Research Article

Agrobacterium Mediated Transformation of OsTZF8 Gene in Oryza sativa for Drought Stress Tolerance

Muhammad Medrar Hussain*, Asad Jan* and Sayyar Khan Kazi

Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Postal Code 25130, Pakistan.

Received | January 30, 2020; Accepted | May 13, 2020; Published | June 01, 2020

*Correspondence | Muhammad Medrar Hussain and Asad Jan, Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, Khyber Pakhtunkhwa, Pakistan, Postal code 25130; Email: medrarbiotech@gmail.com; janasad@aup.edu.pk

Citation | Hussain, M.M., A. Jan and S.K. Kazi. 2020. Agrobacterium mediated transformation of OsTZF8 gene in Oryza sativa for drought stress tolerance. Sarhad Journal of Agriculture, 36(2): 702-713.

DOI | http://dx.doi.org/10.17582/journal.sja/2020/36.2.702.713

Keywords | OsTZF8, Transformation, PCR, Drought stress, Chlorophyll

Introduction

Different biotic and abiotic stresses seriously affect plants which ends in reduced productivity (Kim et al., 2014). Drought is a serious abiotic problem related to decline crop production in different regions of the world. Non-availability of water hampers plant development and growth due to low photosynthesis rate and decline of essential nutrients (Chinnusamy et al., 2004). Worldwide, area affected by drought is gradually increasing and more than 20% of rice growing area is suffering from drought stress (Swamy and Kumar, 2013). At seedling stage, drought stress can slow down plant establishment and decrease seedling survival rate. At vegetative stage, drought can cause decrease in yield by lowering the leaf formation rate and panicle per plant, while at reproductive stage, drought results in increased grain sterility and decreased grain per panicle (Pantuwan et al., 2002).

Drought stress alters the expression of various genes that play a significant part in stress response and tolerance (Yamaguchi-Shinozaki and Shinozaki, 2006; Todaka et al., 2015). There are two main groups of stress inducible genes. The 1st group consists of those genes which encode proteins required for cellular stress tolerance such as molecular chaperons, antifreeze proteins, soluble sugars, water channel proteins and detoxifying enzymes. The 2nd group comprises of those genes which encode proteins which are linked to signal transduction and expression of abiotic stresses related genes (Shinozaki and Shinozaki, 2007) which includes calmodulin binding proteins, components of ABA (abscisic acid) signaling, enzymes involved in metabolism, kinase proteins and transcriptional factors (Yamaguchi-Shinozaki and Shinozaki, 2006).

The plant hormone ABA has a key role in plant adaptation during drought stress. Under drought stress, ABA accumulates due to induction of ABA biosynthetic genes (Iuchi et al., 2001). The expression of many genes are regulated by ABA that leads to important changes which assist plant survival during drought stress (Umezawa et al., 2010). Under drought stress, genomic and molecular investigation have shown the presence of both ABA-dependent and ABA-independent signal transduction pathways (Yamaguchi-Shinozaki and Shinozaki, 2006). During drought stress, several transcription factors function in ABA-dependent regulation of gene expression while some function in ABA-independent regulation of gene expression (Nakashima et al., 2009).

Upon exposure of plants to abiotic stresses, a range of different genes with various functions like production of tolerance proteins etc., are either up regulated or down regulated (Lata and Prasad, 2011). Transcriptional factors are essential regulatory proteins which direct the expression of several defense genes (Chen and Zhu, 2004). These transcriptional factors act on conserved cis acting sequences in the promoter region of many stress linked genes and increase the expression of a complex gene network connected with conferring tolerance against various stresses. In genetic engineering, these transcriptional factors are powerful tools for the overexpression of complete set of genes. A number of transcriptional factors are associated with the expression and regulation of many stress responsive genes. For instance, about 1500 different genes code for different transcriptional factors in Arabidopsis (Riechmann et al., 2000). Several large groups of transcriptional factors are classified in to different families like bZIP, AP2/ERF, zinc finger, MYB, NAC and MYC (Umezawa et al., 2006).

It has been reported that transcription factors are involved in different physiological activities under drought and other abiotic stresses. For instance, ZAT18 (C2H2 zinc finger protein) was transcriptionally induced by drought stress in Arabidopsis. In comparison to wild type, ZAT18 overexpression plants displayed lower content of ROS (Reactive Oxygen Species), less leaf water loss, higher antioxidant enzyme activities and higher leaf water content after drought treatment (Yin et al., 2017). Another C2H2 zinc finger protein called IbZFP1 was reported in drought-tolerant sweet potato (Ipomoea batatas (L.) Lam.) line Xu55-2. Analysis showed that significant increase of soluble sugar, total chlorophyll content, ABA, proline, and reduction of malonaldehyde content and H2O2 were detected under drought and salt stresses in the transgenic plants. Furthermore, the rates of water loss and electrolyte leakage in transgenic plants were reduced (Wang et al., 2016). Similarly, ZAT6 (C2H2-type zinc finger protein) was highly enhanced by drought and salt stress in leaves of in Arabidopsis. Seed germination was greatly improved in overexpressed ZAT6 transgenic plants under stress conditions (Liu et al., 2013)

A diverse transcriptional factor family known as zinc finger proteins, are reported to be involved in several mechanisms like regulation of transcription, RNA binding and protein-protein interactions related to the growth and development of plants (Yilmaz and Mittler, 2008). In these cellular mechanisms of plants, different zinc finger families like ERF, LIM, WRKY, RING-finger and DOF are involved (Lijavetzky et al., 2003).

One of these important zinc finger proteins families is C3H zinc finger protein family. In rice, full genome investigation of C3H zinc finger proteins has revealed 67 genes (Wang et al., 2008). It has been identified that some members of these families are involved in stress tolerance against abiotic stresses. For example, a C3H zinc finger protein gene OsTZF1 (Ubi: OsTZF1-OX), exhibited better tolerance to drought and high NaCl conditions (Jan et al., 2013). Likewise, OsC3H47, (C3H zinc finger protein gene) was reported to induce strongly under ABA, NaCl, PEG and osmotic stresses. The over expression of this gene in rice seedlings exhibited enhanced tolerance to salt and drought stresses (Wang et al., 2015).

A small subfamily of zinc finger proteins called Tandem C3H Zinc Finger (TZF) proteins is characterized by the presence of Tandem Zinc Finger domains which are separated by 17 amino acids. These 17 amino acids are actually involved in RNA binding process. TZF proteins had been reported as RNA-binding proteins having significant role in RNA processing (Jeong et al., 2010). Characterization of these genes specified their role in giving tolerance against various abiotic stresses like cold, drought, salt etc. Some C3H zinc finger proteins members have been identified to have essential roles in plant growth and architecture (Wang et al., 2008; Zhang et al., 2015; Liu et al., 2017; Yin et al., 2017).

Through genetic engineering, rice varieties tolerant to abiotic stresses can be produced by incorporating genes involved in abiotic stresses. For improved tolerance to adverse environmental conditions, such genes could be incorporated into plants through different gene transfer methods like particle bombardment, electroporation and Agrobacterium-mediated transformation. The most extensively practiced gene transfer method in rice is Agrobacterium-mediated transformation (Gelvin, 2003). In Agrobacterium based transformation, one or few copies of desired gene is transferred with high efficiency into plant genome (Wu et al., 2003). Fruitful attempts have been made to infect monocotyledons with Agrobacterium. Successful transformation of cereal crops via Agrobacterium has been successfully carried out in maize, wheat and rice (Shen et al., 1999).

Most of the members of TZF gene family in rice have not been functionally characterized. One uncharacterized member of C3H zinc finger protein genes is OsC3H10 which is also known as OsTZF8 having a unique TZF motif C-x10-C-x5-C-x3-H along with another common motif C-X5-C-X4-C-X3-H. In this research study, we have evaluated the role of OsTZF8 gene in conferring drought stress tolerance. OsTZF8 gene was over-expressed in rice indica variety Swat-1. Agrobacterium mediated transformation was used for OsTZF8 gene transfer into rice calli. Chlorophyll content and morphological parameters of transgenic rice were also evaluated.

Materials and Methods

Plant material and growth conditions

Seeds of Oryza sativa subspecies indica variety Swat-1 was used in the present study. Seeds were acquired from the Plant Genetic Resource Institute (PGRI), National Agriculture Research Council (NARC), Islamabad. For experimental purposes rice seeds were incubated for 3 days at 42°C in oven (Hanumanthappa et al., 2016).

Transformation of pBIG binary vector having Ubi-OsTZF8 construct into Agrobacterium

Agrobacterium mediated transformation was used for transferring OsTZF8 gene (pBIG-Ubi-OsTZF8) into rice for overexpression (Rashid et al., 1996; Toki, 1997). Agrobacterium tumefaciens strain EHA-105 was used for rice transformation. Binary plant expression vector pBIG (12.7kb) was used as backbone for construction of binary vector containing OsTZF8 coding region under the maize ubiquitin (Ubi) promoter and hygromycin phosphotransferase (HPT) marker gene. Freeze/thaw method was used for the transformation of Agrobacterium tumefaciens (EHA-105) with pBIG-Ubi-OsTZF8 construct (Wise et al., 2006). Plasmid DNA (1 µg) was added to 1.5 ml eppendorf tube containing Agrobacterium competent cells and mixed by tapping. Tubes were frozen in liquid nitrogen for 2 min and then thawed at 37oC for 5 min. Thawed bacterial cells were then mixed with 1 ml LB media and incubated in shaking incubator at 28 oC for 2 hrs. Centrifugation was done at 4000 rpm for 5 min and supernatant was discarded. Pellet was re-suspend in 100 µl LB. The bacterial suspension was spread on LB media plats having kanamycin (100 mg/L), hygromycin (50 mg/L) and rifampicin (10 mg/L) and incubated at 28oC for 2 days.

Confirmation of transformed Agrobacterium colonies

After 2 days of selection on antibiotics supplemented LB media, Colony PCR was performed for confirmation of pBIG-Ubi-OsTZF8 construct in colonies of Agrobacterium (Sheu et al., 2000). PCR was done using construct specific forward (Ubi-F1) and reverse (OsTZF8-R200) primers.

Rice calli infection with transformed Agrobacterium tumefaciens

For the transformation of rice calli with pBIG-Ubi-OsTZF8 construct, mature and healthy seeds of Swat-1 indica rice were dehusked and washed with tape water, 0.2% MgCl2 solution (3 times) and sterile distilled water (5 times) for 1 minute each. For the purpose of calli induction, fourteen seeds were cultured on callus induction media (N6) (Chu et al., 1975) for 21-28 days at 25±3 ºC. The procedure of Rashid et al. (1996) and Toki et al. (1997) was followed for rice transformation. Previously transformed Agrobacterium tumefaciens strain EHA-105 was used for the transformation of induced rice calli. Agrobacterium culture in a 50 ml falcon tube containing 30 ml AA-medium (Toriyama and Hinata, 1985) supplemented with acetosyringone (10 mg/L) was used. Before infection, viable rice calli were collected in a sterilized sieve and infected with the above prepared Agrobacterium suspension. After 90 seconds, the calli were dried and placed on co-cultivation media plates and were incubated for 3 days at 28 oC in dark. Co-cultivation medium was prepared by using N6 medium (Chu et al., 1975) constituents and supplementing it with acetosyringone (10 mg/L). After 3 days of co-cultivation, calli were washed with sucrose solution (3%) having 250 mg/ml carbencillin and then with sterile distilled water. Calli were then shifted to selection media plates and incubated at 28oC for 10 days and sub-cultured after every 10 days. For selection medium, N6 medium was prepared having kinetin and 2,4-D as growth regulators. After addition of 1% agar, the media was autoclaved. The medium was then supplemented with 250 mg/L carbencillin and 30 mg/L hygromycine. After selection, calli were shifted to regeneration medium for regeneration. The ingredients of regeneration medium were MS salts with vitamins (Murashige and Skoog, 1962) 4.43g/L, sucrose and D-sorbitol 30g/L each, casamino acid 1g/L, NAA (naphthalene acetic acid) 0.02 mg/L, kinetin 4 mg/L, hygromycine 30 mg/L, carbencillin 500 mg/L and agar 3 g/l (solidifying agent). In regeneration medium calli were permitted to regenerate in light at 28oC (16 hrs light and 8 hrs dark photoperiod). Certain embryogenic rice calli turned green from which small plantlets regenerated.

PCR confirmation of transformed rice calli

For DNA extraction from putative transformed rice calli, Phillips et al. (2001) protocol was followed. Genomic DNA was isolated by taking 0.1 g of calli in tube and crushing in liquid nitrogen and transferred to eppendorf tubes. To each tube, 400 µl EB2 (extraction buffer) solution was added and mixed vigorously for 5 min. Then 200 µl (20%) sodium dodecyl sulphate (SDS) was added to each sample and was mixed for 3 minutes. About 1 µl RNase was added to each tube and was incubated at 37°C for 30 min. To each sample, 400 µl phenol mix was added and centrifuged at 14000 rpm for 5 min. To a fresh tube, supernatant was transferred and 400ul isopropanol was added and centrifuged at 10,000 rpm for 1 min. The pellet containing DNA was washed with 70% ethanol and then dried. The pellet was suspended in 10-30 µl TE (Tris EDTA) buffer for further use. Concentration and quality of isolated DNA was verified through Nano drop (2000/2000C, Thermo scientific, USA). For the confirmation of amplified region in calli DNA, PCR investigation was performed. The PCR product was examined using 1% agarose gel with 1kb plus Ladder and visualized in UV trans-illuminator.

Establishment and acclimatization of transgenic indica rice

Small plantlets were regenerated from selected green calli in regeneration media plates and jars. Then these rice plantlets were shifted for establishment to full and half MS media (Murashige and Skoog, 1962) supplemented with hygromycin (30 mg/L). Plantlets were first shifted to small pots having sterile soil and kept under standard growth conditions at 28°C and 55% humidity and then to green house for acclimatization and maturity.

PCR confirmation of transformed indica rice plants

Molecular level confirmation of the transgenic rice plants having OsTZF8-OX was carried out using PCR. DNA was extracted from the young transgenic rice leaves using Phillips DNA extraction protocol (Phillips et al., 2001). PCR confirmation of OsTZF8 and HPT genes were performed using the DNA extracted from transgenic rice lines.

Drought stress treatment of indica OsTZF8-OX transgenic rice

Rice plant seedlings of OsTZF8-OX transgenic indica rice were grown for two weeks in soil and then subjected to dehydration stress. After the dehydration stress treatment, plants were permitted to recover in normal growth conditions (28°C and 55% humidity). The number of plants survived and continued to grow after two weeks were counted (Jan et al., 2013).

Chlorophyll and Morphological investigation of indica OsTZF8-OX transgenic rice

Chlorophyll content of OsTZF8-OX transgenic rice leaves was measured through Spad meter (Spectrum technologies, USA). To examine the effects of overexpression of OsTZF8 gene, chlorophyll content of OsTZF8-OX transgenic and control rice plants was compared. For this purpose, chlorophyll content was measured from upper, middle and lower portion of first four leaves of rice plants (Wang et al., 2016).

Morphological data like height of the plant, number of tillers, number of total grains, number of filled grains and % yield was collected from OsTZF8-OX transgenic rice plants (Ogunbayo et al., 2005).

Statistical analysis

For the experiments of drought stresses, chlorophyll content etc, the mean values were calculated and diagrammatically represented as bar charts using Microsoft Excel program. To know the treatment efficiency, t-test was applied to various experiments as described by Steel and Torrie (1960); P < 0.05 was considered the minimum value for statistical significance.

Results and Discussion

pBIG-Ubi-OsTZF8 construct map

For the OsTZF8 gene overexpression in rice, a suitable construct was prepared using pBIG binary vector. The construct have OsTZF8 gene under ubiquitin promoter (Ubi) and hygromycin phosphotransferase (HPT) gene under Nos promoter (Figure 1).

Agrobacterium tumefaciens transformation and colony PCR confirmation

Competent cells of Agrobacterium tumefaciens were transformed with pBIG-Ubi-OsTZF8 construct. Few transformed Agrobacterium colonies were obtained on LB selection media plates having kanamycin, hygromycin and rifampicin antibiotics. For the confirmation of transformed colonies, Colony PCR was performed. For this purpose, five colonies of Agrobacterium tumefaciens were selected and there DNA was subjected to PCR. PCR was performed using construct specific forward (Ubi-F1) and reverse (OsTZF8-R200) primers (Table 1). After Colony PCR, three colonies exhibited positive bands on 1% agarose gel, confirming the primers specific amplification of the OsTZF8 gene with band size of approximately 300 bp (Figure 2). Colony PCR confirmed Agrobacterium colonies were selected and successfully revived.

Agrobacterium mediated transformation of rice calli with OsTZF8 gene and its PCR confirmation

About 14 seeds of indica rice were used per callus induction media plate (total seeds used were 397). A total of 280/360 calli were found suitable for transformation. After successful transformation, 24 calli were selected having optimum regeneration characteristics. After several plantlets germination, out of 9 germinated transgenic plants 2 lines were selected for the current study (Table 2). Genomic DNA from indica rice calli was isolated and analyzed through PCR using construct specific forward (Ubi-F1) and reverse (OsTZF8-R200) primers (Table 1). Results of amplified samples on PCR along with positive control were observed on 1% agarose gel. Seven samples (1 to 7) showed amplification of OsTZF8 gene inserted into rice calli. The band size of samples and positive control was approximately 300 bp (Ladder size = 1kb plus) using PCR specific primers (Figure 3). The amplification of DNA bands confirmed the successful transformation of OsTZF8 gene into rice calli.

Table 1: List of primers used for confirmation of OsTZF8 and HPT genes.

Gene name	Primer type	Primer name	Primer sequence
OsTZF8	Forward	Ubi-F1	5´-TGCTCTAACCTTGAGTACCTATTAT-3´
	Reverse	OsTZF8-R200	5´-GGGCACGACGTCCACTCGTGCGCCGCGA- 3´
Hygromycin phosphotransferase (HPT)	Forward	HPT-F	5´-ATGAAAAAGCCTGAACTCACCGCGA-3´
	Reverse	HPT-R	5´-GCAAAGTGCCGATAAACATAACGATC-3´

 

Plants regeneration and acclimatization

After PCR confirmation of OsTZF8 in rice calli, transformed rice plants were regenerated from these calli on full MS and half MS media having hygromycin antibiotic as selection marker. The plants were first grown on media plates, then transferred to media jars and then gradually transferred to small and large pots having sterile soil. Finally, the mature transgenic plants were grown successfully in green house under control conditions (Figure 4).

Molecular level confirmation of OsTZF8-OX and HPT gene in transgenic rice lines

Genomic DNA was isolated from indica OsTZF8-OX transformed plantlets. By using Ubi-F1 and OsTZF8-R200 primers (Table 1), PCR confirmation was performed. Transformed rice plants DNA showed positive bands at 300 bp along with positive control (Figure 5A). The amplification of DNA bands indicated the successful transformation of OsTZF8 gene into indica rice plants.

Table 2: Summary of transformation and regeneration of rice calli.

Seeds per plate (callus induction media)	14
Total seeds used for calli formation	397
Total number of calli regenerated	360
Number of calli used for transformation	280
Number of transformed calli having regeneration capacity	24
Number of germinated transgenic plants	9
Rice transgenic lines selected for research work	2

 

After confirmation of OsTZF8 gene in rice, the inserted transcript was also confirmed for the presence of HPT on PCR using specific primer (Table 1). Positive bands were detected around 200 bp along with positive control (Figure 5B). This result gives an additional confirmation of successful transformation of the transgenic pBI vector into rice.

Drought stress analysis of indica OsTZF8-OX transgenic lines

For the evaluation of drought stress tolerance, two weeks old rice seedlings of OsTZF8-OX transgenic lines were exposed to drought stress. Plants were recovered under normal growth conditions after stress treatment. OsTZF8-OX transgenic line A and B exhibited 69% (25/36) and 64% (23/36) survival rates respectively in comparison to 33% (12/36) of control (Figure 6). These findings confirmed that OsTZF8-OX line A and B revealed considerable level of tolerance to drought stress compared to control rice plants.

Chlorophyll content study of OsTZF8-OX transgenic rice lines

Chlorophyll content measurement was carried out at 4 and 8 weeks after heading (WAH). After 4 WAH,

Table 3: Morphological investigation of mature indica OsTZF8-OX transgenic rice line A, B and

control. Results are the mean ± SD of at least three replicates.

Rice lines	Plant height (cm)	± SD	Tillers per plant	± SD	Grains per panicle	± SD	Filled grains per panicle	± SD	% Yield	± SD
OsTZF8-OX A	84	1.37	18	1.79	110	5.39	97	2.73	88.18	1.92
OsTZF8-OX B	86	0.89	16	0.52	96	5.09	88	3.61	91.66	2.98
Control	95	2.73	15	2.68	94	4.50	79	3.58	84.04	2.23

 

the chlorophyll content of four leaves (top to bottom) of OsTZF8-OX A and B transgenic lines were observed to be high compared to control. Similarly, After 8 WAH, chlorophyll content of OsTZF8-OX transgenic line A and B was high in comparison to control (Figure 7).

Morphological investigation of mature plants

To know the effects of OsTZF8 over-expression, morphological investigation of OsTZF8-OX transgenic rice plants was performed. OsTZF8-OX transgenic line A and B showed plant height of 84±1.37 cm and 86±0.89 cm respectively compared to 95±2.73 cm of control plant. There were 18±1.79 and 16±0.52 tillers in indica OsTZF8-OX transgenic rice line A and B, respectively compared to 15±2.68 of control plant. The % yield was recorded to be 88.18±1.92 and 91.66±2.98 for indica OsTZF8-OX transgenic rice line A and B compared to 84.04±2.23 of control respectively (Table 3).

Rice is a widely consumed staple food crop grown over more than 162 million hectares worldwide with 759.6 million tons annual production (FAO, 2018). It is a common cash crop nourishing more than half of the human population (Benjamin and Nielsen, 2006). Different abiotic stresses pose severe threats to rice crop by affecting plant growth, plant productivity and biomass production. Drought is one such environmental stress effecting rice productivity (Chinnusamy et al., 2004; Rodziewicz et al., 2014). In sever abiotic stresses, plants usually survive and grow by developing several molecular, physiological, biochemical and morphological changes. Upon exposure of plants to these stresses, various genes with different functions are either up regulated or down regulated (Shinozaki and Shinozaki, 2007; Lata and Prasad, 2011). Transcriptional factors are essential regulatory proteins having key roles in the expression of several defense genes (Chen and Zhu, 2004). The zinc finger protein genes is one of the major transcription factors family which is mainly involved in various mechanisms of plant growth and development such as protein-protein interactions, RNA binding and Transcription regulation (Yilmaz and Mittler, 2008). CCCH zinc finger protein family is an important tandem zinc finger (TZF) proteins family having key roles in abiotic stress tolerance. In rice, full genome study of CCCH zinc finger proteins have displayed 67 genes (Wang et al., 2008). One uncharacterized member of CCCH zinc finger protein genes family is OsTZF8 which is also called OsC3H10. In the present research work OsTZF8 genes was transferred to indica rice through Agrobacterium tumefaciens to find out its role in drought stress tolerance.

To know the role of OsTZF8 gene in drought stress tolerance, a suitable construct pBIG-Ubi-OsTZF8 was prepared (Figure 1). pBIG-Ubi-OsTZF8 construct was used for the overexpression of OsTZF8 in rice. First of all the competent cells of Agrobacterium were prepared and transformed with the pBIG-Ubi-OsTZF8 construct. Then the transformed colonies were confirmed through colony PCR (Figure 2). Successful Agrobacterium mediated transformation of rice calli with OsTZF8 was carried out through co-cultivation, selection and regeneration steps using different media supplemented with antibiotics such as carbencillin and hygromycin. The transformed rice calli were confirmed through PCR (Figure 3). Agrobacterium mediated transformation has been effectively used in producing transgenic rice lines previously (Hiei et al., 1994; Dong et al., 1996; Jan et al., 2013). Rashid et al., (1996) successfully applied this method to produce transgenic indica rice (Basmati cultivars). Similarly, Aldemita and Hodges (1996) reported transformation in young embryos of japonica and indica varieties using of Agrobacterium tumefaciens. Furthermore, rice calli has been used successfully to transfer desired genes through Agrobacterium. For instance, Jan et al. (2013) used japonica rice calli to transfer OsTZF1gene using Agrobacterium-mediated transformation. Hiei et al. (1994) also used Agrobacterium-mediated transformation in japonica rice calli to produce transgenic plants which was confirmed at molecular level. PCR level confirmation is a reliable technique and used in routine for the confirmation of genes transformation. For example, PCR confirmation of transgenic calli was carried out for manganese superoxide dismutase (Mn-SOD) gene in rice responsible for salt stress tolerance (Wang et al., 2007). Similarly engineered cysteine proteinase inhibitor (Oryza cystatin-IDD86) for nematode resistance in transgenic rice plants were confirmed through PCR (Vain et al., 1998). After successful Agrobacterium mediated transformation of OsTZF8 gene into rice calli, we regenerated and established transformed rice plants in green house (Figure 4).

Molecular level verification is a key step in confirmation of successful transfer of desired gene into the host cells (Sarker et al., 2015; Cui et al., 2017; Pradhan et al., 2017). In the present study genomic DNA from transformed indica rice plants was extracted through Phillips et al. (2001) method and confirmation of pBIG-Ubi-OsTZF8 construct along with HPT gene was performed through PCR (Figure 5A and B). The positive DNA bands amplification indicated the successful transformation of OsTZF8 gene into rice plants. Genomic DNA of transgenic plants has also been used previously in PCR to confirm successful integration of transgene. For instance, gene insertion of AtDREB1A gene (responsible for gene regulation in drought stress) in rice plants genome was also confirmed through PCR (Hussain et al., 2014). Similarly, OsCYP2 gene responsible for root development in rice was also confirmed from genomic DNA through PCR (Cui et al., 2017). Constitutive expression of OsDof4 gene which codes a C2-C2 zinc finger transcription factor was also confirmed initially through PCR using genomic DNA (Wu et al., 2017).

Drought is an abiotic stress which severely damage crops production in different regions of the world (Chinnusamy et al., 2004). CCCH zinc finger proteins is a transcriptional factor family which conferred tolerance to many abiotic stresses like drought (Zhang et al., 2013; Pradhan et al., 2017). Therefore, we evaluated OsTZF8 gene role in drought stress tolerance in rice. For this purpose, two weeks old rice seedlings were subjected to dehydration stress. OsTZF8-OX transgenic line A and B exhibited 69% and 64% survival rates, respectively compared to 33% in control (Figure 6). Our results suggests the positive role of OsTZF8 gene in conferring drought stress tolerance. In line to our results, previous findings also suggests key roles of stress inducible genes in drought stress tolerance. For example, overexpression of a CCCH gene called OsTZF1in japonica rice showed improved tolerance to drought stress. Japonica OsTZF1-OX line #6 and #9 exhibited 69% and 63% survival rates respectively compared to 38% in control (Jan et al., 2013). Likewise, the overexpression lines #2 and #3 of ONAC045 gene in rice showed 90% and 70% survival rate respectively after recovery in comparison to 35% in control (Zheng et al., 2009). In another study OsDREB1F gene over-expression showed high drought stress tolerance in Arabidopsis and rice transgenic lines compared to controls (Wang et al., 2008).

Measuring chlorophyll content also gives an indication of gene role in phenomenons such as delayed leaf senescence, drought stress tolerance etc. (Wang et al., 2005). To evaluate the effects of OsTZF8 gene on chlorophyll, chlorophyll content of transgenic lines OsTZF8-OX A, OsTZF8-OX B and control were measured for top four leaves after 4 and 8 WAH. It was observed that all four leaves of transgenic lines retain a high chlorophyll content compared to control plants (Figure 7). These results indicated the positive role of OsTZF8 gene in chlorophyll content of OsTZF8-OX transgenic leaves. Previous studies showed that certain genes have roles in increasing or stabilizing chlorophyll content. For example, the AtTZF1 transgenic Arabidopsis lines showed high levels of chlorophyll content compared to control lines (Han et al., 2014). In another study the TaSP gene which was cloned in Arabidopsis for salt stress tolerance also showed high chlorophyll content compared to control plants (Ma et al., 2015). Our findings confirms the link between over-expression of OsTZF8 gene and tolerance to drought stress along with chlorophyll content stability/increase, which suggests that OsTZF8 gene might have an essential role in inducing tolerance to drought stress in rice and perhaps other crops. Overall, our findings provide evidence that OsTZF8 gene was involved in drought stress tolerance in rice.

Conclusions and Recommendations

OsTZF8-OX transgenic indica rice lines were successfully developed through Agrobacterium mediated transformation. After biochemical and Molecular confirmation, the OsTZF8-OX transgenic lines were subjected to drought stress. Drought stress analysis indicated that OsTZF8-OX transgenic lines were tolerant to dehydration stress compared to control. Chlorophyll analysis also supports the current findings. Microarray and Real time PCR studies are suggested for further confirmation of OsTZF8 gene overexpression role in drought stress tolerance. Furthermore, the transgenic lines shall be tested against other abiotic stresses.

Novelty Statement

Drought stress is a major problem for crops especially in rice. In the current study, OsTZF8-OX transgenic rice lines were developed for the first time in local indica rice variety of Pakistan to know its role in drought stress tolerance. Using Agrobacterium mediated transformation, we successfully developed OsTZF8-OX transgenic indica lines which exhibited tolerance against drought stress.

Author’s Contribution

MMH performed the experimental work and wrote the manuscript. AJ designed and supervised the study. SKK helped in supervision and critical revision of the data and manuscript. All the authors read and approved the final manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Aldemita, R.R. and T.K. Hodges. 1996. Agrobacterium tumefaciens- mediated transformation of japonica and indica rice varieties. Planta. 199(4): 612-617. https://doi.org/10.1007/BF00195194

Benjamin, J.G. and D.C. Nielsen. 2006. Water deficit effects on root distribution of soybean, field pea and chickpea. Field Crops Res., 97(2-3): 248-253. https://doi.org/10.1016/j.fcr.2005.10.005

Chen, W.J. and T. Zhu. 2004. Networks of transcription factors with roles in environmental stress response. Trends Plant Sci., 9(12): 591-597. https://doi.org/10.1016/j.tplants.2004.10.007

Chinnusamy, V., K. Schumaker and J.K. Zhu. 2004. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. J. Exp. Bot., 55(395): 225-236. https://doi.org/10.1093/jxb/erh005

Chu, C.C., C.C. Wang, C.S. Sun, H. Chen, Y.K. Chu, C.C. Yin and B.F. Yun. 1975. Establishment of an efficient medium for another culture of rice through comparative experiments on the nitrogen sources. Sci. Sin. 18(1): 659-668.

Cui, P., H. Liu, S. Ruan, B. Ali, R.A. Gill, H. Ma and W. Zhou. 2017. A zinc finger protein, interacted with cyclophilin, affects root development via IAA pathway in rice. J. Integr. Plant Biol., 59(7): 496-505. https://doi.org/10.1111/jipb.12531

Dong, J., W. Teng, W.G. Buchholz and T.C. Hall. 1996. Agrobacterium mediated transformation of Javanica rice. Mol. Breeding. 2: 267-276. https://doi.org/10.1007/BF00564204

FAOUN, 2018. Food and Agriculture Organization of United Nations. Rice market monitor. 2018. 21(1): 1-35. http://www.fao.org/economic/RMM.

Gelvin, S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev., 67(1): 16-37. https://doi.org/10.1128/MMBR.67.1.16-37.2003

Han, G., M. Wang, F. Yuan, N. Sui, J. Song and B. Wang. 2014. The CCCH zinc finger protein gene AtZFP1 improves salt resistance in Arabidopsis thaliana. Plant Mol. Biol. 86: 237-253. https://doi.org/10.1007/s11103-014-0226-5

Hanumanthappa, D., S.N. Vasudevan, K. Maruthi, J.B. Maruthi and A. Sebastian. 2016. Efficacy of dormancy breaking methods in paddy genotypes. J. Appl. Nat. Sci., 8(2): 634-639. https://doi.org/10.31018/jans.v8i2.850

Hiei, Y., S. Ohta, T. Komari and T. Kumashiro. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271-282. https://doi.org/10.1046/j.1365-313X.1994.6020271.x

Hussain, Z., S. Ali, Z. Hayat, M.A. Zia, A. Iqbal and G.M. Ali. 2014. Agrobacterium mediated transformation of “BREB1A” gene for improved drought tolerance in rice cultivars (Oryza sativa L.). Aust. J. Crop Sci. 8(7): 1114-1123.

Iuchi, S., M. Kobayashi, T. Taji, M. Naramoto, M. Seki, T. Kato, S. Tabata, Y. Kakubari, K. Yamaguchi-Shinozaki and K. Shinozaki. 2001. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27: 325-333. https://doi.org/10.1046/j.1365-313x.2001.01096.x

Jan, A., K. Maruyama, D. Todaka, S. Kidokoro, M. Abo, E. Yoshimura, K. Shinozaki, K. Nakashima and K.Y. Shinozaki. 2013. OsTZF1, a CCCH-Tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes. Plant Physiol. 161(3): 1202-1216. https://doi.org/10.1104/pp.112.205385

Jeong, M.S., J.E. Kim and S.B. Jang. 2010. Expression of RNA binding of human zinc finger antiviral protein. Biochem. Biophys. Res. Commun. 396(3): 696-702. https://doi.org/10.1016/j.bbrc.2010.04.164

Kim, J.S., H.M. Park, S. Chae, T.H. Lee, D.J. Hwang, S.D. Oh, J.S. Park, D.G. Song, C.H. Pan, D. Choi, Y.H. Kim, B.H. Nahm and Y.K. Kim. 2014. A pepper MSRB2 gene confers drought tolerance in rice through the protection of chloroplast-targeted genes. PLoS One, 9(3): e90588. https://doi.org/10.1371/journal.pone.0090588

Lata, C. and M. Prasad. 2011. Role of DREBs in regulation of abiotic stress responses in plants. J. Exp. Bot. 62(14): 4731-4748. https://doi.org/10.1093/jxb/err210

Lijavetzky, D., P. Carbonero and J.V. Carbajosa. 2003. Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol. Biol. 3: 1-17. https://doi.org/10.1186/1471-2148-3-17

Liu, X.M., X.C. Nguyen, K.E. Kim, H.J. Han, J, Yoo, K. Lee, M.C. Kim, D.J. Yun and W.S. Chung. 2013. Phosphorylation of the zinc finger transcriptional regulator ZAT6 by MPK6 regulates Arabidopsis seed germination under salt and osmotic stress. Biochem. Biophys. Res. Commun. 430: 1054-1059. https://doi.org/10.1016/j.bbrc.2012.12.039

Liu, D., L. Yang, M. Luo, Q. Wu, S. Liu and Y. Liu. 2017. Molecular cloning and characterization of PtrZPT2-1, a ZPT2 family gene encoding a Cys2/His2-type zinc finger protein from trifoliate orange (Poncirus trifoliata (L.) Raf.) that enhances plant tolerance to multiple abiotic stresses. Plant Sci. 263: 66-78. https://doi.org/10.1016/j.plantsci.2017.07.012

Ma, X., W. Cui, W. Liang and Z. Huang. 2015. Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana. Plant Physiol. Biochem. 97: 187-195. https://doi.org/10.1016/j.plaphy.2015.10.010

Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio assays with Tobacco tissue cultures. Physiol. Plant. 15(3): 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Nakashima, K., Y. Ito and K. Yamaguchi-Shinozaki. 2009. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol. 149: 88-95. https://doi.org/10.1104/pp.108.129791

Ogunbayo, S.A., D.K. Ojo, G.R. Guei, O.O. Oyelakin and K.A. Sanni. 2005. Phylogenetic diversity and relationships among 40 rice accessions using morphological and RAPDs techniques. Afr. J. Biotechnol. 4 (11): 1234-1244.

Pantuwan, G., S. Fukai, M. Cooper, S. Rajatasereekul and J.C.O’Toole. 2002. Yield response of rice (Oryza sativa L.) genotypes to drought under rainfed lowland: 3. Plant factors contributing to drought resistance. Field Crops Res. 73(2-3): 181-200. https://doi.org/10.1016/S0378-4290(01)00194-0

Phillips, N., C.M. Smith and C.W. Morde. 2001. An effective DNA extraction protocol for brown algae. Phycol. Res. 49: 97-102. https://doi.org/10.1111/j.1440-1835.2001.tb00239.x

Pradhan, S., C. Kant, S. Verma and S. Bhatia. 2017. Genome-wide analysis of the CCCH zinc finger family identifies tissue specific and stress responsive candidates in chickpea (Cicer arietinum L.). PLoS One. 12 (7): e0180469. https://doi.org/10.1371/journal.pone.0180469

Rashid, H., S. Yokoi, K. Toriyama and K. Hinata. 1996. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep. 15: 727-730. https://doi.org/10.1007/s002990050108

Riechmann, J.L., J. Heard, G. Martin, L. Reuber, C.Z. Jiang, J. Keddie and R. Creelman. 2000. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science. 290: 2105-2110. https://doi.org/10.1126/science.290.5499.2105

Rodziewicz, P., B. Swarcewicz, K. Chmielewska, A. Wojakowska and M. Stobiecki. 2014. Influence of abiotic stresses on plant proteome and metabolome changes. Acta Physiol. Plant. 36(1): 1-19. https://doi.org/10.1007/s11738-013-1402-y

Sarker, S., R.S. Tammi, S. Biswas, S. Zaman and Z.I. Seraj. 2015. Cloning and characterization of a tissue specific promoter GluB-1 from Nipponbare by transformation in rice. Biores. Commun. 1(2): 69-75.

Shen, W.H., J. Escudero and B. Hohn. 1999. T-DNA transfer to maize plants. Mol. Biotechnol. 13: 165-170. https://doi.org/10.1385/MB:13:2:165

Sheu, D.S., Y.T. Wang and C.Y. Lee. 2000. Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR. Microbiology. 146(8): 2019-2025. https://doi.org/10.1099/00221287-146-8-2019

Shinozaki, K. and K.Y. Shinozaki. 2007. Gene networks involved in drought stress response and tolerance. J. Exp. Bot. 58(2): 221-227. https://doi.org/10.1093/jxb/erl164

Steel, R.G.D. and J.H. Torrie. 1960. Principles and procedures of statistics with special reference to the biological sciences. McGraw-Hill, New York.

Swamy, B.P.M. and A. Kumar. 2013. Genomics-based precision breeding approaches to improve drought tolerance in rice. Biotechnol. Adv. 31(8): 1308-1318. https://doi.org/10.1016/j.biotechadv.2013.05.004

Todaka, D., K. Shinozaki and K. Yamaguchi-Shinozaki. 2015. Recent advances in the dissection of drought-stress regulatory networks and strategies for development of drought-tolerant transgenic rice plants. Front. Plant Sci. 18: 684. https://doi.org/10.3389/fpls.2015.00084

Toki, S., 1997. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol. Biol. Rep. 15(1): 16-21. https://doi.org/10.1007/BF02772109

Toriyama, K. and K. Hinata. 1985. Cell suspension and protoplast culture in rice. Plant Sci. 41: 179-183. https://doi.org/10.1016/0168-9452(85)90086-X

Umezawa, T., M. Fujita, Y. Fujita, K.Y. Shinozaki and K. Shinozaki. 2006. Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future. Curr. Opin. Biotechnol. 17: 113-122. https://doi.org/10.1016/j.copbio.2006.02.002

Umezawa, T., K. Nakashima, T. Miyakawa, T. Kuromori, M. Tanokura, K. Shinozaki and K. Yamaguchi-Shinozaki. 2010. Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol. 51: 1821-1839. https://doi.org/10.1093/pcp/pcq156

Vain, P., B. Worland, M.C. Clarke, G.R.M. Beavis, H. Liu, A. Kohli, M. Leech, J.S.P. Christou and H. Atkinson. 1998. Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IDD86) for nematode resistance in transgenic rice plants. Theor. Appl. Genet. 96: 266-271. https://doi.org/10.1007/s001220050735

Wang, F.Z., Q.B. Wang, S.Y. Kwon, S.S. Kwak and W.A. Su. 2005. Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J. Plant Physiol. 162: 465-472. https://doi.org/10.1016/j.jplph.2004.09.009

Wang, Y., M. Wisniewski, R. Meilan, S.L. Uratsu, M.G. Cui, A. Dandekar and L. Fuchigami. 2007. Ectopic expression of Mn-SOD in Lycopersicon esculentum leads to enhanced tolerance to salt and oxidative stress. J. Appl. Hort. 9: 3-8. https://doi.org/10.37855/jah.2007.v09i01.01

Wang, D., Y. Guo, C. Wu, G. Yang, Y. Li and C. Zheng. 2008. Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice. BMC Genomics. 9: 44-54. https://doi.org/10.1186/1471-2164-9-44

Wang, Q., Y. Guan and Y. Wu. 2008. Over-expression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice. Plant Mol. Biol. 67(6): 589-602. https://doi.org/10.1007/s11103-008-9340-6

Wang, W., B. Liu, M. Xu, M. Jamil and G. Wang. 2015. ABA-induced CCCH tandem zinc finger protein OsC3H47 deceases ABA sensitivity and promote drought tolerance in Oryza sativa. Biochem. Biophys. Res. Commun. 464(1): 33-37. https://doi.org/10.1016/j.bbrc.2015.05.087

Wang, F., W. Tong, H. Zhu, W. Kong, R. Peng, Q. Liu and Q. Yao. 2016. A novel Cys 2/His 2 zinc finger protein gene from sweet potato, IbZFP1, is involved in salt and drought tolerance in transgenic Arabidopsis. Planta. 243(3): 783-797. https://doi.org/10.1007/s00425-015-2443-9

Wise, A.A., Z. Liu and A.N. Binns. 2006. Three methods for the introduction of foreign DNA into Agrobacterium. Thods. Mol. Biol. 343: 43-53. https://doi.org/10.1385/1-59745-130-4:43

Wu, H., C. Sparks, B. Amoah and H.D. Jones. 2003. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep. 21(7): 659-668. https://doi.org/10.1007/s00299-002-0564-7

Wu, Q., X. Liu, D. Yin, H. Yuan, Q. Xie, X. Zhao and D. Li. 2017. Constitutive expression of OsDof4, encoding a C2-C2 zinc finger transcription factor, confesses its distinct flowering effects under long-and short-day photoperiods in rice (Oryza sativa L.). BMC Plant Biol. 17(1): 166-179. https://doi.org/10.1186/s12870-017-1109-0

Yamaguchi-Shinozaki, K. and K. Shinozaki. 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 57: 781-803. https://doi.org/10.1146/annurev.arplant.57.032905.105444

Yilmaz, S. and R. Mittler. 2008. The zinc finger network of plants. Cell. Mol. Life Sci. 65: 1150-1160. https://doi.org/10.1007/s00018-007-7473-4

Yin, M., Y. Wang, L. Zhang, J. Li, W. Quan, L. Yang and Z. Chan. 2017. The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress. J. Exp. Bot. 68(11): 2991-3005. https://doi.org/10.1093/jxb/erx157

Zhang, C., H. Zhang, Y. Zhao, H. Jiang, S. Zhu and B. Cheng. 2013. Genome-wide analysis of the CCCH zinc finger gene family in Medicago truncatula. Plant Cell Rep. 32(10): 1543-1555. https://doi.org/10.1007/s00299-013-1466-6

Zhang, Y., H. Lan, Q. Shao, R. Wang, H. Chen, H. Tang and J. Huang. 2015. An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa). J. Exp. Bot. 67(1): 315-326. https://doi.org/10.1093/jxb/erv464

Zheng, X., B. Chen, G. Lu and B. Han. 2009. Overexpression of a NAC transcription factor enhances rice drought and salt tolerance. Biochem. Biophys. Res. Commun. 379: 985-989. https://doi.org/10.1016/j.bbrc.2008.12.163