Conserving yacon (Smallanthus sonchifolius) through cryopreservation using the PVS2 droplet vitrification method

Proceedings of Cryo2022 Joint Meeting of SLTB and Society for Cryobiology, 19-22 July 2022, Dublin, Irelan

Avaliação de variação somaclonal em plantas derivadas de Arracacia xanthorrhiza por morfogênese indireta

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.O objetivo deste trabalho foi induzir e detectar a variação somaclonal em plantas de batata-baroa (Arracacia xanthorrhiza) regeneradas por morfogênese indireta, para avaliar o potencial dessa técnica em produzir novos genótipos para o melhoramento dessa cultura. Calos foram induzidos em segmentos de pecíolos no meio de Murashige & Skoog (MS) suplementado com 0,1 mg L-1 de ácido 2,4-diclorofenoxiacético. A regeneração de plantas pela morfogênese indireta foi realizada em meio de cultura MS meia-força, sem reguladores de crescimento de plantas. Quinze plantas escolhidas aleatoriamente foram submetidas à citometria de fluxo e à análise entre sequências simples repetidas (ISSR). O nível de ploidia manteve-se estável em todas as plantas regeneradas (2n=4x=44), sem mudanças no genoma. Dezoito iniciadores ISSR produziram um total de 1.584 fragmentos em todas as amostras. Dois iniciadores ISSR produziram quatro fragmentos polimórficos em 26,7% das amostras testadas. A variação somaclonal entre plantas de batata-baroa é resultante da regeneração de plantas pela morfogênese indireta e pode ser detectada por meio de marcadores ISSR

IN VITRO PROPAGATION OF DROSERA INTERMEDIA AS INFLUENCED BY CYTOKININS, PH, SUCROSE, AND NUTRIENT CONCENTRATION

This study was aimed to optimize in vitro propagation of Drosera intermedia for commercial and conservation purposes. The effect of concentration of MS nutrients (⅛ MS, ¼ MS, ½ MS and, MS), various pH (3.7-7.7), sucrose concentration (10-40 g l -1 ) and cytokinins (0.1-3 mg l -1 ), namely BA (N6-benzyladenine), kinetin and zeatin were evaluated. After 60 days of shoot cultivation, growth and developmental characteristics (plant height, number of shoots per explant, diameter of rosette, number of roots per explant, length of roots) were recorded. No significant differences were found for various levels of pH and sucrose. On the contrary, plant height was negatively influenced by an increase of nutrients in the medium. The plants on 1/8 MS medium were significantly taller, and displayed higher proliferation capacity compared with those cultivated on full-strength MS medium. Shoot multiplication and growth was suppressed by supplementation of BA and kinetin, regardless of concentration used. Zeatin at the lowest concentration (0.1 mg l -1 ) provided the best results for shoot proliferation of all 26 treatments and can be recommended for micropropagation of D. intermedia

Assessment of somaclonal variation in somatic embryo-derived plants of yacon [ Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry

Background: Yacon ( Smallanthus sonchifolius ) is a root crop native to the Andean region. Low sexual reproductive capacity is amajor constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and flow cytometry. Results: Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Conclusions: Our findings show that indirect somatic embryogenesis could be used in yacon improvement to widen genetic variability, especially when low sexual reproductive capacity hinders classical ways of breedin

Simplified in vitro propagation protocol for Tacca leontopetaloides (L.) Kuntze and assessment of genetic uniformity of regenerated plantlets

Tacca leontopetaloides is a tuberous species with nutritional, medicinal, and industrial potential. The aim of the present study was to develop a simplified micropropagation protocol for this species, combining phases of in vitro propagation and rooting, and to assess the genetic stability of in vitro regenerants using ISSR and flow cytometric analysis. In the first step, the effect of four cytokinins (BA, zeatin, kinetin, thidiazuron) at concentrations of 0.1-0.7 mg l-1 on shoot organogenesis was tested. The highest number of shoots was achieved on MS medium supplemented with 0.1 mg l-1 zeatin (4.11 ± 0.35 shoots/explant). Thereafter, this treatment was combined with auxins (NAA, IAA) at concentrations of 0.01-0.07 mg l-1 to study shoot organogenesis and in vitro rooting. A combination of plant growth regulators provided unchanged (high) shoot organogenesis, while the root number per explant increased. The highest number of roots of all tested treatments was produced on MS medium supplemented with zeatin (0.1 mg l-1) in combination with NAA (0.05 mg l-1) (5.00 ± 0.38 roots/explant), suggesting that this medium may be used for both propagation and rooting. Well-rooted plants were transferred ex vitro, with a 97.78% survival rate. Ten randomly chosen in vitro regenerants were subjected to ISSR and flow cytometric analysis. Eleven ISSR primers produced 40 clear and reproducible bands per sample. All amplified products were monomorphic; no polymorphism was detected. Similarly, the results of flow cytometric analysis showed no variation in ploidy level. Thus, the micropropagation method optimized here can be used for highly effective production of true-to-type plants of T. leontopetaloides

Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry

Background: Yacon (Smallanthus sonchifolius) is a root crop native to the Andean region. Low sexual reproductive capacity is a major constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and flow cytometry. Results: Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Conclusions: Our findings show that indirect somatic embryogenesis could be used in yacon improvement to widen genetic variability, especially when low sexual reproductive capacity hinders classical ways of breeding

Efficient slow-growth conservation and assessment of clonal fidelity of Ullucus tuberosus Caldas microshoots

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