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1. Details of Module and its Structure
Module Detail
Subject Name Botany
Paper Name Plant Genetic Engineering
Module Name/Title The improvement of crop yield and quality Module Id
Pre-requisites Basic knowledge about plant genetic engineering
Objectives To understand different stratagies to increase crop yield and quality using plant genetic engineering.
Keywords Plant nutraceuticals, crop yield, flavrsavr tomato, Golden rice, Nitrogen assimilation,
Structure of Module/Syllabus of a module (Define Topic / Sub-topic of module) Strategies forAbiotic
Stress Tolerance in Plants
<Sub-topic Name1>
2. Development Team
Role Name Affiliation
Subject Coordinator Dr. Sujata Bhargava Savitribai Phule Pune University, Pune.
Paper Coordinator Dr.Rohini Sreevathsa ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi
Content Writer/Author (CW)
Dr.Vitthal Barvkar Department of Botany, Savitribai Phule Pune University, Pune.
Content Reviewer (CR) Dr. Rohini Sreevathsa Language Editor (LE) Dr. Rohini Sreevathsa
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TABLE OF CONTENTS 1. Introduction
2. Improvement of crop yield 2.1 Photosynthesis
2.2 Plant developmental features 2.3 Nitrogen assimilation
2.4 Seed growth
3. Improvement of crop quality
3.1 Modification of Plant Nutritional Content
3.2 Modification of Food Plant Taste and Appearance 3.3 Genetic Manipulation of Flower Pigmentation 3.4 Modified Plant Oils
3.5 Pharmaceutical Products
1. Introduction
In 21st century genetic engineering is considered as ahope to overcome the limiting crop yield potential. Genetic engineering provides a wide rangeof strategies not only for reducing yield losses (byincreasing resistance to pests and diseases and providing tolerance to abiotic stress) but also by increasing the intrinsic yield potential of the plants. Understanding the complexity of yield potential trait using individual gene expression has led to negative view about technology because of non- significant increase in yield potential. However, there are a few examples where physiological research has led to improved crop cultivars with increased yield. Increased yield should not be compromised with food quality in terms of nutritional aspects hence, a group of researcher all over globe are also addressing this issue by genetic transformation. This module is focused on various traits or candidate genes used for improvement of crop yield and quality.
2. Improvement of crop yield 2.1Photosynthesis
Crop yield has direct connection with photosynthetic assimilation of CO2. Hence increasing net photosynthetic rate, translocation and storage is important to increase crop yield. Scientists are
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trying to modify the enzymes involved in photosynthesis related reactions. Currently many attempts are going on to introduce the precursor pathway for organicacid fixation of CO2 (C4 pathway) into C3 species.Adaptability of plant to light and shade condition was accelerated by increasing amount of photosystem II subunit in tobacco (Kromdijket al,2016).In another study, overexpression of genes involved in NPQ (Non photochemical quenching) lead to increase in photosynthetic efficiency in shade condition. Two transgenic lines of tobacco expressing Rubisco from cyanobacterium Synechococcuselongatus were developed while rubisco in these plants was knocked out. This improved photosynthetic efficiency in transformed lines (Lin et al, 2014).
Figure 1 Photosynthesis from light harvesting to grain production. A flow diagram to indicate the principal subsystems that connect to convert sunlight into crop yield. The influence of internal regulatory mechanisms (dashed lines), environmental factors (dotted lines) and development/acclimation alter both the rate and capacity of the material and energy flux through the whole system. (Adopted from Peter Horton 2000).
Table 1 Description of the processes (Figure 1) where losses in photosynthesis may occur in field‐grown rice growing in the tropics. (Adopted from Peter Horton 2000).
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2.2 Plant developmental features
Plant agronomic traits such as plant height, branching pattern, vasculature, flowering pattern influence overall crop performance and yield. Hence there is a scope to manipulate these traits genetically to improve crop features.
Figure 2 Plant developmental features relevant to crop biomass and yield.
Developmentalfeatures, such as architectural traits, leaf andvasculature morphology, predominantlydetermine plant physiology, including theperception of light, photosynthesis, transport ofphotosynthates and source-sink relationship.These physiological parameters in turndetermine crop yield and biomass. Thedevelopmental features are further regulated byphytohormones, genetic and environmentalfactors. (Adopted from Mathan J. et al. 2016)
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Plant height & branching: GA, Brasinosteroids are known to be involved in determination of plant height. Expression of genes involved in biosynthesis or signalling of these hormones can be manipulated to achieve increase/reduction in plant height. Similarly lateral branching/tillering, meristem function is largely regulated by auxins and cytokinins. Several genes encoding transcription factors in signalling of these hormones have been identified.
Leaf morphology: Leaf morphological features such as size, shape, thickness, no. of stomata directly affect photosynthesis hence influence crop yield. Genes governing these characters are very important in crops where leaf is commercially important.
Flowering time: Complex trait such as flowering time in crops is influenced by various factors such as phytohormones, developmental stage, environmental condition etc. Several QTLs have been identified in Arabidopsis, rice, wheat influencing flowering time.
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Further functional genomics approaches such as QTL mapping, GWAS, transcriptome profiling can help in finding out candidate genes regulating plant development in various crops.
Table 2 Key genes associated with plant architecture in crops (Adopted from Mathan J. et al.
2016)
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2.3 Nitrogen assimilation
Large amount of nitrogen fertilizers are used every year. They have many limitations like they cause soil and water pollution, excessive weed growth in field, they show toxic effect on ecosystem components. Also their use is limited by high cost. Hence there is a need of improving nitrogen use efficiency of crop plants which ultimately should be reflected in crop yield.
Plants absorb nitrogen in the form of nitrates and ammonium. This inorganic form of nitrogen is converted into nitrogenous organic compounds like amino acids. This process involves several enzymatic steps. Glutamine synthetase(GS1) and NADH-dependent glutamine-2-oxoglutarate aminotransferase (NADH-GOGAT) are the key enzymes in this process. (Andrews et al, 2004).Genes encoding cytosolic GS and the corresponding enzyme activity is positively correlated with QTL for yield and its components. (Hirel et al., 2001). When Phaseolus vulgaris GS1 gene was overexpressed in wheat, yield and nitrogen content of grains increased (Habashet al., 2001).
Overexpression of GS2 gene increased growth rate in N. tabaccumand increased photorespiration and drought tolerance in O. sativa(Hoshidaet al., 2000; Miggeet al., 2000)
Along with the genes encodingamino acid biosynthesis, genes involved in their transport have been characterized. Overexpression of Nia gene (encoding NR apoenzyme) does not cause any yield increase.(Reviewed by Pathak et al., 2009)
It is said that transformation in nitrogen assimilation related genes will not alter basic metabolism (Lawlor, D. W. 2002).Hence to increase nitrogen assimilation, data coming from various studies like genomics, transcriptomics, physiology etc. must be compiled and used.(Masclaux-Daubresseet al, 2010).
2.4 Seed growth
Yield in cereals depends upon seed number and seed weight. Transformation of wheat with a gene (Sh2r6hs from maize) encoding altered subunit of AGP (ADP-glucose pyrophosphorylase) enzyme increased seed wight by 38% (Smidanskyet al., 2002). Similar experiment was also performed in rice however there was overall increase in growth of plant hence plant harvest index remained same.
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Figure 3 Structure of the expression construct pSh2r6hs. The Sh2 promoter, modified Sh2 coding region, Sh1 intron 1 cassette, and nopaline synthase polyadenylation region are shown as open boxes drawn approximately to scale. (Adopted from Smidanskyet al., 2002)
3. Improvement of crop quality
3.1 Modification of Plant Nutritional Content
Plants are the major source of food for human being. Besides energy, they also provide various nutrients, essential for normal metabolism in human body. Engineering plants for improving nutritional quality may provide solution to the problems like malnutrition. Using biotechnological tools, recently many plants have been modified for altered nutritional content. Strategies in this respect include manipulation of genes involved in biosynthesis, transport or degradation and regulation of a particular nutrient etc.
Golden rice: Golden riceproducing high levels of β carotene(Vitamin A precursor) was engineered to overcome vitamin A deficiency. (Ye et al, 2000) This was achievedby co- transformation of genes encoding phytoene synthase(psy) and lycopene β-cyclase (lcy)originated from Daffodil (Narcissus pseudonarcissus,) and phytoene desaturase (crt) originated from Bacterium Erwiniauredovora.
High lysine production: dapA gene from Corynebacterium glutamicum is introduced into maize which encodes dihydrodipicolinate synthase (DHDPS) which is insensitive to lysine feedback mechanism. Free lysine level is also increased by suppression of LKR/SDH which is involved lysine breakdown. (Huang et al, 2005)
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Figure 4 Beta-carotene synthesis pathway and regulating enzymes
Figure 5 Lysine enrichment approach by abolishing the feedback inhibition of DHDPS and suppressing Lysine ketoglutarate reductase/saccharopine dehydrogenase biofunctional enzyme (LKR/SDH) involved to lysine degradation. (Source:http://www.isb.vt.edu).
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Other nutritional aspects: Attempts have been made to increase fructan level in sugar beet, to manipulate amylase to amylopectin ratio in potato, increase vitamin E level in soybean and maize, increase in iron content in rice etc.
3.2 Modification of Food Plant Taste and Appearance
Efforts have been made to improve sweetness, flavor, colour, shelf life and to prevent discoloration in fruits and vegetables. Scientists have characterized the genes governing these traits.
Antisense gene of an enzyme polygalactouronase was transformed into ‘FlavrSavr’ tomato to inhibit pectin degradation in cell wall thereby improve shelf life.
Various genes encoding sweet proteins such as monellins are identified and transformed into tomato (Reddy et al, 2015), lettuce(Lola peet al, 1992), strawberry (Min et al., 2015) etc.
Transformation of genes encoding fructans (polymers of fructose) accumulated fructan in the tap root of sugarbeet(Sévenieret al,1998).
Cloning of antisense transcripts of polyphenol oxidase genes under the control of CaMV 35S and GBSS promoter in potato and apple has shown to decrease discolouration.
Figure 6
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Figure 7FlavrSavrTomato producingstrategy (Source: http://vle.du.ac.in/mod/book/view)
Figure 8 Construct showing polyphenol oxidase genes cloned in Agrobacterium plasmid (Source:
http://cen.acs.org).
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3.3 GeneticModification Flower Pigmentation
In flower industries, hundreds of new varieties are developed continuously to improve flower colour and cut life. Through the genetic engineering approach many varieties of commercially important flowers like Petunia, Gerbera, Carnation and roses have been developed. Flavonoids such as anthocyanins, chalcones, aurons, carotenoids are important components responsible for pigmentation in flowers. Overexpression or suppression of chalcone synthase (CHS) gene has been studied in various plants. Overexpression of CHS gene in Petuniablocked anthocyanin biosynthesis and lead to white colored flowers (Napoli, 1990). Petunias transformed with dihydoflavonol 4- reductase gene from corn, flowers showed brick red colour.
Table 3Successful adoption of biotechnology for modification(s) in various attributes of ornamental plants. (Adopted fromNoman A. et. al. 2017)
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Table 4Genes involved in synthesis of various enzymes for floral pigment pathways. (Adopted fromNoman A. et. al. 2017)
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Figure 9Biosynthesis of anthocyanidin. CHS catalyze the formation of Tetrahydroxycalchone.
Later on, different enzymes such as CHI, F3H, DFR, ANScatalyze other steps of pigment production. The methyl groups are only added to anthocyanins not to anthocyanidins. The actual pigment color production is notsolely dependent upon the enzyme catalyzing reactions but also depends upon other factors. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol 4-reductase;
ANS, anthocyanidin synthase; MT, methyltransferase, GT, glucosyltransferase;AT, acyltransferase;
FNS, flavone synthase; FLS, flavonol synthase. (Adopted fromNoman A. et. al. 2017)
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3.4 Modified Plant Oils
Oilseed plants stores fatty acids in the form of triglycerol. Engineering oilseed crop aims at improving desired oil content or production of novel fatty acid healthier for humans (Thelen&Ohlorgge, 2002). This is achieved through antisense/RNAi technology or introduction of gene from other source (Cloutieret al, 2014).
Figure 10Enzymes involved in Fatty acid biosynthesis
Two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9- desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase were downregulated using hairpin RNA-mediated gene silencing to alter composition of fatty acid in cotton seed (Green et al, 2002).
Figure 11 Chimeric silensing constructs transformed into cotton under control of soyabean lectin promoter.
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Seed stearate level was increased by transforming seed specific antisense construct of stearoyl- ACP desaturase gene in Brassica rapa.(Knutzonet al., 1992)
Oleic acid content can be increased in soyabean oil by decreasing level of Δ12-desaturase. Gene encoding Δ12-desaturase was repressed using gene silencing. This makes soyabean oil suitable for commercial use.
Increase in short chain fatty acids in canola (B. napus) was achieved through transformation of canola with thioesterase gene originated from Umbellulariacalifornica.
Table 5Transgenic canola varieties with modified seed lipid contents
3.5 Pharmaceutical Products
Plants can produce a variety of biopharmaceuticals. They can be easily transformed and are a cheaper source to obtain desired product. Plants are being transformed to obtain antibodies, antigens, vaccines, proteins and other pharmaceuticals.
To achieve immunity against hepatitis B virus DNA fragment encoding hepatitis B virus surface antigen was transformed into Lupin and Lettuce through Agrobacterium transformation. The successful production of antibodies against recombinant surface antigen was observed in mice and human (Koprowskiet al, 1999). Hepatitis B viral surface antigen (HBsAg) was also transformed into potato (Bapatet al, 2005). Transgenic banana, potato, tomato, cabbage, strawberry producing antibodies against several viral and bacterial diseases (Richter &Kipp, 2000).
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Advantages of edible vaccines:
They are easy for administration
Easy for production and transportations.
They show reduced risk since they are free from pathogen.
Limitations of edible vaccines:
Dose determination is difficult.
Some individuals may develop immune tolerance against protein/peptide.
Table 6Some of the therapeutic agents produced in transgenic plants (Source: Glick, Bernard R by Molecular Biotechnology Chapter 20)
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Table 7Some recombinant vaccine antigens expressed in plants (Source: Glick, Bernard R by Molecular Biotechnology Chapter 20)
Table 6 Some antibodies and antibody fragments that have been produced in plants (Source: Glick, Bernard R by Molecular Biotechnology Chapter 20)
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SUGGESTED READING
1.S.B. Primrose and R.M. TwymanPrinciples of Gene Manipulation and Genomics 2006 Blackwell Publishing Seventh edition
2. Glick, B. R. and Patten C. L. Molecular Biotechnology Chapter 20 ASM Press American Society for Microbiology 1752 N St. NW Washington, DC Fourth Edition.
3. Ainsworth E. A. et. al. (2012) Accelerating yield potential in soybean: potential targets for biotechnological improvementPlant, Cell and Environment 35, 38–52
4. Noman A. et. al. (2017) Biotechnological Advancements for Improving Floral Attributes in Ornamental PlantsFront. Plant Sci. 8:530.doi: 10.3389/fpls.2017.00530
5. Sinclair T. R. (2004) Crop transformation and the challenge to increase yield potentialTRENDS in Plant Science Vol.9 No.2 70-75
6.Mathan J. et. al.(2016) Enhancing crop yield by optimizing plant developmental features Development 143, 3283-3294 doi:10.1242/dev.134072
7. P. Sharma-Natu* and M. C. Ghildiyal(2005) Potential targets for improving photosynthesis and crop yield Current Science, VOL. 88, NO. 12, 25
8. Peter Horto (2000) Prospects for crop improvement through the genetic manipulation of photosynthesis: morphological and biochemical aspects of light capture Vol 51 pp 475-485
