Onozuka r 10

Ten successive internode cross sections were used to test two different digestion procedures followed by FASGA staining in order to use the developed workflow. The first digestion procedure is the application of 3 mL of a cellulase solution 1 g/L (« Onozuka R-10 from Trichoderma viride»; Serva; 10 mg ref 16,419.03) at 40 °C on previously water rinsed cross sections (H₂0 during 3 h at room temperature). The second digestion procedure is the application of the cellulase solution mentioned above 1 g/L 3 mL at 40 °C on previously NaOH (0.1 M) treated cross sections during 1 h and rinsed (H₂0 during 3 h at room temperature). NaOH (0.1 M) treated and rinsed (H2O during 3 h) at room temperature cross sections were also FASGA stained and analyzed using the workflow.

Meristematic cells of root tips were used as a source of mitoses. Fixed seedlings were washed in 0.01 M citric acid/sodium citrate buffer (pH 4.6–4.8) for 20 min prior to enzymatic digestion in a mixture comprising 20% (v/v) pectinase (Sigma), 1% (w/v) cellulase (Calbiochem), and 1% (w/v) cellulase “Onozuka R-10” (Serva) for 1.5–2.5 h at 37 °C. The root tips were squashed in a drop of 45% acetic acid. Cover slips were removed by freezing, and the preparations were post-fixed in chilled 3:1 (v/v) ethanol:glacial acetic acid mixture, followed by dehydration in absolute ethanol and air dried (Hasterok et al. 2001 ). Chromosome preparations made from at least three individual plants from each accession were subjected to FISH analysis. Chromosome analysis was carried out on 5–10 well-spread metaphases. Each chromosomal preparation was derived from a different single root tip, so that each preparation corresponded to one individual.

Seeds of each species were germinated on filter paper in Petri dishes for 3–4 days in the dark. The root tips were immersed in ice-cooled water for 26 h, fixed in ethanol and acetic acid (3:1, v/v), and stored at −20 °C until required. Mitotic chromosome preparations were made from root tips digested in a mixture of enzymes, diluted with 0.01 M sodium citric buffer, containing 20 % (v/v) pectinase (Sigma), 1 % (w/v) cellulose (Calbiochem), and 1 % (w/v) cellulase ‘Onozuka R-10’ (Serva). Meristems were dissected from root tips, squashed in drops of 45 % acetic acid, and the good quality preparation was frozen (Hasterok et al. 2006 (link)).

We adapted the Aufrère protocol [10 (link)] to our lab conditions and to smaller dry matter sample quantity. This adapted protocol is called Protocol 1 (Table 2) for the rest of the article. Briefly, for this Protocol 1, 30 mg of maize dry matter powder were placed in 5 mL tubes before adding 2 mL of HCl 0.1 N solution with 2% of pepsin during 24 h at 40 °C followed. Then, gelatinization step was carried out by placing the tubes during 30 min at 80 °C in a water bath. The tubes were cooled in ice before being centrifuged at room temperature 10 min at 5000 rpm. The supernatant was discarded. Rinsing was then performed by centrifuging twice with 4 mL of water (5000 rmp—10 min at room temperature) the obtained pellet. After the first rinse centrifugation, the supernatant was discarded and the pellet was rinsed a second time with 4 mL of water. Similarly, the supernatant obtained after the second rinse centrifugation was discarded. 4 mL of Enzymatic solution of Cellulase («Onozuka R-10 from Trichoderma viride»; Serva; 10 mg ref 16,419.03) at 1 mg L⁻¹ were then added to the pellet, vortexed and agitated during 24 h at 40 °C. The pellet recovered after centrifugation (5000 rmp—10 min at room temperature) was then freezed at − 80 °C and lyophilized 48 h before final weighing.

Estimation of the IVDMD of the 6 selected samples was performed by the reference laboratory LANO (Laboratoire Agronomique de Normandie http://www.lano.asso.fr/web/index.php) following the initial reference protocol [6 (link), 10 (link)]. Briefly, 50 mL of HCl 0.1 N solution with 2% of pepsin was applied on 500 mg of maize dry matter during 24 h at 40 °C. After that, samples were placed 30 min at 80 °C (gelatinization step). Rinsing was performed before adding 50 mL of enzymatic solution of Cellulase Onozuka («Onozuka R-10 from Trichoderma viride»; Serva; 10 mg ref 16,419.03) at 1 mg mL⁻¹ during 24 h at 40 °C. A final rinsing was then performed before 12 h drying at 103 °C.

Cytogenetic preparations of root meristems were done as described by Jenkins and Hasterok (2007) (link). Excised root tips were digested for 1.5 h at 37 °C in a mixture of enzymes comprising 6 % (v/v) pectinase (Sigma-Aldrich), 1 % (w/v) cellulase (Sigma-Aldrich) and 1 % (w/v) cellulase ‘Onozuka R-10’ (Serva).

The chromosome preparations were made using the methodology described by Jenkins and Hasterok [67 (link)]. The root tips of 3-day-old seedlings were excised, incubated in ice-cold water for 24 h, and fixed in a mixture of methanol and glacial acetic acid at a 3:1 ratio. The material was stored in −20 °C. The root tips were washed in citrate buffer (pH 4.8) for 15 min, and then digested in an enzyme mixture containing 20% pectinase (Sigma), 1% cellulase (Calbiochem, San Diego, CA, USA), and 1% cellulase ‘Onozuka R-10′ (Serva, Heidelberg, Germany) in citrate buffer at 37 °C for 2 h. After digestion, the meristems were dissected from the root tips, placed in a drop of acetic acid on a microscopic slide, covered with a coverslip, and gently squashed. Good quality slides were frozen on dry ice. After removing the coverslips, the slides were air dried and stored at 4 °C until used.