Appl Biochem Biotechnol DOI 10.1007/s12010-014-1286-3

An Efficient Method of Agrobacterium-Mediated Genetic Transformation and Regeneration in Local Indian Cultivar of Groundnut (Arachis hypogaea) Using Grafting Vivekanand Tiwari & Amit Kumar Chaturvedi & Avinash Mishra & Bhavanath Jha

Received: 21 July 2014 / Accepted: 2 October 2014 # Springer Science+Business Media New York 2014

Abstract Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG20 using de-embryonated cotyledon explant. A high regeneration 52.69±2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67±3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L−1, respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65±1.06 % regeneration, of which 3.82±0.6 % explants were survived on hygromycin after selection. Finally, 14.58±2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting. Keywords Agrobacterium . Grafting . Groundnut . Regeneration . Transformation . Tissue culture

Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-1286-3) contains supplementary material, which is available to authorized users.

V. Tiwari : A. K. Chaturvedi : A. Mishra (*) : B. Jha (*) Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G.B. Road, Bhavnagar 364002 Gujarat, India e-mail: [email protected] e-mail: [email protected]

Appl Biochem Biotechnol

Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BAP 6-Benzylaminopurine CM Co-cultivation media EM Elongation media GUS β-Glucuronidase HPT Hygromycin-phosphotransferase IAA Indole-3-acetic acid LB Luria-Bertani broth MS Murashige and Skoog basal salt media NAA α-Naphthaleneacetic acid PGR Plant growth regulator RM Rooting media SLM Selection media SM Shoot induction media

Introduction Groundnut (Arachis hypogaea L.) is one of the major legumes used as the third largest source of edible oil [1]. Apart from the oil production, groundnut is also used as a source of high nutritive supplement. Groundnut seeds contain 40–60 % oil, 20–40 % protein and 10–20 % carbohydrate, several vitamins, minerals and medicinally important compounds [2, 3]. The waste cake, by-product of oil extraction, is used as animal feed and industrial purposes. Even the haulum are nutritionally better compared to those of cereals as cattle feed. Being a legume crop, it improves the atmospheric N2 fixation to the soil and thus increases the soil fertility [3]. The nutritional value and industrial uses of groundnut make it an important industrial crop worldwide. Global annual production of groundnut is 40.16 million metric tons, of which China, India and USA rank first, second and third, respectively [4]. As documented by the ASG [5], the major portion of the groundnut, produced in India, is contributed by three states: Gujarat, Tamil Nadu and Andhra Pradesh, among which Gujarat ranks first in terms of total annual production. A local cultivar of groundnut GG-20 is developed in 1992, and yet today, this cultivar is popular among farmers, because of high oil content and large seed size [6]. The productivity of groundnut is challenged by several biotic and abiotic stresses. The cultivated taxa of groundnut, Arachis hypogaea L. is an autogamous allotetraploid (2n=4x=40), which behaves as diploid and contains AABB type genome organization derived from Arachis duranensis (A genome) and Arachis ipaënsis (B genome) [7]. Some of the QTL mapping and marker-assisted selection responsible for biotic or abiotic stress tolerance have been initiated for groundnut improvement, but still, the limitations like tetraploidy and low variability in the genome are the major hurdles for molecular breeding [2]. The use of genetic engineering and molecular breeding and their integration with conventional breeding are the approaches which can assist in achieving the goal of higher productivity or better quality of groundnut [8]. In recent years, there were several reports on genetic engineering of groundnut mediated by Agrobacterium tumefaciens [9–11]. Biolistic method of transformation has certain major drawbacks like transgenic plants with high copy number [12, 13], transgene instability and vector backbone integration along with the T-DNA [14]. Furthermore, the method is costly enough to operate as it requires tungsten or gold particle for the bombardment. The

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Agrobacterium-mediated transformations are considered to be superior to biolistic method due to advantages of low copy transgene events and better stability of transgene expression [15]. But, there was a great inconsistency in the transformation efficiency of different cultivars. Only a few groundnut cultivars showed higher susceptibility to the Agrobacterium-mediated transformation [16]. There are several factors like secondary metabolites and phenolic compounds [17, 18], host defence mechanisms [19, 20] and endogenous phytohormones [21–23], which negatively regulate the Agrobacterium-mediated transformation efficiency in plants. These negative factors varied in different genotypes or cultivars of same species due to differences in host genetic compositions and their biosynthetic pathways. Key factors influencing the transformation efficiencies are bacterial growth phase, use of antioxidants, phenolic compounds, sugars/reducing sugars, pH of co-cultivation media and temperature of co-cultivation [24–27]. Agrobacterium method is frequently used for the engineering plants tolerant to different stresses [28–33], and recently, a transgenic groundnut (Arachis hypogaea) was developed over-expressing SbpAPX gene [33]. In the present study, a stable, efficient and rapid method of transformation and regeneration was reported using de-embryonated cotyledon explants. Study included optimization of factors affecting the Agrobacterium virulence and transformation efficiencies. Effects of exogenous and endogenous phytohormones on transformation efficiencies were also studied. A stable transgenic groundnut of the cultivar GG-20 was developed using the optimized method followed by grafting.

Materials and Methods Plant Materials and Explants Preparation Dry and mature groundnut seeds of local cultivar GG-20 were procured from Gujarat Seed Corporation Ltd., Sihor, Gujarat, India. The groundnut seeds were washed briefly (1 min) with 70 % (v/v) ethanol and surface sterilized with 0.1 % mercuric chloride for 10 min with gentle shaking. The seeds were washed with sterile water for five to six times and soaked in the water for 3 h. The seed coats and embryo axes were removed from the cotyledons. The deembryonated cotyledon explants were cut into two vertical halves and used as explants for regeneration and transformation (Supplementary Fig. 1). Optimization of Regeneration For regeneration of groundnut, Murashige and Skoog (MS) salts [34] with combination of Gomborg’s B5 vitamins [35] were used in distilled water, supplemented with 3 % (w/v) sucrose and 0.8 % (w/v) agar (pH 5.8) with different combinations of phytohormones. De-embryonated cotyledon explants were used for the regeneration by direct organogenesis. For this, type and concentration of phytohormones as suggested by three different reports [36–38] were screened comparatively. The shoot induction media used in above three reports were named as shoot induction media (SM1), SM2 and SM3, respectively, while the elongation media were named as elongation media (EM1), EM2 and EM3, respectively. Percent regeneration efficiency and number of shoot buds regenerated per explants were recorded. All cultures were maintained under controlled laboratory conditions at 25±2 °C under a 16/8-h light/dark photoperiod with white fluorescent lamp of 60 μmol m−2 s−1 light intensity. The media (SM and EM) that exhibited the best efficiency was used for the regeneration. Elongated shoots of approximately 2–3 cm, with well-developed stem were sub-cultured in the rooting

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media. The rooting media were designated as rooting media (RM1), RM2 and RM3, respectively. The cultures were sub-cultured two to three times with 2-week interval. Rooted plants were transferred to Soilrite (Keltech Energy Ltd., Bengaluru, India) and covered with polyethylene for 2 weeks to maintain high humidity. The cover was removed slowly day-by-day for acclimatization to the low humidity environment. Finally, after 2 weeks of acclimatization, polyethylene covers were removed and plantlets were transferred to the greenhouse condition for further growth. Determination of Lethal Dose of Hygromycin The lethal dose of hygromycin as selective agent was determined by culturing the SM3 regenerated shoot buds on EM3 media supplemented with different concentrations of hygromycin 0, 10, 20, 30, 40 and 50 mg L−1 and named as selection media (SLM)-1, SLM2, SLM-3, SLM-4, SLM-5 and SLM-6, respectively. Shoot buds were excised and cultured in the SLM media for 21 days. Twelve regenerated shoot buds were used in each group with triplicate culture jars containing a particular hygromycin combination and four explants in each culture jar. After 21 days, numbers of surviving explants were calculated in percentage. The combination showing 50 % death of shoots is considered as lethal dose (LD50), and this concentration was used for the selection of transformed explants. Optimization of Transformation Protocol The plant transformation vector pCAMBIA1301 was mobilized into Agrobacterium tumefaciens strain EHA105 and allowed to grow on LB plate supplemented with 25 mg L−1 rifampicin and 50 mg L−1 kanamycin at 28 °C. Transformation methods, AT1 and AT2 reported by Sharma and Anjaiah [37] and Tiwari and Tuli [26], respectively were evaluated using deembryonated cotyledon explant, and transformation efficiency was calculated. Simultaneously, whole cotyledon was also screened for transformation following AT2 method. Additionally, different parameters like optical density of bacterial culture (OD600 of 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8) and different concentrations of acetosyringone (0, 50, 100, 150, 200 and 250 μM) and Lcysteine (0, 50, 100, 200, 300, 400 and 500 mg L−1) in co-cultivation media were optimized. Effect of Phytohormone on Transformation and Regeneration Different concentrations of BAP alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) were used in co-cultivation media, and its effect on transformation efficiency was studied. Further, transformation efficiency was correlated with the regeneration efficiency of transformed explants. The co-cultivation media that contained MS salts, B5 vitamins, 3 % (w/v) sucrose, 100 mg L−1 myo-inositol, 50 mg L−1 L-cystein, 200 μM acetosyringone, pH 5.8 and 0.6 % (w/v) agar was used with the different combination of BAP and 2,4-D (CM1 to CM20; Table 1). Transformation was performed with optimized method, and the transformants were co-cultivated for 5 days in the dark at 21 °C. Transformation efficiencies were screened by histochemical β-glucuronidase (GUS) staining of the randomly selected 10 transformed explants of each combination. The experiments were performed twice, and average transformation efficiency was recorded. For regeneration efficiencies, explants were cultured on the SM3 media and after 2 weeks subsequently transferred to the SM1 media for the next 2 weeks [26]. The number of explants showed that regeneration was recorded and the average of two independent experiments was calculated.

Appl Biochem Biotechnol Table 1 Co-cultivation media and PGRs used in the study S. no.

Co-cultivation medium

BAP (μM)

2,4-D (μM)

S. no.

Co-cultivation medium

BAP (μM)

2,4-D (μM)

1

CM1

11.1

4.53

11

CM11

11.1

1.13

2

CM2

22.2

4.53

12

CM12

22.2

1.13

3

CM3

44.4

4.53

13

CM13

44.4

1.13

4

CM4

66.6

4.53

14

CM14

66.6

1.13

5

CM5

88.8

4.53

15

CM15

88.8

1.13

6

CM6

11.1

2.26

16

CM16

11.1

0

7

CM7

22.2

2.26

17

CM17

22.2

0

8 9

CM8 CM9

44.4 66.6

2.26 2.26

18 19

CM18 CM19

44.4 66.6

0 0

10

CM10

88.8

2.26

20

CM20

88.8

0

BAP 6-benzylaminopurine, 2,4-D 2,4-dichlorophenoxyacetic acid, CM co-cultivation media

Phytohormone Analysis of the Transformed Explants For phytohormone analysis, untransformed de-embryonated cotyledons were cultured on CM1, 3, 5, 16, 18 and 20 media for 5 days in the dark at 21 °C. Then, explants were washed with sterile water, dipped in liquid nitrogen and stored at −80 °C. The endogenous indole-3acetic acid (IAA) was extracted from the explants according to Pan et al. [39]. The HPLC analysis and detection were performed according to Gupta et al. [40]. The resultant peaks were compared with the peaks obtained from a standard IAA solution of known concentration (100 ppm). The hormone was quantified by comparing the peak area of the sample with peak area of the standard. Genetic Transformation and Regeneration of Transgenic Plants Explants were transformed using the optimized parameters and co-cultivation media in different batches, each containing approximately 1,000 explants. After co-cultivation, explants were washed with sterile distilled water, blotted dry on a sterile paper towel and transferred to SM3 media supplemented with 400 mg L−1 cefotaxime for 2 weeks. Further, they were subcultured in SM1 media supplemented with 400 mg L−1 cefotaxime and incubated for next 2 weeks in 16/8-h light/dark cycle. A number of explants showing shoot bud initiation were recorded, excised from cotyledon explants and then cultured on SLM-3 media for four subcultures, each at 2-week interval. Explants that survived with initiating shoot buds are considered to be positively transformed and allowed to get proliferated after sub-culturing on the same media. Shoots were removed from the nodal part of explants and cultured on the media EM4, supplemented with hormone kinetin (2.325 μM) and BAP (2.22 μM) with 20 mg L−1 hygromycin. Nodal parts of selected explants were again cultured on the SLM-3 medium which resulted in the proliferation of numerous new buds. Each and every bud was sub-cultured on the EM4 medium for the elongation of the shoot buds. For rooting, elongated shoots of 2–3 cm were transferred to RM3 media supplemented with 10 mg L−1 hygromycin. These shoots were also used for grafting on non-transformed 1-week old stocks germinated on Soilrite. For grafting, a “V-shaped” incision was prepared in stocks, while transgenic shoots were cut in a shape complementary to the incision and inserted into the incision. Grafted plants were covered with polyethylene to maintain high humidity, until new

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leaves emerge. Polyethylene covers were removed, and the plants were acclimatized to laboratory condition and then transferred to containment facility (greenhouse condition). Confirmation of the Transgene Integration The transgene integration into the groundnut genome was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Total genomic DNA from the hardened T0 transgenic lines and Wt plants was isolated from expanded leaves. The PCR reactions were performed to screen the presence of hptII and gus genes using specific primer pairs hptF-hptR and GusF-GusR, respectively (Supplementary Table 1). Each PCR reaction was performed in 25 μl of the reaction volume with 1× reaction buffer supplemented with 1.5 mM MgCl2, 0.2 mM dNTPs, 5 pmol of each primer, 1.25 U of Taq DNA polymerase and 200 ng of plant genomic DNA. The amplicons were electrophoresed on a 1.0 % (w/v) agarose gel, detected by ethidium bromide and photographed into Bio-Rad Gel-doc system (Bio-Rad, USA). The Southern hybridization was performed following alkaline capillary transfer method [41–44]. About 25 μg of genomic DNA was digested with HindIII and blotted onto a Hybond (N+) membrane (Amersham Pharmacia, UK). For positive control, 2.5 ng of purified pCAMBIA1301 vector was used. The gus gene-specific DIG-11-dUTP-labelled DNA probe was prepared from pCAMBIA1301 vector by using GUSQF-GUSQR primer pair (Supplementary Table 1) and PCR DIG Probe Synthesis kit (Roche Applied Science, Germany). The hybridization and detection were performed using the CDP-Star detection kit (Roche Applied Science, Germany). The signals were visualized on the Kodak X-ray films after 30-min exposure. The developed X-ray films were scanned with GS-800 Calibrated Densitometer (Bio-Rad, USA). Transgene Expression Analysis in the Transgenic Lines Transgene expression study in the transgenic lines was performed by reverse transcriptase PCR (RT-PCR). Total RNA was isolated from leaf tissues of transgenic lines and Wt plants using the RNeasy Plant Mini Kit (Qiagen, Germany). The complementary DNA (cDNA) was prepared by using ImProm-II™ Reverse Transcriptase (Promega, USA) according to the manufacturer’s protocol and used for PCR reactions containing 100 ng cDNA, 10 pmol of gus primer pair GUSQF-GUSQR or Ah-actin primers Ah-actinF-Ah-actinR, 200 μM dNTPs and 2.5 U Taq DNA polymerase in a 50-μl reaction (Supplementary Table 1). The PCR products were analysed on 1.5 % (w/v) agarose gel electrophoresis. Transient GUS expression in de-embryonated cotyledon explants just after co-cultivation and stable expression in leaves were assessed by using β-Glucuronidase Reporter Gene Staining Kit [45]. Transformed explants after co-cultivation and leaves of hardened putative transgenic plants were washed with water, blotted and dipped into the staining buffer overnight at 37 °C in the dark. The chlorophyll pigments were de-stained using 70 % (v/v) ethanol, and leaves were photographed. Statistical Analyses All the experiments were carried out twice with each 10 biological replicates. The analysis of variance (ANOVA) was performed, and significance was determined at P