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Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

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.

Supplementary information

The online version contains supplementary material available at 10.1007/s13205-021-02645-7.

SUBMITTER: Sreelekshmi R

PROVIDER: S-EPMC7840827 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A dual in vitro regeneration system consisting of indirect organogenesis and somatic embryogenesis (SE), applicable to several varieties of tomato-Solanum lycopersicum (cv. Riogrande, cv. Roma, hybrid 17905 and model cv. M82) has been established. This system is both improved and highly reproducible compared to current methods. Callus initiation, plant regeneration and SE was developed for one-week-old cotyledon explants. Indirect organogenesis via callus induction (CI) was developed for all four varieties of tomato used in this study. One-week-old tomato seedlings were used as a source of cotyledon and hypocotyl segments as explants. The explants were subsequently cultured on Murashige and Skoog (MS) medium supplemented with different combination and concentrations of plant growth regulators (PGRs). Substantial trends in regeneration and propagation response were observed among the varieties and treatments. For commercial varieties cvs. Riogrande and Roma, maximum CI was observed at 2 weeks in CIMT9 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT12 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4 mg/L KIN). However, cv. M82 responded after 4 weeks to a combination of treatments CIMT9 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT13 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA) for the production of calli. Subsequent shoot and root organogenesis were optimized for all four varieties. Cv. Riogrande, exhibited fastidious in vitro regeneration potential and selected for induction of somatic embryos via SE involving novel structure: rhizoid tubers (RTBs). Numerous fine hair like rhizoids (~23/explants) were first developed from cotyledon and hypocotyl explants cultured on MS medium supplemented with 0.5 or 2 mg/L NAA at pH 4.0 in dark conditions. Further incubation of each rhizoid under light conditions on MS media supplemented with 5 mg/L TDZ or BAP at pH 4.0 led to the formation of a novel structure-rhizoid Tubers (RTBs). Thus, as evident from histology, SE in Riogrande tomato species requires a medium with pH of (4.0) and higher concentration of cytokinins (BAP/TDZ) to form on average 40-45 RTBs from both explants. Histological and morphological studies revealed that RTBs develop through different stages of embryogenesis to multiple plantlets, on MS medium with 5 mg/L TDZ/BAP at normal pH (5.8). The results obtained indicated that the induced somatic embryos of tomato with lower pH are a more efficient mode of propagation than the organogenesis with or without callus formation. The RTBs led to a complete plantlets regeneration in 45 days compared to indirect organogenesis at 60 days.

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Effective reversal of hyperhydricity leading to efficient micropropagation of Dianthus chinensis L.

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