Plant Biotechnology Journal (2015) 13, pp. 147–162

doi: 10.1111/pbi.12339

Review article

Progress in genetic engineering of peanut (Arachis hypogaea L.)—A review Gaurav Krishna1,*, Birendra K. Singh2, Eun-Ki Kim2, Vivek K. Morya2 and Pramod W. Ramteke1 1

Jacob School of Biotechnology & Bioengineering, Sam Higginbottom Institute of Agriculture, Technology & Sciences (Formerly Allahabad Agricultural Institute),

Deemed University, Allahabad, Uttar Pradesh, India 2

Department of Biological Engineering, Inha University, Incheon, Republic of Korea

Received 10 October 2014; revised 27 November 2014; accepted 17 December 2014. *Correspondence (Tel +91 9718491338/ +91 532 2684281; fax +91 0532 2684394; email [email protected])

Keywords: peanut (Arachis hypogaea L.), genetic engineering, biotic stress, abiotic stress, transformation efficiency, oral vaccine.

Summary Peanut (Arachis hypogaea L.) is a major species of the family, Leguminosae, and economically important not only for vegetable oil but as a source of proteins, minerals and vitamins. It is widely grown in the semi-arid tropics and plays a role in the world agricultural economy. Peanut production and productivity is constrained by several biotic (insect pests and diseases) and abiotic (drought, salinity, water logging and temperature aberrations) stresses, as a result of which crop experiences serious economic losses. Genetic engineering techniques such as Agrobacterium tumefaciens and DNA-bombardment-mediated transformation are used as powerful tools to complement conventional breeding and expedite peanut improvement by the introduction of agronomically useful traits in high-yield background. Resistance to several fungal, virus and insect pest have been achieved through variety of approaches ranging from gene coding for cell wall component, pathogenesis-related proteins, oxalate oxidase, bacterial chloroperoxidase, coat proteins, RNA interference, crystal proteins etc. To develop transgenic plants withstanding major abiotic stresses, genes coding transcription factors for drought and salinity, cytokinin biosynthesis, nucleic acid processing, ion antiporter and human antiapoptotic have been used. Moreover, peanut has also been used in vaccine production for the control of several animal diseases. In addition to above, this study also presents a comprehensive account on the influence of some important factors on peanut genetic engineering. Future research thrusts not only suggest the use of different approaches for higher expression of transgene(s) but also provide a way forward for the improvement of crops.

Introduction Legumes are third largest family of higher plants, wherein peanut (Arachis hypogaea L.) is one of the most important species. It is one of the choicest world agriculturally economic important crop not only in vegetable oil but also provide a source of protein, calcium, iron and vitamin B complex such as thiamine, riboflavin, niacin and vitamin-A. It is widely grown in the semi-arid tropics, China contributes the highest share and India ranks second by 41.6% and 12.5% in world production, respectively (USDA Foreign Agricultural Service, 2014). In Third World countries, the peanut has shown greater potential to reduce hunger and malnutrition as it is a good source of protein, calories, vitamins and minerals (Enserink, 2008). It is often grown on marginal soils with lesser inputs and usually intercropped with cereals in many of the developing countries. Its production and productivity is constrained by several biotic and abiotic factors. Among the biotic stresses, insect pests and diseases caused by fungi, viruses, etc. are major; whereas among the abiotic stresses drought, salinity, water logging and temperature aberrations cause serious economic losses in peanut production and productivity. Landrace/ wild species of peanut although provide genetic diversity not present in cultivated species are often associated with undesirable agronomic traits (Muehlbauer, 1993). Attempts to develop peanut cultivars resistant to such biotic and abiotic stresses by

conventional breeding methods have had limited success due to narrow genetic variability among the germplasm accessions. In addition, incompatibility problems of breeding associated with wild species necessitates exploring alternative approaches. Presently, the genetic engineering techniques such as Agrobacterium tumefaciens or DNA-bombardment-mediated transformation are used as powerful tools that complement the conventional breeding and expedite rapid progress in peanut improvement by the introduction of agronomically important desirable traits under a high-yield background. Genes that have been successfully incorporated into the genomic DNA of peanut for developing resistance to biotic and abiotic stresses are listed in Table 1.

Biotic stresses Insect pests Among the key pests of peanut, white grubs (Holotrichia spp.) tobacco caterpillar (Spodoptera litura Fabricius), gram pod borer (Helicoverpa armigera Hubner) and leaf folder (Anarsia ephippias Meyrick) are categorized as chewing pests; and jassids (Empoasca spp.) and thrips (Scirtothrips dorsalis Hood and Frankliniella schultzer Trybom) are categorized as sucking pests (Wightman and Amin, 1988). In the semi-arid tropics, peanut production suffers significant losses predominantly due to chewing pests. The cowpea trypsin inhibitor (CpTI) gene has been incorporated in

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

147

C58 –

Coty

Coty

E Cal

ICGV89104

JL24

Gerogia

AT120

Runner

E Ax

–

pKYLX80-N11

pRTL2 pCB13

– –

pCB13

–

E Cal

pBI121-pBTex

LBA 4404

I Coty

E Ax

TMV2

pOxOx

–

Gaja

E Cal

Wilson

pOxOx

–

Georgia

E Cal

Perry

pCAMBIA1300

GV2260

pOxOx

–

resistance

Coty N

JL24

I Coty

E Cal

NC-7

pCAMBIA1302

pCAMBIA2300

C58

EHA105

pCAMBIA1302

pCAMBIA1302

Florunner

Coty

JL24

C58

Viral

Leaf

JL24

green

pCAMBIA2300 pRT66

C58

Coty

CaMV35S

CaMV35S

CaMV35SDE

CaMV35S

CaMV35S

CaMV35SDE

CaMV35SDE

CaMV35S

CaMV35SDE

CaMV35S

CaMV35S

CaMV35SDE

CaMV35S

CaMV35S

CaMV35S

CaMV35S

ICGV86031

LBA 4404

–

Coty

CaMV35S (EN4)

CaMV35S (EN4)

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S:(r)PR1a

CaMV35S

CaMV35S

CaMV35S

CaMV35S

Promoter

pB1333-EN4

Golden

LBA 4404

Coty N

pB1333-EN4

pPK202

Golden

LBA 4404

GV2260

pH602+pSG3525.

–

BARI-2000

Coty N

Coty N

pBinBt8

pH602+pSG3525.

– EHA105

pFZY1

pBinBt8

pPK202

pCAMBIA1300

LBA 4404

EHA105

GV2260

EHA101

pCP203

pH602+pSG3525.

EHA101

–

resistance

JL24

I Coty

Luhua14

Toalson

Epicotyl

K134

I Coty

Seedling

JL24

AM

Coty N

JL24

TMV2

DEC

JL24

MARC-I

I Coty

DEC

Florunner

pSN8E

pSN8E

Plasmid

AZ

AZ

strain

Agrobacterium

Fungal

resistance

Fungal

Insect +

resistance

E Ax

E Ax

Baisha1016

Baisha1016

Insect

tissue

Genotype

Trait

Explant

Table 1 Traits improved in peanut (Arachis hypogaea L.)

N

N

PStV CP2

N

CHI

oxidase

Barley oxalate

oxidase

Barley oxalate

SniOLP+Rs-AFP2

oxidase

Barley oxalate

Rchit

BjD

cpo-p

Rchit

Rchit

Rchit

Bchit

RCG-3

RCG-3

cry1E+Chi11

Bt cry1A(c)

cry1X

Bt cry1A(c)

CpTI-uidA

cry1AcF

cry1EC

cry1EC

cry1EC

Bt cry1A(c)

cry8Ea1+MARs

cry8Ea1

gene

Transformed

Tiwari et al. (2011) Beena et al. (2008)

–

31.00

90.0

63.0

90.0

40.0

–

–

–

–

70.0

–

–

60.0–88.0

40.0

55.0

–

41.0

32.0

–

83.0

63.1

83.0

12.4

Yang et al. (1998)

Higgins et al. (2004)

Yang et al. (1998)

Sankara (2001)

Rohini and

et al. (2005)

Livingstone

et al. (2005)

Livingstone

and Kirti (2012)

Vasavirama

et al. (2005)

Livingstone

Prasad et al. (2013)

Anuradha et al. (2008)

Niu et al. (2009)

Bhatnagar et al. (2010)

Prasad et al. (2013)

Prasad et al. (2013)

Iqbal et al. (2011)

Iqbal et al. (2012)

Iqbal et al. (2012)

Beena et al. (2008)

Singsit et al. (1997)

Entoori et al. (2008)

Singsit et al. (1997)

Qiusheng et al. (2005)

et al. (2013)

Keshavareddy

Tiwari et al. (2008)

– 35.0

Singsit et al. (1997)

–

Geng et al. (2013)

Geng et al. (2012)

References

83.0

61.0

61.0

efficiency (%)

Transformation

148 Gaurav Krishna et al.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

Florunner

Herbicide

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

Salinity

Drought

Coty

Coty

Coty

JL24

JL24

New Mexico

E Ax

Coty

K134

BARI-2000

Valencia A

Coty

Coty

I Emb

JL24

JL24

SA

TMV2

JL24

SA

TMV2

SA

Leaf

NA

Oral

E Cal

E Ax

E Ax

NA

green

resistance

vaccine

Gerogia

Herbicide

resistance

Florigiant

E Ax

VC1

Viral +

Hypocotyl

E Cal

Okrun

NC-7

Valencia A

Leaf

New Mexico

pWRG2114 pWRG2114

– –

LBA 4404

LBA 4404

EHA104

C58

C58

C58

pGNFA- (pAHC17)

pCAMBIA1301

pSARK

CaMV35S

CaMV35S

SARK

CaMV35S

rd29A

– pBI29

rd29A

CaMV35S

CaMV35S

AtNHX1

PDH45

IPT

DREB1A

DREB1A

DREB1A

Zmpsy1

BTV-VP2

RPVH

RVPH

– CaMV35S

UreB

HN

Bcl-xL

tswv-np +gus+bar

tswv-np +gus+bar

N

PStV CP4

PStV CP

N+GUS

N

N

TSV-CP

TSV-CP

TSV-CP

TSV-CP

IPCVcp

gene

Transformed

Oleosin

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35SDE

CaMV35S

CaMV35SDE

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

CaMV35S

Promoter

pBI29

pCAMBIA2300

pCAMBIA2301

– C58

pBI121

pBI121

pBI121

pBI121

EHA 105

EHA 105

EHA105

GV3101

pRT66

pKYLX80-N11

–

–

pKYLX71 pRTL2

LBA4404

pGA306

–

–

EHA105

–

pCB13

–

pCAMBIA1305.1

pCAMBIA1305.1

pCAMBIA1305.1

pCAMBIA1305.1

pROKII:IPCVcp

Plasmid

LBA 4404

I Coty

IL

K6

LBA 4404

LBA 4404

–

DEC

MARC-I

IL

K134

K6

LBA 4404

C58

strain

Agrobacterium

MARC-I

DEC

Coty

tissue

Explant

K134

JL24

Viral

resistance

Genotype

Trait

Table 1 Continued

78.0

–

–

70.0

–

70.0

66.0–100.0

12.4

67.0

–

–

–

87.1

8.8

8.8

77.0

60.0

–

1.5

–

90.0

11.3

34.0

2.2

16.5

55.0

efficiency (%)

Transformation

Asif et al. (2011)

et al. (2014)

Manjulatha

Qin et al. (2011)

et al. (2007)

Bhatnagar-Mathur

et al. (2009)

Bhatnagar-Mathur

et al. (2007)

Bhatnagar-Mathur

Bhatnagar et al. (2010)

Athmaram et al. (2006)

et al. (2003a)

Khandelwal

et al. (2003b)

Khandelwal

Yang et al. (2011)

et al. (2011)

Khandelwala

Chu et al. (2008)

Brar et al. (1994)

Brar et al. (1994)

Magbanua et al. (2000)

Higgins et al. (2004)

Franklin et al. (1993)

Li et al. (1997)

Yang et al. (2004)

Yang et al. (1998)

Mehta et al. (2013)

Mehta et al. (2013)

Mehta et al. (2013)

Mehta et al. (2013)

Anjaiah (2000)

Sharma and

et al. (2000)

Magbanua

References

Research progress of peanut genetic engineering 149

Valencia A

immature leaf; SA, shoot apices.

Qin et al. (2013) –

AM, apical meristem; Coty, cotyledon; Coty N, cotyledonary node; DEC, de-embryonated cotyledon; E Ax, embryo axes/embryonic axes; E Cal, embryogenic callus; I Coty, immature cotyledons; I Emb, immature embryo; I Leaf,

Asif et al. (2011) 83.0

AVP1

AtNHX1 CaMV35S

CaMV35S pPZP212

pGNFA- (pAHC17)

Coty New Mexico

LBA 4404 Coty Golden

runner458

LBA 4404

Banjara et al. (2012) – AtNHX1 CaMV35S pBISN1 GV3101 Flavour

+ salinity

Coty

efficiency (%) gene strain

Drought

Explant

tissue Genotype

Table 1 Continued

Trait

Agrobacterium

Plasmid

Promoter

Transformed

Transformation

References

150 Gaurav Krishna et al. peanut for developing resistance to H. armigera (Qiusheng et al., 2005). While studies on cry1Ec gene alone and in association with rice chitinase gene (Chi 11) for resistance to S. litura coupled with fungal pathogen, Phaeoisariopsis personata have been carried out for developing peanut transgenics (Beena et al., 2008; Tiwari et al., 2008). To promote high rate of pathogenic response with cry1Ec gene, the promoter PR-1a was placed at the downstream of CaMV35S promoter (resulting CaMV35S: PR-1a). Use of this promoter has also witnessed high levels of transgenic expression during insect bites and the presence of salicylic acid in peanut resulting in complete larval mortality at all the stadia of S. litura (Tiwari et al., 2011). Additionally, Bacillus thuringiensis toxin, cry1X coding gene has been used for developing transgenic plants for resistance to S. litura and H. armigera, which witnessed 51.0–52.0% larval mortality in bioassays after 96-h incubation, while the remaining larvae displayed severe stunted growth that was attributed due to low levels of toxin accumulation as result of fewer intake of feed (Entoori et al., 2008). Apart from this, the lesser cornstalk borer [LCB; Elasmopalpus lignoscellus (Zeller)] is another key pest of peanut causing greater economic losses in the USA. A codon modified Bt cry1A(c) gene has been introduced in peanut through genetic engineering, whose bioassays revealed not only complete mortality but also 66.0% reduction in larval weights (Singsit et al., 1997). Specificity of Bt toxins to certain groups of lepidopterous insects has restricted their usage as cry genes, but pyramiding of receptorspecific toxins in a single gene has always broadened the range of susceptibility. In this direction, improvement in the insecticidal property of a chimeric Bt gene with cry1Ac and cry1F domains resulting cry1AcF has been developed in peanut transformation for conferring resistance to S. litura (Keshavareddy et al., 2013). Plants exhibiting positive results by molecular analysis in insect bioassays using leaf tissue, under laboratory conditions, witnessed highest mean larval mortalities of 80.0% and 85.0% coupled with average mean larval mortality of 16.3% and 26.0% in the subsequent consecutive two generations. At the same time, a substantial size reduction of larvae has also been noticed in some events that was reasoned as reduction in toxin accumulation in insects due to lesser feeding conjoined with olfactory responses. Influence of olfactory and gustatory responses on larval colonization among various transgenic events of cry genes has been investigated in pigeon pea through free choice insect bioassay (Krishna et al., 2011). Thus, such chimeric genes approach needs to be explored for improving other traits. White grub (H. parallela) larvae feed on the roots of peanut plants causing not only mortality but also facilitate entry of other pathogens, thereby affecting an estimated annual loss of $8001000 million in India and Australia. A codon optimized synthetic cry8Ea1 gene was transformed among the roots of peanut revealed undamaged stronger roots in bioassay studies of larvae. In contrast, the nontransgenic roots of peanut plants suffered variable degrees of damage associated with increased weight of larvae (Geng et al., 2012). Few transgenic events of cry8Ea1 expressed diverse degrees of root damage, which phenomenon was commonly attributed to different levels of transgenic expression due to position effects and chromosomal differences among the integration sites. In addition, transgene silencing has also been found to be responsible for the inactivation of transgenics shortly after incorporation into plant chromosomes. As an outcome, many transformed plant cell lines are bound to be killed during antibiotic selection resulting in low recovery of transgenic tissues. In this direction, DNA sequences called matrix

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

Research progress of peanut genetic engineering 151 attachment regions (MARs) have been found responsible for promoting the levels of transgene expression and consequent line-to-line variation. Matrix attachment regions are AT-rich DNA sequences that bind to the cell’s proteinaceous nuclear matrix to form DNA loop domains (Spiker and Thompson, 1996). Transgenes flanked with MARs were found responsible in forming their own chromatic domain and thus become insulated from the influence of factors in the chromatin adjacent to site of insertion that ascribed to reduce the effect of gene silencing and causing enhanced levels of transgene expression (Hall et al., 1991). Expression cry8Ea1 gene flanked with tobacco-derived two MARs sequences increased the transcription of mRNA and expression of transgenic proteins to an extent of 9.0 and 2.5 times, respectively, in peanut (Geng et al., 2013). These studies imply that MARs sequences are more potential for promoting the transcription rates at level of mRNA than protein synthesis that might be due to their involvement in T-DNA region to form more accessible chromatin structure leading to RNA polymerase and associated transcription factors to bind DNA for transcription initiation.

Diseases Fungi Among the biotic stresses, fungal diseases have always been devastating peanuts grown in both the tropic and semi-arid tropic regions. Several genes have been tested for developing resistance to fungal diseases in peanut. Among them, chitinases play a significant role in hydrolysing the cell wall component of phytopathogenic fungi by the N-acetylglucosamine polymer chitin. Under in vitro conditions, plant chitinases depict different degrees of antifungal activity against different fungal strains. Expression of cloned chitinase genes in transgenic events have been found associated with plant defence. The level of protection observed in these transgenic plants is influenced by specific activity of the enzymes, their expression, transgene protein concentration within the cell, characteristics of fungal pathogens and nature of host–pathogen interactions (Punja and Zhang, 1993). Two chitinase genes (Rchit and CHI) causing resistance to Fusarium wilt and tikka disease (Cercospora arachidicola) have been tested for inheritance in transgenic events of peanut (Iqbal et al., 2011, 2012; Rohini and Sankara, 2001). Recent development of transgenic events using bacterial chitinase (Bchit) and rice chitinase (RCG-3) genes showed higher expression of enzyme activity conjoined with varied levels of resistance to C. arachidicola (Iqbal et al., 2011, 2012). The variability of pathogen resistance between transgenic events may be due to the localization of chitinase enzymes at the tissue and cellular levels (Grison et al., 1996; Leeuwen et al., 2001). Further use of rice chitinase gene (Rchit) in peanut transgenics displayed longer incubation and latent periods, lower infection rating, fewer lesions against late leaf spot (LLS) and rust diseases (Prasad et al., 2013). Pathogenic-related proteins such as PR proteins and defensins are potent antifungal proteins which are present in traces. Discovery of this class of protein in plants evolves a new avenue for developing resistance in transgenic plants through overexpression. As these genes are native to plants, they have high potential to obtain biosafety clearance for commercialization. Transgenic peanuts expressing defensin gene (BjD) from mustard demonstrated increased levels of resistance to late leaf spot (Anuradha et al., 2008). Using a combination of PR genes such as SniOLP (Solanum nigrum osmotin-like protein) and Rs-

AFP2 (Raphanus sativus antifungal protein-2) enhanced disease resistance was also observed in term of reduced number and size of lesions and the onset of late leaf spot disease when tested using fungal conidia of Phaeoisariopsis personata (Vasavirama and Kirti, 2012). Sclerotinia blight is caused by Sclerotinia minor in peanut. Based on the pathogenic reaction of oxalic acid coupled with Sclerotinia species, transgenics were developed using cDNA sequence of barley oxalate oxidase for protein expression that degrades the oxalic acid content coinciding with resistance to Sclerotinia blight (Livingstone et al., 2005). These transgenics tested over a 5-year period witnessed greater reduction in area spread under the disease progress curve and expressed a yield potential ranging 488–2755 kg/ha compared with nontransgenic counterparts (Partridge-Telenko et al., 2011). Aspergillus flavus and A. parasiticus are held for the production of aflatoxin, known as human carcinogen, in shelled peanuts (Jelinek et al., 1989; Wilson, 1995). An outbreak of aflatoxin accounted on an average $26 million annual losses towards peanut production during 1993 to 1996 at USA (Lamb and Sternitzke, 2001). Although peanut germplasm with reduced preharvest aflatoxin contamination has been found suitable for breeding, attempt was also made through genetic engineering for developing peanut transgenic exhibiting resistance to A. flavus using bacterial chloroperoxidase gene from Pseudomonas pyrrocinia (cpo-p). Antifungal activity with leaf and callus extracts showed 20.0% and 50.0% reduction in A. flavus colonization, respectively, under in vitro conditions; and in situ inoculation of A. flavus (70-GFP) strain showed 50.0–80.0% reduction in the colonization of fungal hyphal growth on the cotyledon of transgenics (Niu et al., 2009). In addition, the use of rice chitinase gene (Rchit) tested with in vitro seed inoculation bioassay showed lower rate of infection at 7day after incubation (Prasad et al., 2013). These results encourage probing further studies on revealing mode of action of cpo-p and Rchit genes behind driving greater levels of resistance to A. flavus.

Viruses In peanut, the bud necrosis disease is caused by serologically two distinct virus types viz., Bud necrosis virus (BNV) and Tomato spotted wilt virus (TSWV). Incorporation of viral nucleocapsid protein coding gene tswv-np in peanut has resulted in developing resistance to TSWV (Brar et al., 1994). In addition, transgenic plants harbouring sense and translationally defective or antisense N gene (nucleocapsid protein) have also been developed and tested for expression through ELISA in progenies (Li et al., 1997; Yang et al., 1998). RNA expression and production of nucleocapsid protein from sense N gene among the progenies of transgenic peanut plants witnessed a delay in symptom development by 10–15 days after mechanical inoculation with the donor isolate of TSWV. This was attributed to hybridization of sense N gene, RNA with the genomic RNA of incoming virus causing inhibition of viral RNA replication after introduction into the plant cells. Additionally, the transgenic progenies of the cultivar, MARC-1 expressing antisense N gene tested by mechanical inoculation under controlled environmental and field conditions showed significantly lower incidence of spotted wilt in comparison with nontransgenic wild types, consistently over a 3-year period (Yang et al., 2004). In another report, transgenic plants developed against the same virus using antisense N gene had also been field evaluated for the expression and noted significant

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

152 Gaurav Krishna et al. effect up to 76.0% symptomless plants for TSWV infection after 10 and 14 week of sampling points (Magbanua et al., 2000). These investigations strongly support the use of sense and antisense orientations of the N gene for protection from TSWV and other tospovirus infections. In both cases, the gene orientation did not exhibit any type of effect on the level of protection in peanut. Thus, both the strategies can further be implemented for virus protection in other plant species. Another viral disease of peanut, clump disease is caused by Peanut clump virus (PCV), which affects the lack pod emergence and >60.0% yield loss at early and late infection. RNA enabled genetic material of PCV is commonly vectored by plasmodiophoromycete fungus and transmitted by seed as well as in soil. In this direction, Sharma and Anjaiah (2000) produced transgenic peanuts carrying coat protein gene (IPCVcp) in the genomic DNA for resistance to Indian peanut clump virus (IPCV). However, no attempts were made using RNAi-mediated resistance that has been found highly effective in controlling RNA virus infections. The pollen grains of widespread weed, Parthenium hysterophorous, act as a symptomless carrier for peanut stem necrosis through thrips infection. Subsequent development of transgenic peanut harbouring antisense coat protein gene for Tobacco streak virus (TSV-CP) witnessed resistance with traces or no systemic accumulation of the virus. Underlying resistance mechanism was further confirmed in the RNA transcripts and protein expression analysis up to T3 generation of peanut to determine stable lines (Mehta et al., 2013). Usage of sense gene approach for Tobacco streak virus-coat protein (TSV-CP) needs to be explored because of its suitability in peanut. During 1983, Peanut stripe virus (PStV) was first reported in China that spread to major peanut growing areas in south-east Asia, including India and Indonesia. Its viral genome had a linear positive sense ssRNA which spread through seed, cell sap and aphids (Aphis craccivora) feeding in a nonpersistent manner. Biologically distinct PStV strains cause a wide range of symptoms such as striping, discontinuous vein banding along the lateral veins and okra leaf mosaic. The virus remains infective in buffered plant extracts after dilution to 10 3, 3-day storage at 20 °C and heating for 10 min only at 60 °C. In view of lack of resistance sources in peanut germplasm, preliminary attempts were made to develop transgenic plants using PStV coat protein gene (Franklin et al., 1993). Later, successful attempts were made to develop transgenic plants carrying two coat protein genes untranslatable full-length sequence (CP2) and translatable CP gene with an Nterminal truncation (CP4) of PStV, which offered resistance to viral inoculation under glasshouse conditions. Interestingly, both the transgenic lines carrying untranslatable CP2 and translatable CP4 witnessed no detectable protein on viral challenge inoculation that suggested underlying resistance via an RNA-mediated mechanism (Higgins et al., 2004).

Abiotic stresses Abiotic stresses comprise multiple factors such as drought, salinity, water logging, high temperate and chilling. These factors are a major constraint to crop productivity predominantly in the semi-arid tropics, where leguminous crops are cultivated. In case of biotic stresses, sources of resistance are available to reduce damage. However, abiotic stresses are beyond the control of farming community. Around 10.0% of the global arable land exposes to one or other abiotic stresses. Moreover, crops that are grown in abiotic stress are usually more susceptible to weeds,

insect pests and diseases, which directly promote to increased losses affecting production and productivity. Among all the abiotic stresses, Sharma and Lavanya (2002) reported an economic loss of $500 million annually. Moreover, the prediction in climate aberrations indicate the occurrence of extreme drought conditions in tropical and subtropical regions of the World in future, which clearly signify the upcoming limitation of irrigation sources and challenges for crop production including peanut (Battisti and Naylor, 2009; Lobell et al., 2008).

Drought Under drought conditions, Bhatnagar-Mathur et al. (2007) attempted to develop transgenic plants by increasing water-use efficiency. The transcription factor, DREB1A, specifically interacts with the dehydration responsive element and induces the expression of genes involved in environmental stress (Kasuga et al., 2004). A T-DNA constructs carrying a DREB1A as a candidate gene driven under control of stress-inducible promoter rd29A had been transformed in a drought susceptible cultivar of peanut for developing transgenic plants. No retardation was noticed by some transgenic lines tested under drought conditions. RT-PCR analysis showed that DREB1A gene driven under rd29A promoter induced only 5 day after irrigation withdrawal to plant which indicates its strong correlation with a fraction of transpirable soil water. This also reveals the significance of transgene in balancing the transpiration rate to a well-water situation, under drought conditions. The effect of DREB1A gene on antioxidative machinery, in transgenic plants, was associated for the antioxidant accumulation, under drought stress, with higher levels of superoxide dismutase (SOD), ascorbate peroxidase (APOX) and glutathione reductase (GR) enzymes as compared to control plants. In addition, significant increases in proline and malondialdehyde contents, which are end products of lipid peroxidation, have also been noticed in transgenic plants under similar conditions. Moreover, the above antioxidants accumulation showed a negative correlation with the fraction of transpirable soil water thresholds, where the normalized transpiration rate started decreasing in transgenic plants (Bhatnagar-Mathur et al., 2009). In another study, DREB1A exhibited resistance in transgenics at 1 °C over a period of 10 days, and this mechanism plays a significant role in screening transgenic events for low temperate shocks. Other transcription factors such as DREB2A could also be tested for stress response in peanut that are involved in the regulation of both dehydration-inducible and heat shock-related genes. Among the phytohormones, cytokinins play a major role in plant development and are plentiful in proliferating tissues. Although the actual cytokinin biosynthesis site is not well known, in the tissues, high level of cytokinin is considered as its sites of synthesis; however, the site of synthesis might differ from the accumulation site. Variation in the levels of cytokinin depends not only in the cell cycle and developmental stages but also during environmental stress conditions, while the levels have been reported to decline with leaf senescence (Nood
en et al., 1990). As the isopentenyl transferase (IPT) gene plays an important role in cytokinin biosynthesis, its overexpression in peanut transgenics not only provided drought tolerance characteristic but also showed higher seed production, transpiration, photosynthesis, stomatal conduction and biomass (Qin et al., 2011). These physiological traits confer greater potential of IPT gene, whose utility can be explored among other leguminous crops. Endo et al. (2001) recommended the use of IPT gene as a selective

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

Research progress of peanut genetic engineering 153 marker in model plant system. Another transferase gene, Zmpsy1 (phytoene synthase) from maize conferring resistance to abiotic stress, which exhibit active involvement in carotenoid biosynthesis, has also been used in peanut transformation, but the related traits have not been assessed in the progenies (Bhatnagar et al., 2010).

Salinity Plant response of maintaining cellular level ions homeostasis against abiotic stress particularly, salinity, is due to harmonized function of various biochemical pathways. Improvement of salinity tolerance in crop plants thus necessitates the identification of tolerant effectors and regulator components that maintain cellular level ions homeostasis during stress conditions. Protein synthesis is reduced by stress condition affecting the cellular gene expression machinery, evincing the involvement of molecules involved in nucleic acid synthesis. Helicases (protein motor) are ATP-dependent enzymes that are commonly present with a function to unpacked nucleic acid particularly DNA, RNA or DNA– RNA hybrid through regulatory processes involving replication, transcription, translation, repair/recombination and ribosome biogenesis (Tuteja et al., 2012). Under stress conditions, helicases have been studied in stabilizing plant growth by modifying abiotic stress-induced pathways for exploring drought and salinity tolerance. The pea DNA helicase, PDH45 isolated by Pham et al. (2000), was homologous to translation initiation factor as well as stimulus for topoisomerase-I activity. In a model system, PHD45 gene under saline conditions in the progeny of transgenic plants yielded not only normal viable seeds but resulted in low levels of Na+ ion accumulation among the leaves by expressing delayed senescence (Sanan-Mishra et al., 2005). This helicase gene has also been tested for salinity tolerance in peanut and other crop plant species (Amin et al., 2012; Manjulatha et al., 2014; Sahoo et al., 2012; Sanan-Mishra et al., 2005). The overexpression of PDH45 gene in transgenic peanut showed 27.0% and 17.2% increased yield under stress and nonstress conditions, respectively. These transgenics also witnessed an increased stability of chlorophyll content and its reduced retardation as well as PEG stimulated dehydration. Thus, the transformation of helicase gene through cellular level tolerance demonstrated the feasibility of pyramiding the traits associated with high-yield and different mechanisms of drought tolerance in peanut. Additionally, the progenies of transgenic plants also showed increased root growth rate under desiccation conditions (Manjulatha et al., 2014). The direct role of helicase gene creates a possible avenue to explore tolerance for salinity and drought conditions in other plant species. Higher concentration of salts in soil entail water deficit conditions leading to accumulation of Na+ and Cl ions which are detrimental to crops plant species including peanuts. But the responses vary at high salt concentrations among the susceptible and tolerant genotypes. Susceptible plants confine the uptake of salts in plant and adjust their cellular osmotic pressures by the synthesis of compatible solutes or signalling molecules such as proline, glycine betaine and sugars. Whereas the tolerant plants confiscate and accumulate salts in cell vacuoles for regulating salt concentrations in the cytosol as well as ensuing maintenance of high cytosolic K+ : Na+ ratio in plant cells (Glenn et al., 1999; Tal and Shannon, 1983). The ion exclusion mechanism provides protection to plants for salinity only at low concentrations of the solvent, thus leading to inhibition of key metabolic processes and concomitant growth under high concentration of salts.

Yamaguchi and Blumwald (2005) suggested two possible strategies for the maintenance of high K+ : N+ ion ratio in the cytosol or intracellular fluid of plant cells under salinity conditions that involve ion extrusion in the cytosol or intracellular fluid of plant cells under salinity conditions with Na+ extrusion out of the cell vacuolar compartment. Proton electrochemical gradient (PEG) across the plasma and tonoplast membranes is the driving force for Na+ ion inflow into vacuole through the activity of Na+/H+ antiporter, which is the molecular basis for the AtNHX1 overexpression that leads to increased salt tolerance in transgenic plants (Blumwald, 2000). Similarly, the overexpression of most abundant vacuolar Na+/H+ antiporter (AtNHX1) was instrumental in developing salt tolerance in A. thaliana, other monocot and dicot plant species (Chen et al., 2007; Ohta et al., 2002; Soliman et al., 2009; Sottosanto et al., 2007; Tian et al., 2006; Xing et al., 2010; Xue et al., 2004; Yin et al., 2004; Zhang and Blumwald, 2001; Zhang et al., 2001). Peanut transgenics carrying AtNHX1 gene in the genomic DNA also confirmed drought tolerance. Levels of salt tolerance have been targeted by exposing plants at 200 mM of NaCl for 21 days that caused significantly higher levels of tolerance at increased levels of Na+ : K+ ratio as well as the proline content in leaf tissue of transgenic plants. Nevertheless, proline accumulation in leaves is purely a stress response through interactions of tolerant effectors. Accordingly, increased proline content in transgenic leaves was attributed to activation of tolerant effectors through AtNHX1 gene in peanut. These transgenics withstand the drought shock over a fortnight period of water deficit conditions as against wild types, wilted in 5 days and turned brown leading to plant mortality (Asif et al., 2011). Evaluation of Na+/H+ antiporter gene in transgenic peanuts confirmed less damage, increased rate of biomass, chlorophyll, photosynthesis, stomatal conductance, transpiration and CO2 assimilation, under salt stress conditions (Banjara et al., 2012). Maintenance of Na+ ion concentration in the plant cell vacuole is promoted by the proton electrochemical gradient (PEG), which is highly influenced by vacuolar adenosine triphosphatase (V-ATPase) and H+ pyrophosphatase enzymes. Increased activity of these metabolic enzymes has led to boost salinity tolerance in transgenic Arabidopsis (Gaxiola et al., 2001) and cotton (Pasapula et al., 2011). Subsequently, overexpression of another Arabidopsis vacuolar H+ pyrophosphatase gene (AVP1) was found to enhance drought tolerance associated with increased yield in peanut transgenics (Qin et al., 2013). In addition, these transgenics also expressed increased rate of obtaining higher biomass. Interestingly, AVP1 transgenic plants also witnessed an outrate yield increase over wild type plants. Thus, these metabolic enzymes hold greater promise for biofuel crops in changing climate, where realizing the biomass is principal baseline factor for energy generation. Other abiotic stresses involve intense heat, cold, UV-radiation, wound etc. causing programmed cell death due to plasma membrane blebbing, cell shrinkage, nuclear condensation and nucleosomal DNA fragmentation. This unique feature of programmed cell death (apoptosis) in plants was associated with these abiotic stresses (Gechev et al., 2006; Hansen, 2000; Hofius et al., 2007; Watanabe and Lam, 2004). Plant cell organelles such as mitochondria and chloroplasts also play an instrumental role in regulating the programmed cell death through signalling pathways, as they are involved in energy production as well as increases in intracellular reactive oxygen species. Reactive oxygen species are also known as toxic metabolic products. Increased level of reactive oxygen species and other death activating

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

CaMV35S x 2 CaMV35S 9 2 Phaseolin ACT2 Emu CaMV35S CaMV35S

– – – – – – –

E Ax

E Cal

E Cal

Leaflets

Leaflets

Leaflets

Valencia A

2S2

– A281

Coty

Stem internode

ATC1

CaMV35S

–

Coty

CaMV35S

–

Tatu

CaMV35S

–

Embryonic leaflets

Embryonic leaflet

Tatu

CaMV35S

LBA4404

ATC1

CaMV35S

CaMV35S

CaMV35S

Tamnut 74

Leaf

TAG-24

ASE1

Hypocotyl A208

LBA4404

Hypocotyl

Stem internode

EHA101

Hypocotyl

Penapo

Okrun

CaMV35S

E Cal EHA105

CaMV 35S

–

E Cal

Leaf section

CaMV 35S

–

Mature seeds

NC-7

New Mexico

CaMV35S

–

I Emb

Luhua 9

GK7

CaMV35S

–

E Ax

ACT2

– CaMV35S

CaMV 35S

–

E Cal –

CaMV 35S

–

E Cal

E Ax

CaMV35S

–

Mature seeds

Gerogia green

CaMV35S

–

E Cal

E Ax

MAS CaMV35S

EHA101 –

EAx

EAx Coty

Georgia Runner

Gajah

Florunner

CaMV35S:(r)PR1a CaMV35S

EHA101 –

DEC

pTiBo542

pFAD6, pEA18

pEA1,

pFAD6, pEA18

pEA1,

pGUSINT

pRT99

pBI121

pTiT37

pKYLX71

pKYLX71

pKYLX71

pBI121

pGN1+pHygr

pDO432+pHygr

pGN1, pHygr

pDO432,

pCAMBIA1301

pTRA140

pEmuGN

pAC2GUS

pPPCH-V-Arah1_si

p524EGFP.1+

p524EGFP.1

pEYFP-C

pEGFP-C

pAC2MR

pGN1+pHygr

pDO432+pHygr

pGN1, pHygr

pDO432,

pH602

pWRG2114

pMON9793

pWRG2114

pCAMBIA1300

pBI121

pRD400

– CaMV35S

Plasmid

Promoter

EAx

C58

Coty

Florigiant

GV2260

Coty N

JL24

strain

Explant tissue

Genotype

Agrobacterium

Table 2 Transformation study on peanut (Arachis hypogaea L.)

GUS

2S albumin

gus, 2S, crsl

GUS

GUS

GUS

GUS

GUS

GUS

GUS

GUS

GUS

Luc

luc, uid A

uid A

GUS

GUS

GUS

Ara h1

GFP

EGFP

EYFP

EGFP

merA

GUS

Luc

luc, uid A

GUS

tswv-np +GUS

GUS

tswv-np +GUS

gus

uid A

GUS

gene

Transformed

Lacorte et al. (1991)

– –

–

–

–

57.1

Lacorte et al. (1991)

Lacorte et al. (1997)

Lacorte et al. (1997)

Clemente et al. (1992) De Pa^adua et al. (2000)

Eapen and George (1994)

Franklin et al. (1993)

– 7.6

Franklin et al. (1993) Franklin et al. (1993)

–

Cheng et al. (1996)

Livingstone and Birch (1999)

Livingstone and Birch (1999)

Livingstone and Birch (1999)

Yang et al. (2001)

Kim et al. (1999)

Kim et al. (1999)

Kim et al. (1999)

Chu et al. (2013)

Chu et al. (2013)

Joshi et al. (2005)

Joshi et al. (2005)

Joshi et al. (2005)

Yang et al. (2003)

Livingstone and Birch (1999)

–

0.2–0.3

50.0

57.0

–

1.6

36.0

15.0

15.0

–

–

81.0

59.0

32.0

–

50.0

Livingstone and Birch (1999)

Livingstone and Birch (1999)

– 57.0

Ozias-Akins et al. (1993)

Brar et al. (1994)

McKently et al. (1995)

Brar et al. (1994)

Tiwari et al. (2011)

Sharma and Anjaiah (2000)

Anuradha et al. (2006)

References

0.0

–

9.0

–

–

55.0

31.0

efficiency (%)

Transformation

154 Gaurav Krishna et al.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

80.0 Leaf

Coty, cotyledon; Coty N, cotyledonary node; DEC, De-embryonated cotyledon; E Ax, embryo axes/Embryonic axes; E Ax Coty, embryonic axes with cotyledon; E Cal, embryogenic callus; I Emb, immature embryo.

GUS pBI121 R1000

I Emb YueYou 116

CaMV35S

CaMV35S

CaMV35S

EHA 105

–

Axillary shoot Valencia A

ATC1

CaMV35S

A281

Embryonic leaflets UPL PN 4

–

Stem internode Tupa

1.6

Kim et al. (2009)

Cheng et al. (1997b)

uid A

Yang et al. (2001)

– GUS pBI121

pCAMBIA1301

Lacorte et al. (1991)

Clemente et al. (1992)

–

–

GUS

GUS

pTiBo542

pRT99

Venkatachalam et al. (2000)

Ozias-Akins et al. (1993) 1.0

CaMV35S

CaMV35S

LBA 4404

– E Cal

A208

Toalson

Coty

47.0 uid A

GUS

uid A

pBI121

– GUS pTiT37

pKIWI105 E Ax TMV-2

ATC1

LBA 4404

Stem internode Tatui

CaMV35S

pH602

Lacorte et al. (1991)

– pTiBo542

3.3

Freitas et al., (1997)

Lacorte et al. (1991)

2.0 GUS

GUS

p35GUSINT CaMV35S

ATC1 A281

EHA101

Stem internode

efficiency (%)

Coty

Tatu branco

Transformed

Transformation

References gene Plasmid Promoter strain

Agrobacterium

Explant tissue Genotype

Table 2 Continued

Rohini and Rao (2000)

Research progress of peanut genetic engineering 155 proteins leads to stimulation of this signalling pathway for programmed cell death. The depression of these stimulating factors escorts not only to overcome programmed cell death but also resulting in tolerance to abiotic stresses. Anti-apoptotic genes originating from mammals, nematodes or viruses have genetically been transformed into plants that witnessed an increase in host tolerance to a broad range of biotic and abiotic stresses (Awada et al., 2003; Li and Dickman, 2004; Mitsuhara et al., 1999; Xu et al., 2004). In addition, some of these anti-apoptosis genes have been used for tolerance testing against herbicide application and wounds (Chen and Dickman, 2004; Qiao et al., 2002). In peanut, a human anti-apoptosis gene Bcl-xL was tested for tolerance to herbicides and salinity stresses. It has been reported that 5 lM of paraquat (methyl viologen dichloride) @ 0.1% of Tween-20 solution could not affect the level of chlorophyll in transgenic plants, while the seeds of transgenic lines did not germinate due to lack of salinity tolerance, when cultured aseptically on medium supplemented with 100 mM NaCl. Transgenic plants expressing resistance to herbicides but not exhibiting tolerance to salinity have been held due to multiple integration of Bcl-xL gene coupled with its deleterious expression (Chu et al., 2008). Peanut plants having bialophos resistance gene (bar) integrated in genomic DNA confirmed resistance @ 500 ppm concentration of the herbicide BASTA (Hoechst) compared with wild type nontransgenic plants (Brar et al., 1994). This signifies a potential to generate single-copy events to test for salt tolerance. There is ample scope for other anti-apoptotic genes originating from plants, viruses and mammals for testing their suitability in peanut genotypes.

Oral vaccine Expression of antigens coding gene in transgenic plants is increasing slowly in the form of edible vaccines to perform oral immunization. This technology of vaccination is gaining importance because of its suitability to express immunogenic proteins against viral pathogens. As a step towards development of a thermo stable subunit vaccine, peanut has been used for the expression of RPVH gene against cattle plague disease (Khandelwal et al., 2003a) caused by Rinderpest virus. The expression and inheritance study for RPVH demonstrated stable integration in T1 generation plants, which have been used for the oral administration of cattle for vaccination resulting into hemagglutinin-specific antibody production (Khandelwal et al., 2003b). Further, BTV-VP2 (bluetongue VP2 gene) has also been expressed in peanut for resistant to bluetongue virus (BTV) which is a causal agent of bluetongue of disease of sheep (Athmaram et al., 2006). Recently, HN gene expressing hemagglutinin– neuraminidase protein of Peste des petits ruminant virus (PPRV) in peanut plants has also been tested for oral administration in vaccination of sheep (Khandelwala et al., 2011). Helicobacter pylori (previously named Campylobacter pylori) is a Gram-negative, microaerophilic bacteria found in human stomach, its colonization is the main cause of peptic ulcers and gastric cancer. Infection of these bacteria is a serious threat to human population in developing countries. It is considered as Class I carcinogen of gastric cancer by World Health Organization (WHO), whose treatment is expensive and less effective for some resistant strains, and thus mostly dependent on antibiotics in combination with proton pump inhibitor (Bazzoli et al., 2002). Urease is an essential protein for the survival and pathogenesis of H. pylori under acidic conditions. UreB is the most potential and

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

156 Gaurav Krishna et al. common immunogen for all the strains of H. pylori in stimulating immune response against infections in mice (Ferrero et al., 1994; Giudice et al., 2001). Plant-derived human vaccines are especially attractive, as they are free from human diseases and enable to reduce virus and bacterial toxins screening cost. Moreover, the expressions of vaccines in plant tissue also offer a heat stable environment and enable oral delivery by preventing injectionrelated hazards. In addition to these facts, the major merit of oral vaccination is that the chance of acquiring mucosal immunity from which generally infectious agents enters the body (Gu et al., 2006). Peanut has been used for the expression of a full-length sequence from ZJC02 stain of H. pylori, urease subunit B gene (UreB) under control of seed-specific promoter, oleosin. These plants tested for its expression as well as transcript analysis were found suitable for using as a vaccine against H. pylori infection in human system (Yang et al., 2011). Among all, the plant species used for oral vaccination against H. pylori such as tobacco, rice and carrot (Gu et al., 2005, 2006; Zhang et al., 2010), peanut have advantages of relatively high level of target antigen present in seeds which can be eaten raw by reducing chances of degradation while cooking or processing.

Genetic transformation First attempt to develop transformation protocol through Agrobacterium tumefaciens was initiated by Lacorte et al. (1991). The advantages of Agrobacterium-meditated transformation include its straightforward methodology, familiarity to researchers, minimal equipment cost and reliable insertion of a single copy or low copy of transgene. Emphasis was laid on the transformation studies linked to the use of a scoreable marker, Agrobacterium strain, vector, explant type, promoter and selectable markers system. Agrobacterium–host compatibility studies have been analysed using explants of stem internodes from different peanut genotypes against various Agrobacterium strains. A summary of various transformation studies carried in peanut along with various factors having potentiality to influence transformation efficiency is presented in Table 2.

Tissue culture protocols To achieve successful and efficient transformation system, adoption of a suitable tissue culture protocol is a baseline factor, as it facilitate most of the in vitro steps of transformation particularly the selection of transformed cells after transgene delivery to explants. Regeneration of transgenic plants through somatic embryogenesis although had several merits over organogenesis, the latter method has always been indispensible due to its shorter duration in plantlet development. Other than these tissue culture-dependent protocols, in-planta transformation (tissue culture-independent protocol) has also been successfully attempted for the transgenic events generation using embryonic axes, apical meristem and seedling explants (Entoori et al., 2008; Keshavareddy et al., 2013; Manjulatha et al., 2014; Rohini and Rao, 2000; Rohini and Sankara, 2001). Due to robustness of Agrobacterium and microprojectile bombardment-mediated gene transfer in plants, they have been equally exploited in peanut transformation. Transformation efficiency linked to tissue culture protocols that are specific to variable procedures adopted for transgenic development, resulting in calculation of variability in terms of explants numbers vs number of antibiotic selected shoot number, transgene expressions, confirmation through PCR and southern hybridization tests. In addition, the biolistic mediated of

transformation calculates sometime on the basis of cotransformation of both selectable marker and transgene (Brar et al., 1994; Chu et al., 2008; Higgins et al., 2004; Iqbal et al., 2012; Singsit et al., 1997; Yang et al., 1998). Thus, studies revealing low transformation efficiency cannot be left a side while choosing a protocol for transforming gene of interest in peanut. In case of somatic embryogenesis, older age of cell lines is highly transformable (Magbanua et al., 2000). In conclusion, although Agrobacterium-mediated protocol reflects low transformation efficiency than biolistic protocol (Asif et al., 2011; Bhatnagar et al., 2010 Yang et al., 1998) which requires large number of explants or cell lines to obtain a low-copy event (Singsit et al., 1997; Yang et al., 1998), the prior method has high potential in achieving single-copy stable lines.

Explants Development of peanut transgenics has been explored through organogenesis and somatic embryogenesis using various explants types (cotyledonary node, cotyledon, de-embryonated cotyledon, embryogenic callus, immature embryo, embryonic axes, immature cotyledons, shoot apices, immature leaf and cotyledon with intact embryo). Among them, cotyledon explants are mostly preferred in transformation via organogenesis and somatic embryogenesis. But they are less productive because of longer duration and low regeneration frequencies in comparison with other explants (Venkatachalam et al., 2000). Thus, stabilization of transformation amenable regeneration method for explants other than cotyledon may greatly aid in achieving rapidly and more number of genetically transformed peanut plants.

Genotypes Although 37 peanut genotypes have been used for genetic transformation, nine types (BARI-2000, Florunner, Georgia Runner, Gerogia green, Golden, JL24, MARC-I, Toalson and VC1) were found compatible exhibiting >75.0% efficiency. In contrast, four genotypes (YueYou116, Toalson, Tatu and Luhua9) showed higher susceptibility to genetic transformation due to their recalcitrant nature. This reflects the development of transformation amenable tissue culture protocol had high potential for these genotypes compared with other genotypes expressing low transformation efficiency.

Transformation protocols Two methods are witnessed for relatively greater success to produce completely stable-transformed plants include as follows: particle bombardment and A. tumefaciens-mediated transformation system. Use of a biological vector, A. tumefaciens, disarmed (nontumorigenic) strain facilitates transfer of desirable transgene engineered into host plant genome. Advantages of Agrobacterium-meditated transformation are not only limited to incorporation of large segments of DNA in single- or low-copy fashion, rare transgene rearrangement, reduced abnormal transgene expression, maintenance of fertility in transgenics lines but also transmission of inserted segment in Mendelian style. On the other hand, particle bombardment has possibility of transferring genes to any type of cell/tissue independent of genotype and without considering the compatibility between the host and bacterium as a prerequisite for the Agrobacterium system. Particle bombardment method, although faster and expensive, has got limitations of multiple transgene copies insertion into the host genome. Diverse biotypes of Agrobacterium such as octopine, nopaline and agropine have been used in peanut transformation studies.

ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 13, 147–162

Research progress of peanut genetic engineering 157 Among them, octopine and nopaline have been used to achieve higher transformation efficiency. Comparative study of nopaline (C58/LBA4404) and agropine (EHA101) strains showed higher gene transformation efficiency with EHA101 under defined culture conditions (Egnin et al., 1998; Franklin et al., 1993). Although Agrobacterium biotypes have been tested individually for the transformation studies in peanut, extensive comparative studies are lacking with different strains comprising a single binary vector in a highly potent genotype. Such studies in peanut would greatly aid in rapid research of peanut improvement through genetic engineering. So far, a number of laboratories have pursued Agrobacteriummediated plant transformation for which different genotypes and/or explants material have been found contributory to varied regeneration responses associated with transgenic development. The gestation period of a transgenic via in vitro-based protocol ideally takes >4–5 months to obtain putative plantlets. To avoid interference of tissue culture-based regeneration, a nontissue culture-based in-planta transformation method has been attempted by Rohini and Rao (2000) using embryonic axes with one cotyledon excised from mature seeds, and Agrobacterium octopine strain (LBA4404) carrying reporter gene. Using similar protocol, tobacco chitinase (CHI), cry1X and cry1AcF genes were utilized for developing transgenic plants resistant to leaf spot, and S. litura either alone or in combination of H. armigera (Entoori et al., 2008; Keshavareddy et al., 2013; Rohini and Sankara, 2001). Abiotic stress tolerant peanut transgenic plants have also been developed using in-planta transformation protocol (Manjulatha et al., 2014).

Transformation vectors The role of transformation vectors has gained importance due to essential features such as bacterial origin of replication, left and right border sequences, vir gene, multiple cloning sites, reporter gene and selectable markers. A binary vector system consists of two plasmids. The helper constituted by the Agrobacterium Ti plasmid without T-DNA carries the vir genes that are necessary for the transfer of T-DNA into the host genome and a binary plasmid derived from the commonly used E. coli cloning vectors carrying genes of interest, flanked by 25-bp terminal repeats, designated as the right and left border sequences (Lee and Gelvin, 2008). The binary cloning vector is a standard molecular tool in the Agrobacterium-mediated transformation of higher plants, because of its amenability to manipulate DNA of host plant genome. In peanut transformation, different genotypes have been used with diverse binary vectors as the prime reason behind achieving variable efficiencies in transgenic production. Plasmid uptake into the Agrobacterium cell is another reason for difference in transformation frequencies. Despite using pTiBo542 and pTiT37, comparative studies utilizing various vectors need to be explored for selecting the most suitable vector for gene transfer in peanut (Lacorte et al., 1991). In addition, exploration of the superbinary vector that carries additional vir genes in the plasmid comprising T-DNA region, besides the native vir genes present in the acceptor vector can also be tested in peanut. Superbinary vector with supervirulence has already been reported for the high transformation efficiency in other crops (Cheng et al., 1997a; Ishida et al., 1996; Ramesh et al., 2004). In peanut, a comparative study of transformation has been conducted using different vectors in single experiment or as a set in case of cotransformation (Chu et al., 2013; Joshi et al., 2005; Kim

et al., 1999; Lacorte et al., 1991; Livingstone and Birch, 1999). The particle bombardment method of genetic transformation although reliable for delivering genes of interest into various species, low-copy number event has always been a limiting factor in its application. During cobombardment with two separate plasmids containing a green fluorescent protein gene and gene of interest with selectable marker were tested for its efficiency on transgene incorporation in peanut plant tissue. While observing fluorescence in bombarded embryogenic tissues, 4- to 6-fold improvement in transformation efficiency was reported in stabletransformed peanut lines using protamine instead of conventional spermidine in the bombardment mixture (Chu et al., 2013).

Promoters Expression of transgene is highly dependent on the promoter selection for protein synthesis. As the primary transformant regeneration is highly dependent on the selection for gene expression, thus the strategy of selection gene expression had similar potential to the gene of interest likely to be placed or expressed for improving traits in any species. In peanut, several promoters of various origins have been tested for transgene expression other than CaMV35S and double enhance CaMV35S. Comparative study of three promoters for the expression of gus gene showed that synthetic maize Emu exhibited more blue spots in a short incubation period as compared to Rice-Actin2 and CaMV35S in peanut, whereas Rice-Actin2 expressed highest GUS activity among all the three promoters. Further, Arabidopsis actin2 (ACT2) promoter has been used for driving mercuric ion reductase gene (merA) to develop alternative selectable marker for peanut transformation (Yang et al., 2003). Moreover, the transgene expression has been observed in vegetative tissues but not in somatic embryos. This implies that ACT2 promoter had a large potential for its use as a regulatory element for marker genes in a transformation system where selection occurs postembryonically or during organogenesis. This promoter also had a large potential for conducting tissue-specific study during various stages of plant development. In another comparative study of CaMV35S and 2S2 promoter for the gus gene expression in immature and mature peanut seed, tissues revealed that transient expression of gene has been found higher in both the cassettes. However, the GUS expression has been found to be higher in immature tissues as compared to matured or stored seed. This type of tissue-specific expression by 2S2 promoters has been reported at late stage of seed development in pigeon pea (Krishna et al., 2010). In addition, the exploitation of promoter vspB from soybean resulted in higher levels of gene expression in the leaves and stem over roots in peanut (Wang et al., 1998). Studies on the effect of stress-inducible promoter, rd29A gene, a transcription factor DREB1A from A. thaliana was responsible for inducing transgene expression only at 5th day of withdrawal of irrigation to transgenic plants (Bhatnagar-Mathur et al., 2007). Another maturation- and stress-induced promoter stress (SARK) has been tested for controlled expression of IPT displayed delay in stress-induced plant senescence (Qin et al., 2011). Towards studying the insect resistance, a pathogenesis responsive promoter PR-1a in combination with CaMV35S has been used for enhancing the resistance in transgenic peanuts. This fusion promoter has resulted in complete mortality of larvae of S. litura associated with high-level expression of cry1EC during each bite. Such type of promoter has greater scope for insect resistance in plant species which are prone to S. litura damage, including peanuts (Tiwari et al., 2011).

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158 Gaurav Krishna et al.

Future research thrusts In view of increasing importance for enhancing the production and productivity of peanut, newer challenges are encountered for a sustainable peanut cultivation in the next millennium. The success of transformation in peanut is still inadequate due to its genotype, explants and protocol dependency. Moreover, focused and intensified research warrants efforts to develop genotypeindependent approaches for obtaining stable in vitro genetic transformants. Optimization of various factors influencing the genetic transformation protocols would possibly improve the efficiency of transgenic development over a short span of time with more precision and reproducibility. However, the conversion rate of somatic embryos into normal plantlets remains low in peanut after transgene incorporation either through Agrobacterium or particle bombardment methods. Thus, future research thrusts must focus on enhancing conversion frequency of somatic embryos after transformation into normal plantlets regeneration. In-planta transformation also needs to be emphasized further which may have a great promise in peanut genetic engineering. Despite substantial advances biotic management strategies, the global production is still threatened by a multitude of insect pests and pathogens. This changed scenario warrants us to respond more efficiently and rapidly to tackle these problems of peanut. The situation demands judicious blending of conventional, unconventional, frontier and modern crop improvement technologies. Pathogenesis-related proteins were effective in arresting fungal infections, but the underlying mechanisms need to be explored. Development of transgenic lines expressing translated/untranslated sequences of nucleocapsid and coat proteins for resistance to potyviruses infection demonstrates high potential for other viral disease such as Peanut mottle virus and Peanut green mosaic virus. Development of genetically transformed peanuts with resistance to other abiotic constraints such as temperature aberration has tremendous impact in peanut productivity in the resource-poor agricultural systems of the semiarid tropics. In addition, the peanut genotypes have proved as a potential factory to the production of edible vaccines for cattle and human beings. Plant made vaccines not only attractive but also free from diseases, heat stable environment and enable oral delivery. Thus, many other oral vaccine-associated genes can also be tested as immunization agent. Peanut genotypes reported for low transformation efficiency through Agrobacterium can be worthy for testing with other methods such as particle bombardment due to genotype independency, ability to target any cell type and in intact organized tissues. In addition, the genotypes that is less responsive to DNA uptake by particle bombardment can as well be subjected to Agrobacterium-based transformation system either under in vitro or in-planta for improvement. Selection of supervirulent strains of Agrobacterium with improved vir gene, use of acetosyringone, thiol compounds and osmotic agent supplementation to the culture medium, including their interactions may improve the transformation efficiency in peanut. Development of highly virulent strains comprising superbinary vector(s) with additional vir gene can be tested for enhancing the transformation efficiency of peanut. So far, a comparative study of various binary vectors and Agrobacterium strain for identifying potential of transformation system has not been attempted. In peanut, most Agrobacterium transformation studies are governed using the direct organogenesis, and the possibility exists to work on the exploration of potential of somatic embryogenesis.

Most widely used technology for the generation of engineered plants employs A. tumefaciens worldwide. However, to commercialize a product developed through Agrobacterium-based technology had lot of obstacles due to a series of key patents on transformation technology. To overcome this, many other bacterium-based transformation methods such as transbacters and Ensifer adhaerens have supported the generation of commercial products (Toni et al., 2012). Other technologies such as agrolistic transformation approach that combines the advantages of efficient biolistic delivery and the precision of the Agrobacterium T-DNA insertion mechanism may have greater potential in peanut improvement as it allows the integration of genes of interest, avoiding the undesired vector sequence (Hansen and Chilton, 1996). Auxin biosynthesis, translocation and its signalling network during fruit development had a greater potential in the form of realizing higher yields, not only in peanut but also in other crop species. Moreover, no work has been carried towards peanut oil fortification although genetic engineering which had a bright scope in improvement. After the development of transgenics, markers genes such as kanamycin, hygromycin and herbicides are usually become redundant. Although, there is no evidence that selectable marker genes are unsafe for consumers and the environment, it is desirable for their elimination from the end product of transgenic events, more so in edible crops. Thus, marker-free approaches for genetic transformation of peanut have greater potential in terms of regulatory approval for commercialization. The marker-free approaches using Agrobacterium as a transformation source for the introduction of gene of interest and plant selectable marker gene present in two different Agrobacterium cells individually carrying two different plasmids or single Agrobacterium cells carrying two plasmids or single Agrobacterium cells carrying plasmid with multiple T-DNA borders can be tested in peanut, as these approaches showed greater success both in monocot and dicot species (Manimaran et al., 2011). Other marker-free approaches such as site-specific recombination and transposon-based selectable marker gene elimination from nuclear or chloroplast genome could also be employed. Low level or line-to-line expression of transgenes is a common phenomenon in peanut transgenics. To increase the levels of expression promoters such as synthetic maize, Emu and RiceActin2 need to be tested for increasing the levels of transformation. In addition, codon optimization of a transgene using legume cDNA database could provide a suitable solution, to minimize codon bias in host plants. Moreover, AT-rich DNA sequences called MARs have been found responsible for increasing the levels of transgene expression due to expression of more mRNA than proteins and reduce line-to-line level of transgene expression both in animal and plants cells. Use of such type of DNA sequences can be tested for RNA-targeted viral gene in developing complete knock-down effect of viral infection in transgenic events of peanut as well as other legumes. Newly emerged targeted genome editing technology of functional genes promises to be a powerful tool in accelerating varietal improvement of plants. Zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs) which require two DNA binding proteins flanking a sequence of interest have proven to be effective in plant-targeted genome editing (Cermak et al., 2011; Li et al., 2012; Lloyd et al., 2005; Mahfouz et al., 2011; Wright et al., 2005). In addition, recently prokaryotic immune system-based high-throughput genome editing technol-

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Research progress of peanut genetic engineering 159 ogy CRISPRs (clustered regularly interspaced short palindromic repeats) have been also found successful in different plant species (Jiang et al., 2013). Unlike ZFNs and TALENs, this system requires one single noncoding RNA sequence namely single-guide RNA (sgRNA) for target specificity. The sgRNA guides Cas9 endonuclease to recognize and cleave/correct targeted DNA in a precise manner. Indeed, these emerging technologies have dramatically expanded the ability to manipulate genome not only in monocots but also in dicotyledonous plant system, and thus, genome editing technologies provide a promising hope for further peanut improvement.

Acknowledgement Authors extend their thanks to Dr. B. Upendranath Singh (Former Principal Scientist, Directorate of Sorghum Research, Rajendranagar, Hyderabad–500030, Andhra Pradesh, India) for providing necessary guidance, valuable suggestions and all the help extended in editing of the manuscript. Authors BKS, EKK and VKM are thank full to Inha University for providing necessary environment during study.

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