Received Date: 03/12/2018; Accepted Date: 27/12/2018; Published Date: 05/01/2019
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Groundnut, the third largest producing oilseed crop grown in arid and semiarid regions worldwide. The crop growth under the rainfed conditions often exposed to the abiotic stresses that severely affects crop production and yield. Introduction of stress resistance traits into crop plants may improve the tolerance capability and mitigates yield losses under stress conditions. The success of breeding approach for developing crop varieties for stress tolerance determined by the efforts of several research fields including plant physiology, molecular biology, and genetics. Also, the other major limitations for breeding programs are the species barriers and genomic incompatibilities. Hence, use of novel molecular biology tools for revealing the important mechanisms of stress tolerance and engineering the stress tolerant crops by the overexpression of stress-specific genes through transgenic approach remains a viable option. This review focuses on the groundnut transgenics developed against various abiotic stresses like drought, salt and oxidative stress by overexpression of candidate genes involved in the regulation of metabolic pathways, cellular components, and stress-responsive pathways. This review includes discussion on the significance of transgenic groundnut events and the importance of trait ability to enhance the abiotic stress tolerance. This review also emphasizes the new progress in the transgenic event developments in groundnut and the novelty of multigene transgenics in comparison with single gene transgenics for the enhanced abiotic stress tolerance.
Groundnut, Transgenics, Abiotic stress, Co-expression, Multigene transgenics
Abiotic stresses are the major constraints for crop plants grown in arid and semiarid regions that alters the plant’s internal homeostasis and adversely affect the growth and productivity [1-3]. Amongst the abiotic stresses, drought and salinity were more prevalent in natural and agricultural systems and severely affects the crop quality thereby causes significant yield losses; hence, attained significant attraction from the researchers [4,5]. Semiarid tropics like Asia, Africa, South and North America are the leading groundnut cultivation regions with ~60% global production where drought and salinity are major stressors affecting both productivity and quality of groundnut [6-8]. Plants have adapted different tolerance mechanisms against abiotic stresses to cope with changing environmental conditions such as maintenance of cell turgor by changing cell metabolisms, osmotic adjustment , differential expression of genes encoding Transcription factors (TFs) , key enzymes in biosynthetic pathways [11,12] and proteins involved in stress perception and cell signaling [13,14].
Groundnut (Arachis hypogaea L.) is the third largest oilseed crop after soybean and rapeseed with a global production of 60.66 million tons under 32.20 million hectares of cultivation area worldwide with an average production of 1.88 tons/ha. India is the second largest producer of groundnut after China and produces 6.85 million tons with 5.8 million hectares of cultivation area . It is one of the major edible oil seed crops and contains 40-50% of oil, proteins (22-30%), minerals (P, Ca, Mg and K), carbohydrates and vitamins (E, K and B group) and also used as feed for livestock [16-19]. Groundnut is cultivated extensively in arid and semiarid regions of the world under rainfed conditions; as its production depends on annual rainfall of a particular region which varies from time to time it is more prone to abiotic stresses like drought, salinity, high temperatures and heavy metal stress [20-22].
In this scenario, there is a need to improve the groundnut productivity under abiotic stress conditions using reliable technologies for achieving maximum success rate to cope up with these adverse conditions. Conventional techniques have many limitations like species barriers, genomic incompatibilities, pollination, etc. makes them impotent for developing crops with desired agronomic traits [23,24]. Genetic engineering approaches have proven to be more versatile and comparatively fast over conventional methods and help to overcome all the barriers in developing crop plants with desired traits [25-27]. Several researchers [28-36] successfully developed transgenic groundnut against different biotic and abiotic stresses by the introgression of different genes including TFs and key enzymes of metabolic pathways through genetic engineering approach.
The abiotic stress tolerance is a multigenic trait and involves different signaling cascades and mechanisms. Even though the transgenic plants overexpressing single gene were successful, it is inadequate to achieve desired crop tolerance against abiotic stress as it is a complex phenomenon hence there is a need for co-expression of multiple genes involved in various metabolic pathways and quantitative traits . From the last two decades researchers were focusing on developing transgenics with more tolerance traits by introducing more than one gene (multigene transfer, MGT) instead of single gene transfer [38-41]. Co-expression of multiple genes in model plants like Arabidopsis , Tobacco [43,44] have shown improved abiotic stress tolerance compared to single gene transgenics and similar results were reported in crop plants like maize , sugarcane  and groundnut [47,48].
Previous review reports on groundnut transgenics tolerant to abiotic stresses so far have focused on drought and salinity . Hence, there is a requisite to document and summarize the information on other abiotic stress tolerant events and recent achievements in groundnut transgenics. This review summarizes the current progress in technology and traits used to develop the groundnut transgenics for the improvement of abiotic stress tolerance using the examples of research focused on drought, salinity, high temperature, and oxidative stress. The review is given specific attention to the advancement in transgenic technology in developing the multigene transgenics which involves the insertion of more than one gene to produce the groundnut transgenics with enhanced abiotic stress tolerance. In total seventeen groundnut transgenics, developed by manipulating with single and multiple genes which are reported to be involved in various abiotic stress tolerance mechanisms were discussed in this review (Table 1).
|Gene Source||Agrobacterium strain||Plasmid||Transformed gene||Promoter||Trait||Mechanism involved||Reference|
|1. Groundnut transgenics manipulated with a single gene against abiotic stresses|
|Macrotyloma uniflorum||EHA105||pCAMBIA2301||MuWRKY3||CaMV35S||Drought||Low MDA, H2O2, superoxides, more proline, sugars and anti-oxidative enzymes|||
|Arabidopsis thaliana||EHA105||pSARK||IPT||SARK||Drought||Increases in photosynthesis, stomatal conductance and transpiration and transpiration|||
|Arabidopsis thaliana||C58||pBI29||DREB1A||rd29A, CaMV35S||Drought||Transpiration efficiency, root development||[51,52]|
|Arabidopsis thaliana||-||pCAMBIA2300||DREB1A||rd29A||Drought||Higher osmolyte accumulation, proline; high RWC; Low EL; less chlorophyll reduction; root:shoot ratio, root volume;|||
|-||LBA4404||pCAMBIA1301||PDH45||CaMV35S||Drought||Improvement of cellular level tolerance|||
|E. coli||-||pCAMBIA1380||mtlD||CaMV35S||Drought||Increased accumulation of mannitol and ROS scavenging activity|||
|Arabidopsis thaliana||LBA4404||pBinAR||AtNAC2||CaMV35S||Drought||Higher CSI, RWC, and reduced RWL;|||
|Macrotyloma uniflorum||EHA105||pCAMBIA2301||MuNAC4||CaMV35S||Drought||Proliferate lateral root growth, enhancement of antioxidative enzyme regulation, osmotic adjustment and reduced membrane damage|||
|Arthrobacter pascens||LBA4404||pHS724||COX||2XCaMV35S||Salinity||Glycine betaine synthesis|||
|Salicornia brachiata||LBA4404||pCAMBIA1301||pAPX||CaMV35S||Salinity||Higher Chlorophyll content, RWC, shoot length, shoot and root weight; reduced electrolyte leakage;|||
|Arabidopsis thaliana||EHA105||pGreen0029, pSoup||HDG11||rd29||Drought & Salinity||Higher antioxidative enzymes, chlorophyll content Proline; longer root system; increased specific leaf area; reduced stomatal density; higher photosynthetic rates; increased intrinsic WUE|||
|Arabidopsis thaliana||LBA4404||pPZP212||AVP1||CaMV35S||Drought & Salinity||Biomass and higher photosynthetic rate|||
|NHX1||CaMV35S||Drought & Salinity||Salt and proline accumulation|||
|Arabidopsis thaliana||GV3101||pBISN1||NHX1||CaMV35S||Drought & Salinity||High chlorophyll content; large photosynthetic surface area, photosynthetic rate|||
|Salicornia brachiate||-||pCAMBIA1301||ASR1||CaMV35S||Drought & Salinity||Higher Chlorophyll, RWC; Lower electrolyte leakage and MDA content; Proline, sugars and Starch accumulation; Lower H2O2, O2 radicals; high SOD transcript levels|||
|Brassica carinata||-||pBinAR||ZAT12||LEA||Drought & Salinity||Delayed wilting of leaves, enhanced osmotic adjustment, improved water and chlorophyll retention, less electrolyte leakage.|||
|Pennisetum glaucum||LBA4404||pGreen0229||eIF4A||rd29A||Drought, Salinity & oxidative stress||Superior growth performance,
Chlorophyll retention and free radical scavenging.
|2. Groundnut transgenics manipulated with a multiple genes against abiotic stresses|
|Arabidopsis thaliana||LBA4404||pKM12GW||DREB2A, HB7, ABF3||CaMV35S||Drought & Salinity||Improved cellular level tolerance by increased expression of detoxifying enzymes, increased cell membrane and chlorophyll stability,
ROS scavenging and Osmotic adjustment.
|Drought & Oxidative stress||Higher root growth, cool crop canopy, higher RWC, Enhanced expression of HSPs, RBX1, Aldo reductases, LEA5 and PRP2 stress responsive genes. tolerance to ethrel, methyl viologen|||
Table 1: Groundnut transgenics developed against different abiotic stresses using genetic engineering approach.
Drought stress resistance
Isopentenyl transferase (IPT), the key enzyme of cytokinin biosynthetic pathway also reported to be involved in drought stress tolerance. The groundnut transgenics overexpressing Arabidopsis IPT gene under maturation and stress-inducible promoter, SARK  resulted in higher photosynthetic efficiency which is correlated with improved biomass when compared to wild type plants under drought stress in laboratory and field conditions. These transgenics also showed higher stomatal conductance and transpiration efficiency under reduced irrigation . Bhatnagar et al.  developed groundnut transgenics by overexpressing a stress inducible transcription factor gene DREB1A under two different promoters; a). constitutive CaMV35s promoter and b). rd29A a stress inducible promoter showed different growth patterns after four weeks of seed germination. The transgenics showed higher transpiration efficiency than wild type plants under water stress conditions with higher yield . Overexpression of DREB1A under rd29A promoter also enhanced harvest index in correlation with higher root to shoot ratio  and shown better root system in deep soil layers in lysimeter system . AtDREB1A improved water retention capacity, enhanced chlorophyll content osmotic adjustment by the accumulation of proline when overexpressed in a drought susceptible groundnut variety GG20 . Manjulatha et al.  overexpressed a plant DNA helicase gene PDH45 and reported increased intrinsic cellular level tolerance in transgenic groundnuts under water stress and also reported the stay green nature of transgenics by maintaining superior mesophyll efficiency and low Δ13 C which can be used as a measure of water use efficiency (WUE). Overexpression of a NAC transcription factor gene, AtNAC2 in groundnut resulted in enhanced water retention capacity with stay green nature and reduced membrane damage than wildtype under water stress conditions . MuNAC4 overexpressing transgenic groundnut revealed increased lateral root development, osmotic adjustment and better antioxidative defense mechanisms than wild type plants and thereby conferring tolerance to drought .
Mannitol, a compatible solute reported to be accumulated during water deficit stress in plants . Bhauso et al.  transformed a bacterial mtlD gene which encodes mannitol 1-phosphate dehydrogenase, which converts mannitol 1-phosphate to mannitol  into groundnut. Overexpression of mtlD gene exhibited 1.3-1.8 fold increase in mannitol biosynthesis in transgenics when compared to wild type that were positively correlated with the drought stress tolerance. These mtlD overexpressing groundnut transgenics were also reported to be maintaining better photosynthetic machinery and reduced oxidative damage in comparison with wildtype plants in dry-down experiments . A novel plant specific transcription factor WRKY3 was isolated from horsegram (Macrotyloma uniflorum) and was used to develop groundnut transgenics. MuWRKY3 transgenics showed improved cellular level tolerance through the accumulation of osmolytes, ROS scavenging system and antioxidative machinery under drought stress conditions .
Salt stress resistance
There are only two reports of groundnut transgenics against salt stress. Vadawale et al.  overexpressed a functional enzyme choline oxidase (COX), that is reported to be involved in glycine betaine biosynthesis. Glycine betaine is one of the osmoprotectants synthesized in plants under abiotic stresses majorly under saline stress . They reported the survival of transformed plants under 100 mM NaCl stress whereas untransformed plants showed curling and burning of leaves from the margins. They correlated these results with COX gene overexpression and increased synthesis of glycine betaine . AtNHX1 expressing groundnut plants maintained better growth, higher photosynthetic rate, stomatal conductance and transpiration rates in 150 mM NaCl stress . Overexpression of peroxisomal ascorbate peroxidase (pAPX) gene from halophyte Salicornia brachiate in groundnut resulted in higher chlorophyll and RWC with reduced electrolyte leakage than WT under 150 mM NaCl stress .
Resistance to multiple abiotic stresses
Drought and Salinity are the principle abiotic stresses; approximately 830 million ha. were affected by soil salinity  and it affects ~7.61 million ha in India  and in the world 20% of the land surface affected by drought at any point of time . Hence the development of groundnut transgenics with multiple abiotic stress tolerance has a significant relevance for the crop improvement.
NHX1 is a Na+/H+ antiporter found on a vacuolar membrane and is involved in the compartmentalization of Na+ ions into vacuole which is detrimental to the plant cells . Overexpression of this NHX1 gene from Arabidopsis in groundnut improved drought and salt tolerance through sequestration of more Na+ in vacuole and accumulation of proline than wild type plants under stress conditions. The transgenics also survived under high concentration of NaCl (200 mM) stress and drought stress where WT plants were failed to survive . Another vacuolar membrane protein from Arabidopsis, H+- pyrophosphatase, a proton pump encoded by AtAVP1 was transferred to groundnut by Agrobacterium mediated gene transformation, transgenics maintained the morphological traits under both drought and salt stress compared to wildtype. These results were positively supported by higher chlorophyll content, photosynthetic efficiency and transpiration rate in transgenics under stress conditions . Tiwari et al.  developed groundnut transgenics by overexpressing SbASR-1 gene, cloned from Salicornia brachiata which codes for abscisic acid stress ripening 1 (ASR-1) protein a group7 LEA family protein. In comparison with wildtype plants, transgenics showed less reduction in chlorophyll content, higher water retention ability and accumulation of more compatible solutes like proline, soluble sugars, etc. under salt and drought stress conditions. Overexpression of Prd29A: AtHDG11 gene cassette in groundnut resulted in upregulation of several abiotic stress tolerance genes  that were responsible for expression of antioxidative enzymes, accumulation of compatible solutes, ROS scavenging enzymes and genes involved in improving intrinsic water use efficiency under drought and salt stress conditions. These findings were positively correlated with healthier morphological traits and yield under drought and salt stress conditions .
Groundnut transgenics overexpressing BcZAT12 under LEA promoter is reported to be conferring multiple abiotic stress tolerance by the accumulation of more proline and improved osmotic adjustment and water conservation ability under NaCl and PEG stress. These transgenics also showed the increased expression of antioxidant enzyme system and better phenotypic traits like less wilting of leaves which is correlated with less reduction of chlorophyll under stress conditions . A eukaryotic translation initiation factor gene (eIF4A) from Pennisetum glaucum transformed into groundnut and reported to confer abiotic stress tolerance by Santhosh Rama Bhadra Rao et al. . The transgenics maintained the higher chlorophyll retention, increased expression of anti-oxidative enzymes and improved water conservation by maintaining higher membrane integrity and superior phenotypic characters under simulated stress conditions.
Abiotic stress tolerance is a complex phenomenon and involves the participation of many genes and their products [75,76]. Scientists developed hundreds of transgenics across several plant species by manipulation of single genes involved in biochemical pathways or regulation of downstream genes. These transgenics were successfully imparted tolerance against specific stress but not up to the desired levels of multiple stress tolerance . To attain the multiple stress tolerance in crop plants, researchers concentrated on introduction of multiple genes simultaneously into plant systems. There are three multigene transfer approaches mainly used by researchers, conventional method, retransformation and co-transformation among which co-transformation is the reliable and time saving option . Co-transformation involves the introduction of two or more genes, each carried by a separate vector simultaneously or all the genes carried by a single vector using multisite cloning approach. In multisite cloning strategy, two or more genes linked together on a single vector either by classical restriction digestion and ligation reaction or advanced gateway cloning technology involving recombination. There are several reports of multigene transfer in crop plants, here we review groundnut transgenics developed through multigene transfer against abiotic stresses.
Pruthvi et al.  engineered a groundnut cultivar TMV2 by co-expression of three Arabidopsis TF genes AtDREB2A, AtHB7 and AtABF3 under constitutive promoter CaMV35s through a modified GATEWAY cloning technology  and conferred improved drought and salt tolerance in transgenic plants. Co-expression of these TFs genes facilitated the reduced membrane damage and maintained higher chlorophyll content. They also altered the expression of several downstream genes involved in acquired stress tolerance (GRX, Aldehyde reductase, Serine threonine kinase like protein, Rbx1, Proline amino peptidase, HSP70, DIP and Lea4) and osmolyte production revealed the improved cellular level tolerance under stress conditions. Another multigene groundnut transgenics were developed by simultaneous expression of Alfin1, PgHSF4 and PDH45 genes cloned from different source plants and overexpressed under three different expression elements. The transgenics showed improved stress tolerance by maintaining the higher growth and productivity than wildtype plants under water limited conditions. Transgenic plants also upheld the increased root growth, cooler canopy temperature with higher RWC under moisture stress. The transgenics exhibited improved oxidative stress tolerance induced by ethrel and methyl viologen and downstream regulation of several stress responsive genes by TFs revealed the enhanced drought tolerance by reducing oxidative damage in transformed plants .
The current review summarizes groundnut transgenics overexpressing genes coding for enzymes, osmoprotectants and transcription factors which have a regulatory role in modifying the pathways involved in abiotic stresses like drought, salinity and oxidative stress. Transgenic plants expressing multiple genes were proven to have higher ability to withstand abiotic stresses compared to the single gene expressing transgenics. Therefore, multigene transgenic approach is the viable option for improving the abiotic stress tolerance in semiarid crops like groundnut to overcome adverse conditions in the climate changing scenario. Stacking and expression of multiple genes responsible for several traits also provides tolerance to multiple abiotic stresses. Further, multigene strategy was advantageous as it is cost-effective, time saving approach and reduces the use of several selectable markers, also stacking of multiple genes together minimizes the chances of segregation of genes in the subsequent generations.
BV greatly acknowledges CSIR-SRF fellowship (No: 09/383(0051)/2016-EMR-I) from CSIR-INDIA and CS acknowledges DBTGOI for financial support (BT/PR15503/AGR/02/913/2015. dated: 09-05-2017). AMAJS acknowledges Dr. DS Kothari Post-Doctoral Fellowship (BL/16-17/0364) from UGC, New Delhi.