Micropropagation, plantlets production estimation and ISSR marker-based genetic fidelity analysis of Guadua magna and G. angustifólia

Abstract

The bamboo species Guadua magna and G. angustifolia have been propagated nearly exclusively by conventional techniques of vegetative propagation. Micropropagation is a promising technique and an alternative to conventional ones. This study aimed to micropropagate plants, estimate the plantlets production and analyze the genetic fidelity of G. magna and G. angustifolia by ISSR molecular markers. Mother plants of both species were cultivated in a greenhouse, and either sprayed or not with fungicide. In the laboratory, microcuttings were disinfested and established in MS culture medium with 3.0 mL L-1 of Plant Preservative Mixture (PPM®) and 1 mL L-1 of Carbendazin®. The contamination-free shoots were multiplied in liquid or semi-solid MS medium with 3.0 mg L-1 of BAP for five subcultures. Rooting was performed in liquid or semi-solid MS1/2 medium, plus 3.0 mg L-1 of IBA. Acclimatization was performed on a commercial substrate, in a growth chamber, and the genetic fidelity of the clones produced was analyzed via ISSR markers. The addition of fungicide and PPM® to the medium reduced the contamination in G. magna, but not in G. angustifolia. The liquid medium was more efficient than the semi-solid one for the multiplication of both species, which showed production potentials between 760 and 920 plants per initial microcutting, after five subcultures. Rooted plants exhibited a survival rate of up to 100 % in acclimatization. No polymorphic regions were found in the clones analyzed by ISSR at the end of the fifth subculture, suggesting that micropropagation is a safe technique for the large-scale multiplication of bamboos.

A Concise Review of Dendrocalamus asper and Related Bamboos: Germplasm Conservation, Propagation and Molecular Biology

“…The incorporation of BAP into the medium was found to be the most effective for the bud breaking and shoot proliferation of several bamboo species. These findings are consistent with the previous research on the in vitro propagation of various bamboo species, such as Guadua angustifolia [147], Dendrocalamus giganteus [159], Guadua magna [160], Bambusa glaucescens [161], Bambusa balcooa [162],Gigantochloa atroviolaceae [150] and Dendrocalamus hamiltonii [91], in which BAP are widely used and proven to be effective. In intact plants, high levels of cytokinins have been shown to adversely affect growth by promoting programmed cell death in cell cultures, the necrosis of leaves, reduced root growth and promote ethylene biosynthesis.…”

Somatic embryogenesis and plant regeneration in Piper aduncum L

“…The extraction of genomic DNA followed the modified methodology of Doyle and Doyle (1990). For the analysis, the methodology used was the one developed by Nogueira et al (2019). In all, 57 primers (Sigma-Aldrich®) were tested, and 20 were selected to be used in the analyses.…”

Efecto de biocidas y consistencia del medio de cultivo para el establecimiento in vitro de Guadua angustifolia

“…Durante la esterilización superficial se eliminó los extremos de los explantes y luego se sembraron en medio de cultivo MS (Murashige y Skook 1962) suplementado con 20 g L -1 de sacarosa, 50 mg L -1 de ácido ascórbico, 0,5 mg L -1 ácido nicotínico, 100 mg L -1 myo-inositol, 50 mg L -1 sulfato de gentamicina (Pasqualini et al 2019a, Nogueira et al 2019; adicionalmente contenían distintas concentraciones del biocida Plant Preservative Mixture (1, 2 y 4 mL L -1 PPM TM ). El pH fue ajustado (NaOH y / o HCl) a 5,7 y, para el medio de cultivo semisólido se agregó 6 g L -1 de agar, luego se esterilizó en autoclave a 120 °C (~1 kgf cm -2 ) por 20 min.…”

“…Esta problemática ha derivado en el desarrollo de diversos protocolos de esterilización superficial con desinfectantes, antibióticos y fungicidas comunes que no son totalmente eficientes (Ray y Ali 2016). Al mismo tiempo, diversos investigadores (Bakshi et al 2015, Saini et al 2016, Nogueira et al 2019 informan que las soluciones a base de cloruro de mercurio (HgCl 2 ) son eficientes en los protocolos de esterilización. Sin embargo, el uso descontrolado e inadecuada manipulación de este producto pueden traer graves consecuencias debido a su alta toxicidad (Branco et al 2017), además de elevar los riesgos de contaminación ambiental producto de los residuos químicos generados en el cultivo in vitro (Ornellas et al 2019).…”