Fig. 2

In-situ chloroplast lysis. a Illustration of in-situ chloroplast lysis. b In-gel chloroplast and its lysis. The Et-S buffer at 35 °C/2.5 h lysed labels 1 and 2 representing the gel blank control −ck (no chloroplast). Label 3 designates the un-lysed in-gel chloroplast control (ck). Labels 4 and 5 depict in-gel chloroplast lysis by Et-S buffer at 35 °C/2.5 h. c Lysate efficacy of the Et-S buffer in the In-gel chloroplast. Comparison of the Et-S buffer’s ability to process in-gel chloroplast by a temporal gradient at 35 °C before the pK buffer washed the agarose matrix and the NDS-1 buffer released the histone of cpDNA. The lysate efficacy of the Et-S buffer with the temporal gradient (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h) from lane 1 to lane 6 that shown in an autoradiogram from 1% PFGE-gel analysis (Reverse Voltage Gradient: 4.5 V/cm, Int. Sw. Tm: 60 s, Fin. Sw. Tm: 90 s, included angle: 120°, Run Time: 24 h, Ramping factor: a=: ENTER). The lanes (7, 8), as the +ck, were the Et-S buffer lysed the In-gel DH 5α (E. coli) for 2.5 h at 35 °C. Lane 9, as the −ck, was the chloroplast gel without lysis buffer for 2.5 h at 35 °C. The results demonstrate that the Et-S buffer has the best lysate effectiveness in In-gel chloroplast (Lane 5, red arrowhead). The HMW lambda ladder is loaded into the far-left corner. d Analysis of crop's in-situ chloroplast lysis by PFGE-gel (Reverse Voltage Gradient: 6 V/cm, Int. Sw. Tm: 1 s, Fin. Sw. Tm: 25 s, included angle: 120°, Run Time: 16 h, Ramping factor: a=: ENTER). The circular cpDNA cannot be separated as discrete bands under PGFE conditions, showing the target band (red arrowhead) in PFGE-gel. Lane 1, Wheat circular cpDNA. Lane 2, Maize circular cpDNA. Lane 3, Soybean circular cpDNA. Lane 4, Barley circular cpDNA. The HMW lambda ladder is loaded into the far-left corner. Attention Circular DNA size cannot be marked up with a linearized DNA ladder because of the circular topology properties, whereas it can serve as a relative reference standard