Tissue culture of saffron (Crocus sativusL.): Somatic embryogenesis and shoot regeneration

Abstract Background Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. Results This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Conclusions The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

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Rejuvenation of chicory and lettuce plants following phase change in tissue culture

BMC Biotechnology ◽

10.1186/s12896-019-0557-z ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Anthony J. Conner ◽

Helen Searle ◽

Jeanne M. E. Jacobs

Keyword(s):

Tissue Culture ◽

Shoot Regeneration ◽

Seed Set ◽

Genetic Manipulation ◽

Adventitious Shoot ◽

In Vitro Flowering ◽

Phase Changes ◽

Adventitious Shoot Regeneration ◽

Cell And Tissue Culture

Abstract Background A frequent problem associated with the tissue culture of Compositae species such as chicory (Cichorium intybus L.) and lettuce (Lactuca sativa L.) is the premature bolting to in vitro flowering of regenerated plants. Plants exhibiting such phase changes have poor survival and poor seed set upon transfer from tissue culture to greenhouse conditions. This can result in the loss of valuable plant lines following applications of cell and tissue culture for genetic manipulation. Results This study demonstrates that chicory and lettuce plants exhibiting stable in vitro flowering can be rejuvenated by a further cycle of adventitious shoot regeneration from cauline leaves. The resulting rejuvenated plants exhibit substantially improved performance following transfer to greenhouse conditions, with increased frequency of plant survival, a doubling of the frequency of plants that flowered, and substantially increased seed production. Conclusion As soon as in vitro flowering is observed in unique highly-valued chicory and lettuce lines, a further cycle of adventitious shoot regeneration from cauline leaves should be implemented to induce rejuvenation. This re-establishes a juvenile phase accompanied by in vitro rosette formation, resulting in substantially improved survival, flowering and seed set in a greenhouse, thereby ensuring the recovery of future generations from lines genetically manipulated in cell and tissue culture.

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The effect of auxin 2,4-D and cytokinin 2-ip on direct somatic embryogenesis formation of Coffea arabica L. leaf explant

Pelita Perkebunan (a Coffee and Cocoa Research Journal) ◽

10.22302/iccri.jur.pelitaperkebunan.v27i2.146 ◽

2011 ◽

Vol 27 (2) ◽

Author(s):

Rina Arimarsetiowati

Keyword(s):

Tissue Culture ◽

Somatic Embryogenesis ◽

Coffea Arabica ◽

Callus Formation ◽

Culture Technique ◽

Plant Production ◽

Direct Somatic Embryogenesis ◽

Arabica Coffee ◽

Randomized Design ◽

Coffea Arabica L

One of the propagation technique for coffee plant production is tissue culture. Tissue culture technique for Coffea arabica L. faces some problems, mainly in the planlet formation regenerated from explants. The objective of this experiment was to examine the effect 2,4-D and 2-ip combination on the formation of direct somatic embryogenesis of Coffea arabica L. in leaves explant. Auxin (2,4-D) and cytokinin (2-ip) concentrations of, respectively, 1; 5 µM and 5; 10; 15; 20 were used as treatments. This research was conducted using completely randomized design with 10 replications. Observation to induce somatic embryos was done by quantitatively on number of callus from explant and number of embryogenic callus. Beside that, observation by qualitative descriptive was also done on deve lopment of embryogenesis. The results showed that Arabica coffee leaves explant of AS 2K clones could be induced in all medium combination except 5µM 2,4-D and 20µM 2-ip combination. Arabica coffee leaves explant of S 795, Sigararutang and AS 1 varieties could be induced in all medium combination. The highest frequency of callus formation was found in AS 2K, Sigararutang and AS 1 varieties on medium containing 1µM 2,4-D in combination with 10µM 2-ip, whereas for the S 795 variety on medium containing 5µM 2,4-D in combination with 10µM 2-ip. The highest frequency of embriogenic callus in all Arabica coffee variety could be reached on medium containing 5µM 2,4-D in combination with 15µM 2-ip. Key words : Coffea arabica L., somatic embryogenesis, 2,4-D, 2-ip, tissue culture, leaves, callus embryogenic.

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Pengaruh Sitokinin, Jenis Eksplan, dan Genotipe terhadap Embriogenesis Somatik Kakao

Jurnal Tanaman Industri dan Penyegar ◽

10.21082/jtidp.v3n2.2016.p71-82 ◽

2016 ◽

Vol 3 (2) ◽

pp. 71

Author(s):

Nur Ajijah ◽

RR. Sri Hartati

Keyword(s):

Tissue Culture ◽

Somatic Embryogenesis ◽

Somatic Embryo ◽

Theobroma Cacao ◽

Somatic Embryos ◽

Interaction Effects ◽

Embryo Formation ◽

Primary Somatic Embryogenesis ◽

Primary Somatic Embryos

Information on the effect of cytokinins on cacao (Theobroma cacao L.) primary somatic embryogenesis and its interaction with explant types and genotypes is not yet known. This study aimed to evaluate the effect of cytokinins and its interaction with explant types and genotypes on cacao somatic embryogenesis. The study was conducted at tissue culture laboratory of IAARD, Bogor from April until December 2012 and October 2014 until February 2016. Three types of cytokinins i.e. kinetin (0.58, 1.16, and 2.32 μM), thidiazuron (0.01, 0.02, and 0.04 μM) and benzylaminopurine (0.55, 1.11, and 2.22 μM) in combination with 9 μM 2,4-D were tested for their effectiveness in inducing somatic embryogenesis from petals and staminoid explants of Cimanggu 1 genotype. Furthermore, three levels of kinetin (0.58, 1.16, and 2.32 μM) also in combination with 9 μM 2,4-D were evaluated for their influences on the somatic embryogenesis from petals and staminoid explants of three cacao genotypes i.e. Sulawesi 02, ICCRI 04 and Cimanggu 3. The result demonstrated that 2.32 μM kinetin and staminoids explant were more effective to induce cacao somatic embryogenesis of Cimanggu 1 genotype (7%, 0.23 embryos/explant). Additionally, there were interaction effects between the level of kinetin with explant types and genotype on the percentage of explants forming embryo at 12 weeks after culture. The highest percentage of somatic embryo formation was shown by ICCRI 04 genotype with the use of petals explant and a kinetin level of 1.16 μM (31.85%), but not significantly different from the level of kinetin 2.23 μM (25.55%). The formation of primary somatic embryos of cacao is largely determined by the type and level of cytokinins, type of explant, and genotype.

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The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii

Plants ◽

10.3390/plants10122620 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2620

Author(s):

Dmitry Miroshnichenko ◽

Anna Klementyeva ◽

Sergey Dolgov

Keyword(s):

Tissue Culture ◽

Somatic Embryogenesis ◽

Callus Induction ◽

Copper Ions ◽

Induction Medium ◽

Tissue Cultures ◽

Triticum Timopheevii ◽

Dark Light ◽

Green Plants ◽

Callus Induction Medium

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

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