Inea . Fresh leaf samples of D. ferruginea subsp. ferruginea (100 mg) had been ground to a fine powder making use of liquid nitrogen with mortar and pestle. Total RNA isolation was carried out with GeneJET Plant RNA Purification Kit (ThermoFischer Scientific, Waltham, MA, USA). Samples were treated with RNase free DNase I to remove genomic DNA contamination. Single strand cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) working with SuperScriptTM III RT-PCR kit based on the instruction encouraged by manufacturer (ThermoFischer Scientific, Waltham, MA, USA). Purified total RNA up to 5 was employed to synthesize cDNA. PhusionHigh-Fidelity DNAInt. J. Mol. Sci. 2021, 22,16 ofpolymerase (NEB, Ipswich, MA, USA) was utilized for amplification with the 3-HSD, P5R1 and P5R2 genes by using gene precise primers as follows: 3-HSD forward primer: five GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTCGTCAAAGCCAAGGTTGG-3 , 3-HSD reverse primer: 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTTCTAACGCAC GACGGTGAAGC-3 , P5R1 forward primer: 5 -GGGGACAAGTTTGTACAAAAAAGCA GGCTTAATGAGCTGGTGGTGGGC-3 , P5R1 reverse primer: five -GGGGACCACTTTGTA CAAGAAAGCTGGGTTAGGAACAATCTTGTAAGCTTTTGCCT-3 , P5R2 forward primer 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTATACCGACACAACGACTTG G-3 and P5R2 reverse primer: 5 -GGGGACCACTTTGTACAAGAAGCTGGGTTAGGGA CAAATCTATAAGTTCTCACTTTGT-3 . Primers utilised inside the present study are also summarized in Supplementary Table S1. The amplified items had been confirmed on 1 agarose gel stained with EtBr and further confirmed by sequencing. For building of final plastid transformation vector, Gatewaycloning was employed. The genes 3-HSD, P5R1 and P5R2 have been cloned into pDONR221 by BP recombination reaction which resulted in entry vectors pENTR-3-HSD, pENTR-P5R1 and pENTR-P5R2. An LR recombination reaction was carried out between pENTR-3-HSD, pENTR-P5R1 and pENTR-P5R2 and pDEST-PN-T in separate reaction for every entry vector and final plastid expression vectors pEXP-PNT-3-HSD, pEXP-PN-T- P5R1 and pEXP-PN-T-P5R2 were obtained. A plastid particular Gatewaycompatible destination vector, pDEST-PN-T , was utilized for the LR reaction. It contained the cassette of aadA gene beneath the manage of psbA promoter (PpsbA), the 5 UTR of tobacco psbA gene (5 psbA) along with the three UTR from large subunit of ribulose-bisphosphate carboxylase gene (rbcL) from Chlamydomonas reinhardtii. The expression of transgene was below the handle of constitutive PrrnPEPNEP promoter, which consisted of the nuclear encoded polymerase (Prrn-62NEP) promoter  fused downstream for the plastid-encoded polymerase (PEP) promoter Prrn16 . Figure 1 shows the vector construction steps. The Gatewaycloning kit was bought from (ThermoFischer Scientific, Waltham, MA, USA) and all cloning reactions have been carried out by following the guidelines of manufacturer. 4.2. Plastid Transformation of Tobacco and Regeneration of Transformed Plants The plastid transformation was carried out by following the procedure as described previously . Briefly, seeds of N. 2-Hexyl-4-pentynoic acid Purity & Documentation tabacum (Nt) cv. Petit Havana have been grown in vitro at 26 C on agar solidified MS  medium containing 3 sucrose. The expression constructs, pEXP-PN-T-3-HSD, pEXP-PN-T-P5R1 and pEXP-PN-T-P5R2, had been coated onto gold particles of 0.six and bombarded on 2 weeks old tobacco leaves by bombarding DNA coated gold particles using particle gun (SRTCX1002 Epigenetics PDS1000He; Bio-Rad, Hercules, CA, USA). Just after bombardment, leaves had been sliced into smaller pieces of five mm and placed on RMOP media conta.