reviewarticle - hindawi publishing...

Review ArticleGenetic Modification for Wheat ImprovementFrom Transgenesis to Genome Editing

Nikolai Borisjuk1 Olena Kishchenko12 Serik Eliby3 Carly Schramm3

Peter Anderson3 Satyvaldy Jatayev4 Akhylbek Kurishbayev4 and Yuri Shavrukov 3

1 School of Life Science Huaiyin Normal University Huaian China2Institute of Cell Biology and Genetic Engineering Kyiv Ukraine3College of Science and Engineering Biological Sciences Flinders University Bedford Park SA Australia4Faculty of Agronomy S Seifullin Kazakh AgroTechnical University Astana Kazakhstan

Correspondence should be addressed to Yuri Shavrukov yurishavrukovflinderseduau

Received 2 November 2018 Revised 8 February 2019 Accepted 21 February 2019 Published 10 March 2019

Academic Editor Pulugurtha Bharadwaja Kirti

Copyright copy 2019 Nikolai Borisjuk et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To feed the growing human population global wheat yields should increase to approximately 5 tonnes per ha from the current33 tonnes by 2050 To reach this goal existing breeding practices must be complemented with new techniques built upon recentgains from wheat genome sequencing and the accumulated knowledge of genetic determinants underlying the agricultural traitsresponsible for crop yield and quality In this review we primarily focus on the tools and techniques available for accessing genefunctions which lead to clear phenotypes in wheat We provide a view of the development of wheat transformation techniquesfrom a historical perspective and summarize how techniques have been adapted to obtain gain-of-function phenotypes by geneoverexpression loss-of-function phenotypes by expressing antisense RNAs (RNA interference or RNAi) and most recently themanipulation of gene structure and expression using site-specific nucleases such as CRISPRCas9 for genome editing The reviewsummarizes recent successes in the application of wheat genetic manipulation to increase yield improve nutritional and health-promoting qualities in wheat and enhance the croprsquos resistance to various biotic and abiotic stresses

1 Introduction

Cereals are a key component of human diets providing asignificant proportion of the protein and calories consumedworldwide While rice and maize dominate global cerealproduction wheat is another vital crop consumed by humanscontributing to approximately 20of our energy needs (calo-ries) and 25 of our dietary proteinTheGreen Revolution ofthe 1970s achieved enormous yield gains via the introductionof disease resistant semidwarf high yielding wheat varietiesdeveloped byDrNEBorlaug and colleagues Since that timehowever global wheat production has stagnated and currenttrends show that yields will not be sufficient to meet growingmarket demands

According to the United Nationsrsquo Food and AgricultureOrganization (FAO) over 756 million tonnes of wheat grainwas harvested from over 220 million ha of arable land

in 20162017 (wwwfaoorgfaostat) Despite this wheat lagsbehind other major cereals such as maize and rice bothin terms of yield and the application of genomic toolsfor its improvement [1] While the average worldwide yieldgrew almost 3-fold during the Green Revolution driven bythe expansion of irrigation intensive use of fertilisers andadvanced breeding [2] the current average global wheat yieldof sim3 tonnes per hectare is far below the croprsquos potential [3]In order to feed the population of 9 billion people predictedfor 2050 wheat yield should grow by over 60 while stillmaintaining andor improving its nutritional characteristics[3 4] To achieve this goal without increasing the area ofcultivated land which is simply not available emphasis mustbe concentrated on key traits related to plant productivityand adaptation to environmental challenges A deficit in thiskey staple crop could present a serious threat to global foodsecurity so improved molecular-based breeding and genetic

HindawiBioMed Research InternationalVolume 2019 Article ID 6216304 18 pageshttpsdoiorg10115520196216304

2 BioMed Research International

engineering techniques are necessary to break through thecurrent yield ceiling

Existing modern breeding efforts now need to be comple-mented with advanced crop functional genomics which canprovide insights into the functioning of wheat genetic deter-minants The available tools for wheat genetic modificationprovide the experimental means to functionally characterizegenetic determinants by suppressing or enhancing geneactivities This knowledge can then be used for targetedimprovements tailored to the specific needs of the diverse andchanging environments in which wheat is grown across theworld This offers the potential to tackle yield gaps whereverthey exist for a variety of causes enabling this global crop tofinally reach its full potential

2 Progress in Wheat Genetic Transformation

Bread wheat (Triticum aestivum L) the most widespread ofall wheat species is an annual herb belonging to the familyGramineae or Poaceae Wheat was domesticated around8000 years ago [29] and has since undergone hybridizationand genome duplication events to generate its hexaploidgenome (2n = 6x = 42 AABBDD) which is more thanfive times larger than the human genome It was previouslyestimated that the genome of common wheat contained over128000 genes [30] with over 80 of the genome consistingof repetitive sequences of DNA [31] However more recentestimates suggested a total of 107891 high-confidence geneswith over 85 repetitive DNA sequences representing athreefold redundancy due to its hexaploid genome [32]

Genetic transformation the fundamental tool of geneticengineering allows the introduction and expression ofvarious genes of interest in the cells of living organismsbypassing when desirable the barriers of sexual incompat-ibility that exist in nature Despite the considerable effortsof the international research community development ofwheat genetic engineering lags behind that of the other keyagricultural crops like rice and maize This may be attributedto the genetic characteristics of wheat including its very large(17000Mbp) and highly redundant complex genome as wellas the relative recalcitrance ofmost varieties to in vitro cultureand regeneration (reviewed recently in [33])

The first successful genetic transformation of commonwheat was conducted at Florida University USA [34] usingbiolistics and financed by a research grant from MonsantoScientists fromMonsanto were also the first to report the gen-eration of transgenic wheat using Agrobacterium-mediatedtransformation [35]

21 Wheat Transformation Methods Presently biolisticsand Agrobacterium-mediated transformation using imma-ture embryos as explants remain the main methods forgenetic engineering of wheat Each method has its ownadvantages and drawbacks (Table 1) The main advantages ofAgrobacterium transformation are the relatively high ratio ofsingle copy gene inserts and relative simplicity of the transfor-mation procedure In contrast biolistics offer benefits in theircapacity to transform organelles and deliver RNA proteinsnanoparticles dyes and complexes into cells Utilization

of linear Minimal Expression Cassettes (MECs) in biolistictransformation enables the production of plants carryingmuch simpler patterns of transgene integration compared toplasmid bombardment with a higher proportion of singlecopy inserts [36ndash38]

In addition biolistic delivery ofMEC simplifies the simul-taneous cotransformation of several genes and in contrastto Agrobacterium does not introduce vector backbone DNAor repetitive border sequences flanking the T-DNA intothe transformed plant cells In experiments with the wheatcv V12 transformation with MECs instead of plasmidsimproved transformation frequency (TF) almost threefoldfrom 04 t] 11 [39] A simplified method for DNAgoldcoating was described by Ismagul et al [38] for the high-throughput biolistic production of single copy transgenicwheat utilizing diluted MECs This method involves theapplication of PM solution (42 PEG 2000 and 560 mMMgCl

2) instead of the spermidine and CaCl

2used in the

standard Bio-Rad procedureBiolistics allow for the transfer into wheat of relatively

large DNA fragments Partier et al [40] conducted successfulbiolistic transformation of wheat with a 53 Pb linear cassettewhich contained a 44 Kb fragment of an Arabidopsis geneflanked by selection and reporter genes The intact cassettewas detected in f

1and f

2generation plants The main

disadvantages of both Agrobacterium and biolistic transfor-mation methods in wheat are genotype dependency and therequirement for lengthy periods of aseptic tissue culture

22 Wheat Transformation Frequencies Until recently theTF for most tested genotypes of common wheat remainedquite low at a level mostly below 5 [8] Many researchgroups use model wheat varieties such as Bobwhite SH98-26 and Fielder in their experiments due to their amenabilityto transformation via published protocols [8 9] The cultivarBobwhite SH98-26 was among 129 sister lines made fromcrosses of cultivars AuroraKalyanBluebird 3Woodpeckerat CIMMYT and selected for its high TF of over 70by biolistic transformation [9] Such high transformationefficiency is yet to be reproduced by other researchers andalthough the reason for this remains unclear it is possible thatnot all of the finer details of the transformation protocol couldbe described in the report The success rate of this high TFmay be explained by the particular hybrid genotype used orsimply the advanced skills and experience of the techniciansat CIMMYT Since publishing [9] A Pellegrineschi has beenemployed by two big Biotech companies Pioneer Hi BreedandDuPont which indirectly confirms that he has developeda reliable wheat transformation protocol that allowed himto produce the published results The authors of the revieware therefore not sceptical however the high TF presented in[9] must be reproduced by other researchers in future if thisprotocol is to be widely adopted The cultivar Fielder whichis the model variety in Agrobacterium transformation waspreselected by the researchers of the JapanTobaccoCompanywhere the detailed protocol ldquoPureWheatrdquo was developed [8]that allows TFs of 40ndash90 In this protocol positive selec-tion through the application of phosphomannose isomerase

BioMed Research International 3

Table1Com

paris

onof

parameterso

fagrob

acteria

land

biolistictransfo

rmation

Parameters

Agrobacterium-m

ediated

transfo

rmation

Biolistictransfo

rmation

References

Genotyped

ependency

High

Less

[5]

Stability

ofexpressio

nof

transgenes

High

HighforM

inim

alEx

pressio

nCassette

s(M

EC)lowe

rfor

plasmids

[67]

Cop

ynu

mbero

finserts

Aroun

d50sin

glec

opy

Depends

onthes

trainand

transfo

rmationcond

ition

s

lt50D

epends

onthe

amou

ntof

DNAsho

tMore

multic

opyinserts

[78]

Integrationof

then

ewgenes

Rand

omM

orethanon

elocus

Rand

omO

ftenmanyatthe

samelocus

[6]

Maxim

umtransfo

rmationfre

quency

(TF)

forw

heat(per

100em

bryostreated)

Upto

90

gt70

[89]

Com

plexity

ofthetransform

ation

procedureinwheat

SimplerU

suallyrequ

ires

aseptic

cond

ition

s

Morec

omplexR

equires

aseptic

cond

ition

sand

aBiolisticGun

[810]

Mainexplantsin

wheat

Immaturee

mbryos

Immaturee

mbryos

[810]

Com

plexity

ofvector

construct

preparationco-tr

ansfo

rmation

Morec

omplex

Simpler

[11]

Maxim

umsiz

esof

transfe

rred

inserts

publish

edUpto

200Kb

150ndash

164Kb

[12ndash14]

Transfe

roff

-DNAbo

rders

Yes

No(fo

rMEC

)[15]

Transfe

rofvectorD

NA

Possible

No(fo

rMEC

)Yes(for

plasmids)

[15]

Transfe

rofb

acteria

lchrom

osom

alDNA

Possible

No(fo

rMEC

)[16]

Markerfreetransform

ationin

wheat

Possible

Possible

[17ndash19]

Inplanta

transfo

rmationin

wheat

Possible

Possible

[20ndash

22]

Deliveryof

RNAproteinsnano

particles

anddyes

No

Possible

[23]

Transfo

rmationof

chloroplastsand

mito

chon

dria

No

Possible

[2425]

Transie

ntgene

expressio

nin

different

tissues

andorgans

ofplants

Efficientfor

limited

numbero

fplant

species

Efficient

[26ndash

28]


(PMI) which converts mannose-6-phosphate into fructose-6-phosphate was found to facilitate a relatively highTF (20)in biolistic transformation of the spring wheat line UC703[41]

23 Agrobacterium-MediatedTransformation In recent yearsseveral groups have reported efficient Agrobacterium trans-formation of a number of wheat cultivars [8 42ndash44] Henselet al [43] developed a protocol to transform the modelgenotype Bobwhite SH98-26 with TFs up to 15 In theexperiments of Richardson et al [42] using the PureWheattechnology with the cv Fielder selection on 5-10 mgl ofphosphinothricin resulted in a TF of 410Without selectivepressure the TF was only around 23 Similar results wereobtained for Chinese commercial cultivars of wheat byWanget al [44] whodemonstratedTF of 377 for cvbj037 227for cv Kenong 199 and 453 for the model cv Fielder Itwas shown by Ishida et al [8] that centrifugation of immatureembryos before infection with Agrobacterium was one ofthe critical requirements for successful transformation whileheat shock contrary to findings in other cereals was notefficient At present Agrobacterium strains EHA101 EHA105[8] AGL0 AGL1 [43 45] GV3101 [46] C58C1 [47 48] andLBA4404 [49] are the most popular in wheat transformation

Transformation using mature wheat embryos is currentlycharacterized by relatively low TFs Wang et al [48] trans-formed longitudinally cut mature embryos and observed TFsof 006 067 and 089 for the cultivars Bobwhite Yumai66 and Lunxuan 208 respectively Aadel et al [50] foundthat with the application of 200 120583V acetosyringone the TFsfor the genotypes Rajae and Amal were 066 and 100Theprotocol ofMedvecka andHarwood [51] using BobwhiteSH98-56 allows production of transformants at a TF of 22More information and a simplified protocol can be found in[52]

In contrast to in vitro methods the in planta approachhas the potential to overcome the problem of high genotypedependency seen with the existing transformation meth-ods There have been several publications on the in plantaproduction of transgenic wheat most involving the directinjection of Agrobacterium Supartana et al [49] reportedagrobacterial transformation of wheat cv Shiranekomugiusing seeds soaked overnight in water Transformation wasachieved by double piercing the area where a shoot wouldlater emerge with a needle dipped into Agrobacterium inocu-lum This method used the Agrobacterium strains LBA4404and an M-21 mutant strain and no tissue culture steps wereused at any stage The plants obtained were analysed forantibiotic resistance and by PCR Southern hybridizationand plasmid rescue to confirm their transgenic status Zhao etal [53] produced Agrobacterium-mediated transgenic wheatby adding inoculum to an incision made at the base of wheatseedlings Their tissue culture-free method was reportedlysuccessful in transferring a powdery mildew-resistance genewith a TF of up to 982 Similarly in the experimentsof Razzaq et al [54] with the wheat cv GA-2002 theapical meristems of imbibed wheat seeds were wounded andinoculated with the Agrobacterium strain LBA4404 contain-ing the binary vector pBI121 (35S-GUS pNOS-NPTII) The

kanamycin resistant plants produced were analysed by PCRand GUS histochemical staining of the embryos Risacher etal [20] developed an efficient semi-in planta transformationprotocol (US patent 7803988 B2 2010) that involves inplanta agrobacterial infection of immature embryos withindeveloping seeds Spikes of the spring wheat line NB

1were

harvested 16 to 18 days after anthesis and Agrobacteriuminjected into the base of each spikelet The spikes with theirflag leaves still attached were incubated in low light for 2 to 3days before embryos were isolated and cultured in vitro Theprotocol achieved an average TF of 5 with 30-50 of plantscarrying a single transgene insertion

The practice of dipping floral buds in a suspension ofAgrobacterium (Floral dip) is a routine and highly efficientmethod of transformation in Arabidopsis but it is rarelysuccessful in other plant speciesHowever Zale et al [21] wereable to generate three independent transgenic lines of thespringwheat line Crocus by utilizing the floral dip approachThe transformants were studied thoroughly at the molecularlevel for three to six generations In their most recentpublication Hamada et al [22] trialled a biolistic methodfor in planta transformation They found that bombardingthe exposed shoot apical meristems of the wheat cultivarsFielder and Haruyokoi using 06 120583m gold particles and 1350psi pressure resulted in the integration of the GFP reportergene into the germline cells in 62 of regenerated plants(transformants) including possible chimeric individuals Thefull potential of the wheat in planta procedures published todate is yet to be fully realized

Microspore transformation using immature pollen grainsis a method that generates doubled haploid homozygouswheat plants in a single generation The great advantage ofthis technique is that it by-passes the several years requiredby other transformation methods to develop true-breedingtransformant lines Current protocols for microspore-basedtransgenic wheat production through electroporation biolis-tics and Agrobacterium-mediated transformation are pre-sented in [55ndash57] Various wheat genotypes have been used asdonors Express Chris Farnum Hollis Louise Perigee andWestBred 926 have all performed successfully in microsporetransformation Four genes were targeted for microsporetransformation including 120573-glucuronidase (GUS) uidAbialaphos resistance Bar Trichoderma harzianum endochiti-nase gene En42 and Bacillus subtilis 14-120573-xylanase gene[55ndash57] In general Agrobacterium-mediated microsporetransformation produces the best outcome generating stablehomozygous plants and fewer chimeric plants Given thevalue of doubled haploid lines in simplifying and acceleratingwheat breeding it is likely that microspore methods will beoptimized in the future

Recent advances in high-throughput sequencing tech-nologies have allowed for more detailed information tobe obtained about the composition and fate of transgenesintroduced into plant cells through Agrobacterium-mediatedtransformation In one report close to 04 of transgenicArabidopsis lines examined contained insertions of Agrobac-terium chromosomal DNA [16] Singer et al [58] showedfor the first time the presence in transgenic plant cells ofthe extra-chromosomal circular T-DNA that they named


ldquoT-circlesrdquo In rice 26 of 331 analysed transgenic linescontained the transposon Tn5393 which was transferredfrom the Agrobacterium strain LBA4404 [59] This transpo-son was not detected in the strains EHA105 and GV3101These data reflect the importance of detailed molecularanalysis of transgenic lines and careful selection of theappropriate agrobacterial strains for transformation A betterunderstanding of the biological mechanisms of the transferand integration of transgenes during bacterial infection willalso permit the creation of new more efficient strains ofAgrobacterium [5 60] or utilization of bacterial species thatare not pathogenic in nature [61]

24 Optimization of Tissue Culture Conditions Tissue cultureconditions are important factors that impact the TF ofall plants including wheat Below we present informationreflecting the current trends inwheat tissue culture thatmightbe of interest to researchers working in the field Changingthe composition of the macrosalts in the growth mediumcan positively affect the TF Callus induction on 6x DSEMmedium with an increased concentration of ammoniumnitrate (6256mM) as the sole nitrogen source resulted in thesevenfold improvement of TF during biolistic transformationof the elite wheat cv Superb [62] The authors point out thatthismodification of the mediumbrought about an increase inthe number of somatic embryoids and possibly also reducedstress during the bombardment of the cells due to the elevatedosmolarity Ishida et al [8] identified some critical points inthe transformation process and developed a detailed protocolfor Agrobacterium-mediated transformation for both imma-ture and mature wheat embryos from amenable genotypesThe authors however did not discuss any work on mediaimprovements

Antioxidants such as ascorbic acid glutathione lipoicacid selenite and cysteine were found to decrease necrosisand darkening of the tissues improving plant regenerationand TF during genetic transformation [63] Lipoic acid at25ndash50 120583V led to a twofold improvement in agrobacterialtransformation of the wheat cv Bobwhite [64]

Arabinogalactan-proteins at a concentration of 5 mglimproved regeneration of bread (cv Ikizce-96) and durum(cv Mirzabey) wheat from 7777 and 7211 to 9486and 8973 respectively [65] Kumar et al [66] were able toachieve close to 100 plant regeneration from calli inducedfrom wheat mature and immature embryos on 20 mglpicloram using a regeneration medium with 01 mgl 24-D 50 mgl zeatin and 15 mgl CuSO

4 Miroshnichenko et

al [67] developed a protocol for the regeneration of einkornwheat (f monococcum L) in which a combination of 30mgl dicamba 500mgl daminozide (an inhibitor of ethylenesynthesis) and 025 mgl thidiazuron in the regenerationmediumwas the most efficientThe authors presume that thisprotocol may be useful for other wheat species as well

25 Transient Transformation Transient transformationwith the use of reporter genes such as GUS [68] and GFP[69] is helpful for optimization of transformation conditionsas well as the analysis of promoters and protein expressionTransgene expression that is tightly targeted to specific

tissues and developmental stages is often desired for directedmodification of morphogenic traits It can also be beneficialfor avoiding feedback mechanisms transgene silencing orother unforeseen effects that can arise from constitutivetransgene expression Transient assays using protoplasts canalso facilitate efficient analysis of gene regulatorymechanismsthrough cotransformation of enhancerrepresser elementsand promoter-reporter gene constructs [11] Viral inducedgene silencing (VIGS) offers a fast and rapid transient assayfor silencing of gene expression VIGS can be achievedthrough a simple vacuum-aided cocultivation of germinatedwheat seeds with Agrobacterium carrying an appropriateVIGS construct [70] However results from VIGS arenot comparable to results from stable transformationexperiments but this does not mean that the VIGS findingsare false or of no value Like RNAi VIGS findings are usefulfor increasing our understanding of gene function and maylead to the development of a strategy that is more successfulthan simple constitutive transgene overexpression

26 Future Directions Gene Stacking and Plant ArtificialChromosomes Gene stacking or pyramiding (defined as thestacking of multiple transgenes at a single chromosomal loca-tion) greatly expands the potential for genetic engineering oftraits in wheat [71] Applications of this technology includemodifying complex and multigenic traits or inserting entirebiosynthetic pathways with integration at a single locusThis significantly simplifies the subsequent breeding processAlternatively for disease resistance traits transgene stackscan aid in generating broad spectrum resistance to multiplethreats or more long-lasting defence to a single pathogenvia genes with differing modes-of-action Construction ofa transgene stack is most often achieved using type IIrestriction enzymes in the Golden-Gate cloning systemor by Gibson Assembly The integration of longer DNAfragments carries certain risks however such as reducedTF fragmentation leading to only partial integration andtransgene silencing if promoters or other regulatory elementsare used repeatedly The magnitude of these risks differsaccording to the nature of the vectors or transformationcassettes the transgene products and the recipient genotypeMuch of the more recent work involving stacked transgenesuses site-specific nucleases so that the transgene stack canbe directed to a favourable locus in the genome Site-specificnucleases will be covered in a later section of this review

Plant artificial chromosomes or minichromosomes werealso developed for multigene transfer [72] The main advan-tage is that they generate a new genetic locus that segregatesindependently of endogenous chromosomes thus lesseningthe disruption of existing genomic regions Minichromo-somes have been developed in mammalian cells througha process named ldquoTelomere-mediated chromosome trun-cationrdquo that targets highly conserved telomeric repeatsequences Analogous sequences have been identified in Ara-bidopsis but progress in developing plant minichromosomesstill lags behind the work in mammalian cells Minichromo-somes have the potential to act as ldquosuper-vector platformsrdquofor the organization and expression of foreign genes and mayeven be designed with Crelox recombination sites to accept


the introduction of new genes This presents the option toldquoadd onrdquo additional alleles or genetic elements at a later dateCurrently the reliable transmission of minichromosomes tothe progeny of primary transformants presents the greatestbarrier to the application of this technology However initialwork has suggested that wheatrsquos allopolyploid nature makes itmore tolerant to chromosomal truncation than that of diploidspecies making this a very promising future prospect forwheat genetic engineering [73]

3 Manipulating Agronomic Traits byTransgene Expression in Wheat

Built on the progress made on techniques for genetic manip-ulation of wheat overexpression of endogenous and foreigngenes has tremendously enriched our understanding of thefunctionality of numerous genes contributing to the gener-ation of many new and improved agronomically importanttraits This improved understanding of gene function hasled to the development of specific promoters to drive geneexpression in response to environmental stimuli andor ina tissue organ or developmental stage-specific manner [7475] Various signals for directing expressed proteins to par-ticular cellular compartments have also been used [76] Alsothe field received another major stimulus through the useof transcription factors that function as major regulators ofgene networks involved in numerousmetabolicphysiologicalpathways Transcription factors are often highly conservedbetween different plant species so the information obtainedfrom studying model plants like Arabidopsis or rice can beapplied to other plants such as wheat [77ndash79]

In pursuit of improved crop performance wheat geneticmanipulations made by transgenic gene expression havetargeted all major agronomic traits including yield grainquality and tolerance to abiotic and biotic stresses The last10 years have been characterized by acceleration in this fieldand an increasing number of publications In this review wewill focus primarily on attempts to improve yield and grainquality the two areas that have not been sufficiently reviewedby other authors For readers interested in improving wheatstress responses we refer to recent comprehensive reviewson the topics of pathogen resistance [80ndash82] and abioticstress tolerance [83 84] as well as a recent review oftransgenicwheat engineeredwith various genes of agronomicimportance [33]

31 Yield Yield is determined by the number and size ofgrains produced by the crop The major genes controllingyield-related traits in wheat have been identified by geneticand genomics techniques as reported in recent reviews [85ndash87]

Grain size (GS) has long been a focus of wheat selectionand modern breeding [88] Progress in comparative wheatgenomics has led to the identification of TaGW2 [89] awheat homolog of a rice gene that negatively influencesGS through regulation of cell division within the spikelethull [90] The first experiments aimed at downregulatingTaGW2 in wheat have shown some controversial resultsBednarek et al [91] demonstrated that expression of a specific

RNAi to suppress three TaGW2 homologs A B and D ofbread wheat cv Recital led to a decrease in grain size andweight In contrast Hong et al [92] in a similar series ofexperiments demonstrated a significant increase in the grainwidth and weight of a Chinese bread wheat cv Shi 4185 that istypically characterized by small grains Such differences in thephenotypes obtained in these two studies may be explainedby a cultivar-specific reaction to experimental interventionandor by the application of different transformation proto-cols as has been previously reported for barley [93]

Plant growth and productivity to a large extent relies onthe uptake of carbon nitrogen and phosphorus Ribulose-15-bisphosphate carboxylaseoxygenase (RuBisCo) themostabundant protein on Earth is the major enzyme assimilatingatmospheric carbon in the form of CO

2into organic com-

pounds in photosynthetic organisms However the enzymehas relatively low efficiency and a slow turnover rate [94]Despite significant progress in characterizing the enzymersquosproperties [95] and improving RuBisCo efficiency in a rangeof plant species [96 97] little success has been achievedso far in commercial crops including wheat Another areafor optimizing carbon assimilation in crops is to introducecomponents of the C

4photosynthetic apparatus into C

3

plants like wheat and rice C4plants such as maize dis-

play definite advantages over plants with C3photosynthesis

especially under high temperature and limited water con-ditions [98] These advantages rely on a number of specificenzymes associated with RuBisCo which provide higherphotosynthetic efficiency and CO

2assimilation for the C

4

plants Transferring genes encoding the C4-specific enzymes

phosphoenolpyruvate carboxylase (PEPC) and pyruvateorthophosphate dikinase (PPDK)has led to promising resultsin rice [99] and wheat [100 101] As an example Zhang et al[101] generated transgenic wheat plants overexpressing PEPCand PPDK both separately and simultaneously in the sametransgenic lines The authors showed a positive synergisticeffect onwheat photosynthetic characteristics and yield in thelatter design The follow-up study on wheat expressing maizePEPC demonstrated that in addition to increased yield thetransgenic lines had improved drought tolerance linked toelevated expression of proteins involved in photosynthesisand protein metabolism [102]

The maize gene encoding the transcription factor Dof1known to upregulate the expression of PEPC was introducedinto wheat by Agrobacterium-mediated transformation [103]Expression ofZmDof1under the control of the light-inducibleRuBisCo promoter led to increased biomass and yield com-ponents in transgenic wheat while constitutive expressionresulted in the downregulation of photosynthetic genes anda corresponding negative impact on crop productivity

The Nuclear Factor Y (NF-Y) transcription factors arerecognized as important regulators of many plant develop-mental and physiological processes [104] NF-Ys are com-posed of protein subunits from three distinct transcriptionfactor families (NF-YA NF-YB and NF-YC) with eachof them represented by multiple members [105] TaNFY-A-B1 a low-nitrogen- and low-phosphorus-inducible NF-YA transcription factor was overexpressed in wheat Thisled to a significant increase in nitrogen and phosphorus


uptake and grain yield in a field experiment [106] Theauthors suggested that increased nutrient uptake resultedfrom stimulated root development and upregulation of bothnitrate and phosphate transporters in the roots of the trans-genic wheat Our own work showed the positive role of thesecond wheat gene TaNF-YB4 in wheat grain yield [107]Constitutive overexpression of the gene under the controlof the maize ubiquitin promoter in transgenic wheat cvGladius resulted in the development of significantly morespikes and a 20ndash30 increase in grain yield compared to theuntransformed control plants

Overexpression of another wheat transcription factorTaNAC2-5A which plays a role in nitrogen signallingenhanced root growth and the nitrate influx rate conse-quently increasing the rootrsquos ability to acquire nitrate fromthe soil Transgenic wheat lines revealed higher grain yieldand higher nitrogen accumulation in aerial parts of the plantwhich was subsequently allocated to grains [108]

Recently a US research group reported on the expressionof a rice gene encoding a soluble starch synthase gene (OsSS-I) with increased heat stability This lead to a significant 21-34 yield increase in T

2and T

3generations of transgenic

wheat under heat stress conditions [109] The expression ofOsSS-I also prolonged the duration of the photosyntheticgrowth period in bread wheat Similarly overexpression ofan endogenous gene coding for the chloroplastic glutaminesynthase gene (TaGS2) in wheat led to prolonged leaf pho-tosynthesis and an increased rate of nitrogen remobilizationinto grains which translated to higher spike number grainnumber per spike and total yield [110] Taken togetherthese reports on the modulation of carbon and nitrogenpathways once again illustrate the tight link between nitrogenassimilation and carbon metabolism [111] and resulting cropproductivity

32 Grain Quality Grain is the harvested part of the wheatplant and its nutritional and health properties are determinedby its biochemical composition Starch making up 55ndash75of total dry grain weight and storage protein 10ndash15 arethe main reserves of the wheat seed Therefore starch andprotein greatly affect the quality of the products made fromwheat flour Along with optimized starch and protein anadequate level of essential elements like iron zinc calciumphosphorus and antioxidants is also essential for healthyand balanced wheat products Many of those quality traitshave been addressed in recent years by means of transgenicinterventions

A number of studies have focused on the biosyntheticpathways and composition of starch as reviewed by Son-newald and Kossmann [112] and more recently by Kumar etal [113] Based on the insights gained a number of biotech-nological interventions have been undertaken in order toboth increase the amount of starch and modulate its qualitywith mixed outcomes In an attempt to increase the levelof precursors for starch synthesis in wheat Weichert etal [114] overexpressed the barley sucrose transporter gene(HvSUT1) which led to enhanced sucrose uptake and proteincontent in wheat grains but no significant modification

to starch levels Expression of an optimized maize ADP-glucose pyrophosphorylase (ZmAGPase) resulted in elevatedyield and enhanced photosynthetic rates in the transgenicwheat lines [115] Downregulation of the transcription factorTaRSR1 a wheat homolog of Rice Starch Regulator (OsRSR1)whichwas shown tonegatively regulate the gene expression ofsome starch synthesis-related enzymes in wheat grains [116]resulted in a significant 30 increase in starch content andalso a sim20 increase in yield in terms of 1000-kernel weight[117] The increases in starch and yield were underpinned bythe marked induction of expression of many key-genes insugar metabolism and starch biosynthesis

Starch consumer quality depends mostly on the ratioof amylose to amylopectin the two main macromoleculesforming starch Starch with increased amylose has attractedmuch interest because of its contribution to resistant starch(RS) in food which confers beneficial effects on humanhealth There is evidence that RS can provide protectionfrom several health conditions such as diabetes obesityand cardiovascular diseases [118] A number of experimentsfocused on downregulation of starch branching enzymesSBEIIa and SBEIIb which led to substantially increasedamylose levels in wheat [119 120] High amylose starch hasdemonstrated positive health-related effects in rats [119] andmore recently in a study involving obesity in humans [121]

Nitrogen is not only the most important plant nutrientcontributing to crop yield but also plays a significant rolein defining the accumulation and to some extent the com-position of storage protein in wheat grains [122] Nitrogen issupplied to the grain by two major pathways remobilizationfrom the canopy (leaves and stems) and root uptake fromthe soil In their experiments Zhao et al [123] identifieda novel wheat gene TaNAC-S a member of the NACtranscription factor family that showed decreased expressionduring leaf senescence but significant expression in a stay-green phenotype Transgenic overexpression of the gene inwheat resulted in delayed leaf senescence and increasedprotein concentration in grains while the croprsquos biomass andgrain yield remained unaffected Another group of Chineseresearchers overexpressed a tobacco nitrate reductase gene(NtNR) in two commercial winter wheat cultivars ND146and JM6358 following Agrobacterium-mediated transforma-tion [124] Constitutive overexpression of the gene remark-ably enhanced foliar nitrate reductase activity and resulted insignificantly augmented seed protein content and 1000-grainweight in the majority of the T

1offspring analysed

The main component of wheat storage proteins glutenprimarily determines the viscoelastic properties of wheatdough [125] Glutens consist of gliadins and glutenins whichtogether comprise 70-80 of the total flour protein There-fore genes encoding different classes of storage proteins havebeen targeted in efforts to improve both the nutritional valueand bread-making quality of wheat In fact the genes cod-ing for high-molecular glutenin subunits (HMW-GS) wereamong the first introduced into wheat [126ndash129] with the aimof improving dough functions following the first reports of amethod for wheat transformation Specifically introductionof the subunits 1Ax1 and 1Dx5 into several common wheatcultivars by genetic transformation has demonstrated the


potential of the transgenes to enhance dough quality tovarious extents [130ndash132] In follow-up studies the HMW-GSgenes were introduced into selected wheat cultivars mostlyvia the easily transformed cv Bobwhite and then transferredinto selected elite commercial varieties Improvements indough properties were obtained demonstrating the feasibil-ity of utilizing transgenic lines in wheat breeding programs[133 134]

In contrast to HMW glutenins which form complexpolymer structures and are strongly correlated to dough elas-ticity gliadins are monomeric components that contributemainly to the extensibility and viscosity of the dough [135]Based on their electrophoretic mobility gliadins are groupedinto three structural types 120572- 120574- and 120596-gliadins eachencoded by tightly-linked multigene clusters The interestin genetic modification of gliadins has been stimulated notonly by their contribution to dough quality but also becausethey include the majority of immunogenic epitopes relatedto immune conditions such as wheat-dependent exercise-induced anaphylaxis (WDEIA) and coeliac disease [136 137]

Iron and zinc are essential micronutrients for humannutrition According to the World Health Organization overa billion people suffer from iron-deficiency anaemia andZn-deficiency is estimated to be associated with an annualdeath rate of almost half a million children under the age offive Modern elite wheat cultivars usually contain suboptimalquantities of micronutrients [138] and because most of it isaccumulated in the outer husk aleurone and embryo themicronutrients are lost during milling and polishing [139]Another problem is that phytic acid a major antinutritionalfactor for iron and zinc uptake in the human digestive tract iscodeposited with the minerals in aleurone storage vacuoles

Biofortification the enhancing of crop nutritional qualityis considered a promising approach to alleviatemicronutrientdeficiency Transgenic studies have made significant headwayin the development of strategies aimed at improving the avail-able content of micronutrients such as iron in wheat grainsPlants store iron primarily in the form of ferritin structuresaccumulated mainly in nongreen plastids etioplasts andamyloplasts Expression of a gene coding for an Aspergillusniger phytase a phytic acid degrading enzyme targeted to thewheat aleurone [140] and endogenous [141] or soybean [142]ferritin genes in wheat endosperm were the first successfulattempts to transgenically biofortify wheat grains with ironRecently Connorton et al [143] have isolated characterizedand overexpressed two wheat Vacuolar Iron Transporter(TaVIT) genes under the control of an endosperm-specificpromoter in wheat and barley They reported that the intro-duction of one of the genes TaVIT2 resulted in a greater thantwofold increase in iron in the flour prepared from transgenicwheat grains without other detected changes

4 RNA Interference Applications in Wheat

RNA interference (RNAi) is a common regulatory mecha-nism of gene expression in eukaryotic cells that has becomea powerful tool for functional gene analysis and the engi-neering of novel phenotypes The technique is based onthe expression of antisense or hairpin RNAi constructs or

other forms of short interfering RNA molecules to directposttranscriptional gene silencing in a sequence specificmanner

The vernalisation gene TaVRN2 was the first wheatgene to be targeted by RNAi in transgenic wheat plants[144] TaVRN1 mRNA in transgenic plants was reduced by60 which led to much earlier flowering In another studyLoukoianov et al [145] suppressed expression of TaVRN1 byup to 80 delaying flowering time by 14 to 19 days andincreasing the number of leaves relative to the nontransgeniccontrols These two breakthrough studies provided essentialevidence for understanding the molecular mechanisms offlowering timing and vernalisation requirements in wheatwhich may assist in diversifying the environments in whichwheat can be grown

The application of RNAi has made a solid contributionto manipulating wheat grain size [146ndash148] and quality [119149ndash151] For example Alterbach and Allen [152] used RNAisilencing to suppress the expression of 120596-gliadins associatedwithWDEIA in the US wheat cv Butte 86They later demon-strated that transgenic wheat lines deficient in 120596-gliadinshad no changes in patterns of other grain proteins andeven showed increased protein stability with improved doughproperties under various growth conditions [153] SimilarlyGil-Humanes et al [154] downregulated the 120574-gliadin genesin the wheat cv Bobwhite and then transferred the trait intothree commercial wheat cultivars by conventional crossing

The reduction of 120574-gliadins in wheat grains was com-pensated by an increased amount of glutenins which ledto stronger dough with better over-mixing-resistance Ina recent comprehensive study Barro et al [149] used acombination of sevenRNAi expressing plasmids to selectivelytarget 120572- 120574- and 120596-gliadins and Low Molecular Weight(LMW) glutenins in the wheat cv Bobwhite with the goalto reduce gluten epitopes related to coeliac disease (CD)The protein analyses showed that three RNAi plasmid com-binations resulted in total absence of CD epitopes from themost immunogenic 120572- and120596-gliadins in the transgenic wheatlinesThese very promising results pave theway to developingwheat varieties with nonallergenic properties

However the major application of RNAi technology forwheat has been in pathogen and pest control using virus-and host-induced gene silencing platforms [155] A recentstrategy referred to as host-induced gene silencing (HIGS)has been developed to silence pest or pathogen genes byplant-mediated RNAi during their feeding or attemptedinfection thereby reducing disease levels This strategyrelies on the host-plantrsquos ability to produce interfering RNAmolecules complementary to targeted pestpathogen genesThese molecules are then transferred to the invader causingsilencing of the targeted gene In wheat HIGS has beenmost widely applied to control fungal and insect diseases Asan example silencing of fungal glucanosyltransferase genes(GTF1 and GTF2) and the virulence effector gene Avra10affects wheat resistance to the powdery mildew fungusBlumeria graminis [156] Three hairpin RNAi constructscorresponding to different regions of the Fusarium gramin-earum chitin synthase gene (Chs3b) were found to silenceChs3b in transgenic F graminearum strains Coexpression


of these three RNAi constructs in two independent elitewheat cultivar transgenic lines conferred high levels of stableand durable resistance to both Fusarium head blight andFusarium seedling blight [157] Stable transgenic wheat plantscarrying an RNAi hairpin construct against the 120573-1 3-glucansynthase gene FcGls1 of F culmorum or a triple combinationof FcGls1 with mitogen-activated protein kinase (FcFmk1)and chitin synthase V myosin-motor domain (FcChsV) alsoshowed enhanced resistance in leaf and spike inoculationassays under greenhouse and near-field conditions respec-tively [158]

In other examples of RNAi targeting the myosin-5 gene(FaMyo5) from F asiaticum provided disease resistance inwheat [159] In further examples wheat transformed witha vector expressing a double-stranded RNA targeting themitogen-activated protein kinase kinase gene (PsFUZ7) fromPuccinia striiformis f sp tritici exhibited strong resistance tostripe rust [160] Transgenic wheat plants expressing siRNAstargeting the catalytic subunit protein kinase A gene fromP striiformis f sp Tritici displayed high levels of stable anddurable resistance throughout the T

3to T4generations [161]

Stable expression of hairpin RNAi constructs with a sequencehomologous to mitogen-activated protein-kinase from Ptriticina (PtMAPK1) or a cyclophilin (PtCYC1) encodinggene in susceptible wheat plants showed efficient silencing ofthe corresponding genes in the interacting fungus resultingin disease resistance throughout the T

2generation [162]

The grain aphid (Sitobion avenae) chitin synthase 1 gene(CHS1) was also targeted with HIGS After feeding on therepresentative T

3transgenic wheat lines CHS1 expression

levels in grain aphid decreased by half and both total andmoulting aphid numbers reduced significantly [163] Othertarget genes used for the control of grain aphids by RNAiin transgenic wheat were lipase maturation factor-like2 genefrom pea aphid Acyrthosiphon pisum [164] carboxylesterasegene [165]Hpa1 [166] and a gene encoding a salivary sheathprotein [167] Feeding on these transgenic plants resulted insignificant reductions in survival and reproduction of aphidsIt seems that the scope of RNAi-induced pest and diseaseresistance is as broad as the number of different pathogen andpest species that infect and cause damage to wheat

5 Site-Specific Nucleases for Targeted GenomeModifications in Wheat

Targeted genome engineering using nucleases such as zincfinger nucleases (ZFNs) and TAL effector nucleases (TAL-ENs) were developed in the late 20th century as innovativetools to generate mutations at specific genetic loci [168]Nuclease-based mutagenesis relies on induced site-specificDNA double-strand breaks (DSBs) which are either repairedby error-prone nonhomologous end joining (NHEJ) or high-fidelity homologous recombination (HR) The former oftenresults in insertions or deletions (InDels) at the cleavagesite leading to loss-of-function gene knockouts whereas thelatter leads to precise genome modification In 2012 thefield of eukaryotic genome editing was revolutionized by theintroduction of CRISPRCas9 (bacterial Clustered Regularly

Interspaced Short Palindromic Repeats) technology [169]This technology confers targeted gene mutagenesis by a Cas9nuclease that is guided by small RNAs (sgRNAs) to thetarget gene through base pairing This is in contrast to theDNA-recognition protein domain that must be specificallytailored for each DNA target in the case of ZFNs and TAL-ENs Because of its universality and operational simplicitycompared to ZFNs and TALEN genome editing systemsCRISPRCas9 has rapidly superseded these earlier editingsystems and been adopted by the majority of the scientificcommunity [170 171]

In wheat the principal applicability of CRISPRCas9 wasdemonstrated in protoplasts and suspension cultures wheremultiple genes were successfully targeted in the year follow-ing the publication of the original CRISPRCas9 principle[172ndash174] Original methods for plant genome editing rely onthe delivery of plasmids carrying cassettes for the coexpres-sion of Cas9 and sgRNA either by Agrobacterium or particlebombardment For gene editing in wheat a Cas9 proteincontaining one or more signals for nuclear localization isexpressed from a codon optimized gene under the control ofRNA polymerase II promoters such as CaMV35S or ZmUbiwhile the sgRNA is usually expressed from a polymeraseIII promoter (most commonly rice or wheat U6 and U3promoters)

One of the additional advantages of the CRISPRCas9system is its potential for multiplexing ie the simultaneoustargeting of several genes with a single molecular constructMultiple sgRNAs can be introduced either as separate tran-scription units or in polycistronic form [175] In bread wheatediting has been reported in PEG-transfected protoplasts[172 173 176ndash178] electroporated microspores [179] andcell suspension cultures transformed by Agrobacterium [174]Edited wheat plants have been regenerated from immatureembryos immature embryo-derived callus or shoot apicalmeristems transformed via particle bombardment [176 177180ndash184] or Agrobacterium [185 186]

Recently protocols for DNA-free editing of wheat bydelivering in vitro transcripts or ribonucleoprotein com-plexes (RNPs) of CRISPRCas9 by particle bombardmenthave been developed [176 177] The authors claim thatthese methods not only eliminate random integration ofthe CRISPRCas9 coding DNA elements into the targetedgenome but also reduce off-target effects Thus theseadvances allow for the production of completely transgene-free mutants in bread wheat with high precision The mainlimitation of these transgene-free protocols is the lack ofselection in the transformation and regeneration processAnother optimized delivery system has been developed byGil-Humanes et al [177] Here the authors used replicatedvectors based on the wheat dwarf virus (Geminiviridae)for cereal genome engineering It was shown that due toincreased copy number of the system components virus-derived replicons increase gene targeting efficiency greaterthan 10-fold in wheat callus cells and protoplasts comparedto the nonreplicating control The virus-based CRISPRCas9system also promoted multiplexed gene targeted integrationin different loci of the polyploid wheat genome by homolo-gous recombination


Since the advent of the principle of RNA guided nucleasegenome editing a number of additional tools for genomemodifications and functional genomics studies have beendeveloped [187] The DNA binding ability of Cas9 and Cas12has been used to develop tools for various applications suchas transcriptional regulation and fluorescence-based imagingof specific chromosomal loci in plant genomes Anothernuclease Cas13 has been applied to degrade mRNAs andcombat viral RNA replication [188] Orthologues of Cas9found in other bacterial species such asNeisseriameningitides[189] Staphylococcus aureus [190] and Campylobacter jejuni[191] have different and more complex Protospacer adjacentmotif (PAM) sequences These PAM sequences functionin their native bacterial hosts to direct the CRISPRCas9complex to the target sequence and not to the CRISPRCas9locus Although the use of these orthologous Cas9 proteinsdoes limit the available target sequences for genome editingit also reduces off-target edits Most recently systems fortargeted base editing in wheat have been established by fusinga cytidine deaminase [181] or adenosine deaminase [192] tothe Cas9 nickase for CG to TA or AT to GC conversionIn these systems the efficiency of base editing was enhancedby using a Cas9-based nickase instead of an inactive Cas9As an example the base editor Cas9-APOBEC3A was usedto edit TaMTL (MATRILINEAL) encoding a sperm-specificphospholipase [182] Loss of function ofMTL triggers haploidinduction in maize [193] Ten base-edited wheat mutantswith TaMTL knock-out were identified at a frequency of167 with three being homozygous for all six alleles withoutInDels Functional analysis of wheat mutants with TaMTLknock-out is still to be completed Other nucleases withsimilar editing functions to that of Cas9 have been iden-tified [194] Most notably Cpf1 possesses both DNase andRNase activity and cleaves DNA to generate four to fivebp 51015840-overhangs potentially enhancing insertion of DNAsequences by homologous recombination The Cpf1-basedediting system has been successfully applied in plants but notin wheat at this stage [195 196]

Although many agriculturally important traits of wheathave been targeted by genome editing some of the main onesinclude the following (i) resistancetolerance to biotic andabiotic stresses (ii) yield and grain quality and (iii) malesterility

(i) The first successful experiment using the CRISPRCas9 system in wheat was editing of TaMLO a powderymildew-resistance locus Powdery mildew diseases causedby Blumeria graminis f sp tritici result in significant wheatyield losses and knock-out of the TaMLO leads to diseaseresistance The mutation frequency of TaMLO in protoplastswas 285 [172] Further Wang et al [180] described editingof the TaMLO-A1 allele by the CRISPRCas9 system andsimultaneous editing of three homoeoalleles of TaMLO inhexaploid bread wheat using the TALEN nuclease Themutation frequency of regenerated TaMLO-edited wheat(56) was similar for both editing methods More recentlyZhang et al [174] used CRISPRCas9 technology to generateTaedr1 wheat lines by simultaneous knock-down of the threehomologs of wheat TaEDR1 a negative regulator of powderymildew resistance The mutated plants were resistant to

powdery mildew and did not show mildew-induced celldeath [174]

The lipoxygenase genes TaLpx1 and TaLox2 attractedattention as potential subjects for gene editing in relation toresistance to Fusarium one of the most devastating fungaldiseases in wheat Lipoxygenases hydrolyze polyunsaturatedfatty acids and initiate biosynthesis of oxylipins playing arole in the activation of jasmonic acid-mediated defenceresponses in plants Silencing of theTaLpx-1 gene has resultedin resistance toFusariumgraminearum inwheat [197]TaLpx1and TaLox2 genes were edited in protoplasts with a mutationfrequency of 9 and 45 respectively [173 183] Wheatplants with mutated TaLOX2 were obtained with a frequencyof 95 of which homozygous mutants accounted for 447[198]

The CRISPRCas9 system was also used for editing awheat homolog of TaCer9 (ECERIFERUM9) with the goalto improve drought tolerance and water use efficiency [199]Mutation of the AtCer9 gene in A thaliana encoding an E3ubiquitin ligase causes elevated amounts of 18-carbon-lengthcutin monomers and very-long-chain free fatty acids (C24C26) in cuticular wax both of which are associated withelevated cuticle membrane thickness and drought tolerance[200]

The Cas9 nickase fused to a human cytidine deaminaseAPOBEC3A was used to produce herbicide resistant wheatplants through editing of TaALS [182] ALS encodes acetolac-tate synthase the first enzyme of the branched-chain aminoacid biosynthesis Amino acid substitutions in TaALS conferresistance to the sulfonylurea class of herbicides Wheatplants with a mutated TaALS gene were obtained in highfrequency (225 27120) Among them two plants had sixalleles simultaneously edited and were nicosulfuron resistant

(ii) With the aim of enhancing grain size and yieldseveral genes have been edited by the CRISPRCas9 sys-tem TaGASR7 [176 184 198] TaGW2 [198 199 201] andTaDEP1 [198] TaGASR7 a member of the SnakinGASA genefamily has been associated with grain length in wheat ACRISPRCas9 vector designed to target TaGASR7 was deliv-ered by particle bombardment into shoot apical meristemsEleven (52) of the 210 bombarded plants carried mutantalleles and the mutations of three (14) of these wereinherited in the next generation [184] Transiently expressingthe CRISPRCas9 DNA and using the CRISPRCas9 RNP-mediated method were also highly effective for TaGASR7editing [176 198] The TaGW2 gene encodes a previouslyunknown RING-type E3 ubiquitin ligase that was reportedto be a negative regulator of grain size and thousand grainweight in wheat [198 199 201] Recent studies detailing thefunctionality of the allelic TaGW2 genes through genome-specific knockouts [201] showed that the TaGW2 gene inwheat acts by regulating the gibberellin hormone biosyn-thesis pathway [202] principally confirming the parallelfunctions of these genes in rice and wheat T

1plants carrying

knock-out mutations in all three copies of the TaGW2 gene(genotype aabbdd) showed significantly increased thousandgrain weight (277) grain area (170) grain width (109)and grain length (61) compared to the wild-type cultivar[183]


Another gene editing target is DENSE AND ERECTPANICLE 1 (DEP1) which encodes a G protein gamma-subunit in rice that is involved in the regulation of erectpanicles number of grains per panicle nitrogen uptake andstress tolerance through the G protein signalling pathway[203] Zhang et al [198] applied the CRISPRCas9 system totarget TaDEP1 and TaNAC2 editing in wheat CRISPRCas9-induced mutation of TaNAC2 (a member of a plant specifictranscription factor family) and TaDEP1 in wheat plants wassuccessful in 2 of cases [198] One possible outcome of theloss of TaNAC2 function could be increased size of grainand changes in stress responses TaPIN1 (PINFORMED1)was edited using CRISPRCas9 technology with a frequencyof 1 [198] The plant specific PIN family of efflux car-riers comprises integral membrane proteins and has beenassociated with polar auxin transport during embryogenesisand endosperm development It should be noted that nohomozygous mutants were obtained for TaDEP1 TaNAC2and TaPIN1 by CRISPRCas9 editing [198] probably becausethey were not viable

CRISPRCas9 technology was also successful inobtaining low immunogenic wheat Sanchez-Leon et al[204] have shown that CRISPRCas9 technology can beused to efficiently reduce the amount of alfa-gliadins inthe seeds providing bread and durum wheat lines withreduced immunoreactivity for consumers with coeliacdisease Twenty-one mutant lines were generated (15 breadwheat and 6 durum wheat) all showing a strong reductionin 120572-gliadins Up to 35 of the 45 different genes identifiedin the wild-type were mutated in one of the lines andimmunoreactivity as measured by competitive ELISAassays using two monoclonal antibodies was reduced by85

(iii) Male sterility and the induction of haploids canprovide a powerful tool in cultivar breeding and geneticanalysis Generation of male-sterile and doubled haploidplants can facilitate development of hybrid seed productionin wheat In maize the male-sterile gene 45 (Ms45)encodes a strictosidine synthase-like enzyme that wasshown to be required for male fertility and required forexine structuring and pollen development [185] Geneticanalysis of mutated plants obtained by CRISPRCas9technology demonstrated that all three wheat Ms45homeologues contribute to male fertility Mutant plantsTams45-abd abort pollen development resulting in malesterility

These examples provide an insight into the many waysin which modern genome modification technologies arebeing used to mine the core research findings from modelplant transgenesis and finally harness that understanding todrive essential crop The ability to enact targeted changesto the genome has revolutionized genetic modification forpolyploid crop species such as wheat There are now someenticing indications that the nearest remaining hurdle in theform of low transformation frequencies in favoured breedinggenotypes may soon be overcome What remains is forpolitical entities and the societies they represent to adopta more receptive view to the very real and practical benefitsthese technologies may provide

6 Conclusions and Prospective Developments

Demand for wheat is projected to rise at a rate of 16annually until 2050 as a result of increased population andprosperity Consequently average global wheat yields on a perhectare basis will need to increase to approximately 5 tonnesper ha from the current 33 tonnes [205] Bread wheat has avery complicated hexaploid genome and therefore furtherprogress in breeding of this crop is strongly dependent uponknowledge of functional genomics Thus it is necessaryto identify the most important key-genes their structurerole and function in the development of wheat plants andfinally for higher grain yield and better quality Based on theknowledge of functional genomics plant biologists can alterthe structures and functions of selected key-genes throughldquogenetic manipulationrdquo Genetic transformation is a verypowerful tool for generating scientific proof of the roles andfunctions of key-genes The authors of this review are notin a position to discuss the applications of GM-wheat inworld breeding practice since this is beyond the scope ofthis review However the term ldquogenetic manipulationrdquo is verybroad and includes other molecular approaches that generateproducts that fall outside the traditional definition of ldquoGMrdquoRNA interference and CRISPRCas9 represent modern andvery advanced GM technologies that in a growing numberof countries such as USA and Canada result in productsthat attract the same level of regulation as the products oftraditional breeding techniques Such ldquoend-product-basedrdquorather than ldquoprocess-basedrdquo regulation presents a far morefavourable environment for the progression of molecular-based breeding technologies which can and should changethe future of wheat breeding across the world However alladvanced methods will remain simply ldquolaboratory toolsrdquo iftheir application is not connected with wheat breeders cur-rently working by traditional methods Therefore we see thechance for real progress and positive future prospects througheffective collaborations between plant molecular geneticistsand wheat breeders The application of novel methods andanalysis of genetically manipulated wheat plants for their util-ity in breeding can be translated to the field through the intro-gression of genetic traits into conventional wheat breedingprograms

Awareness and concerns are also growing regardingthe huge economic gaps between developed and develop-ing countries Underdeveloped countries are more relianton agriculture for their overall economies yet they havefewer opportunities to develop or collaborate on projectsinvolving modern technologies for genetic manipulation inwheat and most other crop plants Therefore researchersin developed countries must take the lead and assumeresponsibility for sharing and freely disseminating theirresults and genetically manipulated wheat germplasm acces-sions with breeders in the developing world This actof ldquoresearch donationrdquo can enrich lives and communitieswhere needs are the greatest and uphold the future secu-rity and sustainability of wheat production a key globalcommodity


Conflicts of Interest

The authors declare that the research was conducted in theabsence of any potential conflicts of interest

Acknowledgments

We want to thank the staff and students of Huaiyin NormalUniversity Huaian China Flinders University of South Aus-tralia SA Australia and S Seifullin Kazakh AgroTechnicalUniversity Astana Kazakhstan for their support in thisresearch and help with critical comments to the manuscriptThis research was supported by a personal grant to NB fromthe Huaiyin Normal University Huaian China The Min-istry of Education and Science (Kazakhstan) also providedfinancial support for this research through Research ProgramBR05236500 (SJ)

References

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[2] R E Evenson and D Gollin ldquoAssessing the impact of the greenrevolution 1960 to 2000rdquo Science vol 300 no 5620 pp 758ndash762 2003

[3] P Langridge ldquoWheat genomics and the ambitious targets forfuture wheat productionrdquo Genome vol 56 no 10 pp 545ndash5472013

[4] R J Henry P Rangan and A Furtado ldquoFunctional cereals forproduction in new and variable climatesrdquo Current Opinion inPlant Biology vol 30 pp 11ndash18 2016

[5] F Altpeter N M Springer L E Bartley et al ldquoAdvancing croptransformation in the era of genome editingrdquoePlant Cell vol28 pp 1510ndash1520 2016

[6] F Altpeter N Baisakh R Beachy et al ldquoParticle bombardmentand the genetic enhancement of crops myths and realitiesrdquoMolecular Breeding vol 15 no 3 pp 305ndash327 2005

[7] H Wu F S Awan A Vilarinho et al ldquoTransgene integra-tion complexity and expression stability following biolistic oragrobacterium-mediated transformation of sugarcanerdquo In VitroCellularampDevelopmental Biology - Plant vol 51 no 6 pp 603ndash611 2015

[8] Y Ishida M Tsunashima Y Hiei and T Komari ldquoWheat(triticum aestivum L) transformation using immatureembryosrdquo in Agrobacterium Protocols vol 1223 of Methods inMolecular Biology pp 189ndash198 Springer New York New YorkNY USA 2015

[9] A Pellegrineschi L M Noguera B Skovmand et al ldquoIden-tification of highly transformable wheat genotypes for massproduction of fertile transgenic plantsrdquo Genome vol 45 no 2pp 421ndash430 2002

[10] C Tassy and P Barret ldquoBiolistic transformation of wheatrdquo inWheat Biotechnology Methods and Protocols P Bhalla and MSingh Eds vol 1679 of Methods in Molecular Biology pp 141ndash152 Springer New York New York NY 2017

[11] H D Jones A Doherty and C A Sparks ldquoTransient trans-formation of plantsrdquo in Plant Genomics J M Walker Ed vol513 ofMethods inMolecular Biology pp 131ndash152 Humana PressTotowa NJ USA 2009

[12] A C Brasileiro C Tourneur J Leple V Combes and LJouanin ldquoExpression of the mutant arabidopsis thaliana ace-tolactate synthase gene confers chlorsulfuron resistance totransgenic poplar plantsrdquo Transgenic Research vol 1 no 3 pp133ndash141 1992

[13] J Mullen G Adam A Blowers and E Earle ldquoBiolistic transferof large DNA fragments to tobacco cells using YACs retro fittedfor plant transformationrdquo Molecular Breeding vol 4 no 5 pp449ndash457 1998

[14] Y Wang H Zeng X Zhou et al ldquoTransformation of rice withlarge maize genomic DNA fragments containing high contentrepetitive sequencesrdquoPlant Cell Reports vol 34 no 6 pp 1049ndash1061 2015

[15] S De Buck C DeWildeM VanMontagu andA Depicker ldquoT-DNA vector backbone sequences are frequently integrated intothe genome of transgenic plants obtained by Agrobacterium-mediated transformationrdquo Molecular Breeding vol 6 pp 459ndash468 2000

[16] B Ulker Y Li M G Rosso E Logemann I E Somssich and BWeisshaar ldquoT-DNA-mediated transfer of Agrobacterium tume-faciens chromosomal DNA into plantsrdquo Nature Biotechnologyvol 26 no 9 pp 1015ndash1017 2008

[17] AGadaleta AGiancaspro A Blechl andA Blanco ldquoPhospho-mannose isomerase pmi as a selectablemarker gene for durumwheat transformationrdquo Journal of Cereal Science vol 43 pp 31ndash37 2006

[18] A Gadaleta A Giancaspro A E Blechl and A Blanco ldquoAtransgenic durum wheat line that is free of marker genes andexpresses 1Dy10rdquo Journal of Cereal Science vol 48 no 2 pp439ndash445 2008

[19] K Wang H Liu L Du and X Ye ldquoGeneration of marker-free transgenic hexaploidwheat via an agrobacterium-mediatedco-transformation strategy in commercial Chinese wheat vari-etiesrdquo Plant Biotechnology Journal vol 15 no 5 pp 614ndash6232017

[20] T Risacher M Craze S Bowden W Paul and T BarsbyldquoHighly efficient agrobacterium-mediated transformation ofwheat via in planta inoculationrdquoMethods in Molecular Biologyvol 478 pp 115ndash124 2009

[21] J M Zale S Agarwal S Loar and C M Steber ldquoEvidence forstable transformation of wheat by floral dip in agrobacteriumtumefaciensrdquo Plant Cell Reports vol 28 no 6 pp 903ndash9132009

[22] H Hamada Q Linghu Y Nagira R Miki N Taoka and RImai ldquoAn in planta biolistic method for stable wheat transfor-mationrdquo Scientific Reports vol 7 article 11443 2017

[23] S Martin-Ortigosa and K Wang ldquoProteolistics a biolis-tic method for intracellular delivery of proteinsrdquo TransgenicResearch vol 23 no 5 pp 743ndash756 2014

[24] C Remacle P Cardol N Coosemans M Gaisne and NBonnefoy ldquoHigh-efficiencybiolistic transformation of Chlamy-domonas mitochondria can be used to insert mutations incomplex I genesrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 103 no 12 pp 4771ndash4776 2006

[25] L Cheng H Li B Qu et al ldquoChloroplast transformationof rapeseed (Brassica napus) by particle bombardment ofcotyledonsrdquo Plant Cell Reports vol 29 no 4 pp 371ndash381 2010

[26] Y Gleba V Klimyuk and S Marillonnet ldquoMagnifection - anew platform for expressing recombinant vaccines in plantsrdquoVaccine vol 23 no 17-18 pp 2042ndash2048 2005


[27] S Ueki S Magori B Lacroix and V Citovsky ldquoTransient geneexpression in epidermal cells of plant leaves by biolistic DNAdeliveryrdquo in Biolistic DNA Delivery Methods and ProtocolsMethods inMolecular Biology S Sudowe andA B Reske-KunzEds vol 940 pp 17ndash26 2013

[28] P Krenek O Samajova I Luptovciak A Doskocilova GKomis and J Samaj ldquoTransient plant transformation mediatedby Agrobacterium tumefaciens Principles methods and appli-cationsrdquo Biotechnology Advances vol 33 no 6 pp 1024ndash10422015

[29] N Goncharov and E Kondratenk] ldquoOrigin domestication andevolution of wheatsrdquo Vestnik VOGiS vol 12 pp 159ndash179 2008httpwwwbionetnscruvogispict pdf2008t12 1 2vogis12 1 2 15pdf

[30] J D Montenegro A A Golicz P E Bayer et al ldquoThepangenome of hexaploid bread wheatrdquo e Plant Journal vol90 no 5 pp 1007ndash1013 2017

[31] P L Bhalla A Sharma and M B Singh ldquoEnabling moleculartechnologies for trait improvement in wheatrdquo inWheat Biotech-nology vol 1679 of Methods in Molecular Biology pp 3ndash24Springer New York New York NY USA 2017

[32] R Appels K Eversole C Feuillet et al ldquoShifting the limits inwheat research and breeding using a fully annotated referencegenomerdquo Science vol 361 no 6403 article eaar7191 2018

[33] A K Shrawat and C L Armstrong ldquoDevelopment and appli-cation of genetic engineering for wheat improvementrdquo CriticalReviews in Plant Sciences vol 37 no 5 pp 335ndash421 2018

[34] V Vasil AM CastilloM E Fromm and I K Vasil ldquoHerbicideresistant fertile transgenic wheat plants obtained by micro-projectile bombardment of regenerable embryogenic callusrdquoNature Biotechnology vol 10 no 6 pp 667ndash674 1992

[35] M Cheng J E Fry S Pang et al ldquoGenetic transformation ofwheat mediated by agrobacterium tumefaciensrdquo Plant Physiol-ogy vol 115 no 3 pp 971ndash980 1997

[36] Q Yao L Cong J L Chang K X Li G X Yang and G YHe ldquoLow copy number gene transfer and stable expression in acommercial wheat cultivar via particle bombardmentrdquo Journalof Experimental Botany vol 57 no 14 pp 3737ndash3746 2006

[37] C Tassy A Partier M Beckert C Feuillet and P BarretldquoBiolistic transformation of wheat increased production ofplants with simple insertions and heritable transgene expres-sionrdquo Plant Cell Tissue and Organ Culture (PCTOC) vol 119no 1 pp 171ndash181 2014

[38] A Ismagul N Yang E Maltseva et al ldquoA biolistic method forhigh-throughput production of transgenic wheat plants withsingle gene insertionsrdquo BMC Plant Biology vol 18 pp 135ndash1432018

[39] Q Yao L Cong G He et al ldquoOptimization of wheat co-transformation procedure with gene cassettes resulted in animprovement in transformation frequencyrdquo Molecular BiologyReports vol 34 pp 61ndash67 2007

[40] A Partier GGay C TassyM Beckert C Feuillet andP BarretldquoMolecular and FISH analyses of a 53-kbp intactDNA fragmentinserted by biolistics in wheat (triticum aestivum L) genomerdquoPlant Cell Reports vol 36 no 10 pp 1547ndash1559 2017

[41] M Wright J Dawson E Dunder et al ldquoEfficient biolistictransformation of maize (Zea mays L) and wheat (Triticumaestivum L) using the phosphomannose isomerase gene pmias the selectable markerrdquo Plant Cell Reports vol 20 no 5 pp429ndash436 2001

[42] T Richardson J Thistleton T J Higgins C Howitt and MAyliffe ldquoEfficient agrobacterium transformation of elite wheat

germplasm without selectionrdquo Plant Cell Tissue and OrganCulture (PCTOC) vol 119 no 3 pp 647ndash659 2014

[43] G Hensel C Marthe and J Kumlehn ldquoAgrobacterium-mediated transformation of wheat using immature embryosrdquo inWheat Biotechnology P Bhalla and M Singh Eds vol 1679 ofMethods in Molecular Biology pp 129ndash139 Springer New YorkNew York NY USA 2017

[44] K Wang B Riaz and X Ye ldquoWheat genome editing expeditedby efficient transformation techniques progress and perspec-tivesrdquoe Crop Journal vol 6 no 1 pp 22ndash31 2018

[45] G R Lazo P A Stein and R A Ludwig ldquoA DNA transfor-mation-competent Arabidopsis genomic library in agrobac-teriumrdquo Nature Biotechnology vol 9 no 10 pp 963ndash967 1991

[46] N R Peters S Ackerman and E A Davis ldquoA modular vectorfor Agrobacterium mediated transformation of wheatrdquo PlantMolecular Biology Reporter vol 17 no 4 pp 323ndash331 1999

[47] N V Larebeke G Engler M Holsters et al ldquoLarge plasmidin agrobacterium tumefaciens essential for crown gall-inducingabilityrdquo Nature vol 252 no 5479 pp 169-170 1974

[48] YWangM Xu G Yin L Tao DWang andX Ye ldquoTransgenicwheat plants derived from Agrobacterium-mediated transfor-mation of mature embryo tissuesrdquo Cereal Research Communi-cations vol 37 pp 1ndash12 2009

[49] P Supartana T Shimizu M Nogawa et al ldquoDevelopmentof simple and efficient in planta transformation method forwheat (triticum aestivum l) using agrobacterium tumefaciensrdquoJournal of Bioscience and Bioengineering vol 102 no 3 pp 162ndash170 2006

[50] H Aadel R Abdelwahd S Udupa et al ldquoAgrobacterium-mediated transformation of mature embryo tissues of breadwheat (triticum aestivum L) genotypesrdquo Cereal Research Com-munications vol 46 pp 10ndash20 2018

[51] E Medvecka and W A Harwood ldquoWheat (triticum aestivumL) transformation using mature embryosrdquo in AgrobacteriumProtocols Volume 1 K Wang Ed vol 1223 of Methods inMolecular Biology pp 199ndash209 Springer New York New YorkNY USA 2015

[52] H Chauhan and P Khurana ldquoWheat genetic transformationusing mature embryos as explantsrdquo in Wheat BiotechnologyMethods and Protocols P Bhalla andM Singh Eds vol 1679 ofMethods in Molecular Biology pp 153ndash167 Humana Press NewYork NY USA 2017

[53] T Zhao S Zhao H Chen et al ldquoTransgenic wheat progenyresistant to powdery mildew generated by agrobacteriuminoculum to the basal portion of wheat seedlingrdquo Plant CellReports vol 25 no 11 pp 1199ndash1204 2006

[54] A Razzaq I A Hafiz I Mahmood and A Hussain ldquoDevelop-ment of in planta transformation protocol for wheatrdquo AfricanJournal of Biotechnology vol 10 no 5 pp 740ndash750 2011

[55] S Rustgi N O Ankrah R A T Brew-Appiah et al ldquoDoubledhaploid transgenic wheat lines by microspore transformationrdquoin Wheat Biotechnology Methods and Protocols P Bhalla andM Singh Eds vol 1679 of Methods in Molecular Biology pp213ndash234 Humana Press New York NY USA 2017

[56] L Folling and A Olesen ldquoTransformation of wheat (triticumaestivum L) microspore-derived callus and microspores byparticle bombardmentrdquo Plant Cell Reports vol 20 no 7 pp629ndash636 2001

[57] R A Brew-Appiah N Ankrah W Liu et al ldquoGeneration ofdoubled haploid transgenic wheat lines bymicrospore transfor-mationrdquo PLoS ONE vol 8 no 11 article e80155 2013


[58] K Singer Y M Shiboleth J Li and T Tzfira ldquoFormationof complex extrachromosomal T-DNA structures in agrobac-terium tumefaciens-infected plantsrdquo Plant Physiology vol 160pp 511ndash522 2012

[59] S Kim and G An ldquoBacterial transposons are co-transferredwith T-DNA to rice chromosomes during Agrobacterium-mediated transformationrdquoMolecules and Cells vol 33 no 6 pp583ndash589 2012

[60] A Pitzschke ldquoAgrobacterium infection and plant defense-transformation success hangs by a threadrdquo Frontiers in PlantScience vol 4 no 519 2013

[61] E Zuniga-Soto E Mullins and B Dedicova ldquoEnsifer-mediatedtransformation an efficient non-agrobacteriumprotocol for thegenetic modification of ricerdquo SpringerPlus vol 4 pp 1ndash10 2015

[62] M S Greer I Kovalchuk and F Eudes ldquoAmmonium nitrateimproves direct somatic embryogenesis and biolistic transfor-mation of Triticum aestivumrdquo New Biotechnology vol 26 no1-2 pp 44ndash52 2009

[63] Y Dan ldquoBiological functions of antioxidants in plant transfor-mationrdquo In Vitro Cellular amp Developmental Biology - Plant vol44 no 3 pp 149ndash161 2008

[64] Y Dan C L Armstrong J Dong et al ldquoLipoic acidmdashanunique plant transformation enhancerrdquo In Vitro Cellular ampDevelopmental Biology - Plant vol 45 no 6 pp 630ndash638 2009

[65] Y Coskun R E Duran C Savaskan T Demirci and MT Hakan ldquoEfficient plant regeneration with arabinogalactan-proteins on various ploidy levels of cerealsrdquo Journal of Integra-tive Agriculture vol 12 no 3 pp 420ndash425 2013

[66] R Kumar H M Mamrutha A Kaur et al ldquoDevelopmentof an efficient and reproducible regeneration system in wheat(triticum aestivum L)rdquo Physiology and Molecular Biology ofPlants vol 23 no 4 pp 945ndash954 2017

[67] D Miroshnichenko I Chaban M Chernobrovkina S Dolgovand R P Niedz ldquoProtocol for efficient regulation of in vitromorphogenesis in einkorn (triticum monococcum L) a recal-citrant diploid wheat speciesrdquo PLoS ONE vol 12 no 3 ArticleID e0173533 2017

[68] R A Jefferson ldquoAssaying chimeric genes in plants the GUSgene fusion systemrdquoPlantMolecularBiology Reporter vol 5 no4 pp 387ndash405 1987

[69] M Hraska S Rakousky and V Curn ldquoGreen fluorescentprotein as a vital marker for non-destructive detection oftransformation events in transgenic plantsrdquo Plant Cell Tissueand Organ Culture vol 86 no 3 pp 303ndash318 2006

[70] J Zhang D Yu Y Zhang et al ldquoVacuum and co-cultivationagroinfiltration of (Germinated) seeds results in tobacco rattlevirus (TRV) mediated whole-plant virus-induced gene silenc-ing (VIGS) in wheat and maizerdquo Frontiers in Plant Science vol8 no 393 2017

[71] S Naqvi G Farre G Sanahuja T Capell C Zhu and PChristou ldquoWhen more is better multigene engineering inplantsrdquo Trends in Plant Science vol 15 no 1 pp 48ndash56 2010

[72] W Yu Y Yau and J A Birchler ldquoPlant artificial chromosometechnology and its potential application in genetic engineeringrdquoPlant Biotechnology Journal vol 14 no 5 pp 1175ndash1182 2016

[73] J Yuan Q Shi X Guo et al ldquoSite-specific transfer of chromo-somal segments and genes in wheat engineered chromosomesrdquoJournal of Genetics and Genomics vol 44 no 11 pp 531ndash5392017

[74] A Furtado R J Henry and A Pellegrineschi ldquoAnalysis ofpromoters in transgenic barley and wheatrdquo Plant BiotechnologyJournal vol 7 no 3 pp 240ndash253 2009

[75] S Alotaibi C Sparks M Parry A Simkin and C RainesldquoIdentification of leaf promoters for use in transgenic wheatrdquoPlants vol 7 no 2 p E27 2018

[76] G Hensel A Himmelbach W Chen D K Douchkov andJ Kumlehn ldquoTransgene expression systems in the triticeaecerealsrdquo Journal of Plant Physiology vol 168 pp 30ndash44 2011

[77] N Borisjuk M Hrmova and S Lopato ldquoTranscriptionalregulation of cuticle biosynthesisrdquo Biotechnology Advances vol32 no 2 pp 526ndash540 2014

[78] H Wang H Wang H Shao and X Tang ldquoRecent advancesin utilizing transcription factors to improve plant abiotic stresstolerance by transgenic technologyrdquo Frontiers in Plant Sciencevol 7 no 67 2016

[79] H Bi J Shi N Kovalchuk et al ldquoOverexpression of theTaSHN1 transcription factor in bread wheat leads to leafsurface modifications improved drought tolerance and noyield penalty under controlled growth conditionsrdquo Plant Cellamp Environment vol 41 no 11 pp 2549ndash2566 2018

[80] R P Singh P K Singh J Rutkoski et al ldquoDisease impact onwheat yield potential and prospects of genetic controlrdquo AnnualReview of Phytopathology vol 54 pp 303ndash322 2016

[81] A E Ricroch ldquoWhat will be the benefits of biotech wheat foreuropean agriculturerdquoMethods of Molecular Biology vol 1679pp 25ndash35 2017

[82] B Keller T Wicker and S G Krattinger ldquoAdvances in wheatand pathogen genomics implications for disease controlrdquoAnnual Review of Phytopathology vol 56 pp 67ndash87 2018

[83] H Budak B Hussain Z Khan N Z Ozturk and NUllah ldquoFrom genetics to functional genomics improvement indrought signaling and tolerance in wheatrdquo Frontiers in PlantScience vol 19 no 1012 2015

[84] S Khan M Li S Wang and H Yin ldquoRevisiting the role ofplant transcription factors in the battle against abiotic stressrdquoInternational Journal of Molecular Sciences vol 19 no 6 pE1634 2018

[85] Y Yu D Zhu C Ma et al ldquoTranscriptome analysis revealskey differentially expressed genes involved in wheat graindevelopmentrdquoe Crop Journal vol 4 no 2 pp 92ndash106 2016

[86] A Nadolska-Orczyk I K Rajchel W Orczyk and S GasparisldquoMajor genes determining yield-related traits in wheat andbarleyrdquoeoretical andAppliedGenetics vol 130 no 6 pp 1081ndash1098 2017

[87] R J Henry A Furtado and P Rangan ldquoWheat seed transcrip-tome reveals genes controlling key traits for human preferenceand crop adaptationrdquo Current Opinion in Plant Biology vol 45pp 231ndash236 2018

[88] V C Gegas A Nazari S Griffiths et al ldquoA genetic frameworkfor grain size and shape variation in wheatrdquoe Plant Cell vol22 no 4 pp 1046ndash1056 2010

[89] Z Su C Hao L Wang Y Dong and X Zhang ldquoIdentificationand development of a functional marker of TaGW2 associatedwith grain weight in bread wheat (triticum aestivum L)rdquoeoretical and Applied Genetics vol 122 pp 211ndash223 2011

[90] X Song W Huang M Shi M Zhu andH Lin ldquoAQTL for ricegrain width and weight encodes a previously unknown RING-type E3 ubiquitin ligaserdquoNature Genetics vol 39 no 5 pp 623ndash630 2007

[91] J Bednarek A Boulaflous C Girousse et al ldquoDown-regulationof the TaGW2 gene by RNA interference results in decreasedgrain size and weight in wheatrdquo Journal of Experimental Botanyvol 63 no 16 pp 5945ndash5955 2012


[92] Y Hong L Chen L Du et al ldquoTranscript suppression ofTaGW2 increased grain width and weight in bread wheatrdquoFunctional amp Integrative Genomics vol 14 no 2 pp 341ndash3492014

[93] W Zalewski W Orczyk S Gasparis and A Nadolska-OrczykldquoHvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different pheno-typesrdquo BMC Plant Biology vol 12 no 206 2012

[94] P J Andralojc E Carmo-Silva G E Degen and M A JParry ldquoIncreasing metabolic potential C-fixationrdquo Essays inBiochemistry vol 62 pp 109ndash118 2018

[95] A Prins D J Orr P J Andralojc M P Reynolds E Carmo-Silva and M A Parry ldquoRubisco catalytic properties of wildand domesticated relatives provide scope for improving wheatphotosynthesisrdquo Journal of Experimental Botany vol 67 no 6pp 1827ndash1838 2016

[96] M T Lin A Occhialini P J Andralojc M A Parry andM R Hanson ldquoA faster Rubisco with potential to increasephotosynthesis in cropsrdquoNature vol 513 no 7519 pp 547ndash5502014

[97] E Carmo-Silva J C Scales P J Madgwick and M A ParryldquoOptimizing Rubisco and its regulation for greater resource useefficiencyrdquo Plant Cell amp Environment vol 38 no 9 pp 1817ndash1832 2015

[98] R A Richards ldquoSelectable traits to increase crop photosynthesisand yield of grain cropsrdquo Journal of Experimental Botany vol 51pp 447ndash458 2000

[99] Y Taniguchi H Ohkawa CMasumoto et al ldquoOverproductionof C4 photosynthetic enzymes in transgenic rice plants anapproach to introduce the C4-like photosynthetic pathway intoricerdquo Journal of Experimental Botany vol 59 no 7 pp 1799ndash1809 2007

[100] L Hu Y Li W Xu et al ldquoImprovement of the photosyntheticcharacteristics of transgenic wheat plants by transformationwith the maize C4 phosphoenolpyruvate carboxylase generdquoPlant Breeding vol 131 no 3 pp 385ndash391 2012

[101] H Zhang W Xu H Wang et al ldquoPyramiding expressionof maize genes encoding phosphoenolpyruvate carboxylase(PEPC) and pyruvate orthophosphate dikinase (PPDK) syner-gistically improve the photosynthetic characteristics of trans-genic wheatrdquo Protoplasma vol 251 no 5 pp 1163ndash1173 2014

[102] N Qin W Xu L Hu et al ldquoDrought tolerance and proteomicsstudies of transgenic wheat containing the maize C4 phospho-enolpyruvate carboxylase (PEPC) generdquo Protoplasma vol 253no 6 pp 1503ndash1512 2016

[103] P A Pena T Quach S Sato et al ldquoExpression of themaize Dof1transcription factor in wheat and sorghumrdquo Frontiers in PlantScience vol 8 no 434 2017

[104] Z A Myers and B F Holt III ldquoNuclear factor-Y still complexafter all these yearsrdquo Current Opinion in Plant Biology vol 45pp 96ndash102 2018

[105] M E Zanetti C Rıpodas and A Niebel ldquoPlant NF-Y tran-scription factors key players in plant-microbe interactions rootdevelopment and adaptation to stressrdquo Biochimica et BiophysicaActa (BBA) - Gene Regulatory Mechanisms vol 1860 no 5 pp645ndash654 2017

[106] B Qu X He J Wang et al ldquoA wheat CCAAT box-bindingtranscription factor increases the grain yield of wheat with lessfertilizer inputrdquo Plant Physiology vol 167 no 2 pp 411ndash4232015

[107] D Yadav Y ShavrukovN Bazanova et al ldquoConstitutive overex-pression of the TaNF-YB4 gene in transgenic wheat significantly

improves grain yieldrdquo Journal of Experimental Botany vol 66no 21 pp 6635ndash6650 2015

[108] X He B QuW Li et al ldquoThe nitrate-inducibleNAC transcrip-tion factor TaNAC2-5A controls nitrate response and increaseswheat yieldrdquo Plant Physiology vol 169 pp 1991ndash2005 2015

[109] B Tian S K Talukder J Fu A K Fritz and H N TrickldquoExpression of a rice soluble starch synthase gene in transgenicwheat improves the grain yield under heat stress conditionsrdquo InVitro CellularampDevelopmental Biology - Plant vol 54 no 3 pp216ndash227 2018

[110] MHu X ZhaoQ Liu et al ldquoTransgenic expression of plastidicglutamine synthetase increases nitrogen uptake and yield inwheatrdquoPlant Biotechnology Journal vol 16 no 11 pp 1858ndash18672018

[111] X Zhu S P Long and D R Ort ldquoWhat is the maximumefficiency with which photosynthesis can convert solar energyinto biomassrdquo Current Opinion in Biotechnology vol 19 no 2pp 153ndash159 2008

[112] U Sonnewald and J Kossmann ldquoStarches-from currentmodelsto genetic engineeringrdquo Plant Biotechnology Journal vol 11 no2 pp 223ndash232 2013

[113] R Kumar S Mukherjee and B T Ayele ldquoMolecular aspectsof sucrose transport and its metabolism to starch during seeddevelopment in wheat a comprehensive reviewrdquo BiotechnologyAdvances vol 36 no 4 pp 954ndash967 2018

[114] NWeichert I Saalbach HWeichert et al ldquoIncreasing sucroseuptake capacity of wheat grains stimulates storage proteinsynthesisrdquo Plant Physiology vol 152 no 2 pp 698ndash710 2010

[115] E D Smidansky F D Meyer B Blakeslee T E WeglarzT W Greene and M J Giroux ldquoExpression of a modifiedADP-glucose pyrophosphorylase large subunit in wheat seedsstimulates photosynthesis and carbon metabolismrdquo Planta vol225 no 4 pp 965ndash976 2007

[116] G Kang W Xu G Liu X Peng T Guo and J Bell ldquoCompre-hensive analysis of the transcription of starch synthesis genesand the transcription factor RSR1 in wheat (triticum aestivum)endospermrdquo Genome vol 56 no 2 pp 115ndash122 2013

[117] G Liu Y Wu M Xu et al ldquoVirus-Induced gene silencingidentifies an important role of the TaRSR1 transcription factorin starch synthesis in bread wheatrdquo International Journal ofMolecular Sciences vol 17 no 10 p 1557 2016

[118] M Meenu and B Xu ldquoA critical review on anti-diabetic andanti-obesity effects of dietary resistant starchrdquo Critical Reviewsin Food Science and Nutrition pp 1ndash13 2018

[119] A Regina A Bird D Topping et al ldquoHigh-amylose wheatgenerated by RNA interference improves indices of large-bowelhealth in ratsrdquo Proceedings of the National Acadamy of Sciencesof the United States of America vol 103 no 10 pp 3546ndash35512006

[120] F Sestili M Janni A Doherty et al ldquoIncreasing the amylosecontent of durumwheat through silencing of the SBEIIa genesrdquoBMC Plant Biology vol 10 no 144 2010

[121] C Vetrani F Sestili M Vitale et al ldquoMetabolic response toamylose-rich wheat-based rusks in overweight individualsrdquoEuropean Journal of Clinical Nutrition vol 72 no 6 pp 904ndash912 2018

[122] C Zorb U Ludewig and M J Hawkesford ldquoPerspective onwheat yield and quality with reduced nitrogen supplyrdquo Trendsin Plant Science vol 23 no 11 pp 1029ndash1037 2018

[123] D Zhao A P Derkx D Liu P Buchner M J Hawkesfordand E Flemetakis ldquoOverexpression of a NAC transcription


factor delays leaf senescence and increases grain nitrogenconcentration in wheatrdquoe Journal of Plant Biology vol 17 no4 pp 904ndash913 2015

[124] X Zhao X Nie X Xiao and H Yang ldquoOver-expression of atobacco nitrate reductase gene in wheat (triticum aestivum l)increases seed protein content and weight without augmentingnitrogen supplyingrdquo PLoS ONE vol 8 no 9 article e746782013

[125] F M Anjum M R Khan A Din M Saeed I Pasha andM U Arshad ldquoWheat gluten high molecular weight gluteninsubunitsstructure genetics and relation to dough elasticityrdquoJournal of Food Science vol 72 no 3 pp R56ndashR63 2007

[126] F Altpeter V Vasil V Srivastava and I K Vasil ldquoIntegrationand expression of the high-molecular-weight glutenin subunit1Ax1 gene into wheatrdquo Nature Biotechnology vol 14 no 9 pp1155ndash1159 1996

[127] A E Blechl and O D Anderson ldquoExpression of a novel high-molecular-weight glutenin subunit gene in transgenic wheatrdquoNature Biotechnology vol 14 no 7 pp 875ndash879 1996

[128] F Barro L Rooke F Bekes et al ldquoTransformation of wheatwith high molecular weight subunit genes results in improvedfunctional propertiesrdquo Nature Biotechnology vol 15 no 12 pp1295ndash1299 1997

[129] L Rooke F Bekes R Fido et al ldquoOverexpression of a glutenprotein in transgenic wheat results in greatly increased doughstrengthrdquo Journal of Cereal Science vol 30 no 2 pp 115ndash1201999

[130] M L Alvarez M Gomez J Marıa Carrillo and R H VallejosldquoAnalysis of dough functionality of flours from transgenicwheatrdquoMolecular Breeding vol 8 pp 103ndash108 2001

[131] P R Shewry S Powers J M Field et al ldquoComparative fieldperformance over 3 years and two sites of transgenic wheat linesexpressing HMW subunit transgenesrdquo eoretical and AppliedGenetics vol 113 pp 128ndash136 2006

[132] Y Li QWang X Li et al ldquoCoexpression of the high molecularweight glutenin subunit 1ax1 and puroindoline improves doughmixing properties in durum wheat (triticum turgidum l sspdurum)rdquo PLoS ONE vol 7 no 11 article e50057 2012

[133] S Li N Wang Y Wang et al ldquoInheritance and expression ofcopies of transgenes 1Dx5 and 1Ax1 in elite wheat (triticumaestivum l) varieties transferred from transgenicwheat throughconventional crossingrdquo Acta Biochimica et Biophysica Sinicavol 39 no 5 pp 377ndash383 2007

[134] XMao Y Li S Zhao et al ldquoThe interactive effects of transgeni-cally overexpressed 1Ax1 with various HMW-GS combinationson dough quality by introgression of exogenous subunits intoan elite chinese wheat varietyrdquo PLoS ONE vol 8 no 10 articlee78451 2013

[135] P I Payne ldquoGenetics of wheat storage proteins and the effectof allelic variation on bread-making qualityrdquo Annual Review ofPlant Physiology and Plant Molecular Biology vol 38 pp 141ndash153 1987

[136] K A Scherf P Koehler and H Wieser ldquoGluten and wheatsensitivities ndash an overviewrdquo Journal of Cereal Science vol 67 pp2ndash11 2016

[137] A Juhasz T Belova C G Florides et al ldquoGenome mappingof seed-borne allergens and immunoresponsive proteins inwheatrdquo Science Advances vol 4 no 8 article eaar8602 2018

[138] I CakmakHOzkanH J Braun RMWelch andV RomheldldquoZinc and iron concentrations in seeds of wild primitive andmodern wheatsrdquo Food and Nutrition Bulletin vol 21 no 4 pp401ndash403 2000

[139] R M Welch and R D Graham ldquoA new paradigm for worldagriculture meeting human needs productive sustainablenutritiousrdquo Field Crops Research vol 60 no 1-2 pp 1ndash10 1999

[140] P B Holm K N Kristiansen and H B Pedersen ldquoTransgenicapproaches in commonly consumed cereals to improve iron andzinc content and bioavailabilityrdquo Journal of Nutrition vol 132no 3 pp 514Sndash516S 2002

[141] S Borg H Brinch-Pedersen B Tauris et al ldquoWheat ferritinsimproving the iron content of the wheat grainrdquo Journal of CerealScience vol 56 no 2 pp 204ndash213 2012

[142] X Sui Y Zhao S Wang et al ldquoImprovement Fe content ofwheat (triticum aestivum) grain by soybean ferritin expressioncassette without vector backbone sequencerdquo Journal of Agricul-tural Biotechnology vol 20 pp 766ndash773 2012

[143] JMConnorton E R Jones I Rodrıguez-Ramiro et al ldquoWheatvacuolar iron transporter TaVIT2 transports Fe and Mn andis effective for biofortificationrdquo Plant Physiology vol 174 pp2434ndash2444 2017

[144] L Yan A Loukoianov A Blechl et al ldquoThewheatVRN2 gene isa flowering repressor down-regulated by vernalizationrdquo Sciencevol 303 no 5664 pp 1640ndash1644 2004

[145] A Loukoianov L Yan and A Blechl ldquoRegulation of VRN-1vernalization genes in normal and transgenic polyploid wheatrdquoPlant Physiology vol 138 no 4 pp 2364ndash2373 2005

[146] CUauy ADistelfeld T Fahima A Blechl and J Dubcovsky ldquoANAC gene regulating senescence improves grain protein Zincand iron content in wheatrdquo Science vol 314 no 5803 pp 1298ndash1301 2006

[147] Y Li G Song J Gao et al ldquoEnhancement of grain number perspike by RNA interference of cytokinin oxidase 2 gene in breadwheatrdquoHereditas vol 155 no 33 2018

[148] X Y Zhao P Hong J Y Wu et al ldquoThe tae-miR408-mediatedcontrol of TaTOC1 genes transcription is required for theregulation of heading time in wheatrdquo Plant Physiology vol 170pp 1578ndash1594 2016

[149] F Barro J C Iehisa M J Gimenez et al ldquoTargeting ofprolamins by RNAi in bread wheat effectiveness of sevensilencing-fragment combinations for obtaining lines devoid ofcoeliac disease epitopes from highly immunogenic gliadinsrdquoPlant Biotechnology Journal vol 14 no 3 pp 986ndash996 2016

[150] J R Li W Zhao Q Z Li et al ldquoRNA silencing of waxy generesults in low levels of amylose in the seeds of transgenic wheat(triticum aestivum L)rdquo Acta Genetica Sinica vol 32 no 8 pp846ndash854 2005

[151] S Yue H Li Y Li et al ldquoGeneration of transgenic wheat lineswith altered expression levels of 1Dx5 high-molecular weightglutenin subunit byRNA interferencerdquo Journal of Cereal Sciencevol 47 no 2 pp 153ndash161 2008

[152] S B Altenbach andP VAllen ldquoTransformation of theUS breadwheat rsquoButte 86rsquo and silencing of omega-5 gliadin genesrdquo GMCrops vol 2 pp 66ndash73 2011

[153] S B Altenbach C K Tanaka and B W Seabourn ldquoSilencingof omega-5 gliadins in transgenic wheat eliminates a majorsource of environmental variability and improves doughmixingproperties of flourrdquo BMC Plant Biology vol 14 no 393 2014

[154] J Gil-Humanes F Piston M J Gimenez et al ldquoThe introgres-sion of RNAi silencing of 120574-gliadins into commercial lines ofbread wheat changes the mixing and technological propertiesof the doughrdquo PLoS ONE vol 7 no 9 article e45937 2012

[155] W Lee K EHammond-Kosack andKKanyuka ldquoBarley stripemosaic virus-mediated tools for investigating gene function in


cereal plants and their pathogens virus-induced gene silencinghost-mediated gene silencing and virus-mediated overexpres-sion of heterologous proteinrdquo Plant Physiology vol 160 no 2pp 582ndash590 2012

[156] D Nowara A Gay C Lacomme et al ldquoHIGS host-inducedgene silencing in the obligate biotrophic fungal pathogenBlumeria graminisrdquoe Plant Cell vol 22 no 9 pp 3130ndash31412012

[157] W Cheng X Song H Li et al ldquoHost-induced gene silencingof an essential chitin synthase gene confers durable resistanceto fusarium head blight and seedling blight in wheatrdquo PlantBiotechnology Journal vol 13 no 9 pp 1335ndash1345 2015

[158] Y Chen Q Gao M Huang et al ldquoCharacterization of RNAsilencing components in the plant pathogenic fungus fusariumgraminearumrdquo Scientific Reports vol 5 no 12500 2015

[159] X Song K Gu X Duan et al ldquoA myosin5 dsRNA thatreduces the fungicide resistance and pathogenicity of fusariumasiaticumrdquo Pesticide Biochemistry and Physiology vol 150 pp1ndash9 2018

[160] X Zhu T Qi Q Yang et al ldquoHost-induced gene silencing of theMAPKK gene PsFUZ7 confers stable resistance to wheat striperustrdquo Plant Physiology vol 175 no 4 pp 1853ndash1863 2017

[161] T Qi X Zhu C Tan et al ldquoHost-induced gene silencing of animportant pathogenicity factor PsCPK1 in Puccinia striiformisf sp tritici enhances resistance of wheat to stripe rustrdquo PlantBiotechnology Journal vol 16 no 3 pp 797ndash807 2018

[162] V Panwar M Jordan B McCallum and G Bakkeren ldquoHost-induced silencing of essential genes in Puccinia triticinathrough transgenic expression of RNAi sequences reducesseverity of leaf rust infection in wheatrdquo Plant BiotechnologyJournal vol 16 no 5 pp 1013ndash1023 2018

[163] Y Zhao X Sui L Xu et al ldquoPlant-mediatedRNAi of grain aphidCHS1 gene confers common wheat resistance against aphidsrdquoPest Management Science vol 74 no 12 pp 2754ndash2760 2018

[164] L Xu Q Hou Y Zhao et al ldquoSilencing of a lipase maturationfactor 2-like gene by wheat-mediated RNAi reduces the surviv-ability and reproductive capacity of the grain aphid sitobionavenaerdquo Archives of Insect Biochemistry and Physiology vol 95no 3 article e21392 2017

[165] L Xu X Duan Y Lv et al ldquoSilencing of an aphid car-boxylesterase gene by use of plant-mediated RNAi impairsSitobion avenae tolerance of Phoxim insecticidesrdquo TransgenicResearch vol 23 no 2 pp 389ndash396 2014

[166] M Fu M Xu T Zhou et al ldquoTransgenic expression of afunctional fragment of harpin protein Hpa1 in wheat inducesthe phloem-based defence against English grain aphidrdquo Journalof Experimental Botany vol 65 no 6 pp 1439ndash1453 2014

[167] E Abdellatef T Will A Koch J Imani A Vilcinskas andK Kogel ldquoSilencing the expression of the salivary sheathprotein causes transgenerational feeding suppression in theaphid Sitobion avenaerdquo Plant Biotechnology Journal vol 13 no6 pp 849ndash857 2015

[168] H Puchta and F Fauser ldquoGene targeting in plants 25 yearslaterrdquo e International Journal of Developmental Biology vol57 no 6-7-8 pp 629ndash637 2013

[169] M Jinek K Chylinski I Fonfara M Hauer J A Doudnaand E Charpentier ldquoA programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunityrdquo Science vol 337no 6096 pp 816ndash821 2012

[170] P Mali K M Esvelt and G M Church ldquoCas9 as a versatiletool for engineering biologyrdquo Nature Methods vol 10 no 10pp 957ndash963 2013

[171] Y Demirci B Zhang and T Unver ldquoCRISPRCas9 an RNA-guided highly precise synthetic tool for plant genome editingrdquoJournal of Cellular Physiology vol 233 no 3 pp 1844ndash18592018

[172] Q Shan Y Wang J Li et al ldquoTargeted genome modification ofcrop plants using a CRISPR-Cas systemrdquo Nature Biotechnologyvol 31 pp 686ndash688 2013

[173] Q Shan YWang J Li and C Gao ldquoGenome editing in rice andwheat using the CRISPRCas systemrdquo Nature Protocols vol 9no 10 pp 2395ndash2410 2014

[174] S K Upadhyay J Kumar A Alok and R Tuli ldquoRNA-guidedgenome editing for target gene mutations in wheatrdquoG3 GenesGenomes Genetics vol 3 no 12 pp 2233ndash2238 2013

[175] T Cermak S J Curtin J Gil-Humanes et al ldquoA multi-purposetoolkit to enable advanced genome engineering in plantsrdquo ePlant Cell vol 29 pp 1196ndash1217 2017

[176] Z Liang K Chen T Li et al ldquoEfficient DNA-free genomeediting of bread wheat using CRISPRCas9 ribonucleoproteincomplexesrdquo Nature Communications vol 8 p 14261 2017

[177] J Gil-Humanes Y Wang Z Liang et al ldquoHigh-efficiencygene targeting in hexaploid wheat using DNA replicons andCRISPRCas9rdquo e Plant Journal vol 89 no 6 pp 1251ndash12622017

[178] Y Zhang Y Bai G Wu et al ldquoSimultaneous modificationof three homoeologs of TaEDR1 by genome editing enhancespowdery mildew resistance in wheatrdquoe Plant Journal vol 91no 4 pp 714ndash724 2017

[179] P Bhowmik E Ellison B Polley et al ldquoTargetedmutagenesis inwheat microspores using CRISPRCas9rdquo Scientific Reports vol8 no 6502 2018

[180] Y Wang X Cheng Q Shan et al ldquoSimultaneous editing ofthree homoeoalleles in hexaploid bread wheat confers heritableresistance to powdery mildewrdquo Nature Biotechnology vol 32no 9 pp 947ndash951 2014

[181] Y Zong YWang C Li et al ldquoPrecise base editing in rice wheatand maize with a Cas9-cytidine deaminase fusionrdquo NatureBiotechnology vol 35 no 5 pp 438ndash440 2017

[182] Y Zong Q Song C Li et al ldquoEfficient C-to-T base editing inplants using a fusion of nCas9 and human APOBEC3ArdquoNatureBiotechnology vol 36 pp 950ndash953 2018

[183] W Wang Q Pan F He et al ldquoTransgenerational CRISPR-Cas9 activity facilitates multiplex gene editing in allopolyploidwheatrdquoe CRISPR Journal vol 1 pp 65ndash74 2018

[184] H Hamada Y Liu Y Nagira R Miki N Taoka and RImai ldquoBiolistic-delivery-based transient CRISPRCas9 expres-sion enables in planta genome editing in wheatrdquo ScientificReports vol 8 no 14422 2018

[185] M Singh M Kumar M C Albertsen J K Young and A MCigan ldquoConcurrent modifications in the three homeologs ofMs45 gene with CRISPR-Cas9 lead to rapid generation of malesterile bread wheat (Triticum aestivum L)rdquo Plant MolecularBiology vol 97 no 4-5 pp 371ndash383 2018

[186] R M Howells M Craze S Bowden and E J WallingtonldquoEfficient generation of stable heritable gene edits in wheatusing CRISPRCas9rdquo BMC Plant Biology vol 18 no 215 2018

[187] JMurovec Z Pirc andB Yang ldquoNew variants of CRISPRRNA-guided genome editing enzymesrdquo Plant Biotechnology Journalvol 15 no 8 pp 917ndash926 2017

[188] OO Abudayyeh J S Gootenberg P Essletzbichler et al ldquoRNAtargeting with CRISPRndashCas13rdquo Nature vol 550 no 7675 pp280ndash284 2017


[189] C M Lee T J Cradick and G Bao ldquoThe Neisseria meningi-tides CRISPR-Cas9 system enables specific genome editing inmammalian cellsrdquo Molecular erapy vol 24 no 3 pp 645ndash654 2016

[190] F A Ran L Cong W X Yan et al ldquoIn vivo genome editingusing Staphylococcus aureus Cas9rdquo Nature vol 520 no 7546pp 186ndash191 2015

[191] E Kim T Koo S W Park et al ldquoIn vivo genome editing witha small Cas9 orthologue derived from Campylobacter jejunirdquoNature Communications vol 8 article 14500 2017

[192] C Li Y Zong Y Wang et al ldquoExpanded base editing in riceand wheat using a Cas9-adenosine deaminase fusionrdquo GenomeBiology vol 19 no 59 2018

[193] T Kelliher D Starr L Richbourg et al ldquoMATRILINEAL asperm-specific phospholipase triggers maize haploid induc-tionrdquo Nature vol 542 no 7639 pp 105ndash109 2017

[194] I Fonfara H Richter M Bratovic A Le Rhun and ECharpentier ldquoThe CRISPR-associated DNA-cleaving enzymeCpf1 also processes precursor CRISPR RNArdquo Nature vol 532no 7600 pp 517ndash521 2016

[195] R Xu R Qin H Li et al ldquoGeneration of targeted mutant riceusing a CRISPR-Cpf1 systemrdquo Plant Biotechnology Journal vol15 no 6 pp 713ndash717 2017

[196] X Tang L G Lowder T Zhang et al ldquoA CRISPRndashCpf1 systemfor efficient genome editing and transcriptional repression inplantsrdquo Nature Plants vol 3 article 17018 2017

[197] V J Nalam S Alam J Keereetaweep et al ldquoFacilitationof Fusarium graminearum infection by 9-Lipoxygenases inArabidopsis and wheatrdquo Molecular Plant-Microbe Interactionsvol 28 no 10 pp 1142ndash1152 2015

[198] Y Zhang Z Liang Y Zong et al ldquoEfficient and transgene-free genome editing in wheat through transient expression ofCRISPRCas9 DNA or RNArdquo Nature Communications vol 7Article ID 12617 2016

[199] Z Liang K Chen Y Yan Y Zhang and C Gao ldquoGenotypinggenome-edited mutations in plants using CRISPR ribonucleo-protein complexesrdquo Plant Biotechnology Journal vol 16 no 12pp 2053ndash2062 2018

[200] S Lu H Zhao D L Des Marais et al ldquoArabidopsis ECER-IFERUM9 involvement in cuticle formation and maintenanceof plant water statusrdquo Plant Physiology vol 159 no 3 pp 930ndash944 2012

[201] W Wang J Simmonds Q Pan et al ldquoGene editing andmutagenesis reveal inter-cultivar differences and additivity inthe contribution of TaGW2 homoeologues to grain size andweight in wheatrdquoeoretical and Applied Genetics vol 131 no11 pp 2463ndash2475 2018

[202] Q Li L Li Y Liu et al ldquoInfluence of TaGW2-6A on seeddevelopment in wheat by negatively regulating gibberellinsynthesisrdquo Journal of Plant Sciences vol 263 pp 226ndash235 2017

[203] H Xu M Zhao Q Zhang Z Xu and Q Xu ldquoThe dense anderect panicle 1 (DEP1) gene offering the potential in the breedingof high-yielding ricerdquo Breeding Science vol 66 no 5 pp 659ndash667 2016

[204] S Sanchez-Leon J Gil-Humanes C V Ozuna et al ldquoLow-gluten nontransgenic wheat engineered with CRISPRCas9rdquoPlant Biotechnology Journal vol 16 no 4 pp 902ndash910 2018

[205] ldquoWheat vital grain of civilization and food securityrdquo CGIARResearch Program Wheat Annual Report CGIAR MexicoDF 2013

Hindawiwwwhindawicom

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Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


engineering techniques are necessary to break through thecurrent yield ceiling

Existing modern breeding efforts now need to be comple-mented with advanced crop functional genomics which canprovide insights into the functioning of wheat genetic deter-minants The available tools for wheat genetic modificationprovide the experimental means to functionally characterizegenetic determinants by suppressing or enhancing geneactivities This knowledge can then be used for targetedimprovements tailored to the specific needs of the diverse andchanging environments in which wheat is grown across theworld This offers the potential to tackle yield gaps whereverthey exist for a variety of causes enabling this global crop tofinally reach its full potential

2 Progress in Wheat Genetic Transformation

Bread wheat (Triticum aestivum L) the most widespread ofall wheat species is an annual herb belonging to the familyGramineae or Poaceae Wheat was domesticated around8000 years ago [29] and has since undergone hybridizationand genome duplication events to generate its hexaploidgenome (2n = 6x = 42 AABBDD) which is more thanfive times larger than the human genome It was previouslyestimated that the genome of common wheat contained over128000 genes [30] with over 80 of the genome consistingof repetitive sequences of DNA [31] However more recentestimates suggested a total of 107891 high-confidence geneswith over 85 repetitive DNA sequences representing athreefold redundancy due to its hexaploid genome [32]

Genetic transformation the fundamental tool of geneticengineering allows the introduction and expression ofvarious genes of interest in the cells of living organismsbypassing when desirable the barriers of sexual incompat-ibility that exist in nature Despite the considerable effortsof the international research community development ofwheat genetic engineering lags behind that of the other keyagricultural crops like rice and maize This may be attributedto the genetic characteristics of wheat including its very large(17000Mbp) and highly redundant complex genome as wellas the relative recalcitrance ofmost varieties to in vitro cultureand regeneration (reviewed recently in [33])

The first successful genetic transformation of commonwheat was conducted at Florida University USA [34] usingbiolistics and financed by a research grant from MonsantoScientists fromMonsanto were also the first to report the gen-eration of transgenic wheat using Agrobacterium-mediatedtransformation [35]

21 Wheat Transformation Methods Presently biolisticsand Agrobacterium-mediated transformation using imma-ture embryos as explants remain the main methods forgenetic engineering of wheat Each method has its ownadvantages and drawbacks (Table 1) The main advantages ofAgrobacterium transformation are the relatively high ratio ofsingle copy gene inserts and relative simplicity of the transfor-mation procedure In contrast biolistics offer benefits in theircapacity to transform organelles and deliver RNA proteinsnanoparticles dyes and complexes into cells Utilization

of linear Minimal Expression Cassettes (MECs) in biolistictransformation enables the production of plants carryingmuch simpler patterns of transgene integration compared toplasmid bombardment with a higher proportion of singlecopy inserts [36ndash38]

In addition biolistic delivery ofMEC simplifies the simul-taneous cotransformation of several genes and in contrastto Agrobacterium does not introduce vector backbone DNAor repetitive border sequences flanking the T-DNA intothe transformed plant cells In experiments with the wheatcv V12 transformation with MECs instead of plasmidsimproved transformation frequency (TF) almost threefoldfrom 04 t] 11 [39] A simplified method for DNAgoldcoating was described by Ismagul et al [38] for the high-throughput biolistic production of single copy transgenicwheat utilizing diluted MECs This method involves theapplication of PM solution (42 PEG 2000 and 560 mMMgCl

2) instead of the spermidine and CaCl

2used in the

standard Bio-Rad procedureBiolistics allow for the transfer into wheat of relatively

large DNA fragments Partier et al [40] conducted successfulbiolistic transformation of wheat with a 53 Pb linear cassettewhich contained a 44 Kb fragment of an Arabidopsis geneflanked by selection and reporter genes The intact cassettewas detected in f

1and f

2generation plants The main

disadvantages of both Agrobacterium and biolistic transfor-mation methods in wheat are genotype dependency and therequirement for lengthy periods of aseptic tissue culture

22 Wheat Transformation Frequencies Until recently theTF for most tested genotypes of common wheat remainedquite low at a level mostly below 5 [8] Many researchgroups use model wheat varieties such as Bobwhite SH98-26 and Fielder in their experiments due to their amenabilityto transformation via published protocols [8 9] The cultivarBobwhite SH98-26 was among 129 sister lines made fromcrosses of cultivars AuroraKalyanBluebird 3Woodpeckerat CIMMYT and selected for its high TF of over 70by biolistic transformation [9] Such high transformationefficiency is yet to be reproduced by other researchers andalthough the reason for this remains unclear it is possible thatnot all of the finer details of the transformation protocol couldbe described in the report The success rate of this high TFmay be explained by the particular hybrid genotype used orsimply the advanced skills and experience of the techniciansat CIMMYT Since publishing [9] A Pellegrineschi has beenemployed by two big Biotech companies Pioneer Hi BreedandDuPont which indirectly confirms that he has developeda reliable wheat transformation protocol that allowed himto produce the published results The authors of the revieware therefore not sceptical however the high TF presented in[9] must be reproduced by other researchers in future if thisprotocol is to be widely adopted The cultivar Fielder whichis the model variety in Agrobacterium transformation waspreselected by the researchers of the JapanTobaccoCompanywhere the detailed protocol ldquoPureWheatrdquo was developed [8]that allows TFs of 40ndash90 In this protocol positive selec-tion through the application of phosphomannose isomerase


Table1Com

paris

onof

parameterso

fagrob

acteria

land

biolistictransfo

rmation

Parameters

Agrobacterium-m

ediated

transfo

rmation

Biolistictransfo

rmation

References

Genotyped

ependency

High

Less

[5]

Stability

ofexpressio

nof

transgenes

High

HighforM

inim

alEx

pressio

nCassette

s(M

EC)lowe

rfor

plasmids

[67]

Cop

ynu

mbero

finserts

Aroun

d50sin

glec

opy

Depends

onthes

trainand

transfo

rmationcond

ition

s

lt50D

epends

onthe

amou

ntof

DNAsho

tMore

multic

opyinserts

[78]

Integrationof

then

ewgenes

Rand

omM

orethanon

elocus

Rand

omO

ftenmanyatthe

samelocus

[6]

Maxim

umtransfo

rmationfre

quency

(TF)

forw

heat(per

100em

bryostreated)

Upto

90

gt70

[89]

Com

plexity

ofthetransform

ation

procedureinwheat

SimplerU

suallyrequ

ires

aseptic

cond

ition

s

Morec

omplexR

equires

aseptic

cond

ition

sand

aBiolisticGun

[810]

Mainexplantsin

wheat

Immaturee

mbryos

Immaturee

mbryos

[810]

Com

plexity

ofvector

construct

preparationco-tr

ansfo

rmation

Morec

omplex

Simpler

[11]

Maxim

umsiz

esof

transfe

rred

inserts

publish

edUpto

200Kb

150ndash

164Kb

[12ndash14]

Transfe

roff

-DNAbo

rders

Yes

No(fo

rMEC

)[15]

Transfe

rofvectorD

NA

Possible

No(fo

rMEC

)Yes(for

plasmids)

[15]

Transfe

rofb

acteria

lchrom

osom

alDNA

Possible

No(fo

rMEC

)[16]

Markerfreetransform

ationin

wheat

Possible

Possible

[17ndash19]

Inplanta

transfo

rmationin

wheat

Possible

Possible

[20ndash

22]

Deliveryof

RNAproteinsnano

particles

anddyes

No

Possible

[23]

Transfo

rmationof

chloroplastsand

mito

chon

dria

No

Possible

[2425]

Transie

ntgene

expressio

nin

different

tissues

andorgans

ofplants

Efficientfor

limited

numbero

fplant

species

Efficient

[26ndash

28]







1were










4 Miroshnichenko et















2into organic com-



3





4









2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table1Com

paris

onof

parameterso

fagrob

acteria

land

biolistictransfo

rmation

Parameters

Agrobacterium-m

ediated

transfo

rmation

Biolistictransfo

rmation

References

Genotyped

ependency

High

Less

[5]

Stability

ofexpressio

nof

transgenes

High

HighforM

inim

alEx

pressio

nCassette

s(M

EC)lowe

rfor

plasmids

[67]

Cop

ynu

mbero

finserts

Aroun

d50sin

glec

opy

Depends

onthes

trainand

transfo

rmationcond

ition

s

lt50D

epends

onthe

amou

ntof

DNAsho

tMore

multic

opyinserts

[78]

Integrationof

then

ewgenes

Rand

omM

orethanon

elocus

Rand

omO

ftenmanyatthe

samelocus

[6]

Maxim

umtransfo

rmationfre

quency

(TF)

forw

heat(per

100em

bryostreated)

Upto

90

gt70

[89]

Com

plexity

ofthetransform

ation

procedureinwheat

SimplerU

suallyrequ

ires

aseptic

cond

ition

s

Morec

omplexR

equires

aseptic

cond

ition

sand

aBiolisticGun

[810]

Mainexplantsin

wheat

Immaturee

mbryos

Immaturee

mbryos

[810]

Com

plexity

ofvector

construct

preparationco-tr

ansfo

rmation

Morec

omplex

Simpler

[11]

Maxim

umsiz

esof

transfe

rred

inserts

publish

edUpto

200Kb

150ndash

164Kb

[12ndash14]

Transfe

roff

-DNAbo

rders

Yes

No(fo

rMEC

)[15]

Transfe

rofvectorD

NA

Possible

No(fo

rMEC

)Yes(for

plasmids)

[15]

Transfe

rofb

acteria

lchrom

osom

alDNA

Possible

No(fo

rMEC

)[16]

Markerfreetransform

ationin

wheat

Possible

Possible

[17ndash19]

Inplanta

transfo

rmationin

wheat

Possible

Possible

[20ndash

22]

Deliveryof

RNAproteinsnano

particles

anddyes

No

Possible

[23]

Transfo

rmationof

chloroplastsand

mito

chon

dria

No

Possible

[2425]

Transie

ntgene

expressio

nin

different

tissues

andorgans

ofplants

Efficientfor

limited

numbero

fplant

species

Efficient

[26ndash

28]







1were










4 Miroshnichenko et















2into organic com-



3





4









2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








1were










4 Miroshnichenko et















2into organic com-



3





4









2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







4 Miroshnichenko et















2into organic com-



3





4









2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











2into organic com-



3





4









2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






2and T








1offspring analysed



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



















2generation [162]



















1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











1plants carrying













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018













Acknowledgments


References


























































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

































































































































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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