Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Abstract

As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H.B.K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3,5,6-trichloropicolinic acid), N 6 -benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l 21 (4.52 mM) 2,4-D plus 0.5 mg l 21 (2.22 mM) BA, and 2 mg l 21 (8.88 mM) BA plus 1 mg l 21 (4.14 mM) Picloram with or without 40 mg l 21 (296.08 mM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l 21 (9.05 mM) 2,4-D combined with 0.25 (1.11 mM) or 0.50 mg l 21 (2.22 mM) BA, or 1 mg l 21 (4.52 mM) 2,4-D with 0.50 mg l 21 (2.22 mM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l 21 (1.44 mM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l 21 (9.05 mM) 2,4-D to 62.2 for induction medium containing 2 mg l 21 (8.28 mM) Picloram, 1 mg l 21 (4.44 mM) BA and 40 mg l 21 (296.08 mM) adenine. Regenerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells

“…This concentration of kanamycin is comparable to that utilized previously in some dicots such as papaya (Cai et al 1999) or tobacco (Holmström et al 2000). Studies with other morphogenic callus lines of B. gracilis (Aguado-Santacruz et al 2001a) have also shown the efficiency of the aminoglycoside antibiotic, paramomycin, as a selective agent, at similar concentrations to those reported in this work for kanamycin (150 mg/l).…”

“…Compared to the number of tissue, protoplast or cell-culture methods reported for forage and turf grasses (revised by Green 1978;Ahloowalia 1984;Lee 1996;Chai and Sticklen 1998;Aguado-Santacruz et al 2001a) the species for which transformation systems have been developed is still very low. Among the explanations accounting for this discrepancy are: (1) a slow growth of the target tissue incompatible with genetic transformation systems, (2) loss of the regenerative potential of the totipotent material with time, and (3) technical constraints related to the transformation and/or regeneration systems employed (Green 1978;Chai and Sticklen 1998).…”

Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae)

“…However, in monocots, especially in graminaceous species, the plantlet regeneration was considered mainly performed via somatic embryogenesis (Vasil 1982). Evidences of morphogenic pathways in the same kind of explants under the control of auxins and/or cytokinins were only observed in few species, such as sorghum (Bhaskaran and Smith 1989), Bouteloua gracilis (Aguado-Santacruz et al 2001), minor millet (Paspalum scrobiculatum) (Vikrant 2002(Vikrant , 2003, sugarcane (Lakshmanan et al 2006), Neyraudia arundinacea (Ma et al 2007). Although combinations of plant hormones required for different morphogenic pathway were similar to dicots in those studies, the regulation processes were more complicated and the studies on regulation mechanisms were much fewer.…”

EMBRIOGÉNESIS SOMÁTICA INDUCIDA CON ESTRÉS OSMÓTICO EN Bouteloua curtipendula (Michx.) Torr., ESPECIE RECALCITRANTE Y APOMÍCTICA

“…Las técnicas de cultivo de tejidos in vitro han sido una herramienta valiosa para el desarrollo de protocolos de regeneración en especies forrajeras apomícticas; se han usado en programas de mejoramiento o transformación genética en especies como B. gracilis (Aguado-Santacruz et al, 2001), B. eriopoda (Osuna y Barrow, 2004), Festuca alta (Arundinacea gigantea; Chen et al, 2004), Brachiaria brizantha (Cabral et al, 2011), Paspalum notatum (Mancini et al, 2014), Eragrostis curvula (Zappacosta et al, 2014) y pasto buffel (Cenchrus ciliaris; Carloni et al, 2014).…”