AGRICULTURE BIOTECHNOLOGY

Plant Genetic Engineering

Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or "knocked out", using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine.

Plant genetic engineering is the art and science of changing the traits of plants in order to produce desired characteristics. Plant genetic engineering can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics for propagation, to more complex molecular techniques.

Plant genetic engineering is important for ensuring food security by developing new varieties that are higher-yielding, resistant to pests and diseases, drought-resistant or regionally adapted to different environments and growing conditions.

Genetic modification

Genetic modification of plants is achieved by adding a specific gene or genes to a plant, or by knocking down a gene with RNAi, to produce a desirable phenotype. The plants resulting from adding a gene are often referred to as transgenic plants. Genetic modification can produce a plant with the desired trait or traits faster than classical breeding because the majority of the plant's genome is not altered.

To genetically modify a plant, a genetic construct must be designed so that the gene to be added or removed will be expressed by the plant. To do this, a promoter to drive transcription and a termination sequence to stop transcription of the new gene, and the gene or genes of interest must be introduced to the plant. A marker for the selection of transformed plants is also included. In the laboratory, antibiotic resistance is a commonly used marker.

The construct can be inserted in the plant genome by genetic recombination using the bacteria Agrobacterium tumefaciens or A. rhizogenes, or by direct methods like the gene gun or microinjection. Using plant viruses to insert genetic constructs into plants is also a possibility, but the technique is limited by the host range of the virus. For example, Cauliflower mosaic virus (CaMV) only infects cauliflower and related species.

 

Objectives of Plant Breeding

The prime aim of plant breeding is to improve the characteristics of plants that they become more useful automatically and economically. Some of the objectives may be summarized as follows.

1. Higher Yield:

Higher yield of grain, fodder, fibre, sugar, oil etc. developing hybrid varieties of Jawar, Maize, Bajara, etc.

2. Improved Quality:

The quality characters may vary from one crop to another such as grain size, shape, colour, milling and backing quality of wheat, cooks quality in rice, malting in barley. Size shape and flavour in fruits and keeping quality of vegetables, protein contents in legumes, methionine and tryptophan contents in pulses etc.

3. Disease and Pest Resistance:

Resistant varieties offer the cheapest and most convenient method of disease and pest control. They not only helps to increase the production but also stabilize the productivity e.g. Rust resistance in wheat.

4. Maturity Duration:

It permits new crop rotation and extends crop area. Thus breeding for early maturing varieties suitable for different dates of planting. This enables the farmer to take two-three crops in a year.

5. Agronomic Characters:

Three includes the characters such as dwarf, profuse tillering, branching erect resistance and fertilizer responsiveness.

6. Photo and Thermo Insensitivity:

Development of photo and thermo insensitive varieties in rice and wheat will permit to extend their cultivation to new areas. E.g Cultivation of wheat in Kerala and West Bengal, Cultivation of rice in Punjab and Himachal Pradesh.

7. Synchronous Maturity:

It is desirable in crops like mung ( Vigna radiate) where several pickings are necessary.

8. Non-Shattering Characteristics:

E.g.  Mung, Black Gram, Horse Gram, etc.

9. Determinate Growth Habit:

It is desirable in mung, pigeon pea and cotton, etc.

10. Dormancy:

In some crops, seeds germinate even before harvesting if there are rains at the time of maturity. E.g Mung, barley, etc. A period of dormancy in such cases would check the loss due to germination while in other cases it may be removed it.

11. Varieties for a New Season:

Breeding crops suitable for seasons. E.g Maize (Kharif) which is grown in Rabi and summer also.

12. Moisture Stress and Salt Tolerance:

Development of varieties for a rainfed area and saline soils would help to increase crop production in India.

13. Elimination of Toxic Substance:

It will help to make them safe for consumption E.g Khesari ( Lathyrus sativus) seeds have a neurotoxin causing paralysis.

14. Wider Adaptability:

It helps in stabilizing the crop production over region and seasons.

15. Useful for Mechanical Cultivation:

The variety developed should give response to application of fertilizers, manures and irrigation, suitable for mechanical cultivation etc.

Scope of Plant Breeding (Future Prospects)

I) Genetic manipulation of population by increasing the frequency of desirable alleles in cross pollinated crops and introducing male sterile in self pollinated crops like wheat and Rice.

II) Intensive breeding of pulses and oil seed crops as it was done in cereals and other crops.

III) Proper breeding methods with improved crop management practises.

IV) Use of heritability methods with improved crop management practises.

V) Development of improved high yielding varieties of vegetable and seed crops.

VI) Quality Improvement in Oil seed and Vegetables.

VII) Use of transgenic plants as a medicine. E.g. Potato.

VIII) Development of varieties which are desirable for mechanical threshing and cultivation.

Transgenic Plants

A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species: transgenic Bt corn, for example, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called genetically modified or GM crops, although in reality all crops have been genetically modified from their original wild state by domestication, selection and controlled breeding over long periods of time.

 

Procedure

Introduction to DNA

The underlying reason that transgenic plants can be constructed is the universal presence of DNA (deoxyribonucleic acid) in the cells of all living organisms. This molecule stores the organism's genetic information and orchestrates the metabolic processes of life. Genetic information is specified by the sequence of four chemical bases (adenine, cytosine, guanine, and thymine) along the length of the DNA molecule. Genes are discrete segments of DNA that encode the information necessary for assembly of a specific protein. The proteins then function as enzymes to catalyze biochemical reactions, or as structural or storage units of a cell, to contribute to expression of a plant trait. The general sequence of events by which the information encoded in DNA is expressed in the form of proteins via an mRNA intermediary is shown in the diagram below.

The transcription and translation processes are controlled by a complex set of regulatory mechanisms, so that a particular protein is produced only when and where it is needed.

Locating Genes for Plant Traits

Identifying and locating genes for agriculturally important traits is currently the most limiting step in the transgenic process. We still know relatively little about the specific genes required to enhance yield potential, improve stress tolerance, modify chemical properties of the harvested product, or otherwise affect plant characters. Usually, identifying a single gene involved with a trait is not sufficient; scientists must understand how the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes active in the same biochemical pathway. Public and private research programs are investing heavily into new technologies to rapidly sequence and determine functions of genes of the most important crop species. These efforts should result in identification of a large number of genes potentially useful for producing transgenic varieties.

Designing Genes for Insertion

Once a gene has been isolated and cloned (amplified in a bacterial vector), it must undergo several modifications before it can be effectively inserted into a plant.

Simplified representation of a constructed transgene, containing necessary components for successful integration and expression.

1. A promoter sequence must be added for the gene to be correctly expressed (i.e., translated into a protein product). The promoter is the on/off switch that controls when and where in the plant the gene will be expressed. To date, most promoters in transgenic crop varieties have been "constitutive", i.e., causing gene expression throughout the life cycle of the plant in most tissues. The most commonly used constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Sometimes, the cloned gene is modified to achieve greater expression in a plant. For example, the Bt gene for insect resistance is of bacterial origin and has a higher percentage of A-T nucleotide pairs compared to plants, which prefer G-C nucleotide pairs.
2. The termination sequence signals to the cellular machinery that the end of the gene sequence has been reached.
3. A selectable marker gene is added to the gene "construct" in order to identify plant cells or tissues that have successfully integrated the transgene. This is necessary because achieving incorporation and expression of transgenes in plant cells is a rare event, occurring in just a few percent of the targeted tissues or cells. Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides

Transforming Plants

Transformation is the heritable change in a cell or organism brought about by the uptake and establishment of introduced DNA. There are two main methods of transforming plant cells and tissues:

1. The "Gene Gun" method (also known as microprojectile bombardment or biolistics). This technique, which is shown and explained in the animated demo section of this web site, has been especially useful in transforming monocot species like corn and rice.
2. The Agrobacterium method, which is described below. Transformation via Agrobacterium has been successfully practiced in dicots (broadleaf plants like soybeans and tomatoes) for many years, but only recently has it been effective in monocots (grasses and their relatives). In general, the Agrobacterium method is considered preferable to the gene gun, because of the greater frequency of single-site insertions of the foreign DNA, making it easier to monitor.

Selection and Regeneration

Selection of successfully transformed tissues. Following the gene insertion process, plant tissues are transferred to a selective medium containing an antibiotic or herbicide, depending on which selectable marker was used. Only plants expressing the selectable marker gene will survive, as shown in the figure, and it is assumed that these plants will also possess the transgene of interest. Thus, subsequent steps in the process will only use these surviving plants.

When grown on selective media, only plant tissues that have successfully integrated the transgene construct will survive.

Regeneration of whole plants. To obtain whole plants from transgenic tissues such as immature embryos, they are grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins. This regeneration step has been a stumbling block in producing transgenic plants in many species, but specific varieties of most crops can now be transformed and regenerated.

·  Testing of plant performance is generally carried out first in the greenhouse or screenhouse to determine whether the modified plant has the desired new trait and does not have any new unwanted characteristics. Those that perform well are planted into the field for further testing. In the field, the plants are first grown in confined field trials to test whether the technology works (if the plants express the desired traits) in the open environment. If the technology works then the plants are tested in multi-location field trials to establish whether the crop performs well in different environmental conditions. If the GM crop passes all the tests, it may then be considered for commercial production.

·  Safety assessment. Food and environmental safety assessment are carried out in conjunction with testing of plant performance.

Transgenesis

Transgenesis is the process of introducing an exogenous gene — called a transgene — into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. Transgenesis can be facilitated by liposomes, plasmid vectors, viral vectors, pronuclear injection, protoplast fusion, and ballistic DNA injection.

Transgenic organisms are able to express foreign genes because the genetic code is similar for all organisms. This means that a specific DNA sequence will code for the same protein in all organisms. Due to this similarity in protein sequence, scientists can cut DNA at these common protein points and add other genes. An example of this is the "super mice" of the 1980s. These mice were able to produce the human protein tPA to treat blood clots.

Using plasmids from bacteria

The most common type of transgenesis research is done with bacteria and viruses which are able to replicate foreign DNA.^[1] The plasmid DNA is cut using restriction enzymes, while the DNA to be copied is also cut with the same restriction enzyme, producing complementary sticky-ends. This allows the foreign DNA to hybridise with the plasmid DNA and be sealed by DNA ligase enzyme, creating a genetic code not normally found in nature. Altered DNA is inserted into plasmids for replication.^[2]

Gene transfer technology

DNA microinjection

The Desired gene construct is injected in the pronucleus of a reproductive cell using a glass needle around 0.5 to 5 micrometers in diameter. The manipulated cell is cultured in vitro to develop to a specific embryonic phase, is then transferred to a recipient female. DNA microinjection does not have a high success rate (roughly 2% of all injected subjects), even if the new DNA is incorporated in the genome, if it is not accepted by the germ-line the new traits will not appear in their offspring. If DNA is injected in multiple sites the chances of over-expression increase.^[3]

Retrovirus-mediated gene transfer

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. Retroviruses are used as vectors to transfer genetic material into the host cell. The result is a chimera, an organism consisting of tissues or parts of diverse genetic constitution. Chimeras are inbred for as many as 20 generations until homozygous genetic offspring are born.^[3]

Stem cell transgenesis

There are three types of stem cell transgenesis.

Multipotent stem cell transgenesis

Pluripotent stem cell transgenesis

Totipotent stem cell transgenesis

Applications

Pharming

Pharming is a portmanteau of "farming" and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO).

Pharming Examples:

• Haemoglobin as a blood substitute
• human protein C anticoagulant
• alpha-1 antitrypsin (AAT) for treatment of AAT deficiency
• insulin for diabetes treatment
• vaccines (antigens)- Vaccines produced by genetic engineering offer an advantage that the microbial strains from which the proteins are extracted do not contain complete viruses. And thus, there are no risks of accidental inoculation with live virus.
• growth hormones for treatment of deficiencies
• factor VIII blood clotting factor
• factor IX blood clotting factor
• fibrinogen blood clotting factor
• lactoferrin as an infant formula additive

Research

Genetic engineering is an important tool for natural scientists. Genes and other genetic information from a wide range of organisms are transformed into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research

Medical

Transgenesis can be used to neutralize genes that would normally prevent xenotransplantation. For example, a protein found in pigs can cause humans to reject their transplanted organs. This protein can be replaced by a similar human genome to prevent the rejection.

Industrial

Another benefit of genetic engineering is realized in production of valuable proteins. Recombinant DNA made possible the use of bacteria to produce proteins of medical importance. One such example is that of genetically engineered human insulin which is of great importance and now marketed throughout the world.

Some important genetically engineered proteins include:

Human Insulin
Human insulin or Humulin has great importance. Earlier, patients could not tolerate pig insulin, as it has slightly different amino acid sequence as compared to human. Humulin eventually became cheaper than that extracted from animal pancreas and is now available.

Interferon
Interferon is an antiviral agent which is secreted by cells which are attacked by virus. Several types of genetically engineered interferon are available in market and gives rise to antitumoral effect (thwarting formation of cancerous tumors).

Growth hormone
In humans, growth hormone helps in treatment of hypopituitary dwarfs. Genetically engineered growth hormones may prove useful in the treatment of bone fractures, skin burns and bleeding ulcers of digestive tract. The human hormone is marketed in United States and bovine hormone is expected to yield bigger cattle and thus more beef. Hence growth hormones are commercially very demanding.

Agriculture

1. Improved nutritional quality
2. Better Nitrogen Fixation
3. Disease resistant Plant
4. Enhanced efficiency of minerals used by plants to prevent early exhaustion of fertility of soil.
5. Reduced post harvest losses

Pesticide resistant rape plants

Scientists have transferred a gene to the rape plant which enables the plant to resist a certain pesticide. When the farmer sprays his genetically modified rape crop with pesticides, he or she can destroy most of the pests without killing the rape plants.

Advantages:

    * The farmer can grow a larger crop because it is easier to fight pests.

    * In some cases the farmer can use a more environmentally friendly crop spray.

    * The farmer can also protect the environment by using less crop spray.

Insecticide sweet corn

Scientists have genetically modified sweet corn so that it produces a poison which kills harmful insects. This means the farmer no longer needs to fight insects with insecticides. The genetically modified corn is called Bt-corn, because the insect-killing gene in the plant comes from the bacteria Bacillus thuringiensis.

Advantages:

    * The farmer no longer has to use insecticide to kill insects, so the surrounding environment is no longer exposed to large amounts of harmful insecticide.

    * The farmer no longer needs to walk around with a drum of toxic spray wearing a mask and protective clothing.

Golden rice

Golden rice is genetically modified rice that now contains a large amount of A-vitamins. Or more correctly, the rice contains the element beta-carotene which is converted in the body into Vitamin-A. So when you eat golden rice, you get more vitamin A.

Beta-carotene gives carrots their orange colour and is the reason why genetically modified rice is golden. For the golden rice to make beta-carotene three new genes are implanted: two from daffodils and the third from a bacterium.

Advantages:

    * The rice can be considered a particular advantage to poor people in underdeveloped countries. They eat only an extremely limited diet lacking in the essential bodily vitamins. The consequences of this restricted diet causes many people to die or become blind. This is particularly true in areas of Asia, where most of the population live on rice from morning to evening.

Long-lasting tomatoes

Long-lasting, genetically modified tomatoes came on to the market in 1994 and were the first genetically modified food available to consumers. The genetically modified tomato produces less of the substance that causes tomatoes to rot, so remains firm and fresh for a long time.

Advantages:

    * Because the GM tomatoes can remain fresh longer they can be allowed to ripen in the sun before picking - resulting in a better tasting tomato.

    * GM tomatoes can tolerate a lengthier transport time. This means that market gardens can avoid picking tomatoes while they are green in order that they will tolerate the transport.

    * The producers also have the advantage that all the tomatoes can be harvested simultaneously.

Quality improvements

Plants are remarkable in their capacity to synthesize a variety of organic substances, such as vitamins, sugars, starches and amino acids. As many as 80,000 different substances are synthesized in plants, including macronutrients and micronutrients significant to human health.

Carbohydrates.

Plants manufacture both polymeric carbohydrates, like starch, and individual sugars, like sucrose. Plant starch is used in a wide range of industrial applications such as coatings for paper and textiles and as a gelling agent in the food industry (Heyer et al. 1999). It is now possible to make starches that are free of the amylose fraction, resulting in a gelling agent that is clearer and forms a gel at a lower temperature. Starches with higher levels of amylose are more desirable as coating agents and maintain texture at higher temperatures. For example, “sticky rice” has lower amylose content. The presence or absence of amylose greatly influences the physiochemical properties of starch; genetic engineering will result in specialized starches with higher value for specific applications.

Lipids, fats and oils.

Dietary fats and oils contribute to balanced nutrition, providing energy, fat-soluble vitamins and the essential polyun-saturated fatty acids (linoleic and ?-linolenic) required for growth, cell-membrane function, development and disease prevention. Conversely, fats and oils have been implicated in a diverse group of ailments such as obesity, cancer and heart disease.

A whole range of genetically modified oil seeds may be available in the future to promote health and prevent disease. In addition, specialty seeds with novel oil composition may be available to serve unique nutritional needs.

Proteins.

Plant proteins provide amino acids important for human health. Many plant proteins, like those present in corn seed, do not have the complete complement of essential amino acids. Plant-seed proteins can be modified to express proteins with a more desirable amino-acid composition.

Nutritional components.

Health-conscious consumers are compelling farmers and seed companies to improve the overall nutritional quality of their products. Extensive medical, biochemical and epidemiological research points to specific plant-produced substances (phytochemicals), as well as classes of phytochemicals that offer specific health benefits. Fruits and vegetables are a major source of beneficial phytochemicals.

Flavor and color.

In flowers, the altered expression of the enzymes of flavonoid biosynthesis yielded novel floral pigmentation patterns. Such approaches have not been applied to fruits yet, but the potential exists.

Improved flavor is of major interest to consumers, but it does not receive significant attention from breeders, who work largely to improve production and durability during postharvest distribution. The complexity of flavor — which includes a balance between sweetness and acidity as well as the compounds that give products their characteristic taste — has discouraged the pursuit of biotechnological approaches to flavor improvement. Biotechnological efforts to improve sweetness have met with little success so far.

Fiber quality.

In cotton plants, fiber cells manufacture individual fibers. Plant transformation will make it possible to deliver and express unique genes within these specialty cells to create unique cotton fiber products. Fiber strength can be improved to make cotton stronger and more lightweight.

Genes that express a variety of different colors could be introduced to provide a greater range of naturally colored fibers.

Seedlessness.

Consumers clearly prefer seedless varieties of certain fruits; for example, seedless grapes and watermelons dominate the marketplace. Researchers are exploring novel strategies to convert seeded fruits to seedless versions more quickly and accurately than is possible with conventional breeding.

Shelf life and ripening.

Two tomato products targeted at extending shelf life have met with limited success. One by Calgene reduces polygalacturonase activity to retard softening, while the other from DNA Plant Technology blocks ethylene synthesis to retard overall ripening. These products were not successful due to limitations in the quality of the base germplasm, the development of competitive nontransgenic products and the difficulty of obtaining premium prices when shelf life is not a primary consumer concern.

Productivity and yields

Early flowering.

Plants flower both in rhythm with their own internal development and in response to external signals such as temperature and day length.

For many herbaceous crop plants, altering the expression of genes identified principally through studies of Arabidopsis can produce a range of altered flowering patterns. These include neutral patterns (from previously day-length-dependent flowering time), flowering earlier or later in the growing season or changing the number of flowers per plant. Altered patterns can be obtained by altered expression of genes such as constans and terminal flower, among others. Novel patterns can be exploited to secure better market opportunities or increase seasonal yields.

Self-incompatibility.

Self-incompatibility is a widespread mechanism in flowering plants that prevents self-fertilization, requiring the presence of pollinator plants to ensure cross-pollination for fruit production.

Stress tolerance.

The geographical distribution of growing regions is largely determined by climate, because drought and freezing temperatures stifle crop quality and productivity.

Photosynthetic enhancement and yield increase

There is a persistent hope amongst plant breeders, whether they be concerned with conventional or transgenic varieties, that the photosynthetic efficiency of crops can be improved. After all, the first three priorities for a breeder are yield, yield, and yield, and it is assumed that improved energy capture can be translated into greater harvestable yield. Although there is still little reliable information that relates transgenic modification of specific photosynthetic genes to performance under agricultural conditions, results from many preliminary tests have been published. The tests of crops claimed to have an enhanced level of photosynthesis, and included the more numerous trials of plants with ‘enhanced yield’.

In vitro Propagation

Micro propagation (tissue culture or in-vitro culture) refers to the multiplication of plants, in aseptic condition and in artificial growth medium. From very small plant parts like Meristem tip, callus embryos, anther etc. At present tissue culture finds extensive application in agriculture and horticulture in several countries. Though some achievements have been made but the commercial utilization of the techniques of tissue culture is still lacking behind.

In vitro propagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction.

Methods of Micro-propagation

1. Meristem Culture:

In Meristem culture the Meristem and a few subtending leaf primordial are placed into a suitable growing media. Art elongated rooted platelet is produced after some weeks is transferred to soil when it has attained a considerable height. A disease free plant can be produced by this method. Experimental results also suggest that this technique can be successfully utilized for rapid multiplication of various herbaceous plants.

2. Callus Culture:

A callus is mass of undifferentiated parenchymatous cells. When a living plant tissue is paced tissue is placed in an artificial growing medium with other conditions favorable, callus is formed. The growth of callus varies with the homogenous levels of auxin and Cyotkininn and can be manipulated by endogenous supply of these growth regulators in the culture medium. The callus growth and its organogenesis or embryogenesis can be referred into three different stages.

Stages – I: Rapid production of callus after placing the explants in culture medium
.
Stage – II: The callus is transferred to other medium containing growth regulators for the induction of adventitious organs.

Stage – III: The new plantlet is then exposed gradually to the environmental condition.

3. Embryo Culture:

In embryo culture, the embryo is excised and placed into a culture medium with proper nutrient in aseptic condition. To obtain a quick and optimum growth has growth into a platelets, it is transferred to soil. It is particularly important for the production of interspecific and intergeneric hybrids and to overcome the embryo abortion.

4. Protoplast Culture:

The protoplast are fist culture in liquid medium at 25 to 28 C with a light intensity of 100 to 500 lux or in dark and after undergoing substantial cell division, they are transferred into solid medium congenial or morphogenesis in many horticultural crops response well to protoplast culture.

Stages of In vitro propagation

Establishment

In vitro propagation begins with the selection of plant material to be propagated. The plant tissues are removed from an intact plant in a sterile condition. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent on the type of tissue to be used; including stem tips, anthers, petals, pollen and others plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes, and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easily grown on simple media, but others require more complicated media for successful growth; the plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokinins are used to create branched shoots from plant buds.

Multiplication

Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured.

Pretransplant

This stage involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment.

"Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to the controlled nature of their maturation, the plantlets often do not have fully functional dermal coverings. This causes them to be highly susceptible to disease and inefficient in their use of water and energy. In vitro conditions are high in humidity, and plants grown under these conditions often do not form a working cuticle and stomata that keep the plant from drying out. When taken out of culture, the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question.

Transfer from culture

In the final stage of plant In vitro propagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.

This stage is often combined with the "pretransplant" stage.

 

Merits of In vitro propagation:

1. Tissue culture helps in rapid multiplication of true plats throughout the year.

2. A new plant can be regenerated from a miniature plant part, whereas in conventional methods a shoot of considerable length is required.

3. Large number of plants can be produced in culture tubes in small space with uniform growth and productivity of growing them in large area in nursery.

4. Plants raised by tissue culture are free from diseases.

5. Tissue culture coupled with somatic hybridization helps in evolving new cultivar in a short time.

6. Micro propagation facilitates long distance transport of propagation material and long term storage of clonal materials.

7. Tissue culture methods are not viable (male sterile) or not available easily (e.g. banana ) and in plant where propagation by conventional methods are expensive ( e.g. orchid ).

Demerits of In vitro propagation:

1. The cost involved in setting up and maintenance of laboratory is very high and may not justify their use in all the horticultural plants ordinarily.

2. Tissue culture techniques require skill and manpower.

3. Slight infection may damage the entire lot of plants.

4. Some genetic modification (mutation) of the plant may develop with some varieties and culture systems which may alter the quality of the produce.

5. The seedling grown under artificial condition may not survive when placed under environmental condition directly if thing is not given.

Cytoplasmic inheritance

Extranuclear inheritance or cytoplasmic inheritance is the transmission of genes that occur outside the nucleus. It is found in most eukaryotes and is commonly known to occur in cytoplasmic organelles such as mitochondria and chloroplasts or from cellular parasites like viruses or bacteria.

Mitochondria are organelles which function to produce energy as a result of cellular respiration. Chloroplasts are organelles which function to produce sugars via photosynthesis in plants and algae. The genes located in mitochondria and chloroplasts are very important for proper cellular function, yet the genomes replicate independently of the DNA located in the nucleus, which is typically arranged in chromosomes that only replicate one time preceding cellular division. The extranuclear genomes of mitochondria and chloroplasts however replicate independently of cell division. They replicate in response to a cell's increasing energy needs which adjust during that cell's lifespan. Since they replicate independently, genomic recombination of these genomes is rarely found in offspring contrary to nuclear genomes, in which recombination is common. Mitochondrial disease are received from the mother, sperm does not contribute for it.

Types

Three general types of cytoplasmic inheritance exist.

• Vegetative segregation results from random replication and partitioning of cytoplasmic organelles. It occurs with chloroplasts and mitochondria during mitotic cell divisions and results in daughter cells that contain a random sample of the parent cell's organelles. An example of vegetative segregation is with mitochondria of asexually replicating yeast cells.^[5]
• Uniparental inheritance occurs in cytoplasmic genes when only one parent contributes organellar DNA to the offspring. A classic example of uniparental gene transmission is the maternal inheritance of human mitochondria. The mother's mitochondria are transmitted to the offspring at fertilization via the egg. The father's mitochondrial genes are not transmitted to the offspring via the sperm. Very rare cases which require further investigation have been reported of paternal mitochondrial inheritance in humans, in which the father's mitochondrial genome is found in offspring.
• Biparental inheritance occurs in cytoplasmic genes when both parents contribute organellar DNA to the offspring. It may be less common than uniparental cytoplasmic inheritance, and usually occurs in a permissible species only a fraction of the time. An example of biparental mitochondrial inheritance is in the yeast Saccharomyces cerevisiae. When two haploid cells of opposite mating type fuse they can both contribute mitochondria to the resulting diploid offspring.

One of the earliest and best known examples of cytoplasmic inheritance is that discovered by Correns in a variegated variety of the four-o'clock plant Mirabilis jalapa. Variegated plants have some branches which carry normal green leaves, some branches with variegated leaves (mosaic of green and white patches) and some branches which have all white leaves.

Correns discovered that seed produced by flowers carried on the green branches gave progeny which were all normal green.  It made no difference whether the phenotype of the branch which carried the flower used for pollen was green, white or variegated.  Seed taken from white branches likewise gave all white progeny, regardless of the pollen donor phenotype.  These of course died in the seedling stage.  Seeds from flowers on variegated branches gave three kinds of progeny, green, white and variegated, in varying proportions; again regardless of the pollen donor phenotype.  In other words, the phenotype of the progeny always resembled the female parent and the male made no contribution at all to the character.  The effect is seen quite clearly in the difference which Correns found between reciprocal crosses:

 

Transgressive segregation

In genetics, transgressive segregation is the formation of extreme phenotypes, or transgressive phenotypes, observed in segregated hybrid populations compared to phenotypes observed in the parental lines. The appearance of these trangressive (extreme) phenotypes can be either positive or negative in terms of fitness. If both parents' favorable alleles come together, it will result in a hybrid having a higher fitness than the two parents. The hybrid species will show more genetic variation and variation in gene expression than their parents. As a result, the hybrid species will have some traits that are transgressive (extreme) in nature. Transgressive segregation can allow a hybrid species to populate different environments/niches in which the parent species do not reside, or compete in the existing environment with the parental species.

Causes

Genetic

There are many causes for transgressive segregation in hybrids. One cause can be due to recombination of additive alleles. Recombination results in new pairs of alleles at two or more loci. These different pairs of alleles can give rise to new phenotypes since gene expression has been changed at these loci. Another cause can be elevated mutation rate. When mutation rates are high, it is more probable that a mutation will occur and cause an extreme phenotypic change. Reduced developmental stability is another cause for transgressive segregation. Developmental stability refers to the capability of a genotype to go through a constant development of a phenotype in a certain environmental setting. If there is a disturbance due to genetic or environmental factors, the genotype will be more sensitive to phenotypic changes. Another cause arises from the interaction between two alleles of two different genes, also known as the epistatic effect. Epistasis is the event when one allele at a locus prevents an allele at another locus to express its product as if it is masking its effect. Therefore, epistasis can be related to gene over dominance caused by heterozygosity at specific loci. All of these causes lead to the appearance of these extreme phenotypes and creates a hybrid species that will deviate away from the parent species niche and eventually create an individual "hybrid" species.

Environmental

Other than the genetic factors solely causing transgressive segregation, environmental factors can cause genetic factors to take place. Environmental factors that cause transgressive segregation can be influenced by human activity and climate change. Both human activity and climate change have the capability to force species of a specific genome to interact with other species with different genomes.

Testing for transgressive segregation

There are many ways to test if transgressive segregation occurred within a population. One common way to test for transgressive segregation is to use a Dunnett's test. This test looks at whether the hybrid species' performance was different from the control group by looking whether or not the mean of the control group (parent species) differs significantly from mean of the other groups. If there is a difference, that is an indication of transgressive segregation. Another commonly used test is the use of quantitative trait loci (QTL) to assess transgressive segregation. Alleles with QTL that were opposed (either by overdomiance or underdominance) of the parental parent QTL indicate that transgressive segregation occurred. Alleles with QTL that was the same as the predicted parent QTL showed that there was no transgressive segregation.

Importance

Transgressive segregation creates an opportunity for new hybrid species to arise that are more fit than their ancestors. Transgressive segregation can be used to create a species that is more adaptable and resistant in areas where there is environmental stress. Transgressive segregation can be seen as genetic engineering in the way that the goal for each of these events is to create an organism that is more fit than the last.

Drought resistant plants

 

Breeding for drought stress tolerance is the process of breeding plants with the goal of reducing the impact of drought on plant growth.

In nature or crop fields, water is often the most limiting factor for plant growth. If plants do not receive adequate rainfall or irrigation, the resulting drought stress can reduce growth more than all other environmental stresses combined.

Drought can be defined as the absence of rainfall or irrigation for a period of time sufficient to deplete soil moisture and injure plants. Drought stress results when water loss from the plant exceeds the ability of the plant's roots to absorb water and when the plant's water content is reduced enough to interfere with normal plant processes.

Causes of drought

Aside from the moisture content of the soil, environmental conditions of high light intensity, high temperature, low relative humidity and high wind speed will significantly increase plant water loss. The prior environment of a plant also can influence the development of drought stress. A plant that has been drought stressed previously and has recovered may become more drought resistant. Also, a plant that was well-watered prior to drought will usually survive drought better than a continuously drought-stressed plant.

Effects of drought

. Results in osmotic stress.

. Inhibits photosynthesis.

. Increases the concentration of toxic ions (reactive oxygen species) within the cells.

. Loss of water from the cell causing plasmolysis and f i n a l l y cell death.

Tolerance to osmotic stress

The plant cells are sublected to severe osmotic stress due to water deficit. They however, produce certain compounds, collectively referred to as osmoprotectants ( osmolytes, to overcome the osmotic stress. Osmoprotectants are non-toxic compatable solutes and are divided into two groups.

1. Sugar and sugar alcohols e.g. mannitol, sorbitol, pinitol, ononitol, trehalose, fructans.

2. Zwitterionic compounds : These osmoprotectants carry positive and negative charges e.g.proline, glycine betaine.

Strategies to develop water deficit tolerance plants

As explained above osmoprotectants offer good protection to plants against osmotic stress and therefore water deficit. lt is therefore, logical to think of genetic engineering strategies for the increased production of osmoprotectants.

Some progress has been made in this direction. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding key enzymes isolated. In fact, some progress has been made in the development of transgenic plants with high production of osmoprotectants.

Genetic Engineering Drought Tolerant Plants

Although not a crop plant, Arabidopsis has played a vital role in the elucidation of the basic processes underlying stress tolerance, and the knowledge obtained has been transferred to a certain degree to important food plants¹⁰. Many of the genes known to be involved in stress tolerance have been isolated initially in Arabidopsis. The introduction of several stress-inducible genes into plants by genetic engineering has resulted to increased tolerance of transgenics to drought, cold and salinity stresses^{8, 9}. Some examples are reviewed in the following section.

Genetic manipulation of the stress response to abscisic acid (ABA)

ABA levels in the plant greatly increase in response to water stress, resulting in the closure of stomata thereby reducing the level of water loss through transpiration from leaves and activate  stress response genes. The reaction is reversible: once water becomes available again, the level of ABA drops, and stomata re-opens. Increasing the plant’s sensitivity to ABA has therefore been a very important target for improving drought tolerance.

ERA1, a gene identified in Arabidopsis, encodes the ß-subunit of a farnesyl-transferase, and is involved in ABA signaling. Plants lacking ERA1 activity have increased tolerance to drought, however are also severely compromised in yield. In order to have a conditional, reversible down-regulation of ABA, researchers used a drought-inducible promoter to drive the antisense expression of ERA1, in both Arabidopsis and canola plants. Transgenic plants performed significantly better under water stress, with consistently higher yields over conventional varieties. Multi-location trials have confirmed yield increases due to enhanced protection to drought to be 15-25 percent compared to non-transgenic controls.