Three peanut cultivars Zhonghua 12 Zhonghua 24 and Huayu 23 were used in this study. Zhonghua 12 (var. vulgaris) and Zhonghua 24 (var. hypogaea) are developed by Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, China in 2006 and 2015, respectively. Huayu 23 (var. hypogaea) is developed by Shandong Peanut Research Institute, Shandong Academy of Agricultural Sciences, Qingdao, China in 2004.

A cassette comprising a Nos promoter and 3’ UTR flanking Basta selection marker gene was amplified by PCR from pBinGlyBar1 vector using the following primers with added AscI restriction sites: 5’-CATGGGCGCGCCGCACGCTGCCGCAAGCAC-3’ and 5’-CATGGGCGCGCCCGCGCCGATCTAGTAACA-3’ (the added restriction sites are underlined). Then the AscI digested fragment was inserted into the binary vector pBinGlyRed2 (Huai et al., 2020) containing a 35S promoter-driven DsRed2 gene to generate pBinBarRed ( Figure S1 ).

Protoplasts were isolated from leaves of 14 days old aseptic seedlings of peanut cultivar Zhonghua 12, which were obtained from seeds sterilized with 0.1% (w/v) HgCl₂ and cultured in dark. The leaves were cut into 0.5 mm strips, and then dispersed into 30 mL of enzyme solution (2% cellulose R-10, 1% macerozyme R-10, 0.2% pectolase Y-23, 0.1% BSA, 0.6 M mannitol, 10 mM MES (pH 5.7) and 15 mM CaCl₂) with gently shaking at 40 rpm for 12 hours. After digestion, cells were filtered with a 70 μm nylon meshes, and then washed twice by 30 mL W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl and 2 mM MES, pH5.8). Protoplasts were resuspended in 2 mL W5 solution and stored on ice for 30 mins. Lastly, protoplasts were centrifuged at 100 g for 2 mins, and resuspended in 2 mL MMG solution (0.6 M mannitol, 15 mM CaCl₂ and 4 mM MES). Cell concentration was measured using a hemocytometer and a light microscope.

Totally 2x10⁵ isolated single protoplasts were suspended in 200 μL MMG solution, and stored on ice for 30 mins. Approximately 20 μg plasmid DNA and 220 μL PEG solution (40% PEG4000, 0.6 M mannitol and 100 mM CaCl₂) was added, mixed gently and stored at room temperature for 20 mins. After incubation, 1 mL W5 solution was added and stored on ice for 5 mins. Then cells were centrifuged at 100 g for 2 mins, and washed twice by 300 μL W5 solution. The protoplast pellets were resuspended with 1.5 mL W5 solution, and incubated in the dark at room temperature for 48-72 hours. The protoplast pellets were harvested for further analysis.

The vector pBinBarRed was introduced into Agrobacterium tumefaciens strain GV3101 by electro-transformation. A single transformed colony was inoculated into a flask (50 ml) containing sterile 10 ml LB liquid medium supplemented with 50 mg/L kanamycin. The flask was incubated at 28°C for 24 h in a shaker incubator set at 180 rpm until the OD₆₀₀ reached 0.6-0.8. Bacterial suspension was pelleted by centrifugation for 10 min at 250 g and resuspended the cells in the Agrobacterium infection medium (2.2g/L MS-B5 and 20 g/L sucrose, OD_{600 =} 0.6-0.8).

The A. tumefaciens-mediated transformation was performed as described by Sharma and Bhatnagar-Mathur (2006) with some modifications. Mature seeds from the peanut cultivar Zhonghua 12 were surface sterilized by rinsing in 75% ethanol for 1 min followed by treatment with 0.1% (w/v) HgCl₂ for 4 min and then washed 4-6 times with sterile-distilled water and soaked in sterile water overnight. After removing the seed coat, the embryo was removed and each cotyledon was cut into vertical halves which were used as explants. Explants were immersed in the Agrobacterium infection medium for 2-5 min, and then transferred onto the Co-cultivation Medium (4.4 g/L MS-B5, 20 g/L sucrose, 4 mg/L BAP, 1 mg/L 2,4-D and 8 g/L agar, pH=5.8) maintained in dark for 72 hours at 26°C ( Figure 1A ). Then explants were transferred onto the Shoot Induction Medium (4.4 g/L MS-B5, 20 g/L sucrose, 4 mg/L BAP, 1 mg/L 2,4-D, 300 mg/L Timentin, 1 mg/L Basta and 8 g/L agar, pH=5.8) and kept at 26°C under 16 h day/8 h dark for 14 days. The explants were transferred onto a fresh medium every 14 days until the shoots come out ( Figures 1B–D ). The shoots were transferred onto the Shoot Elongation Medium (4.4 g/L MS-B5, 20 g/L sucrose, 2 mg/L BAP, 300 mg/L Timentin and 8 g/L agar, pH=5.8) and kept at 26 °C under 16 h day/8 h dark for 14 days. The shoots were transferred onto a fresh medium every 14 days until the shoots grew to 3-4 cm high ( Figures 1E, F ). Subsequently, the elongated shoots were transferred onto the Root Induction Medium (4.4 g/L MS-B5, 20 g/L sucrose and 8 g/L agar, pH=5.8) until the roots grew to 4-5 cm long ( Figures 1G, H ). The plants were transplanted into autoclaved sand-soil (1:1) mixture in plastic pots and maintained in a growth cabinet at 26 °C under 16 h day/8 h dark until the seeds mature ( Figure 1I ).

The vector pBinBarRed was introduced into Agrobacterium rhizogenes strain K599 by electro-transformation. A single transformed colony was grown in 10 mL LB liquid medium containing kanamycin at 50 mg/L and incubated overnight at 28 °C with shaking at 180 rpm until the OD₆₀₀ reached 0.6–0.8. The bacterial cells were collected by centrifugation for 10 min at 250 g and resuspended in the A. rhizogenes solutions (2.2 g/L MS, 20 g/L sucrose and 100 μM AS, OD_{600 =} 0.6-0.8).

Mature seeds from the peanut cultivar Huayu 23 were surface sterilized and germinated on ½MS medium ( Figures 2A–C ). After 1 week, the radicle and hypocotyl were cut from each seedling and the remaining portion was used as explant. Explants were dipped in the A. rhizogenes solutions and incubated for 2-5 min, and then transferred onto the co-cultivation medium (4.4 g/L MS, 20 g/L sucrose, 50 μM AS and 8 g/L agar, pH=5.8) maintained in dark at 26°C for 3 days ( Figure 2D ). Subsequently, explants were transferred onto the hairy root induction medium (4.4 g/L MS, 20 g/L sucrose, 300 mg/L Timentin and 8 g/L agar, pH=5.8) and kept at 26 °C under 16 h day/8 h dark until the root grow well ( Figures 2E, F ).

The fluorescence in protoplast cells was examined under a laser-scanning confocal microscope (Olympus FV 10-ASW). The fluorescence in callus and samples form transgenic plants were observed using a green-light hand-held lamp (LUYOR, China), with a red camera filter lens.

Total genomic DNA was isolated from young leaves of transgenic plantlets and wild-type peanut plants using a EasyScript Plant Genomic DNA Kit (Transgen, China). To detect the presence of DsRed2 gene, the following primers were used for PCR amplification: 5’- TTCAAGGTGCGCATGGAG-3’ and 5’-CGTTGTGGGAGGTGATGT-3’. The amplification cycle consisted of denaturation at 94 °C for 1 min, primer annealing at 58 °C for 1 min, and primer extension at 72 °C for 1 min. After 30 repeats of the thermal cycle and final extension 72 °C for 10 min, amplification products were analyzed on 1% agarose gels. The putative PCR product was 577 bp.

Total RNA was extracted from young leaves of transgenic plantlets and wild-type peanut plants using TRIzol reagent (Sigma, USA). Reverse transcription was implemented using SuperScript IV First-Strand Synthesis System (Invitrogen, USA). Real time PCRs were performed on a Bio-Rad CFX96 Real-Time system using SYBR Green as fluorescent dye. The peanut actin gene was used as internal control using primers: 5’- TAAGAACAATGTTGCCATACAGA-3’ and 5’-GTTGCCTTGGATTATGAGC-3’. The primers for DsRed2 gene were: 5’-GTACGGCTCCAAGGTGTACG-3’ and 5’-TAGATGAAGCAGCCGTCCTG-3’.

A high oleate peanut cultivar Zhonghua 24 was pollinated with the pollens from transgenic Line 1 (T₁), which were developed from normal oleate cultivar Zhonghua 12. As the difference between the oleic acid contents in parents were caused by mutations in AhFad2 genes (Chu et al., 2009; Pandey et al., 2014), primers for AhFad2 genes were used for hybrid identification: 5’- CACTAAGATTGAAGCTC-3’ and 5’-CACTAAGATTGAAGCTC -3’. A 500 bp fragment was amplified by PCR and sequenced by Sanger sequencing.

Firstly, the DsRed2 gene was expressed in protoplasts isolated from peanut leaves. After transformation, the protoplasts were centrifuged to the bottom of tubes ( Figure 3A ). Under green light, the transformed protoplasts R-1 and R-2 showed bright red fluorescence compared with CK ( Figure 3B ). Under a laser confocal microscope, red fluorescence was detected in R-1 and R-2, but not in CK ( Figure 3C ). These results indicated that DsRed2 could be used as a reporter in peanut protoplast transformation.

The DsRed2 gene was constitutively expressed in peanut via A. tumefaciens-mediated transformation. Under green light, red fluorescence could be observed at very early stage of callus formation ( Figure 4B ), and became increasingly pronounced with callus age. Upon the emergence of shoots, the transgenic shoots were selected by red fluorescence ( Figure 4D and Figure S2B ). The shoots showing red fluorescence developed into plantlets with red fluorescence ( Figure 4H ). The whole plant exhibited bright red fluorescence under green light at both vegetative and reproductive stages, including the root ( Figure 4F ), leaf ( Figure 4K ), flower ( Figure 4M ), pod shell ( Figure 4O ) and seed ( Figure 4Q ). Under white light, after removal of the seed coat, the color of embryo and cotyledon was red in transgenic seeds, but was white in CK ( Figure 4R ), allowing very easy discrimination of the transgenic seeds with naked eyes. The T₁ progeny of DsRed2 peanut exhibited the same morphological characteristics as T₀ generation, indicating that the fluorescence can be inherited by subsequent generations. Hence, DsRed2 can serve as a stable reporter in A. tumefaciens-mediated peanut transformation.

A total of 52 T₀ transgenic plantlets were obtained, and 15 of which were tested by PCR. As shown in Figure 5A , all the 15 lines harbored the DsRed2 gene. Compared with the transgenic lines without the DsRed2 reporter, the positive rate increased from 56.9% ( Figure S3 ) to 100% ( Figure 5A ). The expression levels of DsRed2 were detected in leaves of the 15 transgenic lines by qRT-PCR. As expected, the expression of DsRed2 was not detectable in the non-transformed control, while was detected in transgenic lines ( Figure 5B ). Hence, the application of DsRed2 in peanut transformation can greatly increase the screening efficiency and improve the positive rate.

The transgenic Line 1 with DsRed2 was used as the male parent (with normal oleate trait) to be crossed with the high-oleate peanut cultivar Zhonghua 24, and 28 F₁ hybrid seeds were obtained. The seed testa color was pink in the female parent Zhonghua 24, while red in the male parent transgenic Line 1. The seed testa color of hybrid was pink, suggesting that the F₁ seeds were obtained from the female parent instead of mixing with seeds from the male parent ( Figure 6A ). The color of embryo and cotyledon was white in the female parent, red in the male parent, while lightly red in the hybrid seeds, indicating that the DsRed2 gene was transferred into hybrid plants ( Figure 6C ). Furthermore, red fluorescence was detected in both hybrid seeds and male parent seeds, but not in female parent seeds under green light ( Figures 6B and D ). Sanger sequencing results revealed that the genotypes of AhFAD2 genes controlling the oleic content in all the 28 red seeds were heterozygous, indicating that they were true hybrids ( Figure S4 ). All these results suggested that DsRed2 could be used as a reliable reporter in peanut hybrid identification.

The selection effect of DsRed2 in A. rhizogenes-mediated peanut transformation was also evaluated ( Figure 7a ). As shown in Figure 7B , red fluorescence can only be observed in induced hairy roots but not in other parts under green light. Twenty-one plantlets with transgenes were selected by red fluorescence, and verified by PCR. The positive rate of transgenic hairy root was also reached to 100% ( Figure 7C ). These results also demonstrated that DsRed2 is also an ideal reporter in A. rhizogenes-mediated peanut transformation.