Raspberry tissue culture and transformation
JHI researchers have made progress in three areas:
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In vitro culture: We can grow, multiply, and root raspberry plants under laboratory conditions, providing material for our experiments (Figure 1).
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Transformation: We have obtained raspberry calli (clusters of plant cells) expressing the RUBY reporter, which produces a visible red colour (Figure 2). Regeneration of whole plants from these calli remains a challenge.
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Protoplasts: We have isolated raspberry protoplasts (cells with their walls removed) and introduced DNA carrying the GFP reporter. We observed green fluorescence, showing transient GFP expression (Figure 3). Regeneration of whole plants from these protoplasts remains a challenge.
In vitro culture
Figure 1: Multiplication of rooted raspberry plants grown in vitro: Representative Skye material after two months on SIM Multiplication medium. (A-C) Regenerated shoots with visible roots. (D) Separated rooted cuttings showing shoot multiplication and developed root systems. (E) Group of regenerated Skye shoots before the next subculture.
Transformation
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Figure 2: Different Glen Dee calli expressing the RUBY reporter. The arrow indicates the red/purple RUBY-positive tissue.
Regeneration
Figure 3: Protoplast isolation and transient GFP expression in raspberry cv. Glen Dee following antioxidant pre-treatment. (A) Leaf strips incubated in the antioxidant pretreatment solution; (B) protoplast pellet before resuspension; (C) protoplast suspension layered onto the 0.6 M sucrose cushion; (D) bright green protoplast band recovered following centrifugation with the sucrose cushion; (E) isolated protoplasts observed in a Neubauer chamber at 10× magnification; (F) isolated protoplasts observed at 40× magnification; (G) GFP fluorescence observed at 40× magnification three days after PEG-mediated transfection; (H) GFP fluorescence observed at 40× magnification five days after transfection.