Plant Transformation in Biotechnology by MEJAST Journal

Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 103-123, July-September 2019

Plant Transformation in Biotechnology Gulshan Atiq1, Nasrullah1, Sonia kanwal1, Muhammad Asif Raheem1 & Rana Khalid Iqbal*1 1

*Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya University, Multan-60800, Pakistan.

Article Received: 21 February 2019

Article Accepted: 15 July 2019

Article Published: 30 August 2019

ABSTRACT

Plant transformation permits the introduction of the gene of interest for producing novel transgenic plants. "When a gene from one species is moved or relocated to another species by using recombinant DNA technology are called genetically modified organisms. Genetic engineering is one way to modify the plants by selecting for desired traits. Genetically modified organisms have foreign genes derive from not only plant source but also from bacteria, viruses, fungi, insects and animals. Transformation is the introduction and addition of the desired gene in plant for the generation of transgenic plant. Plant transformation is a challenging process for scientists. DNA transfer by artificial methods like DNA transfer through physical method is micro-injection, biolistic or gene gun methods, electroporation, silica carbide, microinjection, lipofection, microinjection. DNA also transfers by chemical methods. In natural method like in biological method, Agrobacterium-mediated transfer, Rhizobium, virus-mediated and planta transformation. Plant transformation involves three phases target gene, Plant tissues, vector for successful transformation. Plant transformation offers a momentous means to gain desire character or trait of interest. Plant transformation technique benefit agriculturalists to grow more crops in less area of land. And give more yield at less cost consumption. Plant transformation technology is familiarizing many crops with our desired characters. This review explains the natural method of plant transformation and benefits of transgenic plant. Keywords: Gene expression, Transgenic, Particle bombardment, Agrobacterium tumefaciens-mediated Transformation, Origin of replication, Genetic engineering, Electro transformation, Hygromycin, Transgenic Crops.

INTRODUCTION Transformation states to the introduction of a foreign gene of choice into the genome of the plant to the generation of transgenic plant. Plant transformation is the incorporation of the foreign DNA into a plant genome (nuclear or cytoplasmic). (Wilson and Coverley 2017) Plant transformation involves three phases target gene, Plant tissues, vector for successful transformation. Plant transformation offers a momentous means to gain desire character or trait of interest. (GarcĂa-Sogo et al. 2012) Plant transformation technology is familiarizing many crops with our desired characters. (Nanjundan et al. 2017) Due to plant transformation, breeding of plants occurs, and genetic diversity of plant products with beneficial and our desire character. From conventional breeding, we try to produce highly beneficial character and remove the undesirable character from plants. (Zhuoya Chen et al. 2018) It is also a source of creating diverse genotypes within a species, to develop various genes for beneficial phenotypes, and to eradicate unnecessary sequences from the transformation process. As genetically modified (GM) crops become available, produces and used so it is necessary to safeguard that plants are safe for food, feed, and environmental. Plant transformation methods depend on the method of delivery, incorporation, and expression of genes in plant cells (D. Wang et al. 2017) and nucleus. For successful transfer of desire gene, we need the regulatory sequence of a gene which take part in the expression of a gene, like Promotor, the gene of interest and terminator.

Figure1: Structure of complementary sequence required promoter, transgene with the desired character, Terminator and antibody as a selection marker. (Olieric et al. 2010)

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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 103-123, July-September 2019

The biotechnological tool for the generation of genetically modified plants has generated a range of cultivars with excellent traits. (Nadal et al. 2012) (Lei et al. 2017) Here, we review the different methods of plant transformation. Plant tissue culture and genome editing methods need optimization and interpretation for productivity and minimization of time in culture transporting transgenes to cells. (Abhishek et al. 2017) Of the various methods, microinjection and Agrobacterium tumefaciens are a popular option for delivering exogen into a plant. The acceptance of biotechnology has a very helpful for farm income resulting mostly from a combination of improved productivity and efficiency of gains. (Baranova et al. 2017) Both biosafety and benefits are important to maintain. These are GMOs crops commercially available corn, papaya, alfalfa, (Zhu et al. 2013) sugar beets, summer squash, apple, Potato, Soybeans, Cotton, Canola, sweet corn, pea, rice, wheat, tomatoes, banana, Hawaiian papaya.

Figure 2: Overview of various advantages of gene auditing in the field of agriculture plants to produce transgenic plants. (Nanjundan et al. 2017) (Nanjundan et al. 2017) Plant transformation improved food qualities, struggle to diseases, droughts, (D. Wang et al. 2017) salt, (Fan et al. 2019) herbicides, weeds, insects, fungicides and pesticides and also increase the nutrition value and or productivity of crops. (Konopka-Postupolska et al. 2015) (Fan et al. 2019) Nowadays, it is possible due to new scientific methodologies to insert genes from not only plant source but also from bacteria, viruses, fungi, insects and animals. Genetic engineering in plant transformation is producing more variety and diversity in the plant which is beneficial to survive in hard condition. (Czolkoss et al. 2016) It is one of the greatest powerful and beneficial tool for genome editing in numerous plant species. It increases outstanding facilities in fruit production and enhancement through the edition of exogen which is a source of healthy and disease-free planting material, (Deng et al. 2018) increased efficiency, improvement in fruit quality, (Prabhu et al. 2017) enhancing

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shelf-life, maintenance of biodiversity and Variety, accessibility of biopesticides, biofertilizers, bioinsecticides, (L. Kang et al. 2013) bioherbicides, etc. (Qi et al. 2018) Plant transformation becomes the fastest technology to produce more valuable plants. (Gordon-Kamm et al. 2016) Plants are the basic source of many food webs they are primary producers who control the whole web of the food chain. They are also a source of pharmaceutical products. (Teklehaymanot 2017) So that the important traits and characteristics of the plant types in a network have a bulky effect on the wildlife, animals human and even other organisms that depends on plants. METHODS There are many methods of a direct and indirect way of plant transformation. PHYSICAL METHODS OF PLANT TRANSFORMATION Genetic engineering is used to change the sequence of the genome of plant species. Modification in the construction and composition of genetic material in a living organism. (K. Wang et al. 2016) There are two methods for genetic transformation indirect or direct transformation. (Yao et al. 2018) DIRECT METHODS Direct methods are micro-injection, biolistic or gene gun methods, electroporation, silica carbide, microinjection, lipofection, (Keymer et al. 2017) microinjection, sonification, calcium phosphate method, Transfer of DNA by use of polyethylene glycol. Indirect methods are Agrobacterium-mediated transfer, rhizobium mediated transfer. Still, the desire for higher transformation efficiency has encouraged work on not only improving several methods but also in discovering novel methods. The most published techniques for gene transfer into plant cells are DNA transfer by artificial methods like DNA transfer in physical methods are micro-injection, biolistic or gene gun methods, electroporation, silica carbide, microinjection, lipofection, microinjection. (Narusaka and Narusaka 2004) DNA also transfer by chemicals methods. In natural method like in biological method, Agrobacterium-mediated transfer, Rhizobium, virus-mediated. (Westwood et al. 2013)

Figure 3: There is a list of different methods of the direct and indirect method of plant transformation. (Keshavareddy, Kumar, and S. Ramu 2018) 105 | P a g e

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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 103-123, July-September 2019

IN PLANTA TRANSFORMATION In planta transformation meristem transformation, floral dip transformation method, pollen transformation mostly uses as an advanced technology to produce transgenic plants. Nowadays, mostly all work of gene transformation depends on particle bombardment of DNA or Agrobacterium-mediated transformation for gene transfer to harvest transgenic plants in many plant species. Because these methods are more accurate and give maximum production. (Zhang, Wang, and Meng 2011) DNA TRANSFER BY ARTIFICIAL METHOD By PHYSICAL METHOD MICROINJECTION The microinjection is the procedure in which transporting foreign DNA into the cell through the microneedle and direct DNA inject in the nucleus under microscope. Glass micropipette straightforwardly penetrate in the nucleus of the plant. They have problems because it burst the cell wall of cells. The vacuole of a plant is big which contain 90% water so it also burst the vacuole and then many toxic releases which kill the cell. BIOLISTIC OR MICROPROJECTILE OR PARTICLE BOMBARDMENT Biolistic or microprojectiles for DNA transfer the most common technique for direct transformation is microprojectile or particle bombardment. The expression vector with the target gene (s) is precipitated into tungsten or gold particles which are then transferred to the plant nucleus directly.

Figure 4: Overview of the steps of plant transformation through gene gun bombardment method: (a) separation of the protoplast from leaf, (b) the bombardment of the DNA coated with golden particles into protoplast, (c) transfer to the solid media, (d) Transfer of the transgenic plant in a greenhouse. (Narusaka and Narusaka 2004)

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But some DNA particle not enter into a callus. In a small percentage of the DNA will integrate into the target and show expression. (Ismagul et al. 2018)The main drawback of this technique is that often-multiple copies of the transgene insert. It is also necessary to have an in vitro regeneration system in place. Biolistic or particle bombardment practices enhanced microparticles to transport to the nucleus. The gene gun is a device that accurately integrates DNA into target cells. An explosive force fires the bullet towards the target cells. Not all but some of the coated particles cross the cell wall. It has more advantages not laborious but expensive due to the use of gold and tungsten particles. (Ismagul et al. 2018) (Zhang, Wang, and Meng 2011) PROTOPLAST ELECTROPORATION METHOD In this process, high electric shocks use to generate small holes in the cell membrane of the plant which allow the DNA to enter from these holes. (S. Kang, Kim, and Kim 2015) These holes create for a short time less than 5 seconds. This process needs protoplast formation before transfer DNA because large molecule can't cross the cell membrane of the plant. (Furuhata et al. 2019) Then after passing the pores of cell membrane DNA become the part of the chromosome of the plant nucleus. But it is time-consuming to the production of protoplast. (Potter and Heller 2013) (Mini et al. 2018) SILICON CARBIDE WHISKER-MEDIATED TRANSFORMATION Silicon carbide fibers make them skilled in piercing cells membrane lacking to damage and breakage to the cell membrane. (Nahar et al. 2013) (Mini et al. 2018) In this process, silicon carbide fibers use to break plant cells to produce holes. Silicon carbide fibers and desired plant cells are mixed in a flask. The mechanical force generated by the vortex drive the fibers into the cell. By means of this belonging, produce a lot of novel plants because it is an easy, cheap, and speedy procedure. (Chumakov 2007) ULTRASOUND-MEDIATED TRANSFORMATION The limitations of the beforehand defined methods for transformation interested the search for more efficient, easier and safer techniques for DNA exogenous integration to vegetable cells are ultrasonic waves mediated transformation. (David et al. 2017) Through sonication process sound waves of high-frequency produce which allow the suspension of DNA to cross the nuclear membrane and become the part of uncles. Only ultrasonic wave with more than 20 kilohertz use. (Sivanandhan et al. 2015) LASER MICROBEAMS In modern technologies, one of the new technology is the use of laser beams to the introduction of desired genetic material into plant production. Because laser rays use in every field of life nowadays. Through lasers, holes produce for less than 5 seconds and DNA cross from the cell membrane. It is a quick easy and accurate and time-saving process. But need a lot of care because it also damages the biological material of the cell. It is necessary to control the energy level and pulse rate. So, it is necessary to have a satisfactory laser system (like nitrogen lasers, excimer pumped dye lasers, or titaniumâ&#x20AC;&#x201C;sapphire lasers) that can be in the production of transgenic plants and organisms. UV laser microbeam cell fusion has been induced selectively integration and DNA was introduced into isolated chloroplasts This is a very expensive method due to the use of costly apparatus to consent to focus a laser beam on

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the correct position. It also needs a lot of care because laser radiation can damage biological material, so it is necessary to restrict the beam through a channel and control the energy and pulsation duration with high exactness and reproducibility. (Yao et al. 2018) CHEMICAL METHODS There are two methods of chemicals DNA transformation. DNA TRANSFER BY CALCIUM PHOSPHATE In this method of gene transformation, a proper solution of DNA and of calcium chloride (CaCl) and phosphate(P) is made. Then DNA becomes precipitation with the calcium phosphate. Then precipitation of DNA transfer to the target tissue or cell. (Keshavareddy, Kumar, and S. Ramu 2018) TRANSFER OF DNA BY USE OF POLYETHYLENE GLYCOL (PEG) The first step of gene transfer by polyethylene glycol (PEG) is protoplast preparation in which the removal of cell wall through enzymatic digestion occurs. The protoplast of desire plant treated with polyethylene glycol (PEG) with the solution of DNA. Protoplasts are beneficial fragments for genetic engineering for the reason that they are more totipotent, which have quick ability to grow. In this technique Protoplasts of plants are cultivated in the presence of optimized conditions that are suitable for growing daughter plant with the desired character with the help of callus, shoots, and roots, and redevelop into whole plants PEG induces reversible permeabilization of the plasma membrane. It has very Low transformation efficiency (only 0.0004%). Problematic to redevelop laborious and costly. Protoplasts are â&#x20AC;&#x17E;Naked Plant Cellsâ&#x20AC;&#x; Plant cells with their Cell wall removed. Which cause Serious Drawbacks Very Tedious, Cultivar limitations, Not effective, On-fertile plants. (Masani et al. 2014) (Li et al. 2017) DNA TRANSFER THROUGH NATURAL OR BIOLOGICAL METHODS Natural transformation is a widespread biological belonging common by different strains of bacterial species which take part in the development of producing genetic diversity through horizontal or straight gene transfer. (Dalia et al. 2015) There are many methods to transfer gene through natural methods. (Seitz et al. 2014) CLONING In genetic engineering, cloning and recombinant DNA technology play an important role as natural methods of gene transformation. Cloning is the process in which creation of new mixtures of the DNA sequence which are not present together in nature. (Yakhin et al. 2017) Plant transformation involves three phases target gene, Plant tissues, vector for successful transformation. Major steps of plant transformation through cloning are the isolation of donor(foreign) DNA isolation of plasmid vector DNA. Through restriction enzymes cut the desire genes; restriction enzymes are the scissors of molecular genetic engineering, Joining of the desired gene into a right vector (for construction of recombinant DNA) than the introduction of vector into an organism or plant. (Guo et al. 2015) Plant transformation means the addition of genetic material in nuclear or cytoplasmic. Because foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse plant species which is maintained by the regulatory sequence of the plant. (Wen et al. 2018)

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There are five steps of transformation; 1.Chose the desired gene and cut it with restriction enzymes 2.Chose DNA vector with the regulatory sequence 3.Insert the desired gene into a vector 4.Transform vector into the host cell 5.Separation of genetically modified cells and purification (Cohen 2013) VECTORS To introduce genetic material which has our desire character into plant cells, Agrobacterium relies on a source of carrier vector which is a loop of DNA called a plasmid. Once got the selectable marker gene, then the idea to choose a vector to become easy. A vector is a piece of DNA which is used to transfer DNA from one organism to another, in this case of Agrobacterium it is also playing the role as a vector to transfer desire gene from Agrobacterium to a plant cell. (N. Wang et al. 2018) Vector is one of the most important elements in recombinant DNA (rDNA) technology and in gene cloning. A vector has a replicon (replication sites) that allows it to replicate in host cells. It also has several marker genes like antibiotic resistance (ampicillin, tetracycline) markers. (Mirouze et al. 2018) There are types of the vector which use in genetic engineering or cloning of plant transformation. Virus-associated vector (VAC) Yeast associated vector (YAC) Bacteria associated vector (BAC) PLASMID VECTORS It contains multiple cloning sites which cut by different restriction enzymes and an origin of replication. Introduction of selectable markers like antibiotic resistance (ampicillin, tetracycline) markers. (Vafaee et al. 2014) pBR 322 the most widely used cloning vector. A circular plasmid contains a gene regulating sequence used for studying gene regulation and gene transfer in cells. (Lu et al. 2013) The complication of plasmid construction solved through the development of the â&#x20AC;&#x17E;binary vectorâ&#x20AC;&#x; system in modern technology nowadays. (Himmelbach et al. 2007) EXPRESSION VECTORS Cloned vectors which have exogen next insertion in vivo translate into RNA then transcript into protein. (Kemski, Stevens, and Rappleye 2014) Foreign genes expression may be failed due to the absence of plant transcriptional machinery particularly like promoter sequences which recognize only specific foreign gene. Because in nature specific promotor use for specific gene transcription. (Olieric et al. 2010) Representative plant expression vectors contain any Agrobacterium origin of replication so that the plasmid can be manipulated in Agrobacterium. (Goadsby, Kurth, and Pressman 2016)

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1. A bacterial antibiotic resistance gene, like ampicillin. For the selection of bacteria that contain the plasmid which has desire genes. 2. Constitutive promoters have high gene expression in all types of cells and are used to produce proteins of interest. The 35S CaMV gene consults a high transgenic expression in most cell types. A plant promoter, often Cauliflower Mosaic Virus (CaMV) 35S promoter. (Zhu et al. 2013) Mostly Constitutive 35S promoter is used because it found in the whole plant in every time. (Nadal et al. 2012) (Huang et al. 2018) SELECTABLE MARKERS AND REPORTER GENES Selectable Markers are antibiotics and herbicides. Reporter genes are like GUS (β-glucuronidase gene), (Ho et al. 2019) LUC (luciferase gene), GFP (Green Fluorescent Protein) (Du et al. 2018) CFP (Cyan fluorescent protein), YFP (Yellow Fluorescent Protein). (García-Sogo et al. 2012) (Banerjee et al. 2013) SELECTABLE MARKERS Antibiotics are neomycin, kanamycin, geneticin (G418), paromomycin, neomycin phosphotransferases II, Hygromycin – hygromycin phosphotransferase. (De Vicente et al. 2015) Selectable markers herbicides phosphinothricin (Rustgi et al. 2013) For the construction of the vector by means of typical lab techniques (restriction digests, ligation) the vectors then introduced it in A. tumefaciens. Agrobacterium-mediated transformation depends on bacteria strains, plasmids structure, and optimum culture conditions and selectable markers, Agrobacterium tumefaciens and A. tumefaciens isogenic strains. AGROBACTERIUM-MEDIATED TRANSFORMATION TO GENERATE A TRANSGENIC PLANT Agrobacterium tumefaciens is a direct technique that is best known as one of nature‟s natural agents in producing genetically modified organisms (GMOs). (Yu et al. 2016) (Kimura, Cutler, and Isobe 2015) (Brenna et al. 2014) It attacks only dicotyledonous plants (and not easy on monocots plants). The capability to cause crown gall disease is connected with the presence of the Ti (tumor-inducing) plasmid. (Song et al. 2019) So, Agrobacterium effects only injured plant because the injured plant produces phenolic compounds like acetosyringone. So, it activates the making of the copy of T-DNA in bacteria. Scientist study crown gall tumor of the plant than they found that tumor cell has not any tumor causing hormone to grow callus (auxin and cytokine) and it have same genes as Agrobacterium .So they found the reason that tumor caused by Agrobacterium. (Subramoni et al. 2014) So, the tumor cell has both chromosomal DNA and also plasmid DNA of Agrobacterium bacteria. (Agrobacterium tumefaciens) or hairy root (A. rhizogenic) that are able to produce long hairy roots. A. tumefaciens, which influences crown gall disease in plants in the root and shoot. Agrobacterium tumefaciens and Agrobacterium rhizogenes are pathogenic soil born bacteria that contain a Ti (tumor-inducing) or Ri (root inducing) plasmid. (Mysore et al. 2012) A minor portion of this plasmid, the T-DNA is transferred from the bacteria to the plant and participates steadily into the plant genome. (Mysore et al. 2012)By removing disease cause genes, they it become a vector. Mostly A. tumefaciens and the Ti-plasmid used because due to more mutual and large knowledge. Scientist reported that A. tumefaciens have its plasmid, which contains a series of different eleven vir genes (virG, virE, virA, 110 | P a g e

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virJ,virB, virC, and virD) and transfer DNA (T-DNA) counties. (GĹ&#x201A;owacka et al. 2016) The T-DNA regions have two tumor producing groups of genes contain oncogenic genes Phenolic compounds and sugars projected from the plant root induces vir gene expression so ,the first is responsible for producing opines, so providing a carbon and nitrogen source for A. tumefaciens,, the second group is responsible for the production of auxin as indole-3-acetic acid (IAA) and cytokinin which produce callus-like tumor growth. In other words, this hormone (auxin & cytokinin) are the reasons for abnormal growth. (Subramoni et al. 2014)These are amino acids but they are different in the structure of the plantâ&#x20AC;&#x;s amino acids. So, the plant produces amino acids due to different size unable to use them and thus A. tumefaciens use these amino acids. These specific opines cause the plant to produce tumor and provide amino acid for the growth of bacteria and also these amino acids produce by plant but use these amino acids to bacteria. Crown gall disease is not usually harmful, but it reduces plant strength and crop yield, and crown galls attract other phytopathogens or pests. (Korzun et al. 2014) In 1983 researchers established that they can add new genes of desire with beneficial traits by Agrobacterium tumefaciens. Scientist uses Ti-plasmid to transfer the new desired gene into a plant cell to produce a transgenic plant nowadays. (Hayta et al. 2018) (Hayta et al. 2018)When we transfer gene in A. tumefaciens, we need a regulatory sequence of a gene like Promotor, terminator, complete component of DNA vector, (Benoit et al. 2016) many restrictions site, (Many restriction sites are present in one place so, that to cut vector more than one different enzyme),35s constitutive promoter. (It recovers from cauliflower mosaic virus and very strong promotor), the origin of replication and also Promotor of the host.

Figure 5: Plasmid vector of the Agrobacterium tumefaciens bacteria which contain two equal sequences of left and right border, auxin, and cytokinin, opine, Origen of replication, vir genes, and opine catabolism. Transgene insert between left and right border by disarmed the T-DNA which cause a tumor. But transgene contains our gene of interest. (Hayta et al. 2018) MOLECULAR MECHANISMS Natural tumor-inducing (Ti) plasmid, holding both the virulence (Vir) genes and the transferred DNA (T-DNA), first deactivated the T-DNA with the genes of interest.(Gohlke and Deeken 2014) The limitation of the big size of

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vector cover by a binary vector. The binary vector system has two 25 bp of repeat sequences as Left and Right Border (LB, RB). (Mini et al. 2018) The 25 bp repeats flanking the T DNA mediates the transfer of T-DNA from bacterium to host cell. (Lee et al. 2009) The LB and RB sequences are very similar in nucleotide sequence and gene of interest insert between the left and right border of the plasmid.(Huang et al. 2018) And insert it into Agrobacterium. The first step is the interaction between Agrobacterium and the addition of the bacterium to the surface of the plant cell. A plant cell becomes susceptible to Agrobacterium when it is wounded. The wounded cells release phenolic compounds, such as acetosyringone, that activate the vir-region of the bacterial plasmid. Then wounded plants produce sap with acidic pH (5.0 to 5.8). Then activation of vir genes (infectious genes) occurs and VirG binds to the vir box enhancer elements in the promoters of the virA, virB, virC, virD, virE and virG operons and upregulating transcription. Then production T-stand with the help of virD1 and virD2 occur. Then the formation of T-complex occurs in which one T-strand DNA molecule, one VirD2 protein, and around 600 VirE2 proteins present. VirE2 is responsible for the transformation of the gene of interest. (Nicolia et al. 2017) Transport of the T-complex into the host cell than into the nucleus occurs through a type IV secretion system.(Kado 2014) (Huang et al. 2018) THE “HAIRY ROOT” DISORDER ENCOURAGED BY AGROBACTERIUM RHIZOGENES The process of Agrobacterium rhizogenes is similar to Agrobacterium tumefaciens but it produces hairy roots due to the presence of auxin (IAA). The “hairy root” pattern, characterized by the appearance of adventitious roots at the wound location of infected plants. A. rhizogenes, as the associated Agrobacterium tumefaciens species with the large plasmid, are well known for the dimensions to transfer part of their DNA (Ri, root-inducing) to the plant genome through a natural infection process, foremost to abnormal roots (hairy roots). (Franche et al. 1998) The Ri T-DNA appears to lack a gene for cytokinin biosynthesis, which probably accounts for the “Rooty” morphology of A. rhizogenes-induced tumors. (Sobczak et al. 2012) ADVANTAGES OF AGROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION The genus Agrobacterium takes a wide host range. Agrobacterium tumefaciens-mediated transformation is a controlling tool for inverse genetics and functional genomic examination in an extensive diversity of plants. (Utami, Hariyanto, and Manuhara 2018) This technique shows high expression of desire protein about 40%. Different Agrobacterium strain-specific host range is unknown. (T. Wang et al. 2017) Nowadays it is possible in monocot plants to transformed with desire gene and create transgenic plant from monocots. (Gordon-Kamm et al. 2016) Under lab environments, T-DNA can be transported to yeast, bacteria other fungi, and even animal and human cells. Agrobacterium is now used as a vector for monocots and dicots plant to increase the productivity of commercial crops. (Zhongying Chen et al. 2014) Agrobacterium and the gene gun are practicable procedures to plant gene transfer efficiently and more accurately. Through Agrobacterium transformation, the introduction and addition of a gene of interest into the genome of the plant and confirm for the generation of transgenic plants. (Czolkoss et al. 2016) (D. Wang et al. 2017)Mostly Agrobacterium is selected as a method of choice for plant transformation. Because it is a natural process. (Cairns et al. 2017) And due to the low copy number of the transgene needs, a higher proportion of stable transformants; larger DNA segments can be transferred; more

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time-efficient; more accurate and increase in crop productivity with desire and beneficial trait. Agrobacterium-mediated transformation approved to produce transgenic crops which are improved by the foreign gene of interest such as transgenic corn, transgenic soybean, and cereal crops to improve its products all over the world (Abhishek et al. 2017) (Ahmed et al. 2018). It plays a role in the research field and new production. Although gene transformation in monocots plants possible, but only one or two genes of interest introduce into monocotyledon per species of plants. But more work on monocotyledon plants is done in most academic laboratories. (Allah et al. 2019) For monocotyledons, there have been continuously struggling for progress and development. Since legume crops revolution to that intermediated by Agrobacterium tumefaciens, in addition to modifications in tissue culture procedures and selection approaches. (Nonaka et al. 2017) Agrobacterium transformation system depends on optimizing the introductory examines, including of explant source, growth media, antibiotics and Agrobacterium strains response in the callus of monocots. (Hiei, Ishida, and Komari 2014) Natural Agrobacterium transformation is not affected by highly fragmented and damaged DNA and it is safe to transfer method. (Nielsen et al. 2013) Multiple genome editing in genetic engineering by natural transformation developed in Escherichia coli and has been extensively successful to gain more than one desire and beneficial character in one transgenic plant. (Satoh et al. 2016) PLANT TISSUE CULTURE FOR TRANSFORMATION Plants can also be regenerated from a single cell of plant this capability is called “totipotency. Many, but not all plant cells are totipotent. Tissue culture is an important element for creating transgenic plants. (K. Wang et al. 2016) PLANT TISSUE CULTURE Plant tissue culture is the process of producing artificially daughter plants with the same genotype of their parent plants under the sterile condition and in the presence of micro-molecule and macromolecules. This process is also used in the cloning of plant. Regeneration of cells tissues, organs, and plants from isolated cells or tissues under a controlled, aseptic and optimum condition like PH, Temperature, humidity and in the laboratory to produce plants of the desired character. (Ho et al. 2019) Especially the plant which have not seeded like sugarcane and banana. Callus formation occurs by Intermediate ratio of auxin and cytokinin. Shoot formation occurs by High auxin. Root formation occurs by Low auxin. Produce callus → Transform callus → Stimulate shooting by cytokinin and stimulate rooting by auxin. Through callus production in laboratory clones of desire plants produce in control and sterilized conditions which have similar genetic makeup. (Nonaka et al. 2017) TYPES OF TISSUE CULTURE Cell suspension culture Solid media culture Immature embryo culture

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Pollen culture Ovule culture Single cell suspension (Rustgi et al. 2013) IN PLANTA TRANSFORMATION Although tissue culture is successful plant regeneration method, tissue culture has more steps to produce a plant of the desired character. To overcome this issue, it is necessary to develop transformation that greatly eradicates the prevailing tissue culture steps which are laborious. In planta transformation methods provide such an opportunity. Methods that involve the delivery of transgenes in the form of naked DNA directly with the intact of plants flower parts are called planta transformation methods. (Song et al. 2019) These methods eliminate tissue culture steps, and also simple quick easy time-saving procedure and not more laborious to produce a lot of plants with the desired character. The first plant was Arabidopsis thaliana that was successfully transformed by planta transformation. In modern technologies by planta transformation directly genetic material transfer into the flowering stage, pollen, ovule, and new growing cells. Several planta methods including vacuum infiltration transformation of germinating seeds and floral dipping, (Abhishek et al. 2017) floral spray, pollen transformation, and embryo transformation. There are more outcomes through planta transformation as compare to vacuum infiltration and floral dip methods. COMBINATION OF AGROBACTERIUM AND IN PLANTA TRANSFORMATION METHODS The newest Agrobacterium-based transformation technique in Arabidopsis and some other important plants crops are "floral dip" method. In this, In planta method of transformation, the step of tissue culture stage is eradicated. (Chumakov 2007) As a replacement for the plant is dipped into a suspension of Agrobacterium at the stage when it has the maximum number of unopened flowery outgrowth or bud clusters. (Zhang, Wang, and Meng 2011) The suspension also includes a cleansing agent because it reduces the surface tension, and also some sugar. The bacteria adjustment occurs into the developing flowers and transfers the T-DNA. (Schouten et al. 2017) Then it results in the development of transgenic seeds that can be implanted out on medium with a selectable marker. The plants that survive on the selectable medium becomes transplanted and can be studies or screened. (Kemski, Stevens, and Rappleye 2014) POLLEN TRANSFORMATION For the production of transgenic plant injection of naked DNA into pollen is known as pollen transformation. (Rustgi et al. 2013) PLANT TRANSFORMATION BY CRISPR CRISPR (clustered regularly interspaced short palindromic repeats) /Cas9 from 2013 used as a novel technology to addition, deletion, and insertion of desired traits such as CRISPR/Cas9. (Benoit et al. 2016) It is also used for the transformation of more than one gene at a time in one plant. (L. Chen et al. 2018) Through CRISPR gene editing at a specific target location very precisely and accurately. Cas9 is an enzyme which cut the DNA at very specific

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points with the help of RNA guider genes. It is a very time-saving, quick and reliable method of gene transfer in the plant to produce a genetic plant with the desired character. (Nishihara et al. 2018) (Aman et al. 2018) CONCLUSION Transformation discusses the genetic alteration of a cell resulting from the direct acceptance, incorporation, and expression of exogenous genetic material. (Kemski, Stevens, and Rappleye 2014) Genetic material crosses the cell membrane and becomes the part of the nucleus and chromosomes. The purpose of plant transformation is that plants are the primary source of the food web, without proper production of plants the peace of the world may be destroyed. Plants are a source of food, medicines, energy, shelter and also maintain and clean the environment. (Sundaram et al. 2013) Through plant transformation, new varieties and diversities create in less period of growth with more efficiency. (Schuman, Allmann, and Baldwin 2015) Nowadays through planta transformation, floral dip, floral spray and CRISPR technology used to get a more accurate outcome. (Curtin et al. 2018) More achievements will come from a combination of several prevailing methods, with the most suitable, cheaper, time-saving, quick, precisely and most expected popular gene transfer method having the availability, and simplicity for a large number of plant species nowadays and further continuously work done to develop new amazing technique. (Maenni and Yardley, n.d.) REFERENCES 1. Abhishek, Alok, Tanushri Kaul, Reeva Singh, Arunava Pattanayak, Pranjal Yadava, Pawan K. Agrawal, and Ishwar Singh. 2017. “Advances in Maize Transformation Technologies and Development of Transgenic Maize.” Frontiers in Plant Science 7 (January): 1–12. https://doi.org/10.3389/fpls.2016.01949. 2. Ahmed, Rana Imtiaz, Anming Ding, Minmin Xie, and Yingzhen Kong. 2018. “Progress in Optimization of Agrobacterium-Mediated Transformation in Sorghum (Sorghum Bicolor).” International Journal of Molecular Sciences 19 (10). https://doi.org/10.3390/ijms19102983. 3. Allah, Elsayed Fathi Abd_, Abeer Hashem, Ahmed Al-Harrasi, Tapan Kumar Mohanta, Abdul Latif Khan, and Dhananjay Yadav. 2019. “Genomic and Evolutionary Aspects of Chloroplast TRNA in Monocot Plants.” BMC Plant Biology 19 (1): 1–24. https://doi.org/10.1186/s12870-018-1625-6. 4. Aman, Rashid, Shouwei Ding, Fatimah Aljedaani, Muhammad Zuhaib Khan, Haroon Butt, Zahir Ali, Ahmed Mahas, and Magdy Mahfouz. 2018. “RNA Virus Interference via CRISPR/Cas13a System in Plants.” Genome Biology 19 (1): 1–9. https://doi.org/10.1186/s13059-017-1381-1. 5. Banerjee, Joydeep, Dipak Kumar Sahoo, Nrisingha Dey, Robert L. Houtz, and Indu Bhushan Maiti. 2013. “An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis Thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants.” PLoS ONE 8 (11). https://doi.org/10.1371/journal.pone.0079622. 6. Baranova, Ekaterina N., Irina N. Berdichevets, Dmitry G. Shpakovski, Andrey V. Aralov, Svetlana V. Kubrak, Ivan Yu. Slovokhotov, Svetlana G. Spivak, et al. 2017. “A Key Enzyme of Animal Steroidogenesis Can Function

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