The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A
600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.
Protocorm-like bodies (PLBs) of Dendrobium orchids are emerging as a potential source of valuable secondary metabolites. This study examined the effect of four additives namely 1-naphthaleneacetic acid (NAA), kinetin, thidiazuron (TDZ), and activated charcoal (AC) used in culture medium on genetic variability in PLBs of Dendrobium Sabin Blue. Nine ( 9) ISSR primers and eleven (11) DAMD primers were used to assess the genetic variability of PLBs that were subcultured over a period of two years. We confirmed that the use of kinetin in culture medium for two years resulted in the highest rate of somaclonal variation in PLBs. On the other hand, TDZ and activated charcoal registered the lowest genetic variability in PLBs. The findings of this study suggest the importance of selecting additives used in the culture medium to maintain stable genetic lines of PLBs. We recommend that the assessment of somaclonal variations should be performed for long term maintenance of tissue cultures.
The present study was conducted to develop targeted region amplification polymorphism (TRAP) and start codon targeted polymorphism (SCoT) DNA markers for the identification of somaclonal variation in cryopreserved Dendrobium Bobby Messina. With reference to previous orchid cryopreservation via PVS2 (plant vitrification solution) vitrification methods, regenerated explants were assessed in order to determine the genetic similarity in comparison to the mother plant. 3 different samples were selected involving stock culture PLBs (protocorm-like bodies), non-cryopreserved PLBs and thawed cryopreserved PLBs. During the study, eight pairs of (8) TRAP primers (TRAP CMS TRAP 8-4A, FLS TRAP 2-2A, TRAP 14-5A, TRAP 20-6B, TRAP 16-3B, ChIC TRAP 5-5B, TRAP3-1D and TRAP 9-5A) produced unambiguous and reproducible bands ranging from 100 to 2000 bp. In cryopreserved PLBs, all TRAP primers displayed polymorphic bands. In the non-cryopreserved PLBs, primer TRAP 20-6B produced monomorphism and the remaining 7 showed both complete and partial polymorphism, respectively. SCoT markers indicated that four primers were able to generate reproducible and clear bands at the sizes of 500 to 3000 bp. SCoT primers (S26, S32, S33 and S36) showed polymorphism for both cryopreserved and non-cryopreserved PLBs of Dendrobium Bobby Messina. The TRAP and SCoT DNA markers designed were found to be an efficient tool to evaluate potential rate of somaclonal variations of regenerated PLBs following cryopreservation.
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