Assessment of somaclonal variation in indirect morphogenesis-derived plants of <i>Arracacia xanthorrhiza</i>

The objective of this work was to induce and detect somaclonal variation in arracacha (Arracacia xanthorrhiza) plants regenerated via indirect morphogenesis, in order to evaluate the potential of this technique to produce new genotypes for breeding purposes of this crop. Calli were induced from petiole segments on Murashige & Skoog (MS) medium supplied with 0.1 mg L^-1 2,4-dichlorophenoxyacetic acid. The regeneration of plants via indirect morphogenesis was carried out on half-strength MS medium without plant growth regulators. Fifteen randomly chosen plants were subjected to flow cytometry and “inter-simple sequence repeat” (ISSR) analysis. Ploidy level remained stable in all tested regenerants (2n=4x=44), with no changes in the genome. Eighteen ISSR primers produced a total of 1,584 fragments in all samples. Two ISSR primers produced four polymorphic fragments in 26.7% of the tested samples. Somaclonal variation in arracacha is a result of plant regeneration via indirect morphogenesis and can be detected by ISSR markers.

Index terms:
arracacha; ISSR; plant breeding; ploidy level; somatic embryogenesis

Authorship

Jan Vitamvas

Czech University of Life Sciences Prague, Faculty of Forestry and Wood Sciences, Department of Forest Ecology, Kamýcká 129, Prague 165 21, Czech Republic. E-mail: vitamvas@fld.czu.czCzech University of Life Sciences PragueCzech RepublicCzech Republic Czech University of Life Sciences Prague, Faculty of Forestry and Wood Sciences, Department of Forest Ecology, Kamýcká 129, Prague 165 21, Czech Republic. E-mail: vitamvas@fld.czu.cz

http://orcid.org/0000-0001-8181-8151

Iva Viehmannova ^**Corresponding author: viehmann@ftz.czu.cz

Czech University of Life Sciences Prague, Faculty of Tropical AgriSciences, Department of Crop Sciences and Agroforestry, Kamýcká 129, Prague 165 21, Czech Republic. E-mail: viehmann@ftz.czu.cz, hnkflknk@gmail.comCzech University of Life Sciences PragueCzech RepublicCzech Republic Czech University of Life Sciences Prague, Faculty of Tropical AgriSciences, Department of Crop Sciences and Agroforestry, Kamýcká 129, Prague 165 21, Czech Republic. E-mail: viehmann@ftz.czu.cz, hnkflknk@gmail.com

http://orcid.org/0000-0002-2540-7443

Petra Hlasna Cepkova

Crop Research Institute, Gene Bank, Division of Crop Genetics and Breeding, Drnovská 507/73, Prague 161 06, Czech Republic. E-mail: hlasna@vurv.czCrop Research InstituteCzech RepublicCzech Republic Crop Research Institute, Gene Bank, Division of Crop Genetics and Breeding, Drnovská 507/73, Prague 161 06, Czech Republic. E-mail: hlasna@vurv.cz

http://orcid.org/0000-0001-9663-681X

Hana Mrhalova

http://orcid.org/0000-0002-4883-0818

Katerina Eliasova

The Czech Academy of Sciences, Institute of Experimental Botany, Rozvojová 263, Prague 165 02, Czech Republic. E-mail: eliasova@ueb.cas.czThe Czech Academy of SciencesCzech RepublicCzech Republic The Czech Academy of Sciences, Institute of Experimental Botany, Rozvojová 263, Prague 165 02, Czech Republic. E-mail: eliasova@ueb.cas.cz

http://orcid.org/0000-0002-6606-6577

*Corresponding author: viehmann@ftz.czu.cz

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (2)
Tables (1)

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Figure 1.
Callus induction and plant regeneration from explants of arracacha (Arracacia xanthorrhiza). A, 50-day-old light yellow-green friable callus with meristematic centers on its surface, cultivated on a medium containing 0.1 mg L^-1 2,4-dichlorophenoxyacetic acid; B, histological analysis of the morphogenic callus with meristematic center (MC); C, shoot regeneration from a callus on a medium without plant growth regulators (PGRs); D, histological analysis of shoot regeneration from a callus cultivated on a medium without PGRs, where ST is the shoot tip and VS is the vascular system; E, matured somatic embryo obtained from regeneration MS medium without PGRs; F, histological analysis of early cotyledonary somatic embryo, where Ct is the cotyledon, Hy is the hypocotyl, Ra is the radicle, and SA is the shoot apex; and G, plant recovered from a callus on half-strength MS medium.

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Figure 2.
Molecular, flow cytometry, and karyological analyses of in vitro regenerants of arracacha (Arracacia xanthorrhiza). A, inter-simple sequence repeat profile of in vitro regenerants and a control plant using the UBC836 primer, where R1‒R15are 15 randomly chosen regenerants, C is the control plant, L is the ladder, and arrows indicate the position of the polymorphic bands; B, representative histograms showing relative fluorescence intensity of the control plant (on the left) and of a randomly chosen in vitro regenerant (on the right), with the peak of the internal standard, Bellis perennis, indicated with an asterisk; and C, chromosome counts in root tips of arracacha (2n = 4x = 44).

Thumbnail

Table 1.
Details on the inter-simple sequence repeat (ISSR) analysis of 15 in vitro regenerants and a control plant of arracacha (Arracacia xanthorrhiza).

Figure 1. Callus induction and plant regeneration from explants of arracacha (Arracacia xanthorrhiza). A, 50-day-old light yellow-green friable callus with meristematic centers on its surface, cultivated on a medium containing 0.1 mg L^-1 2,4-dichlorophenoxyacetic acid; B, histological analysis of the morphogenic callus with meristematic center (MC); C, shoot regeneration from a callus on a medium without plant growth regulators (PGRs); D, histological analysis of shoot regeneration from a callus cultivated on a medium without PGRs, where ST is the shoot tip and VS is the vascular system; E, matured somatic embryo obtained from regeneration MS medium without PGRs; F, histological analysis of early cotyledonary somatic embryo, where Ct is the cotyledon, Hy is the hypocotyl, Ra is the radicle, and SA is the shoot apex; and G, plant recovered from a callus on half-strength MS medium.

Figure 2. Molecular, flow cytometry, and karyological analyses of in vitro regenerants of arracacha (Arracacia xanthorrhiza). A, inter-simple sequence repeat profile of in vitro regenerants and a control plant using the UBC836 primer, where R1‒R15are 15 randomly chosen regenerants, C is the control plant, L is the ladder, and arrows indicate the position of the polymorphic bands; B, representative histograms showing relative fluorescence intensity of the control plant (on the left) and of a randomly chosen in vitro regenerant (on the right), with the peak of the internal standard, Bellis perennis, indicated with an asterisk; and C, chromosome counts in root tips of arracacha (2n = 4x = 44).

Table 1. Details on the inter-simple sequence repeat (ISSR) analysis of 15 in vitro regenerants and a control plant of arracacha (Arracacia xanthorrhiza).

Primer code⁽¹⁾	Sequence 5’-3’⁽²⁾	T_an⁽³⁾ (°C)	No. of bands per primer	Total no. of bands	No. of polymorphic bands per primer	PPB⁽⁴⁾ (%)	No. of polmorphic regenerants	Polymorphic regenerants	Range of amplification (bp)
UBC807	(AG)₈T	48	4	64	2	50	2	R14, R15	250-650
UBC809	(AG)₈G	50	4	64	‒	0	0	‒	290‒700
UBC810	(GA)₈T	49	5	80	‒	0	0	‒	600‒1,300
UBC812	(GA)₈A	50	5	80	‒	0	0	‒	380‒1,000
UBC823	(TC)₈C	54	3	48	‒	0	0	‒	600‒1,700
UBC824	(AG)₈YT	55	10	160	‒	0	0	‒	200‒1,900
UBC828	(TG)₈A	48	5	80	‒	0	0	‒	600‒1,300
UBC829	(TG)₈C	50	4	64	‒	0	0	‒	520‒1,900
UBC834	(AG)₈YT	50	5	80	‒	0	0	‒	210‒1,900
UBC835	(AG)₈YC	51	5	80	‒	0	0	‒	450‒1,200
UBC836	(AG)₈YA	48	9	144	2	22	2	R8, R13	200‒1,300
UBC840	(GA)₈YT	52	6	96	‒	0	0	‒	400‒1,000
UBC841	(GA)₈YC	48	6	96	‒	0	0	‒	200‒700
UBC844	(CT)₈RC	52	8	128	‒	0	0	‒	480‒1,300
UBC847	(CA)₈RC	54	3	48	‒	0	0	‒	590‒1,400
UBC851	(GT)₈G	52	7	112	‒	0	0	‒	700‒1,700
UBC855	(AC)₈YT	54	7	112	‒	0	0	‒	450‒1,800
UBC856	(AC)₈YA	49	3	48	‒	0	0	‒	800‒1,600
Total			99	1584	4	4.04	4

⁽¹⁾
Primers obtained from University of British Columbia (UBC). ⁽²⁾Single letter abbreviations for mixed-base positions: Y = (C, T) and R = (A, G). ⁽³⁾T_an, annealing temperature. ⁽⁴⁾PPB, percentage of polymorphic bands.

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Este documento possui uma errata:

10.1590/S1678-3921.pab2019.v54.00301e

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Assessment of somaclonal variation in indirect morphogenesis-derived plants of <i>Arracacia xanthorrhiza</i>

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[1] *Corresponding author: viehmann@ftz.czu.cz