Abstract
The effective reversion of hyperhydricity (HH) in Dianthus chinensis L. facilitated efficient in vitro production of hyperhydricity-free plantlets. Under routine sub-culture practice, the problem of HH arises after third sub-culture in agar (0.85%) gelled Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA). To confirm the role of ethylene on hyperhydricity induction, an ethylene releasing compound ethephon (5 µM) was used in combination with 2.5 µM BA and demonstrated 100% HH with reduced stomatal aperture. Supplementation of 10 µM silver nitrate (AgNO3) to 2.5 µM BA containing medium resulted HH reversion with reduced shoot number (19.0); however, addition of 5 µM cobalt chloride (CoCl2) produced highest microshoots (202.0). The combination effect of AgNO3 (10 µM), CoCl2 (5 µM), and BA (2.5 µM) showed complete HH reversion and upheld normal microshoots (55.0) with reduced relative water content (78.3%). The Ag and Co salts regulate ethylene biosynthesis and thereby 50% reductions in H2O2 content characterized by formation of green healthy shoots with proper stomatal morphology. The gene expression profile of 1-Amminocyclopropane-1-carboxylase synthase (ACS1) and 1-Amminocyclopropane-1-carboxylic acid oxidase (ACO1) showed reduced expression after the retroversion of microshoots in anti-ethylene reversion medium compared to hyperhydric shoot. In vitro raised shoots were rooted (93.3%) ex vitro by 10 mM IBA treatment and 92.2% plants were survived. The genetic stability of micropropagated plants were analyzed and proved that addition of low levels of heavy metal salt in the medium does not cause any variation in banding pattern. The protocol forwards a novel method to revert HH of in vitro cultures by adopting intermittent exposure of anti-ethylene compounds added in the medium and the procedure can be applied to many other plants facing similar HH problems.
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Abbreviations
- MS:
-
Murashige and Skoog
- HH:
-
Hyperhydricity
- CoCl2 :
-
Cobalt chloride
- AgNO3 :
-
Silver nitrate
- RWC:
-
Relative water content
- ISSR:
-
Inter-simple sequence repeat
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Acknowledgements
The authors are grateful to Dr. S Suhara Beevy, Professor and Head, Department of Botany, University of Kerala, Kariavattom, Thiruvananthapuram, for providing the facilities. One of the authors RS also thanks University of Kerala for financial support in the form of Junior Research Fellowship (No. AcVI(1)/715/BOT/13289/2015).
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RS is responsible for designing and conducting the experiments under the advice of EAS. Data analysis and manuscript editing has been performed by EAS.
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Sreelekshmi, R., Siril, E.A. Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.. 3 Biotech 11, 95 (2021). https://doi.org/10.1007/s13205-021-02645-7
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DOI: https://doi.org/10.1007/s13205-021-02645-7