Abstract
Hyoscyamus muticus is cultivated in Egypt, India, Pakistan and USA for the production of the medicinally important tropane alkaloids (TA) hyoscyamine and scopolamine. The TA pathway provides an excellent model to test the feasibility of transgenic strategies to modify secondary metabolic flux. However, H. muticus transformation and regeneration has been proven difficult. We report a successful experience obtaining several independent fertile plant lines, genetically engineered to redirect the TA metabolic flux into alternative directions. cDNA from H. niger encoding tropinone reductase enzymes (TR1 or TR2), which catalyse the reduction of tropinone into scopolamine and hyoscyamine or calystegines, respectively, were incorporated in a silencing vector, followed by Agrobacterium tumefaciens transformation. Most explants produced large amounts of calli. Shoot formation occurred in the majority of the calli, being ready for rooting after an average of 9 months. On average, 45% of the shoots formed roots after 4 months. However, most rooted shoots died during ex vitro acclimatization, regardless of substrate type and cultivation conditions. This problem was overcome by the development of a specialized protocol (cultivation on soil like substrate under sterile conditions) that allowed the recovery of a reasonable percentage (44%) of fertile plants per rooted shoot. In this study were obtained 11 tr2 lines (25 plants), three tr1 lines (3 plants), four empty vector lines (13 plants) and three untransformed-regenerated lines (3 plants); a significant result compared to previous attempts to stable transform H. muticus.
Key message
Twenty-one fertile H. muticus plant lines were regenerated, being the acclimation protocol (cultivation under sterile conditions on high-drainage soil-like substrate and irrigation with Hoagland’s solution) decisive for rooted shoots survival.
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Data availability
The data that support the findings of this study are available from the corresponding author, Nora, L., upon reasonable request.
Abbreviations
- Adc :
-
Adenine carboxylase
- BAP:
-
6-Benzylaminopurine
- CC:
-
Co-culture medium
- CIM:
-
Callus induction medium
- GUS :
-
β-Glucuronidase
- IBA-K:
-
Indole butyric acid
- KIN:
-
Kinetin
- MES:
-
2-(N-morpholino) ethanesulfonic acid
- NAA:
-
1-Naphthaleneacetic acid
- nptII:
-
Neomycin phosphotransferase II
- odc :
-
Ornithyne decarboxylase
- PMT:
-
Putrescine-N-methyl transferase
- PVX:
-
Potato virus X
- RIM:
-
Root induction medium
- RM:
-
Rooting medium
- SCIM:
-
Selective callus induction medium
- SIM:
-
Shoot induction medium
- SRIM:
-
Selective root induction medium
- SSIM:
-
Selective shoot induction medium
- X-Glc:
-
X-Glucoside
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Acknowledgements
The authors thank Mark J. Leech, Pedro J. Rocha Salavarrieta, Leo A. H. Zeef, Anne Edwards, and Steve Mackay for sharing their knowledge of plant transformation and regeneration.
Funding
This research was supported by the Ministry of Education of the Brazilian Government (CAPES - Project no 1802992) and by the John Innes Centre (supported in part by Grant-in-aid from the BBSRC), Norwich, United Kingdom.
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CM directed the study, LN planned and performed the experiments and interpreted the results, FRN contributed to carrying out the experiments. GOD contributed to data summarization and manuscript assembling and submission. All authors provided critical feedback and helped shape the manuscript.
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Nora, L., Dalmazo, G.O., Nora, F.R. et al. Successful regeneration of fertile stably transformed tropane alkaloid-producing plant (Hyoscyamus muticus L.) with PVX-gus-(astr1 or astr2)-nptII constructs. Plant Cell Tiss Organ Cult 145, 517–531 (2021). https://doi.org/10.1007/s11240-021-02023-4
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DOI: https://doi.org/10.1007/s11240-021-02023-4