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Using GFP to Select for Transgenic Sweet Orange
 Derived from Electroporated Protoplasts

UV illuminated mcrografted 'Hamlin' citrus plant regenerated from GPF positive embryoid/p-calli.


Randall P. Niedz, William L. McKendree, and Robert G. Shatters, Jr.
USDA-ARS, U.S. Horticultural Research Laboratory
2001 South Rock Road, Ft. Pierce, FL, USA 34945
RNiedz@ushrl.ars.usda.gov

Background

  • Plants are easily regenerated from embryogenic cell lines of citrus.
  • Though citrus protoplast regeneration systems are well developed, it is not clear why citrus protoplasts have not been more amenable to transformation. One possibility is that the standard selection agents (e.g., kanamycin) provide inefficient selection.
  • Can green fluorescent protein (GFP) be used as a screenable marker to identify transgenic p-calli? Previous efforts in our laboratory resulted only in transient expression of GFP in citrus protoplasts.
  • We report here the routine recovery of transgenic citrus plants obtained via protoplast-mediated transformation and GFP selection.

Objectives

  • To develop a GFP construct that could be used to visually select for transformed calli or embryos derived from protoplasts.
  • To regenerate transgenic plants from selected calli or embryos using GFP as a screenable marker.

Materials and Methods

  • Protoplast Isolation and Culture - Protoplasts were isolated from an embryogenic callus line (H97) derived from Citrus sinensis (L.) Osbeck cultivar 'Hamlin', electroporated with plasmid DNA, and cultured.
Embryogenic cell line used as a protoplast source.
Freshly isolated protoplasts used in electroporation experiments.
4-week old protoplast-derived embryoids/p-calli screened for GFP expression.
Freshly isolated H97 protoplasts used in electroporation experiments. 4-week old protoplast-derived embryoids/p-calli screened for GFP expression.
H97 embryogenic cell line used as a protoplast source.
Selection and Regeneration of Transformed Plants - Forty to sixty days after protoplast isolation and electroporation green fluorescent colonies were selected and plants regenerated.

Analysis of Transformed Plants - Isolated cell lines were analyzed by fluorescence spectrofluorimetry of GFP and protein levels. Plants were analyzed by Southern analysis using a P32-labeled EGFP coding sequence.

Vector Construction

Three variables were considered in the design of two vectors to achieve stable GFP transformation in citrus:

  1. EGFP as the GFP molecule. The highly fluorescent EGFP molecule selected is 35 times brighter than the wtGFP we had used in previous studies.
  2. Double 35S AMV promoter. To ensure adequate expression, we placed EGFP under the control of the double 35S AMV promoter that we had identified earlier in electroporation optimization experiments as a very strong constitutive promoter in citrus.
  3. Targeted and nontargeted expression. To determine if cytoplasmic expression was toxic or inhibitory to cell division, a targeting vector was constructed to direct expression of EGFP into the lumen of the endoplasmic reticulum (ER).
pARS101 - Placed the EGFP coding sequence under the control of the 35S-35S promoter containing 33 bp of the untranslated leasder sequence from AMV.
pARS108 - Same as pARS101 but EGFP was modified adding the 5' terminal signal sequence from an Arabidopsis vacuolar basic chitinase and a C-terminal HDEL sequence for retention in the lumen of the ER.
pARS108 - Same as pARS101 but EGFP was modified adding the 5' terminal signal sequence from an Arabidopsis vacuolar basic chitinase and a C-terminal HDEL sequence for retention in the lumen of the ER.
pARS101 - Placed the EGFP coding sequence under the control of the 35S-35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus.

Results

Regeneration of Transgenic Plants
Fluorescent protoplast 24 housrs after transformation.
Selected fluorescent embryoid/p-calli.
Fluorescent embryogenic callus derived from selected GFP positive embryoid/p-calli.
40 days - selected fluorescent (pARS108) embryoid/p-calli.
6 months - fluorescent (pARS108) embryogenic callus derived from selected GFP positive embryoid/p-calli.
24 hours - fluorescent (pARS108) protoplast.
Brightfield illuminated micrografted plant regenerated from selected GFP positive ebryoid/p-calli.
UV illuminated micrografted plant from selected GFP positive embryoid/p-calli.
4 months - micrografted plant regenerated from selected GFP positive (pARS108) embryoid/p-calli - brightfield illumination. 4 months - micrografted plant regenerated from selected GFP positive (pARS108) embryoid/p-calli - UV illumination. Thirty two and twenty one independent transformants were selected from electroporation experiments with pARS101 and pARS108, respectively.
Southern Analysis




  12,000-



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     bp      1      2    3    4    5    6    7    8    9



 12,000-

3,000-
   2,000-

     bp      1     2   3    4    5    6    7    8    9
Southern analysis of 9 of 32 GFP positive plants. Hybridization using a 1.38 kb EGFP coding sequence. Southern analysis of 9 of 21 GFP positive plants. Hybridization using a 0.97 kb EGFP coding sequence.
Southern analysis of 9 of 32 GFP positive plants. Hybridization using a 1.38 kb EGFP coding sequence.
 Southern analysis of 9 of 21 GFP positive plants. Hybridization using a 0.97 kb EGFP coding sequence.

Conclusions

  • Cell division, colony formation, plant regeneration, and growth were not affected by the presence of GFP, targeted or nontargeted.
  • Targeted and nontargeted GFP are suitable as screenable markers in citrus transformation.

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Last modified: 2/19/2001.   Send comments or questions to Randall Niedz at RNiedz@ushrl.ars.usda.gov