Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa

Abstract

Background: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier.

Results: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T₁ seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot.

Conclusion: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.

Keywords: Agrobacterium-mediated transformation; Floral dip; Rapid-cycling Brassica rapa; Vacuum infiltration; Wisconsin fast plants.

Fig. 1

Kanamycin selection on wild-type rapid-cycling Brassica rapa. The diameters of presented plates were 90 mm. The image was taken 12 days after plating

Fig. 2

Bud dissection of rapid-cycling Brassica rapa. a Intact inflorescence of RcBC 1-33, the standard line of RCBr, prior to dissection. b Floral buds from one inflorescence placed in order of occurrence on the stem. Scale bars 5 mm. c, d and e, Dissection of indicated floral buds from B showing: ① and ②, sealed carpels, diameters of buds were around 2 mm. ③, unsealed carpel (white arrow), the diameter of bud was less than 1 mm

Fig. 3

Stages of rapid-cycling Brassica rapa chosen to transform. a 22-day-old seedlings. b 14-day-old seedlings. c 8-day-old seedlings. Scale bars, 3 cm

Fig. 4

Transformation of rapid-cycling Brassica rapa. a An example of GFP-negative seedlings. Scale bar, 1 mm. b Two examples of GFP-positive T₁ seedlings. c Transformation efficiencies for the different treatments. Mean +/− SE for the data in Table 1. The means are not significantly different (ANOVA, p < 0.05). VI, vacuum infiltration; FD, floral dip

Fig. 5

Verification of rapid-cycling Brassica rapa transformation. a Schematic of T-DNA and flanking region of the circular binary vector p201G, with corresponding aph, GFP and backbone PCR products depicted at bottom. b representative results of PCR assays; +, GFP-positive; −, GFP-negative; Agro, Agrobacterium strain GV3101(pMP90)(p201G); WT, wild-type RCBr plant; VI, T₂ from vacuum infiltration; FD, T₂ from floral dip; M, marker. C58 glyA is an Agrobacterium chromosomal gene. c Southern blot analysis. Left, electrophoresis gel of BamHI-digested plant genomic DNA; right, Southern blot probed with GFP probe indicated in (a)