Influence of the cryopreservation technique, recovery medium and genotype on genetic stability of mint cryopreserved shoot tips

AbstractThe popularity of nanoparticles (NPs) is continuously increasing. To date, however, there has been little research on the application of NPs in plant cryopreservation, i.e. storage of tissues in liquid nitrogen (LN). The aim of this study is to analyze the effect and evaluate the usefulness of gold nanoparticles (AuNPs) in regard to cryobiology studies. In vitro-derived shoot tips of Lamprocapnos spectabilis ‘Valentine’ were cryopreserved with the encapsulation-vitrification protocol. Gold nanoparticles (at 10–30 ppm concentration; 13 nm in size) were added either into the preculture medium; to the protective bead matrix during encapsulation; or to the recovery medium after rewarming of samples. The control plants were produced from cryopreserved explants non-treated with nanoparticles or treated with colloid dispersion medium without NPs. A non-LN-treated standard was also considered. The influence of AuNPs on the cryopreservation efficiency was determined by evaluating the recovery rate of explants and their morphogenic response; the membrane stability index (MSI); the concentration of pigments in shoots; and the antioxidant enzymes activity. The genetic stability of the plant material was evaluated using Start Codon Targeted Polymorphism (SCoT) markers. It was found that 10 ppm of AuNPs added into the alginate bead matrix improved the recovery level of LN-derived shoot tips (70.0%) compared to the non-NPs-treated cryopreserved control (50.5%). On the other hand, the presence of nanoparticles in the recovery medium had a deleterious effect on the survival of explants. AuNPs usually had no impact on the MSI (73.9–85.9%), except for those added into the recovery medium at the concentration of 30 ppm (decline to 55.8%). All LN-derived shoots were shorter and contained less chlorophyll and carotenoids than the untreated standard. Moreover, the application of AuNPs affected the enzymatic activity in L. spectabilis. Minor genetic variation was found in 8.6% of plants if AuNPs were added either into the preculture medium (at 10 and 20 ppm) or to the alginate matrix (at 30 ppm). In conclusion, AuNPs added at a lower concentration (10 ppm) into the protective bead matrix can significantly improve the cryopreservation efficiency in L. spectabilis with no alternation in the DNA sequence.

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Conservation, Regeneration and Genetic Stability of Regenerants from Alginate-Encapsulated Shoot Explants of Gardenia jasminoides Ellis

Polymers ◽

10.3390/polym13101666 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1666

Author(s):

Stefanos Hatzilazarou ◽

Stefanos Kostas ◽

Theodora Nendou ◽

Athanasios Economou

Keyword(s):

Sodium Alginate ◽

Calcium Chloride ◽

Genetic Stability ◽

Nutrient Medium ◽

Shoot Tips ◽

Mother Plant ◽

Short Term ◽

Alginate Encapsulation ◽

Gardenia Jasminoides ◽

Regeneration Response

The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat–perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95–100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

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150. Genetic stability assessment of plants regenerated from wasabi shoot tips cryopreserved for 10 years

Cryobiology ◽

10.1016/j.cryobiol.2009.10.164 ◽

2009 ◽

Vol 59 (3) ◽

pp. 412

Author(s):

Toshikazu Matsumoto ◽

Daisuke Tanaka ◽

Takashi Akihiro ◽

Shinya Maki ◽

Takao Niino

Keyword(s):

Genetic Stability ◽

Shoot Tips ◽

Stability Assessment

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Genetic stability of cryopreserved shoot tips of Rubus germplasm

In Vitro Cellular & Developmental Biology - Plant ◽

10.1007/s11627-009-9265-z ◽

2010 ◽

Vol 46 (3) ◽

pp. 246-256 ◽

Cited By ~ 40

Author(s):

Nina Rosa F. Castillo ◽

Nahla V. Bassil ◽

Sugae Wada ◽

Barbara M. Reed

Keyword(s):

Genetic Stability ◽

Shoot Tips

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Survival and genetic stability of shoot tips of Hedeoma todsenii R.S.Irving after long-term cryostorage

In Vitro Cellular & Developmental Biology - Plant ◽

10.1007/s11627-017-9854-1 ◽

2017 ◽

Vol 53 (4) ◽

pp. 328-338 ◽

Cited By ~ 9

Author(s):

Valerie C. Pence ◽

Megan Philpott ◽

Theresa M. Culley ◽

Bernadette Plair ◽

Suzanne R. Yorke ◽

...

Keyword(s):

Genetic Stability ◽

Shoot Tips

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Effect of Bead Composition, PVS Type, and Recovery Medium in Cryopreservation of Bleeding Heart ‘Valentine’—Preliminary Study

Agronomy ◽

10.3390/agronomy10060891 ◽

2020 ◽

Vol 10 (6) ◽

pp. 891

Author(s):

Dariusz Kulus

Keyword(s):

Calcium Alginate ◽

Dry Weight ◽

Synthetic Seeds ◽

Shoot Tips ◽

Ms Medium ◽

Sterile Water ◽

Murashige And Skoog Medium ◽

Preliminary Study ◽

Lamprocapnos Spectabilis ◽

Recovery Medium

Bleeding heart (Lamprocapnos spectabilis (L.) Fukuhara) is a valuable ornamental and medicinal perennial. To date, there are few studies focused on cryopreservation of this species, although it could be useful in storage and breeding. This research is aimed at analyzing the effect of bead composition, type of plant vitrification solution (PVS), and the recovery medium of cryopreservation of bleeding heart. Shoot tips of L. spectabilis ‘Valentine’ were used in the study. The explants were precultured on modified Murashige and Skoog medium (MS; 1962), supplemented with 9% sucrose, 1.0-mg·L−1 kinetin (KIN), and 2.62-mg·L−1 abscisic acid. Next, in the first experiment, the shoot tips were embedded in 3% calcium alginate, based either on an MS medium or distilled sterile water. The produced synseeds were inoculated on the recovery medium with 3.0-mg·L−1 KIN, 0.5-mg·L−1 6-benzyladenine (BA), or cytokinin–free control. Based on the results of the first study, in the second experiment, precultured shoot tips were embedded in 3% calcium alginate based on MS medium and dehydrated with PVS2 or PVS3 for various durations. The pre-treated explants were plunged in liquid nitrogen and, after rewarming, inoculated on the recovery MS medium with 0.5-mg·L−1 BA. PVS3 was more effective in securing the shoot tips than PVS2. The highest recovery level (68.3%) was reported after a 150-min pretreatment with PVS3. Explants from this experimental combination also proliferated the highest number of shoots, as well as those with the greatest length. On the other hand, a higher share of dry weight was found in PVS2-derived shoots (13.5–18.2%) compared with plants produced after PVS3 treatment (10.6–11.4%). The obtained results here can serve as a good basis for further studies related to synthetic seeds and cryopreservation of bleeding heart.

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