Abstract
Lycium ruthenicum is an excellent eco-economic shrub. Numerous researches have been conducted for the function of its fruits but scarcely focused on the somaclonal variation and DNA methylation. An efficient micropropagation protocol from leaves and stems of L. ruthenicum was developed in this study, in which not only the leaf explants but also the stem explants of L. ruthenicum were dedifferentiated and produced adventitious buds/multiple shoots on one type of medium. Notably, the efficient indirect organogenesis of stem explants was independent of exogenous auxin, which is contrary to the common conclusion that induction and proliferation of calli is dependent on exogenous auxin. We proposed that sucrose supply might be the crucial regulator of stem callus induction and proliferation of L. ruthenicum. Furthermore, results of methylation-sensitive amplified polymorphism (MSAP) showed that DNA methylation somaclonal variation (MSV) of CNG decreased but that of CG increased after acclimatization. Three types of micropropagated plants (from leaf calli, stem calli and axillary buds) were epigenetically diverged more from each other after acclimatization and the ex vitro micropropagated plants should be selected to determine the fidelity. In summary, plants micropropagated from axillary buds and leaves of L. ruthenicum was more fidelity and might be suitable for preservation and propagation of elite germplasm. Also, leaf explants should be used in transformation. Meanwhile, plants from stem calli showed the highest MSV and might be used in somaclonal variation breeding. Moreover, one MSV hotspot was found based on biological replicates. The study not only provided foundations for molecular breeding, somaclonal variation breeding, preservation and propagation of elite germplasm, but also offered clues for further revealing novel mechanisms of both stem-explant dedifferentiation and MSV of L. ruthenicum.
Highlights
On the auxin-free Murashige and Skoog (MS) medium supplemented with 0.44 μM 6-BA, the stem explants of inGdonor and inDdonor produced multiple shoots from axillary buds and nodular calli from cross section enwrapped by the medium (Fig 3D and 3E)
Dedifferentiation and redifferentiation rates of stem explants (Table 1), redifferentiation rates of leaf callus (Table 1), percentage of stem explant producing multiple shoots (Table 1) and rooting rates (Table 1) were all significantly different between the two groups, indicating that stem explant dedifferentiation and redifferentiation, multiple shoots formation from axillary bud, redifferentiation of leaf callus and rooting of L. ruthenicum were all affected by genetic background
We developed a novel efficient micropropagation protocol from leaves and stems of L. ruthenicum and found that stem explant dedifferentiation and redifferentiation, multiple shoots formation from axillary bud, redifferentiation of leaf callus and rooting of L. ruthenicum were all affected by genetic background
Summary
No permits were required for the research. The seeds used in the study were collected from the experimental field of our university (Shenyang Agricultural University). Mature seeds of L. ruthenicum were collected from two plants (PlantD and PlantG) and planted in Shenyang of China (41 ̊ 49’ 25” N; 123 ̊ 34’ 10” E, 60 m above sea level). The seeds were decontaminated with 75% (v/v) alcohol for 30 s, and a 0.1% (w/v) mercuric chloride solution for 2 min, and rinsed 4 times with sterile distilled water [29]. The sterile seeds were horizontally inoculated on half-strength Murashige and Skoog (1/2 MS) medium [30] without any plant growth regulator (PGR). The 1/2 MS medium was supplemented with 2.0% (w/v) sucrose and 0.50%
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