Adama Science and Technology University

Plant biotechnology has existed ever since the beginning of civilization, resulting in remarkable enhancements in plant cultivation through crop domestication, breeding and selection. As enabling and developing technology, plant tissue culture techniques have been developed and used as an innovative means to contribute to plant breeders in crop improvement perspectives. These innovative approaches can be used to either increase the speed and/or the efficiency of breeding process in order to improve the accessibility of existing germplasm and to create new genetic variation for crop improvement.

These include eradication of pathogens from planting materials using meristem culture, elimination of sexual incompatibility by embryo rescue technique, somatic hybridization using protoplast technology, production of haploids via anther culture and most importantly the induction of new genetic variability through somaclonal variation and selection of desirable agronomic traits. Thus, plant tissue culture technology has a vast potential to produce plants of superior quality and selection of useful variants in well adapted high yielding genotypes with better disease resistance and stress tolerance capacities.

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Another innovative area is genetic engineering which facilitate the farming of crops with multiple durable resistances to pests and diseases, improved nutritional values by genetically modifying plants. Hence, crops should be engineered to meet the demands and needs of consumers and the genetic base of crop production should be preserved and widen by an integration of biotechnology tools in conventional breeding.

Plant biotechnology has been around since the start of civilization (Figure 1). It started around 13000 years ago, followed by crop domestication, considerably influencing the morphology and genomes of the plants involved (Meyer et al.

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, 2012).

Plant biotechnology deals with cell and tissue culture, genetic transformation, gene cloning, DNA markers, and other molecular approaches. Genetic modifications are most of the time done at organ, tissue, cell, protoplast, and molecular levels using biotechnological techniques (Bello and Kadams, 2003). These innovative techniques are used, which improves the conventional methods for efficient and precise plant breeding (Kang et al., 2007). It has been possible to produce crop varieties with higher yields, pest and disease resistance, salt, drought, and tolerance using conventional breeding methods Kadams (2000). Though they have limitations such as linkage problem, longer time span for crop maturity, incompatibility, mapping of specific genes and sterility problems are among others. It was only in the past 50 years that techniques became available that permitted the integration of genes into plants from other species through transformation. The last couple of decades have observed the advancement of many tools that permit the precise editing of the genome, providing an opportunity to rapidly integrate new traits and genes into elite cultivars (Baltes and Voytas, 2015).

Plant biotechnology is a growing technology and fast evolving from the laboratory to bring improvement in agricultural activity (Cardwell, 1992). The two major areas of plant biotechnology that deals with crop improvements are: plant tissue culture and genetic engineering (Gautheret, 1985). Plant tissue culture is the in vitro sterile culture of cells, tissues, organs or whole plant under controlled nutritional and environmental surroundings (Thorpe, 2007) often to produce the clones of plants. The plants are derived from stem, root or leaf tissues and the technology mostly helps in mass production of wanted crop varieties (Bachraz, 1998).

Plant tissue culture method holds substantial potential for creation, preservation, and use of genetic variability for improvement of an extensive variety of crop plants. Among these are somaclonal variation, meristem culture for generation of pathogen-free plant material, embryo culture, anther culture and protoplast culture and fusion have direct applications in crop improvement (Kang et al., 2007). Plant tissue culture technique has made it possible to induce somaclonal variation by varying chemical and physical mutagens (Hare et al., 2016). This method enables to expose natural variability of the crops so to use their potential for improvement (Jain, 2001). Somaclonal variation is the variation amid callus-derived plants, an effective method for widening the genetic base, mainly in the vegetatively propagated species (Evans & Sharp, 1986). Using in vitro selection, many million cells/protoplasts can be screened against several biotic and abiotic stress factors in a petri dish, which is more efficient as compared with field screening that needs more time and space (Kaur et al., 2001).

Somaclonal variation in cultured plant cells and tissue culture has been used as a selection method for development of herbicide resistant crops (Kandasamy et al., 2002). Selection for resistance to herbicides inhibiting the enzyme acetolactate synthase (ALS) site of action using callus (maize), microspores and protoplasts (rape seed) and plant cell suspension cultures (maize) have been possible (Chaleff, 1988). Attaining, maintaining and mass propagation of particular pathogen-free plants has been possible by meristem culture technique. Meristem culture has been used effectively in the elimination of viruses from many plants. For example, from potato (Miassar et al., 2011; Mori et al., 1969), dahlia, strawberry, sugar cane and many other plants (Mori et al., 1969). Lizarraga et al. (1986) reported that plant pathogens such as nematodes, fungi, bacteria and viruses, can spread from diseased plants to healthy plants. Though, not all plant cells become diseased; the meristematic tissues are at times disease-free. It is possible to isolate non-infected plants by in vitro meristem culture techniques and grow them into healthy plants.

Embryo culture is one type of in vitro culture technique that is probably of the highest value to breeders and used in solving real-world problems (Dunwell, 1986). Some of its applications reported by Bridgen (1994) are:

  • Dormancy in seeds can be bypassed which will reduce breeding cycle by using embryo culture. Dormancy may be caused by endogenous inhibitors, light requirements, low temperatures, dry storage requirements, and embryo immaturity (Yeung et al., 1981). Reduction of influences by the above factors will help embryos to germinate and grow quickly in less period of time.
  • Embryo rescue plays a vital role in modern plant breeding through the development of many interspecific and intergeneric crop hybrids. Many studies (Ramming, 1990; Sharma and Gill, 1983; Williams and De Lautour, 1980) have reported embryos arising from interspecific hybrids and intergeneric hybrids.
  • Embryo culture has been very useful in determining seed viability. Tukey (1944) reported the use of embryo culture for testing the viability of peach seed.

The use of anther/pollen culture has been evident in the development of haploids (sporophytes with gametophytic chromosome number) and double haploid (DH) (are haploids that have undergone chromosome duplication) (Germana, 2011). He also reported that the use of haploid and DH as a potent breeding tool involves the availability of dependable tissue culture protocols that can overcome numerous methodology difficulties, such as low frequencies of embryo induction, albinism, plant regeneration, plant survival and the genotype and season dependent response, in order to improve the regeneration efficiency in an extensive range of genotype.

Non-haploid (diploid, triploid, tetraploid, pentaploid, hexaploid) embryos and plantlets have also been obtained from anther culture of various genotypes (Dunwell, 2010; D’Amato, 1977). Triploids regenerated from anther culture have been stated in Datura innoxia (Sunderland, 1974) and several other fruit species (Germana, 2006).

Protoplast culture has advantage over other plant tissue culture methods in that any treatment applied has a direct effect on the protoplast (Filippone el al., 1992). They also observed that, the characteristic of protoplast that makes it suitable for genetic manipulation for crop improvement include:

  1. Protoplast, irrespective of their source tend to fuse, when brought together into close contact with each other
  2. Protoplast can uptake DNA, chloroplast, nuclei, virus or even whole bacterium.
  3. Protoplast like its container, the cell, have huge advantage of totipotency

In plants protoplast culture technique has been produced from protoplast in varied crop species such as cassava, yam, sweet potato etc. with induced disease resistance, pest resistance and increased yield (Ng, 1989). Fusion of protoplasts from unrelated plant species have been used to fuse traits from otherwise sexually incompatible species. Further, cybrids (cytoplasmic hybrids) and organelle recombinants, not likely through conventional methods, can also be developed (Hinnisdaels et al., 1988).

Resistance to some diseases like potato leaf roll virus, PVX, and PVY have been incorporated into Solanum tuberosum from Solanum brevidens and Solanum phureja through protoplast fusion (Rokka et al., 1994). Similarly, resistance against Phoma lingam disease in Brassica species (Sjödin & Glimelius, 1989) and tristeza virus (CTV) in citrus (Mendes et al., 2001) were established using protoplast fusion.

High rate loss of plant species has called for germplasm conservation method which is increasingly becoming an essential activity (Filho et al., 2005). The purpose of germplasm conservation is to make sure the ready accessibility of valuable germplasms. In seed propagated crops, seed is widely used for conservation of germplasm using conventional methods (Garcia-Gonzales et al., 2010). Though, numerous plant species produce seeds that do not survive drying and freezing conditions. Therefore, they cannot be kept for long time and in this case tissue culture can be used for plant conservation in vegetative state, often under environments of slow growth (Lambardi et al., 2002) or for cryopreservation (Halmagyi and Pinker, 2006) for advantages of relatively low costs and reduced space usage (Tyagi et al., 2007).

The conservation of plant parts in vitro has a number of advantages over in vivo conservation, e.g., in vitro techniques allow conservation of plant species endangered of being extinct. In vitro storage of vegetativly propagated plants has big advantage when it comes to storage space and time used, and sterile plants that cannot be reproduced generatively can be maintained in vitro. Complete plants have been effectively regenerated from tissues cryopreserved at −196◦C in liquid N2 in many crops for several months to years (Ford et al., 2000; Panis et al., 2001).

Genetic engineering is the process of adding new DNA to an organism. The purpose is to add one or more new traits that are not previously found in that organism (Primrose and Twyman, 2013). Creation of genetically engineered/modified or transgenic organisms requires recombinant DNA. According to (Singh and Singh, 2014), recombinant DNA is a combination of DNA from different organisms or different locations in a given genome that would not normally be found in nature.

Applications for genetic engineering in crop improvement are growing as researchers work on to find the locations and functions of specific genes in the DNA sequence of various organisms (Getahun Bekana, 2017). Once each gene is classified, development of systems to modify these genes is followed to produce disease and pest-resistant crops (Acquaah, 2007).

In its wide sense genetic engineering comprises a deliberate genetic manipulation of species (crops) to produce superior new ones (Mohammed el al., 1977). It is concerned with transfer of transgene from one organism to another in the process called transgenesis. This is possible since every living cell is “totipotent” and has the capability to uptake a purified DNA molecule, virus or bacterium (Fitch, 1990). He further reported that genetic transformation is attained by successful:

  • uptake of exogenous DNA
  • expression of encoded genetic information
  • its assimilation and integration into the host genome. where in, the imported DNA along with the hosts DNA will undergo normal replication. transcription and translation

Biological-based transformation systems resemble natural substances so rely on natural process.

Viral based transformation: offers very high expression temporarily in plant cells. some of plant viral vectors that are commonly used are potato virus x (Ruiz et al.,1998), barely stripe mosaic virus (Holzberg et al., 2002) and geminivirus vector (Kjemtrup et al., 1998). The most effective way to incorporate cDNA derived viral RNA into plants is through direct inoculation (Ahlquist et al.,1984).

Generally, it is the DNA virus that has received most consideration as potential gene vectors. Davies and Stanley (1989) pointed out that DNA is more stable and far less susceptible to error while in replication. Additionally, DNA viruses have a nuclear phase whereas RNA is usually cytoplasmic and are highly likely to mutate during replication. Using viruses as gene vector has one main disadvantage which is they have narrow host range of plant species and cell types.

Agrobacterium mediated transformation: Agrobacterium tumefaciens is a Gram-negative soil pathogen and naturally infects the injured spots in various dicotyledonous plant species (Mubeen et al., 2016). Under ordinary conditions the infection’s consequence is the formation of crown gall tumors (Smith and Towsend, 1907). The bacterium transfers a part of its DNA known as transfer DNA or mobile DNA segment (T-DNA) into the nucleus of infected cells along with virulence proteins coding DNA. It is then stably integrated into the host genome and transcribed (Nester et al., 1984; Binns and Thomashow, 1988).

The bacterium genetically transforms several dicots, some monocots and gymnosperms (DeCleene and DeLey, 1976). Agrobacterium has been used for gene transfer in crops such as cowpea, field beans, tobacco, cotton, mango, aerial yam (Table 1) (IAPTC, 1990).

Non-biological methods use chemical or physical DNA uptake induction into protoplasts, intact cells or whole plants. These methods are potentially suitable for all species and all plant materials and do not require specialized vector systems.

Micro injection: is the injection of DNA into individual cells using a specialized instrument of fine capillary tubes and high power microscope for observation. Micro injection has been successfully used to obtain stable transformation of crops such as tobacco, alfalfa (Reich et al., 1986).

Biolistic: is a method of genetic modification involving the shooting of small particles of metal particles coated with DNA or messenger RNA directly into cells or tissues at high velocity (Bello and Kadams, 2003). It is a technique for gene transfer into a different crop species (Sanford, 1990).

Electroporation: is a method of introducing DNA from one organism into a cell of another using an electric pulse of high voltage. The electric field generates holes in the plasma membrane thus allowing DNA to be taken up by the cell. It has been used for transformation in barley caryopses (Ahokas, 1989).

The potential of genetic engineering has now been widely recognized and extensively implemented in the plant breeding of crops in the following areas:

Wally and Punja (2010) have reported the approaches for increasing disease resistance in transgenic plants such as expressing R-genes, detoxification of pathogen virulence factors, antimicrobial genes have been developed.

Genetic engineering allows for introduction of resistance genes (R-genes which are only useful against biotrophic pathogens) from unrelated plant species, which often remain functional in the new host plant (Collinge et al., 2008). The R-gene Rxo1 from maize was successfully introduced into rice and conferred resistance against bacterial streak disease caused by Xanthomonas oryzae (Zhao et al., 2005). Additional examples of this strategy involve the R-gene RCT1 from Medicago truncatula that was expressed in alfalfa and conferred resistance to Colletotrichum trifolii (Yang et al., 2008).

Detoxification of virulence factors is another mechanism which will hinder the ability of the pathogen to degrade polysaccharides inside the plant cell wall. Polygalacturonase-inhibitory proteins (PGIPs) aid to impede the activity of the fungal cell wall-degrading polygalacturonases (De Lorenzo et al., 2001). Overexpression of PGIPs in transgenic plants has positively reduced disease symptoms due to Botrytis cinerea (Joubert et al., 2007) and Bipolaris sorokininia (Janni et al., 2008).

Another frequently used method for engineering fungal and bacterial resistance in plants is through the expression of antimicrobial peptides. They have a variety of functions, including degrading fungal cell walls, membranes, RNA or are involved in generating secondary metabolites or increasing cell physical barriers. For instance, chitinase overexpression has been moderately successful in increasing tolerance to diseases caused by both biotrophic and necrotrophic fungal pathogens (Punja, 2001). Combined expression of a chitinase and β-1, 3 glucanase often resulted in a synergistic effect, further enhancing the resistance in several plant species (Zhu et al., 1994).

Herbicides normally affect processes like photosynthesis or biosynthesis of essential amino acids (Richard et al., 2006). Some of the approaches using genetic engineering for herbicide resistance in plants reported by Richard et al. (2006) are:

  • The bar gene has been used to engineer glufosinate (non-selective pro-herbicide) tolerance in many crops including corn (Gordon-Kamm et al., 1990), rice (Christou et al., 1991), wheat (Vasil et al., 1992), sugar beet (D’Halluin et al., 1992), oilseed rape and alfalfa (Cobb, 1992) and other crops.
  • Comai et al. (1985) reported the use transformed 5- enolpyruvylshikimate phosphate (EPSP) enzyme genes for glyphosate tolerance development in plants. They created glyphosate resistant transgenic tobacco with a modified EPSP synthase determined by the aroA gene of Salmonella typhimurium in which an amino acid replacement of a proline for serine produced a reduced affinity for glyphosate devoid of disturbing the kinetics of the enzyme. The glyphosate oxidoreductase (GOX) gene copied from Pseudomonas sp. Strain LBr has been used together with the cp4 gene to confer glyphosate resistance in a many commercially accessible crops including soybeans, corn, canola, and cotton (Franz et al., 1997).
  • The gene, tfdA (from bacterium Ralstonia europhus), was shown to encode a 2,4-D dioxygenase, which degrades 2,4-D (the first growth regulator herbicide to be made). Transformation of tobacco with tfdA gene conferred 10-fold resistance to the herbicide related to non-transformed plants (Streber and Willmitzer, 1989). The tfdA gene in cotton, produced plants that were tolerant to three times the field application rate of 2,4-D used in wheat, corn, sorghum and pasture crops (Bayley et al., 1992).

Insect-resistant crops have genes from the soil bacterium Bacillus thuringiensis (Bt). The protein formed in the plant by the Bt gene is toxic to a specific group of insects: for example, European corn borer or corn rootworm but not to mammals (Byrne, 2014). The Bravo Model is an updated version of the model originally proposed by Knowles and Ellar (1987) which shows the three steps of Bt toxin to affect insects (Pigott and Ellar, 2007) (Figure 2).

Genes conferring resistance to insects have been inserted into a wide array of crop plants including maize, cotton, potato, tobacco, rice, broccoli, lettuce, walnuts, apples, alfalfa, and

soybean (Wilson et al., 1992). Insects growth can be hindered if they consume high amount of numerous insecticidal proteins of plant source, for instance lectins, amylase inhibitors, and protease inhibitors (Jongsma & Boulter, 1997).

Based on the need by the end users, producers and agro- based industries, crops can be targeted to improve in their contents including: carbohydrates, proteins, oils, vitamins, iron, and amino acids (Sharma et al., 2002). Genetic engineering has been playing major role in improving nutritional qualities. Such as:

  • The carotene gene has been moved from daphoddils to rice grains (Golden Rice) for increasing Beta-carotene content in grains and for prevention of blindness in children by overcoming lack of vitamin A (Ye et al., 2000).
  • Antisense sterol desaturase gene inserted into sunflower developed high oleic acid containing types of the plant (Naranjo and Vicente, 2008).
  • Transgenic rice with higher levels of iron has been produced using genes involved in the making of an iron binding protein that enables iron accessibility in human diet (Goto et al.,1999).
  • Transgenic alterations have also been used to alter the ratio of amylose to amylopectin in starch (McLaren, 1998).
  • Improving digestibility of food by reducing the amounts of oligosaccharides such as raffinose and stachyose (which most of the time pass undigested through the stomach and upper intestine), and reduction in the amount of flatulence during digestion (McLaren, 1998).
  • transgenic technology can also be used to remove anti-nutritional factors (Kaufman et al., 1998).

Innovative approaches of cellular and molecular biotechnology have emerged as a valuable adjunct to supplement and complement the conventional methods for precise and efficient breeding of a wide variety of crop plants. Biotechnological approaches are now increasingly being used for creation, conservation, characterization, and utilization of genetic variability for germplasm enhancement. Generation of pathogen-free plant material has been possible using meristem culture. Techniques of anther culture, somaclonal variation, embryo culture, and protoplast fusion are being exploited to obtain incremental improvements in plants. Cryopreservation of germplasm at the cellular/tissue/organ level in liquid nitrogen at −196◦C is highly rewarding for establishing germplasm banks, especially in the case of vegetatively propagated and rare, endangered plant species.

Several genes, particularly for insect and disease resistance, have been isolated and cloned from different organisms using various techniques. Several vector and non-vector methods have been developed for genetic transformation of plants. Agrobacterium and ‘particle gun’ methods have become routine and are now being widely used for efficient production of transgenic plants. In fact, transgenesis has emerged as an additional tool to carry out single-gene breeding or transgenic breeding of crop plants. Unlike conventional breeding, only the cloned genes of agronomic importance are introduced without co-transfer of other, undesirable genes from the donor. The transformation method provides access to a larger gene pool as the gene(s) can be acquired from viruses, bacteria, fungi, insects, animals, human beings, unrelated plants, and even from chemical synthesis in the laboratory. Genetically modified foods, such as, golden rice with enhanced vitamin A have been produced and Changing protein levels, composition of fatty acids, vitamins and amino acids are being more and more given focus for value addition.

Generally, plant biotechnology has made tremendous contributions toward increasing crop yields; improving nutritional quality; enabling crops to be raised under adverse conditions; increasing shelf life of plants and developing resistance to pests, herbicides, insect pests and diseases.

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