing through anther, microspores and protoplast etc. in lesser time comparative to conventional breeding.”(Morrison et al.1998)

Haploid plants: those plants which have single set of chromosomes.

Naturally the process by which haploid cells are formed is called meiosis and the haploid cells thus formed are called gametes. While in plants these haploid cells are pollen i.e. male cell and egg i.e. female cell.so will take either egg or pollen and culture it in nutrient media. It will develop into a whole plant which will be haploid i.e. having single set of chromosomes. This plant will be sterile, means it will not be able to produce offspring through pollination as their cells will not undergo meiosis and they will not be able to produce gametes for fertilization. Hence in order to use them for plant breeding practices, diploidy is induced by colchicine treatment and homozygous pure lines can be obtained which are known as double haploid or diploid plants.

Androgenesis: The process of producing haploid plants without undergoing fertilization from pollen cells. In this process we use anther. (sudherson et al 2008)

Pollens can be extracted from anther by these two methods:

Float culture method; in this method the cold treated anthers are surface sterilized and cultured on liquid media. After 2 to 7 days anther will liberate pollens in the culture. Another wall cells are removed as large debris and only pollens will be taken in culture media.
Sucrose density gradient centrifugation: using this method anther is crushed and by repeated filtration pollens are separated out. Filtrate obtained is then layered on 30% sucrose solution and then centrifuged. Androgenic pollen grains form a layer at the top of solution which is then pipetted out for culturing (venal et al. 1975)

This technique can be used to speed up the breeding programs of in bred line and lessens the c of seed dormancy and increases the viability of embryo. Haploid plants produced are resistant to certain biotic and abiotic factors.

Germplasm conservation:

“Preservation and maintenance of germplasm of plants which are vulnerable to insects, pathogens and environmental hazardetc.”

As specie number is decreasing day by day so in order to protect them from extinction germplasm conservation is an important ex situ conservation method.(sengar et al 2010) This is an alternative method of growing plants in fields so less land is used that’s why it is cost effective method. This method is used to keep crops genetic background conserved. As we can protect genetic material and seeds in gene bank named seed banks respectively so genetic background of species which are disappearing can be protected (Filho et al 2008). There are some species which do not produce seed .by conserving their genetic background they can be preserved.

Cryopreservation is an important technique for preservation of plant species. It is the storage of plant at low temperature i.e. -196 Celsius in liquid nitrogen. At low temperature cells metabolic activities will stop and cell will not need energy to live alive. So it can be preserved for long time and when we will provide cell normal temperature and normal conditions it will start regrowing (Harding K 2004).if cell grows at normal rate after cryopreservation then it will be successful cryopreservation. So it can be helpful in crop improvement as more species can be protected and grown by using these techniques. We can conserve embryo tissues using technique of cryopreservation (Corridoira et al 2004)

Synthetic or artificial seeds:

Totipotent cells from plant are taken and encapsulated by chemicals i.e. calcium alginate and grown in soil these will act as subtitles of seed and will be able grow into a whole plant either invitro or invitro. (Stewart et al.)

Examples of crops and plants improved by tissue culture technique

Case study 1:

Moth orchids: These plants are important not only because of their beauty and fragrance but they are also used as medicine for treating diarrhea. They are difficult to grown by vegetative propagation and their growth process is time consuming. They can be regenerated by invitro propagation of callus hence the problem of explant shortage can also be solved. Invitro propagation of callus is usually a quick process so issue of time shortage can also met.MS medium having 3% sucrose ,0.8% agar and BAP AND 2 4-D is maintained and callus is cultured on it. After 30 days callus is subculture for proliferation. Callus showed maximum proliferation when medium was provided with 0.5 mg/I BA. For root and shoot formation medium is provided accordingly. In this way mass production of orchids was obtained which was helpful economically. (Hussain et al.)

Case study 2:

Tobacco tissue culturing:

A large area of Pakistan is covered under cultivation by tobacco. It is also good economically. Leaves and meristems are used as explants and they are cultured on MS medium. Usually Pakistan tobacco board provides the mother plant. Callus shows maximum growth when medium is supplemented with 1.0mg/l 2, 4-D. Then callous was transferred to a medium containing 0.5mg/l BAP in which it showed maximum shoot proliferation. For maximum root induction medium was provided with 2.0mg/l IBA.

Case study 3:

**Honey plant (Stevia rebaudiana Bertoni):**

Seeds were cultured on MS medium for colonel propagation of Stevia rebaudiana (Haberlandt ET al.1902) by placing it in growth room for photoperiod of 16 hrs light and 8 hours dark. Four nodal segment of seedling each having size of 0.5 cm was used as explants. Nodal plants were cultured in media supplemented with 30% sucrose and either alone 1.0, 2.0, 3.0, 4.0, 5.0 mg/l BAP and kinetin or in combination with 0.5 or 0.25mg/l IAA. It showe3d maximum shoot multiplication when medium was supplemented with 2.0mg/l BAP. Highest shoot formation which was about (3.73 _+ 0.14) per micro shoot was observed after 15 days of inoculation when MS medium was supplemented with 0.2 kn and 0.25mg/l IAA. Then for root induction medium was supplemented with 0.25, 0.5, 1.0, and 1.5 mg/l of NAA and IBA separately. In this way Stevia rebaudiana can be obtained for commercial purposes and can be grown in various environmental conditions of Pakistan.

Case study 4:

Potato (Solanum tuberosum):

It is very important crop in world as it is enriched with nutrients and it is cultivated on maximum land and farmers get much benefit in return. For production of disease free potato it is cultured on MS medium. Research has been conducted on three varieties of potato i.e. Desiree, Cardinal and Diamante. For research a company named four brothers agri services Pakistan provided the plant material. This company is working for betterment of crops in Pakistan. A potato was taken and it was washed with detergent for removing any kind of impurities and allowed for sprouting. It was kept to sprout for almost 15 days. Then this sprouted potato was cut in pieces 10mm each having node and was used as explant for culturing. Espinosa medium along with vitamin B5 having concentrations of GA3 and BAP was used. When medium was provided with 0.5mg/l BAP and 0.25mg/l GA3, maximum shoot proliferation was observed.Heighest root induction was observed when medium was supplemented with 0.1mg/l NAA.In this rooted plantlets were acclimatized and supplied to company which were than cultivated. (Hussain et al.)

Case study 5:

Physic nut (Jatropha curcas L.)

In order to get higher yield of seed and oil content, Jatropha curcas is of the main focus. In the propagation of Jatropha curcas is carried out.About seven days old leaf and apical meristems were taken which were used as explants and were induced for propagation.

Murashige and skoog (MS) medium was used for in vitro propagation of Jatropha curcas. Different growth regulators were used. Maximum callus induction was observed when medium was supplemented with 1.0mg/L 2; 4-D. Callus showed faster growth in IBA concentration during 7 to 30 days and then stabilized. Callus obtained was white, friable and soft. For direct shoot regeneration. apical meristems were used. When the medium was provided with 2.0mg/L IBA, it showed maximum root induction.It is then acclimatized and released to fields for further planting.

Conclusion and future perspective:

With increase in population the demand for food is increasing day by day while land available for cultivation is decreasing .S o in order to meet the demand for food we have develop methods which may produce enough food fastly and by using lesser land. Plant tissue may help to produce better quality food by using different methods. In future this technique will be more concerned and valued as food shortage is one of the major issues faced by mankind. This technique may produce disease free plants. Another benefit is that we can produce food irrespective of the season by developing different methods. It can also be used to produce plants which may be used as edible vaccines.

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Plants improvement via plant cell culture

Plants improvement via plant cell culture