Crop modelling for GHG emission estimation

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Role of Biotechnology in Combating Diseases of Crops

Badri Khanal[1]

Abstract:

Plant biotechnologist are facing many challenges in development of disease resistant crop varieties. Unlike many insect resistant and herbicide tolerance cultivars, disease resistant has only been success in few crop cultivars. Lot of efforts have been put by conventional breeding, but genetic engineering (GE) techniques are more effective. Some of the common techniques are: recombinant-DNA, RNA-interference, monococol antibody, use of different proteins phytoalexins, resistance genes and RNA silencing. Each of these techniques utilizes a different approach to deliver DNA into the vicinity of chromosomes into which the DNA may then integrate. These techniques has resulted in successful control of many economically important plant diseases, however these are still to be replicated for commercial application in plant disease control.

Introduction:

Diseases of plants are one of the important threats to world agriculture. Agricultural and horticultural crop species are attack by many pathogens “(see Glossary)”, which cause loss of yield significantly. Fungi alone causes more than 70 % of all major crop diseases [1]. Chemicals are mostly used for control of crop diseases. For some diseases, chemical control is very effective for some of the diseases; whereas it is often non-specific in its many associated effects, which kills beneficial organisms along with pathogens. Health, safety and environmental risks are undesirably affected by chemical control. Many cultivars resistant to various diseases are developed using traditional breeding methods. Nevertheless, this process is time-consuming and this means has left little room for improvements due to limited availability of genetic resources. There are numbers of reasons for the availability of limited genetic resources for breeding. Two most important ones are: i) gene pool loss during the domestication and breeding of plants and ii) many of the gene traits which may be beneficial in one plant tissue like seeds and fruits, may be deleterious in other vegetative tissues [1].

Genetic engineering or biotechnology has been identified as one key approach for increasing agricultural production along with reducing losses due to both abiotic and biotic stresses in the field as well as storage [2]. Since past few decades, breeding possibilities have broadened by genetic engineering and gene transfer technologies including mapping of gene and identification of the sequence of genome of model crops and plants. Transcriptomics, proteomics and metabolomics and similar modern technologies are now proved to be important in knowing plant metabolic pathways and the role of important genes associated with their regulation. This facilitates new ideas into the complex metabolic neighborhoods which give rise to a given phenotype and may modify a given pathway by discovery of new target genes. These new gene can then be subject to different metabolic engineering efforts and applications [3].

Genetic engineering is proved as potential tools to develop a cornucopia of beneficial plant traits, particularly an enhanced capability to resist or withstand attack by different plant pathogens. New biotechnological approaches to plant disease control are very important particularly for pathogens that are difficult to manage or control by existing methods [4]

Glossary Candidate genes: The candidate gene approach to conducting genetic association studies focuses on associations between genetic variation within the pre-specified genes of interest and phenotypes or disease states. Genetic Engineering: GE is the modification of genetic composition of an organism by artificial means, often involving the transfer of specific genes or traits, from one organism into a plant (or animal) of an entirely different species. Genetically Modified Organisms (GMOs): It can be defined as organisms (i.e. plants, animals or microorganisms) in which the genetic material (DNA) has been modified in such a way that does not occur naturally by any mating and/or natural recombination. In-vitro selection:  It is method used to screen large number of plants and/ or cells for a certain characteristics, for example, salt tolerance (before growing in the field). Metabolomics: It is the scientific study of the set of different metabolites present within an organism, cell, or tissue. Pathogens: A pathogen or infectious agent is a biological agent that causes illness or disease to its host. The term pathogen is most often used for agents which disrupt the normal physiology of a multicellular animal or plant. Proteomics: This is the large-scale study of the proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they play role as the main components of the physiological metabolic pathways of cells. Transcriptomics: It is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome, under specific conditions or in a specific cell—using high-throughput methods, for example microarray analysis.

Box1: A simplified model of defense illustrating successful transgenic strategies [5] Strategy 1 related with direct interference with pathogenicity (or inhibition of pathogen physiology). Thus 1a involves in constitutive expression of any type of antimicrobial factors and 1b in pathogen-induced expression of one or more genes in the transgenic plant. Strategy 2 is related with the regulation of the natural induced host defenses. 2a involves altering recognition of the pathogen (e.g., R-genes) and 2b involves downstream regulatory pathways (e.g., SAR), and also includes transcription factors. Strategy 3 is related with pathogen mimicry: the manipulation of the plant to prime recognition of a specific pathogen via pathogen derived gene sequences called genetic vaccination.

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 Resistance against disease is the most effective way of controlling disease. However, there are many such pathogens for which no effective sources of disease resistance identified. Only very few genetically modified (GM) disease resistant cultivars have been introduced to commercial agriculture although genetic engineering has been promoted more than two decades to develop disease resistant plants. This is in stark contrast to the development for two other key disciplines of plant protection, (insect pest and weed control), where Bt1 and herbicide-tolerant crops represent over 90% of all GM crops. The answer to this primarily lies in the complexity of the biology of the traits concerned. Economics has also played a role as the investment for transgenic insect and herbicide resistance was considered safe due to established key technologies concerned. The implementation of new products furthermore delayed as a result of moratoria from negative public opinion for GM plants and expense of commercialization [5].

Various types of plant pathogens has different biological traits creating substantial problems in development of GM resistant plants. Firstly, organisms causing disease are highly diverse taxonomically; major being cellular pathogens such as bacteria, fungi and the algal Oomycetes and molecular pathogens such as viruses. They completely differ physiologically and therefore no single gene product can be toxic effect on all of these pathogens. Another trait is, pathogens use two major life strategies i.e.biotrophy and necrotrophy. Biotrophic pathogens act as a sink for the host’s anabolic assimilates, and keep alive to host. Necrotrophic pathogens in other hand consume the tissues of host as invaded. Hemibiotrophs combine both of these strategies [5]

This review paper highlights recent advances in understanding of role of biotechnology in combating diseases of commercial crops and discuss future prospects for use of biotechnology in field of plant pathology.

Techniques of Biotechnology in Plant Diseases control:

Enhanced disease resistance has been achieved and established using several strategies. These are depicted in Fig 1 “(see Box1)”.  Some of the common and successful techniques are tried to comprehend as below.  

Tissue Culture Techniques:

 Almost all the tissue culture techniques are used in plant pathology. Some of the important one are as follows.

i). Protoplast Fusion: Protoplast fusion is one of the methods that can be used to circumvent related problems in introgression genes to develop resistance. By this, factors that contribute to crossing barriers between species can be avoided and viable hybrids ( called Cybrids) have been recovered even between the distantly related species [3].

ii). Chemically induced fusion: Isolated protoplast fusion can occur in the presence of high pH (9-10) and high CA²⁺ but commonly used chemical (fusogen) is polyethyleneghycol(PEG). Addition of PEG causes adhesion of protoplast to neighbors that can be assessed by microscope [3].

Selection for Disease Resistance:

In-vitro Selection has an advantage over other selection system as it allows significant saving of time, space and money. Plants diseases that are damaged through toxins can have disease resistant genotype by cell selection for toxin resistance in cultures and plans regeneration from descendants of the selected cell.  An example is, disease resistant potato plant have been developed through in vitro selectin to control Phytopthera infestans [3].

Recombinant DNA Technology:

 The r-DNA technology has produced a notable success with regard to viral diseases. For example, the transfer and expression of coat protein genes of tobacco mosaic virus(TMV) and tobacco alfalfa mosaic virus (AMV), resulting in protection against or  disease development delay in transgenic plants [3].

RNA-interference Techniques:

RNA interference defined collectively to diverse RNA based processes that results in sequence-specific inhibition of gene expression at any of the transcription, mRNA stability or translational level. The  unifying feature are the production of small RNAs(21-26 nucleotides(nt)) that act as specific determinants for down-regulating gene expression and required for one or more members of Agronaute protein. RNAi triggers the action of dsRNA intermediates, that processed into RNA duplexes of 21-24 nucleotides by Dicer( a ribonuclease III- like enzyme).After that, the small RNA molecules or short interfering RNAs(siRNAs) are incorporated in to a multi-subunit complex known as RNA induced silencing complex(RISC). This  RISC is formed by an endonuclease and  siRNA among other component [3].

Methods to Induce RNAi in Plants

Agroinfiltration:

The injection of Agrobacterium which carries similar DNA constructs into the leaves intracellular spaces for triggering RNA silencing is called as agroinoculation or agroinfiltration [1]

Micro-Bombardment:

A linear or circular template is transferred by micro-bombardment into the nucleus, in this method. Synthetic siRNAs are delivered into plants by biolistic pressure to silence GFP expression. Bombarding cells with particles which are coated with dsRNA, siRNA or DNA that encode hairpin constructs as well as sense or antisense RNA, plays to activation of  the RNAi pathway [1].

Virus Induced Gene Silencing (VIGS):

RNA is induced in plants using modified viruses as RNA silencing triggers. Many RNA and DNA viruses have been altered to act as vectors for gene expression Some viruses, for example,  Tobacco mosaic virus (TMV) and Potato virus X (PVX) can be used for both gene silencing and protein expression [1].

Monococol Antibodies Technique:

In this technique there is the fusion of cancer cells (mycloma cells) with antibody-body producing while blood cell (B- lymphocytes). The resulting hybrid celled is called a hybridoma. This, techniques have been used to produce large quantities of identical antibodies. [3].

Pathogenesis-related (PR) proteins:

PR protein genes are potential source for candidate genes for resistance of fungi. Host plants contribute an enormous number of resistance genes of diseases such as those encoding pathogenesis-re­lated (PR) proteins, which have been used against fungal diseases.  These proteins are induced during hyper­sensitive response (HR) and also during systemic acquired resistance (SAR). Thus, these are thought to have a role in natural defense or plants resistance against pathogens [6].

Antifungal proteins:

Fungal resistance in plants has been enhanced by introduction of the chitinase gene in tobacco and rice. The major constituents of the fungal cell wall (chitin and α-1, 3 glucan) is degraded by chitinase enzyme. Coexpression of chitinase and glucanase genes in tomato and plants confers a higher level of resistance than that imparted by individual genes[6].

Phytoalexins:

Phytoalexins are low molecular weight compounds, that possess antimicrobial properties and that develops plant resistance to fungal and bacterial pathogens. Active oxygen species (AOS) and hydrogen peroxide, play an important role in plant defense responses to infection of pathogen. Transgenic potato plants which express an H2O2-generating fungal gene for glucose oxidase were found to have resistance against both to fungal and bacterial pathogens, mostly in case of verticillium wilt pathogen [6].

Antimicrobial Proteins:

Botrytis cinerea growth was subsequently reduced when expressed in transgenic geranium with an antimicrobial protein with homology to lipid transfer protein. Antimicrobial peptides are synthesized in the laboratory to develop smaller (10-20 amino acids in length) molecules that have gained potency against fungi [6].

Plant ribosome-inactivating proteins and other peptides:

Plant enzymes with 28S rRNA N-glycosidase activity are ribosome –inactivating proteins which depending on their specificity will inactivate conspecific or foreign ri­bosomes, ultimately shutting down protein synthesis. Plant RIPs cause inactivation of foreign ribosomes of distant spe­cies along with other eukaryotes including fungi. A purified RIP from barley crop inhibits growth of several fungi in vitro. Resistance levels improved when RIP was used in addition to either PR2 or PR3[6].

Resistance genes (R-gene):

The plant disease resistance genes(R genes) help for protein encoding to detect pathogens[7].The R-gene products  are cloned from many crops like to­mato, tobacco, rice and several other plant species, which shares one or more similar motifs: a serine or threonine kinase domain, a leucine zipper,  a nucleotide binding site,  or a leucine-rich repeat region, that may contribute to recognition specificity The plant’s resistance R-gene product plays role  as a signaling receptor for the pathogen’s avirulence (Avr) gene product  when there is presence of resistance-regulating fac­tors such as RAR1 and SGT. This leads to a form of cell death termed hypersensitive response [6].

Degradation of Phytotoxic metabolites:

The penetration of fungal pathogens are blocked by plant cell wall and numerous techniques have evolved to overcome this. Production of phytotoxic metabolites of fungi for example, oxalic acid and mycotoxins, have been shown to facilitate infection of host tissues following the cell death. The enzymes expressed in transgenic plants when degrades these compounds provides an opportunity to develop resistance to disease [6].

RNA silencing:

In all plant diseases caused by viral, fungal and bacterial pathoges, RNA-mediated gene silencing is being tried as a reverse tool for targeting gene. Homology-based gene silencing  that is induced by transgenes (i.e. co-suppression), dsRNA and/or antisense RNAhas been demonstrated in many plant pathogenic fungi, along with  Cladosporium fulvum  Magna­porthae oryzae and many other species. hairpin-vector technology have been used in Venturia inaequalis and is able to induce high frequency silencing of a green fluorescent protein (GFP) transgene along with an endogenous trihydroxynaphthalene re­ductase gene (THN) simultaneously[6].

Conclusion and Future Perspectives

Recent advancement in biotechnological tools has made possible to develop and establish techniques to develop plant resistant to many diseases. Mostly resistance against fungi, bacteria, virus and nematode has been developed. Techniques like tissue culture are equally successful as of recombinant DNA techniques. New techniques like RNA interference has been successfully implemented. RNAi techniques are used either by agrobacterium mediated, bombardment or virus mediated method of gene transformation.  Other successful techniques are use of different kinds of proteins, like antifungal proteins, antimicrobial and plant ribosome inactivating. Gene silencing and use of resistance gene are also used extensively in plant disease control.

Though the use of these techniques are found to be successful technically, commercially they are found less established. Only traits like Insect resistance (IR) and herbicides tolerance (HT) has been commercially produced in case of crops. There may be couple of reasons like (a) Complexity of biology of traits concerned, due to differing biology of the various types of pathogens, no single gene product can be toxic to all types of pathogens. (b) High Investment in IR and HT GMO and technology developed quickly and (c) Public opinion against GMO was high in later years.

Thus successful commercialization of disease resistance crops are needed in future. This is only possible with development of GMO crops and creation of scientific base to make people ensure that GMOs are safe for our health and environment.

References

[1] Wani, S.H. et al. (2010) Biotechnology and plant disease control- role of RNA interference,  American J of Plant Sc, 1, 55-68

[2] Mundembe, R. et al. (2012) Genetic engineering of plants for resistance to viruses, genetic engineering - Basics, new applications and responsibilities, Prof. Hugo A. Barrera-Saldaña (Ed.), ISBN: 978-953-307-790-1

[3] Fagwalawa, L.D. et al. (2013) Current issues in plant disease control: biotechnology and plant Disease, Bayero J of pure and App Sc, 6(2), 121-126

[4] Shamin, Md. et al. (2013) Role of biotechnology in plant diseases management: An overview, J of Gen and Env Res Cons, 1(1),215-220

[5] Collinge, D.B. et al. (2008) What are the prospects for genetically engineered, disease resistant plants? Eur J Plant Pathol,121,217–231

[6] Wani, S.H. (2010) Inducing Fungus-Resistance into Plants through Biotechnology, Notulae Scientia Biologicae, 2(2), 14-21

[7] McDowell , J.M. and Woffenden, B.J. (2003) Plant disease resistance genes: recent insights and potential applications, Trends in Biotechnology, Vol.21 No.4, 178-183

[1] Author: Badri Khanal FVSU, GA, USA

Keywords: plant diseases, biotechnology, recombinant DNA, RNA interference, gene silencing

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Genetics and Mendelian Inheritance

      GENETICS:

Genetics is the study of inheritance, the transmission of traits from parent to offspring and the expression of these traits.  The hereditary material, DNA (deoxyribonucleic acid), found in chromosomes is organized into units called genes. Gene is a segment of the DNA molecule, and its location on specific chromosome is called locus. For any genetic character, the offspring will have two genes, one from one parent and another from another parent for that gene character on the homologous chromosome. Genes often exist in at least two alternate forms known as alleles.

The modern genetics started from Gregor Mendel (1822-1884). Mendel selected strains of garden pea with seven clearly contrasting pairs of traits. He studied only one or two of these pairs of traits at a time. Mendel carried out a series of monohybrid crosses, mating individuals that differed in only in one trait. When crossing dominant (Yellow) with recessive (green), F1 generation or first filial generation gave all dominant seed crops and F2 generation gave 3:1 or (Yellow: Green=3:1). Mendel also used test cross to support his hypothesis. A testcross involves mating an individual with an unknown genotype to a homozygous recessive individual.

Mendel also analyzed a series of dihybrid crosses, mating that involved parents that differed in two independent traits. Phenotypically offspring in F1 was with dominant character but genotypically it was heterozygous for both characters. At F2 generation phonotypical ratio was 9:3:3:1 for four possible combinations of traits. This gave to a principle called principle of independent assortment, which states that members of one gene pair segregate independently from other gene pairs during gamete formation.

There are lot of development and findings after Mendel. Whenever the heterozygous phenotype is intermediate, the genes are said to show incomplete dominance. Sometimes when neither allele is dominant, both alleles are expressed independently in the heterozygote, this condition is known as co-dominance. In Incomplete dominance, the heterozygote shows an intermediate phenotype, but in co-dominance both phenotypes are expressed.

Sometimes more than two alleles exist for a given character. For example in fruit fly, a large number of alleles affect the eye color by determining the amount of pigment produced , this is due to multiple alleles. Often a character is controlled by more than one pair of genes, and each allele has an additive effect on the same character, this is called polygenic inheritance. Chromosome are inherited as units, so genes that occur on one chromosome tend to be inherited together, a condition known as linkage. Because the genes are on the same chromosome. They move together through meiosis and fertilization.

Molecular genetics is the greatest achievement in biology.  It has identified the DNA- the genetic material. Hershey and Chase provided information about gene and Watson and Crick described the structure of DNA in 1953. They suggest that nitrogen bases always pair up in a specific pattern, with one purine base( adenine or guanine) hydrogen bonding to one pyrimidine base(thymine or cytosine).

Genes control for the proteins. The Beadle and Tatum found that, for each individual gene identified, only one enzyme was affected. Their hypothesis was later modified to state that each gene codes for one polypeptide chain.

The sequence of bases in the DNA molecule determines the sequence of amino acids in proteins, but the information in the DNA is not used directly. A molecule of messenger RNA (mRNA) is made as a complimentary copy of a gene, a proportion of one strand of the double helix. The process of RNA synthesis from DNA is called transcription. The enzyme RNA polymerase is responsible for attaching nucleotide together in the sequence of specified by DNA. The molecule of mRNA represents a gene, and each gene in an organism is represented by a different mRNA molecule. Each mRNA contains in its sequence of bases information that will be translated into sequence of amino acids that constitute a specific protein. mRNA has introns (removing segments) and Extron (expressed segments). Each of three consecutive bases of mRNA molecule constitutes a code word, or codon, that specifies a particular amino acid. This genetic code contains a total of 64 codons, with 61 of them coding for amino acids. Three codons do not code for amino acids but act to signal termination (UAA, UAG, and UGA). One codon (AUG) codes for amino acid methionine, acts as a signal to start translation.

The translation of the mRNA codons into an amino acid sequence occurs on ribosomes in the cytoplasm of the cell. In addition to mRNA, two other RNA, rRNA and tRNA function in translation. Ribosomal RNA (rRNA) joins with number of proteins to form ribosomes, the site of protein synthesis. Transfer RNA (tRNA) molecules are transport molecules that carry specific amino acids to a ribosome and align the amino acids to form a polypeptide chain.  In addition to three types of RNA involved in protein synthesis, two additional classes of RNA molecule are also involved i.e. microRNA (miRNA) and small interfering RNA (siRNA).

Mutations are the changes in DNA. Once a DNA sequence has changed DNA replication copies the altered sequence and passes it along to future generations of that cell line. The smallest mutation are called point mutation. A mutation in which a small segment of the DNA is list is known as a deletion and a mutation in which a segment is added is called an insertion. The insertion modify the mRNA reading frame, known as frame shift mutation. Mutations can occur in any cell. If they occur in cells that do not lead to gametes, they are called somatic mutations.

Recombinant DNA entails the introduction of genes from one organism into the DNA of a second organism. The formation of recombinant DNA makes use of proteins called restriction enzymes to cut a gene from its normal location. Transferring the isolated gene to another species requires the use of a vector, usually a plasmid, which is small, circular strand of DNA that also occurs in bacterial cells. The ends of the plasmid join to the ends of the gene, with the result being a recombinant DNA molecule that is transferred to a cell in another organism.

     MENDELIAN INHERITANCE

The concept of heredity started from pangenesis to blending hypothesis of inheritance, according to which, the factors that dictate hereditary traits can blend together from generation to generation. However, the pioneer work of Gregor Mendel would prove instrumental in refuting this viewpoint.

Mendel work started with hybridization concept. When two distinct individuals with different characteristics are bred, or crossed, to each other- a process called a hybridization experiment-their offspring are referred to as hybrids. Mendel chose pea plant as his experimental organism. There were couple of reason for choosing pea. It was easy for Mendel to carry out self-fertilization or cross-fertilization experiments and they were available in several varieties in which a character existed in two distinct variants. There are many laws derived from Mendel.

Law of segregation

Along with qualitative experimentation, Mendel also conducted empirical study (quantitative study). Mendel conducted single-factor crosses in which he followed the variants for single character. The results of his single factor crosses showed that the dominant trait was always observed in the F1 generation and displayed a 3:1 ratio in the F2 generation. Based on the results of his single-factor crosses, Mendel proposed three key ideas regarding inheritance.

i.                    Traits may be dominant or recessive.

ii.                  Genes are passed unaltered from generation to generation.

iii.                Two copies of a given gene segregate (or separate) from each other during transmission from parent to offspring. This third idea is known as law of segregation.

A Punnett square can be used to deduce the outcome of crosses. Mendel’s 3:1 phenotypic ratio is consistent with the law of segregation. Each of the seven character that Mendel studied is influenced by different genetic materials, known as gene.

Law of Independent Assortment:

Mendel investigated the pattern of inheritance by conducting two-factor crosses and proposed the law of independent assortment, which states that two different genes randomly assort their alleles during the formation of haploid cells. A Punnett square can be used to predict the outcome of two factor crosses.  The multiplication method and forked-line method can be used to predict the outcome of crosses involving three or more genes.

Chromosome Theory of Inheritance:

The chromosome Theory of inheritance describes how the transmission of chromosome can explain Mendel’s law. Mendel’s law of segregation is explained by the separation of homologs during meiosis. Mendel’s law of independent assortment is explained by the random alignment of different chromosomes during metaphase of Meiosis. The chromosome theory of inheritance is based on a few fundamental principles.

i.                    Chromosome contains the genetic material that transmitted from parent to offspring and from cell to cell.

ii.                  Chromosome are replicated and passed along, generation after generation, from parent to offspring. They are also passed from cell to cell during development of a multicellular organism. Each type of chromosome retains its individuality during cell division and gamete formation.

iii.                The nuclei of most eukaryotic cells contain chromosomes that are found in homologous pairs-they are diploid. One member of each pair is inherited from the mother, the other from the father. At Meiosis, one of the two members of each pair segregates into the other daughter nucleus. Gametes contain one set of chromosome-they are haploid.

iv.                 During the formation of haploid cells, different types of (nonhomologous) chromosomes segregate independently of each other.

v.                   Each parent contributes one set of chromosomes to its offspring. The material and parental sets of homologous chromosomes are functionally equivalent: each set carries a full complement of genes.

Studying Inheritance Pattern in Humans

Human inheritance pattern are determined by analyzing family trees known as pedigrees analysis. This is commonly used to determine the inheritance pattern of human genetic disease.

Conclusion: Thus heredity and Inheritance has been defined by Mendel and passed through many generation and have many advancement like recombinant DNA techniques, nowadays.

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