Genetic transformation of blue grama grass with the rolA gene from Agrobacterium rhizogenes: regeneration of transgenic plants involves a “hairy embryo” stage

Abstract

Until recently, information about the effects of transforming plants with the rolA gene of Agrobacterium rhizogenes has been restricted mainly to dicots in which a severely wrinkled phenotype, reduced internode distances, and abnormal reproductive development were commonly observed. In this work, we analyzed the effects associated with the expression of this gene in a new genetic context: the forage grass genome. Transgenic P 35S •rolA plants of blue grama grass (Bouteloua gracilis) were obtained by a biolistic approach employing embryogenic chlorophyllic cells as the target material. Four independent transgenic lines with regeneration capacity were recovered, which showed stable integration of this transgene as demonstrated by polymerase chain reaction and Southern blot hybridization. Growth of the rolA-transformed lines under greenhouse conditions provided evidence for a new biotechnological application for the rolA gene in plants, namely, the improvement of biomass production in forage grasses. Additionally, we described here a new phenotypic marker (referred to here as the "hairy embryo" syndrome) that can be instrumental for the early identification of transformation events when transforming grasses with this gene.

Induction of Hairy Roots on Somatic Embryos of Rhizoclones from Typha domingensis Seedlings

“…The SEs of T. domingensis developed typical hairy roots through the transfection of rhizoclones with the A. rhizogenes K599, LBA9402, and A4 strains. The phenotype of these hairy roots was similar to the phenotype of hairy roots reported in the transgenic embryos of Bouteloua gracilis [35]. The virulence was different for each of the three A. rhizogenes strains; however, the K599 strain stood out in terms of its hypervirulence, and the A4 strain stood out in terms of its virulence.…”

“…The biological effect and confirmation for the genetic transformation by the presence of the transferred DNA (T-DNA) of A. rhizogenes to the target plant tissue is still under study, as there are certain limitations in the case of recalcitrant species [39,44]. The infectivity of the strains has been confirmed via morphologic markers of the hairy phenotype in the Agrobacterium-treated SEs [35] as well as by the coculture technique performed in this study. Similar to the study that induced transgenic embryos of Bouteloua gracilis, polymerase chain reaction (PCR), Southern blot, and Northern blot analyses are commonly used to confirm the presence of the rol A genes in hairy SEs; however, these molecular techniques are not infallible for confirming genetic transformation.…”

CRECIMIENTO Y ESTABLECIMIENTO DE PLÁNTULAS DE Bouteloua gracilis (Kunth) Lag. ex Griffiths Y Eragrostis curvula var. conferta Stapf BAJO UN RÉGIMEN SIMULADO DE LLUVIA

“…También Majerus (1975) encontró una clara relación entre potencial hídrico del suelo y el crecimiento de hojas en el pasto B. gracilis, ya que una vez que el potencial hídrico del suelo a 25 cm de profundidad fue menor de -1 bar, el crecimiento de hojas se redujo en cerca de 50 %. La producción de hojas se ha considerado un proceso íntimamente ligado a la formación de vástagos (Aguado-Santacruz et al, 2009). E. curvula formó mayor número de hojas que el zacate navajita azul bajo el régimen simulado de lluvia, lo cual coincide con los resultados reportados por Carrillo et al (2009) quienes observaron una eficiencia superior a 90 % en la producción de hojas en la especie exótica Eragrostis lehmanniana en comparación con la especie nativa B. gracilis, mientras que Arredondo et al (1998) concluyeron que las especies invasoras pueden producir mayor área foliar que las especies nativas.…”

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“…ex Steud.) plants under greenhouse conditions, it was mainly related to shoot growth [32]. Several liquorice Glycyrrhiza glabra L. hairy root clones bearing rolB gene were more branched and showed about 8 fold higher root biomass on solid medium than untransformed ones [33].…”

“…The transformation of dicots with the rolA gene results in the shortened internode length, green darkened leaves, reduced apical dominance, leaf wrinkling, decreased length-to-width leaf ratio, shortened styles, larger flower size, reduced flower number, condensed inflorescences, male sterility, and retarded onset of flowering [68]. The rolA B. gracilis (the important forage grass) lines showed a 2-fold reduced root system, but they produced up to twice as much foliage as control plants due to enhanced height (up 1.3 fold) and increased tillering and leaf number (up to 1.7 and 1.5 fold, respectively) [32].…”