Plant Genetic Transformation and Gene Silencing

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The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena

The Role of Green Fluorescent Protein (GFP) in Transgenic Plants to Reduce Gene Silencing Phenomena

flank the transgene may also minimize silencing (Spiker and Thompson,1996). The results expected that the transgenic soybeans accumulating the modified glycinin V3–1 to have a higher level of methionine than nontransformants (Fig. 4). Up to date, gene silencing was seen as a problem for plant genetic transformation, as it prevented reliable expression of a desired phenotype within transgenic plants (Taylor and Fauquet, 2002). However, with increasing knowledge of the mechanisms underlying this phenomena, and realization that it can be utilized to down-regulate native genes within the plants, and it will become a powerful tool in future transgenic applications (Vance and Vaucheret, 2001; Lessard et al,, 2002).
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The roles of bacterial and host plant factors in Agrobacterium mediated genetic transformation

The roles of bacterial and host plant factors in Agrobacterium mediated genetic transformation

also involved in the host defense against Agrobacterium infection; specifically, small interfering RNAs (siRNAs) specific for the T-DNA sequence are generated by the host plant during Agrobacterium infection (Dunoyer et al., 2006), and plants deficient in siRNA pathways are hypersusceptible to Agrobacterium. Conversely, the miRNA pathway seems to be required for disease development (Dunoyer et al., 2006). This silencing response to the invading bacterial DNA likely contributes to the limited and relatively early timing of high levels of transient T-DNA expression observed in most transformations experiments: this time period may be required for the host to mount the defense reaction, after which RNA silencing takes effect, leading to reduction in T-DNA gene expression. Sup- porting this idea, expression of RNA silencing suppressors encoded by diverse plant viruses, such as P19 of Tomato bushy stunt virus, HcPro of Potato virus Y, or V2 of Tomato yellow leaf curl virus, during Agrobacterium infection, significantly increased the level and duration of transient T-DNA expression (Voinnet et al., 2003; Zrachya et al., 2007). Although, by analogy to plant viruses most of which encode silencing suppressors (Qu and Morris, 2005), evolution of an RNA silencing suppressor in Agrobacterium would make biological sense, such suppressor has not been identified to date. While a decrease of siRNAs corresponding to the T-DNA sequences levels was observed in developing tumors, it was at- tributed to modifications in hormonal status of tumor tissues, rather than to a putative Agrobacterium silencing suppressor (Dunoyer et al., 2006).
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Genetic transformation of major cereal crops

Genetic transformation of major cereal crops

The two most efficient and widely used methods for genetic transformation are the delivery systems mediated by Agrobacte- rium and particle bombardment. Agrobacterium tumefaciens is a soil bacterium known to cause crown gall disease in plants. The unique feature of this pathogen is its ability to deliver and integrate part of its own DNA (T-DNA) into the genome of the infected cells. However, the limitation of this system lies in the susceptibility of the host plant to Agrobacterium and is, hence, highly dependent on plant and genotype. In contrast, transformation via bombardment is a physical delivery method based on the rapid acceleration of DNA-coated metal microprojectiles into target cells. Therefore, it can transform a broader spectrum of plant species and genotypes as long as the target tissue is regenerable. Since its development in mid-1980s (Klein et al., 1987), biolistic bombardment has been used to transform almost all major cereal crops, including maize, rice, wheat, barley, and sorghum. The disadvantages of the bi- olistic delivery are its complex integration patterns and high-copy numbers of transgenes in the transgenic plants, often leading to gene silencing and unstable inheritance (Dai et al., 2001; Hu et al., 2003; Shou et al., 2004; Travella et al., 2005). Currently, if a plant could be transformed by both methods, the Agrobacterium method would be chosen to obtain high numbers of single or low- copy transgene insertion. Nevertheless, both methods deliver DNA into the genome at random positions. The Agrobacterium- mediated method also generates transgenic events that carry DNA fragments outside the T-DNA region (vector backbones) (Shou et al., 2004), which is an undesired effect. To prevent extra DNA segments from being inserted into the genome, some researchers used purified DNA fragments that carry only the cassette with the gene of interest for bombardment (Breitler et al., 2002; Loc et al., 2002), whereas others devised multiple T-DNA borders (Kuraya et al., 2004) or launched T-DNA from the Agrobacterium chromo- some (Oltmanns et al., 2010) to reduce the backbone insertion
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A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica

A virus-induced gene silencing (VIGS) system for functional genomics in the parasitic plant Striga hermonthica

Genetic engineering through cross species RNA inter- ference (RNAi) technology offers great promise in para- sitic plant management [12-19]. However, its applicability in S. hermonthica management has been constrained by lack of methods to deliver the silencing molecules and the lack of candidate genes to target [20]. The recent report on horizontal gene transfer from Sorghum bicolor to S. hermonthica has increased prospects of delivering the silencing RNA molecules from cereal hosts to S. hermonthica [21]. This suggests the possibility of RNAi in some monocots which have been reported to be recalcitrant to transformation. Host-derived resistance using RNAi dependent on efficient delivery of siRNAs from the host to the parasite in order to determine if the gene causes an alteration in the parasites’ phenotype, reviewed in [15]. An alternative approach to determine if a gene has a function on the parasite would be to develop a high throughput genetic transformation protocol for Striga. These two approaches present challenges, as transform- ation of grasses (especially rice, maize wheat and sor- ghum) is recalcitrant [22-26] and no protocols exist for Striga transformation yet.
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RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

Host gene silencing -hair pin RNAi (HGS-hpRNAi) is reported as more stable gene si- lencing method in plants [13]. This method can be employed to increase fungal and bacterial disease resistance by changing the gene expression against pathogens through genetic engineering in the host plant. Flagellin (a bacterial component) can stimulate the expression of specific miRNA to increase disease resistance signaling pathway in Arabidopsis [14]. Over-expression of a gene AtFAAH in Arabidopsis, responsible for fatty acid (N-acylethanolamines) me- tabolism can alter phyto-hormone signals through intersecting with plant defense pathways to increase resistance against bacterial pathogens [15]. In rice, RNAi can knockdown OsSSI2 (OsSSI2-kd), meant for fatty acid desaturase activity that cause increase re- sistance against bacterial pathogen (Xanthomonas ory- zae pv. Oryzae) of leaf blight and blast fungus (Mag- naporthe grisea) [16]. siRNAs proved effective against the crown gall disease in Arabidopsis, Nicotiana and Lycopersicum species caused by a pathogen Agrobacte- rium tumefaciens by transformation of inverted repeats of this pathogen genes ipt and iaaM to encode pre- cursors of biosynthesis for auxin and cytokinin [17]. Gene silencing can be obtained by host-induced gene (Avra10), that results in limited fungal disease attach in wheat (Triticum aestivum) and barley (Hordeum vulgare) through a transient gene expression resistant to RNAi because of silent point mutations. This sug- gests that the transfer of RNA from host plant to fungal pathogen Blumeria graminis, leads to RNAi-based plant protection against these pathogens [17].
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Silencing the HaAK Gene by Transgenic Plant-Mediated RNAi Impairs Larval Growth of Helicoverpa armigera

Silencing the HaAK Gene by Transgenic Plant-Mediated RNAi Impairs Larval Growth of Helicoverpa armigera

the transcript levels of HaAK were distinctly sup- pressed in the third-instar larvae, we did not observe any significant lethal phenotypes. It seemed that the third-instar larvae had developed resistance to the transgenic plants. There are several possible reasons for this observation. First, the transgenic plants may not have produced sufficient dsRNA due to the ex- pression of heterologous genes that were derived from different plant backgrounds [30,31]. Dicer-like enzymes in plants could have decreased the expres- sion levels of the intact dsRNA in the transgenic lines. We found that the higher the concentrations of intact dsRNA that were produced in transgenic plants, the greater the RNAi inhibitory effects. Second, third-instar larvae of this species are large in size. Arabidopsis plants are short-lived, which is adverse for the long-term observational experiments. In our feeding experiments, the first-instar larvae had higher mortality rates. This feeding regime did not seem to meet the need of insects considering their growth rates. There may be a time-lag between gene silencing and larval death. Moreover, we do not know whether there are energy metabolic pathways that may com- pensate for the RNAi targeting HaAK.
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Plants as Digital Things: The Global Circulation of Future Breeding Options and Their Storage in Gene Banks

Plants as Digital Things: The Global Circulation of Future Breeding Options and Their Storage in Gene Banks

The impact of bioinformatics on plant collections is a research issue on its own. Most of the literature on the conjunction of computer tech- nology and life science in general (Beaulieu 2004; Thacker 2004; Howe et al. 2008) or ‘bio-banks’ in particular (Fujurama and Fortun 1996; Gott- weis and Petersen 2008) have focused on the human and on those gene banks that hold “digitalized genotypic (genetic) and phenotypic (envi- ronmental and lifestyle) information” (Ratto and Beaulieu 2007, 176) which have been turned from “well documented, local tissue-sample col- lections to large-scale bioinformatics resources with a national or supra- national scope” (Ratto and Beaulieu 2007, 175) over the last decades. The largest and most prominent collections of that kind are the “Nucleotide Sequence Archive”, produced and maintained by the European Bioin- formatic Institute, established in 1980 in Heidelberg, the “GenBank” hosted by the US-National Institute of Health, opened in 1982, and the “DNA Data Bank” of Japan released in 1986. These bio-banks are under- stood to be repositories that store biological samples, mostly human, for research purposes, chiefly in the field of genomics or personalized medi- cine. Along with the establishment of those large bio-banks STS and His- tory of Science have also gained interest in the practices of collecting and in the role that collections play within the production of scientific knowledge (Bowker 2000a; Strasser 2011). By the impact of molecular
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Manipulating plant RNA silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems

Manipulating plant RNA silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems

have limited its application in stable transgenic plants. The p19-induced leaf alterations are probably caused by its interference with the plant endogenous siRNA and miRNA pathways [30, 38]. While p19 can bind many miR- NAs, it does not bind miR168 that targets AGO1 mRNA, thus reducing the accumulation of AGO1 [30, 39]. In this study, we observed reduced miR168 levels in type I trans- genic plants expressing p19 together with the CRISPR/ Cas9 cassette, but the expression levels recovered in type II and type III plants (Fig. 5g), suggesting a dosage-dependent regulation of miR168 by p19 during the interaction between virus and host. The two CRISPR ele- ments, sgRNA and Cas9, were also regulated by p19 in a dosage-dependent manner. In type I transgenic plants with low p19 expression, showing a WT leaf phenotype, sgRNA and Cas9 transcript levels were lower than in plants transformed with the same CRISPR/Cas9 con- structs without p19 (Fig. 5f, g). As the expression of p19 increased, the leaf phenotype became more accentuated (types II & III) and the transcript levels of sgRNA and Cas9 became higher (Fig. 3c). Considering that plants are continuously evolving counter measures to fight against viral suppressors by producing endogenous VSR recogni- tion receptors [23], we think that the downregulation of miR168 and CRISPR/Cas9 transcripts observed in p19- low-expressing plants may be due to the triggering of the plant defense machinery while high p19 expression even- tually overwhelms the defense response resulting in re- duced transgene silencing.
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Gene Silencing and Homology-Dependent Gene Silencing in Arabidopsis: Genetic Modifiers and DNA Methylation

Gene Silencing and Homology-Dependent Gene Silencing in Arabidopsis: Genetic Modifiers and DNA Methylation

ddm1 C-insert double homozygote there was a consider- The evidence for the involvement of methylation in able increase in CHS mRNA size and abundance com- epimutation, cosuppression, transinactivation, paramu- pared with the C-insert homozygotes, but little intact tation, and other silencing and HDG silencing events CHS mRNA and only low levels of anthocyanin were is strong but largely correlative ( Stam et al. 1997). The found. The other homozygotes contained no detectable molecular mechanisms involved in these phenomena CHS mRNA and little or no anthocyanin. Overall, the have been the subject of a fairly lively debate with no anthocyanin level shows a strong correlation with the clear consensus emerging, even on the most basic points level of intact CHS message. The hog1 and ddm1 muta- ( Matzke et al. 1996; Meyer and Saedler 1996; Stam tions affect both the size and abundance of the CHS et al. 1997). At this point it is unclear whether silencing mRNA, presumably by affecting its transcription and/ and HDG-silencing phenomena are aspects of a single or turnover. The blot was reprobed with an rRNA probe process or a collection of diverse processes with little (Figure 3, center panel), and the loadings were compa- or nothing in common. The study of mutants impaired
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Tissue Culture-based Agrobacterium -mediated and in planta Transformation Methods

Tissue Culture-based Agrobacterium -mediated and in planta Transformation Methods

critical points in floral dip transformation is the use of Silwet L-77 surfactant that dramatically increases the transformation efficiency (Richardson et al. 1998). In addition to Silwet L-77 surfactant, another key ingredient in floral dip transformation is sucrose. Compared to VIAAT, floral dip may result in lower overall transformation rates and higher seed set, but repeated application of Agrobacterium can improve the transformation rate (Clough & Bent 1998). The most important feature in floral dip transformation is the number of seeds produced on an individual plant. Since the efficiency of the floral dip method is about 0.1 to 5%, this method is more efficient in plant species that form more than 100 seeds per re- productive cycle (Tague & Mantis 2006). Although in planta transformation methods were developed with a floral dip method for Arabidopsis in the past decade (Clough & Bent 1998; Kojima et al. 2006), it seems that this method could be used easily in plants of Apiaceae species whose umbrella-like inflorescence can easily be exposed to Agrobacterium suspension (Figure 2). Theoretically, the umbel inflorescences of Apiaceae species host a large number of exposed flowers and produce many seeds (Baranski 2008), but unfortunately there has been no successful report on floral dip in Apiaceae so far. Successful reports on Agrobacterium-mediated gene transformation using floral dip are listed in Table 2.
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Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence

Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence

PCN silencing favors P. brasiliensis susceptibility to killing by macrophages. To assess PCN involvement in the interaction of P. brasiliensis with host cells, the capacity of AsPCN mutant strains to adhere to A549 lung epithelial cells was compared with ability of the WT strain to do the same. After 2 h of cell contact, the efficiencies of yeast cell adherence to epithelial cells, as estimated by CFU counting, did not differ between the AsPCN mutant strains and the WT strain (Fig. 4A). The involvement of PCN in P. brasiliensis interactions with phagocytes was evaluated by coculturing AsPCN1, AsPCN2, AsPCN3, EV, or WT strains with murine peritoneal macrophages. Using optical microscopy to counting yeast cells in a Neubauer chamber, after disruption of macro- phages, we observed that all yeast strains were similarly phagocytosed by macro- phages after 4 h of coculture (Fig. 4B). We conclude that PCN does not play a role in macrophage phagocytosis of P. brasiliensis. To examine whether PCN is relevant in P. brasiliensis sensitivity to macrophage killing, we measured yeast cell recovery using FIG 2 Growth of P. brasiliensis yeasts from WT, EV, and PCN-deficient strains. Yeasts from WT, EV, and PCN-deficient (AsPCN1, AsPCN2, and AsPCN3) strains were cultivated in DMEM at 37°C for 9 days. Growth curve profiles were determined by counting of yeasts by optical microscopy, using Neubauer chambers.
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Higher plant transformation: principles and molecular tools

Higher plant transformation: principles and molecular tools

Inducible promoters are specifically activated in response to external stimuli. In contrast to constitutive promoters, the fused transgenes can be expressed at a distinct developmental stage for a certain duration or in a specific tissue. Additionally, the pro- moters are inactive in the absence of inducers and, therefore, have no negative impact on plant development. The promoter activity can be induced by chemical factors, such as tetracycline, ethanol, steroids, copper ions, and herbicides, or by physical factors, such as heat, cold, and light. Promoters that respond to specific chemical compounds, not found naturally in the organ- ism of interest, are of particular interest in genetic engineering because of the ease of manipulation. Some of the most commonly used chemically inducible promoters in plants (Padidam, 2003) are briefly described. Tetracyclines are particularly attractive as gene expression inducers, because they are small lipophilic com- pounds that enter easily into eukaryotic cells by passive diffusion and they have been routinely used in both human and veterinary medicine with negligible side effects. The tetracycline-inducible system consists of three main components: the transcriptional repressor, the tetracycline-responsive operator, and an antibiotic of the tetracycline family. The tetracycline-inducible system has been used successfully to produce valuable pharmaceutical or industrial proteins in plant cell suspension cultures (Bortesi et al., 2012). In the steroid-inducible systems, heterelogous proteins are fused to a receptor for glucocorticoid or estrogen and induced by steroids. The glucocorticoid receptor-based steroid-inducible sys- tem has significantly advanced the insight into the function of plant transcription factors that control plant developmental pathways (Lloyd et al., 1994; Aoyama et al., 1995). The ethanol-inducible gene expression system is derived from the filamentous fungus Aspergillus nidulans and consists of two elements: the alcohol- regulated transcription factor (ALCR) that binds the alcA-derived promoter that regulates the expression of the transgene (Roslan et al., 2001). The ethanol-inducible system has been optimized for the production of proteins in plants (Dugdale et al., 2013).
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Tissue culture and biolistic–mediated transformation of Impatiens balsamina

Tissue culture and biolistic–mediated transformation of Impatiens balsamina

Generally, there are two types of gene expression in transformation. The transient gene expression is temporary, occurs almost immediately after gene transfer with the higher frequency than stable integration and does not require the regeneration of whole plants. Therefore, transient expression is a rapid and useful method for analyzing the function of gene of interest (Altpeter et al., 2006; Lincoln et al., 1998) and the transient expression frequency provides the most convenient measure of the frequency of introduction of DNA into explants during the optimization of bombardment conditions (Birch and Bower, 1994). In contrast, although the stable expression occurs with the lower frequency, the expression was maintained for long term as the DNA incorporate into the chromosome of the recipient cell (Datta et al., 1997; Mitrovic, 2003).
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Genetic Transformation of Citrus sinensis L  with an antisense ACC oxidase Gene

Genetic Transformation of Citrus sinensis L with an antisense ACC oxidase Gene

This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500 mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration per- centage of 60. The delivery of an antisense ACC oxidase gene into the species C. sinensis mediated by Agrobacterium tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.
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Expression Vector Construction and Genetic Transformation of Paeonia lactiflora Gibberellin 20 Oxidase Gene

Expression Vector Construction and Genetic Transformation of Paeonia lactiflora Gibberellin 20 Oxidase Gene

In conclusion, the overexpression vector of the PlGA 20 ox gene was success- fully constructed and the function was explored in this study, which will un- doubtedly facilitate the genetic transformation of herbaceous peony. However, the genetic transformation system of Paeonia lactiflora has not been established yet, so whether overexpression of PlGA 20 ox gene can cause changes in the phe- notype of peony remains need to be studied. In future, complete transformation system of peony should be constructed for achieving molecular breeding of the famous plant, other members of PlGA 20 ox gene family has not been isolated and the functions have been known completely, so the interaction among family members also needs to be studied. In addition, the key technical obstacles of peony genetic transformation system need to be further solved, which not only helps us have a deeper cognition on the mechanism of PlGA 20 ox , but also im- prove ornamental value and application prospect by genetic engineering.
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Genetic Transformation of Plants

Genetic Transformation of Plants

Electro oration is a method in which presence of electric field creates small pores in the plasma membrane of the cells, which makes it permeable for the substances like piece of DNA, molecular probes or any drug 52 . The pores are transient and after some time reseal automatically and comes to its natural state, this process is known as reversible electroporation 52,53 . For reversible electroporation it is needed to keep the membrane potential below the critical value, but if it exceeds its critical value, irreversible electroporation takes place which cause the damage to cell by permanently making pores in membrane and loss its viability 54 . It can be used to transfer DNA into a large number of cells in a very short period of time 52 . Study using DNA in suspension under an artificial bilipid layer suggests that, when we apply the electric field, DNA will interact with the membrane and promote the formation of pores in the membrane 55 . Larger electric field is required to permeabilize the cells having small radius 56 . Remarkable success has been achieved to transform the cereals like maize, rice and barley 57,58,59 . However, electroporation has a limited use in transformation of some species like wheat because of its lower efficiency 60,43 . Production of fertile transgenic wheat plants depends on the quality of the material used for the transformation 61 .
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Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid

Agrobacterium tumefaciens - mediated transformation of nicotiana benthamiana with dehalogenase gene resistant to monochloroacetic acid

Many farmers used broad spectrum herbicide such as monochloroacetic acid (MCA) which is cost effective and efficient at killings a wide range of weeds. MCA is a phytotoxic chemical that used as broad spectrum of herbicide against broad leaf weeds, grasses and woody plants (Munn et al., 2005). The high concentration of MCA may kill desired plant that lack of herbicide reistance. Unfortunately, MCA and the others broad-spectrum of herbicide can also kill valuable crops and also causing significant losses in agricultural productivity because the herbicides cannot differentiate between plants that are crops and plants that are weeds.
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Genetic Transformation of Plants

Genetic Transformation of Plants

For centuries, people from all over the world have developed different types of new crops by selecting plants from the existing collection. Plant breeding is the technique which involves the identification and selection of desirable parental traits and combining them to obtain a new improved plant variety 1 . Currently, there are lot of changes brought about in the genetic structure of a plant which forms the basis of the plant breeding. Each plant cell has approximately 30,000 genes and these genes codes the information for a plant’s phenotype. Mendel’s laws of genetics proved to be an important area of study in genetics since 1900, and his laws provided the scientific basis for plant breeding. As all traits of a plant are controlled by genes located on chromosome, conventional plant breeding can be considered as the manipulation of the combination of chromosome 2 . Conventional plant breeding is nothing but changing the genes of a plant to develop a variety that is better than the existing ones and new in its own individual way. The aim is to combine those traits that are favourable in both parent plant varieties. Conventional plant breeding may also make use of ‘wider crosses’ that involve crossing species or even genera that are quite unrelated. But these crosses cannot take place without help – and thus a number of sophisticated techniques like genetic transformation were taken in use 3 .
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EFFICIENT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION PROTOCOL FOR RECALCITRANT INDICA RICE (ORYZA SATIVA L.).

EFFICIENT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION PROTOCOL FOR RECALCITRANT INDICA RICE (ORYZA SATIVA L.).

of the Agrobacterium are the most essential steps during a transformation event (Merritt et al., 2007; Uddain et al., 2015). Many studies have taken explant wounding as a routine practice presumably because vir inducers were produced at the host plant wounded sites which acted as chemo-attractants for Agrobacterium attachment (Simoh et al., 2007, Zia et al., 2010). While this might be true, additional wounding of rice shoot apices did not result in high percentage expression of the reporter genes. Excising germinated rice seedlings to expose the shoot apical meristem region was considered as a wounding effect. Deliberate explant wounding have considerably increased the number of explants showing necrotic symptoms which has subsequently led to lower regeneration rates (Rajagopalan and Perl-Treves, 2005). Wounded tissues of monocots were also found to actively differentiate into lignified or sclerified cells which blocked Agrobacterium from invading the wounded sites. This rapid differentiation of wounded tissues left only a handful of cells marginally transformed (Sood et al., 2011). Ethylene production in response to plant wounding, biotic and abiotic stress has been a common phenomenon (Wang et al., 2002) but the production of this simple two-carbon compound has elicited the host plant response to suppress the activation of Agrobacterium’s vir gene. Fortunately, the inhibitory effect of ethylene can be relieved by the supplementation of vir inducers such as acetosyringone (Nonaka et al., 2008). Plants from the Gramineae, including maize also tend to produce 2-hydroxy-4,7-dimethyloxybenzoxazin-3-one (MDIBOA), a major organic exudate of the maize seedling roots, that inhibited the growth of Agrobacterium and the induction of vir gene expression system (Sahi et al., 1990; Zhang et al., 2000; Tzfira and Citovsky, 2002). All these factors could have contributed to the low expression of the reporter genes in scalpel and needle wounded rice shoot apices.
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Molecular Cloning of Sucrose Isomerase Gene and Agrobacterium-Mediated Genetic Transformation of Potato (Solanum tuberosum L.) Plants

Molecular Cloning of Sucrose Isomerase Gene and Agrobacterium-Mediated Genetic Transformation of Potato (Solanum tuberosum L.) Plants

Sucrose isomerase expression under the control of the tuber-specific patatin class I B33 promoter leads to in vivo conversion of sucrose into palatinose, tuber extracts from potatocv. Désiréewere analyzed for their soluble carbohydrate composition using HPLC. The chromatograms assay indicated that there was an additional major peak that was not present in the control and compared to the standard this peak could be set to palatinose(Fig.9). An additional minor peak eluted close to the sucrose signal in extracts from transgenic tubers.Quantitative analysis of non-structural carbohydrates of transgenic tubers has shown accumulation of palatinose in the range of 2.4 mol g-1 FW to 19.5 mol g-1 FW while, sucrose and glucose content was only 1.4 mol g-1 FW and 0.48 mol g-1 FW, respectively, whereas the non-transgenic tubers contained 16.8 mol g-1 FW sucrose and 4.85 mol g-1 FW glucose. The conversion of sucrose into palatinose drastically affected the sucrose and glucose content of transgenic tubers. These results indicate almost quantitative conversion of sucrose into palatinose via palI expressing potato tubers. These results are compatible withRocha- Sosa etal. (1989), they have the sucrose isomerase gene expression under the control of the tuber-specific patatin class I B33 promoter in transgenic potato plants. This protein was combined with the signal peptide of the proteinase inhibitor II, which governs secretion of the enzyme into the apoplasmic space (Von Schaewen et al., 1990).The patatin class I B33 promoter it seems inactive during early tuberization (Tauberger et al., 1999). The apoplasmic localization of the sucrose isomerase leads to accumulation of palatinose. This indicates the presence of sucrose within the apoplast even in later stages of tuber development which have been interpreted as a result of (B)
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