Cry3Aa inheritance in filial generations of Solanum tuberosum

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Cry3Aa inheritance in filial generations of
Solanum tuberosum

• Jeff Skoneczka and Richard Veilleux
• Department of Horticulture
• Virginia Polytechnic Institute State University
• Blacksburg, Virginia

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Purpose

• Incorporation of sexual hybrids into production
  systems
• Easy start-up
• Economical
• Difficulties
• Heterozygosity and polyploidy
• Transgenics

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Objectives

• In a prior study (Johnson 2001), Cry3Aa
  expression in sexual hybrids of potato differed
  depending upon whether the transgene was
  maternally or paternally inherited (using
  independently transformed parents)
• We wanted to verify a difference in transmission
  through the male and female gametophyte based on
  reciprocal hybrids using the same transgenic
  parent

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Construct
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Establishment

• After confirmation of transformation in vitro,
  transfer plants to greenhouse for breeding.
• Established plants were used to create reciprocal
  hybrids

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• Parental Lines
• Atlantic - tetraploid commercial cultivar
• APM-2 heterozygous diploid line with high
  frequency of 2n pollen
• Single insert transgenic lines of the above,
  denoted by TC1

Hybrid Families
Atlantic TC1 X Katahdin
Katahdin X Atlantic TC1
Atlantic TC1 X Atlantic
Atlantic X Atlantic TC1
Atlantic TC1 self
Atlantic TC1 X wt APM-2
Atlantic TC1 X APM-2 TC1

• Katahdin tetraploid commercial cultivar

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Material and methods

• The seedlings were planted in sets of 16 plants
  per family in three different plantings, two in
  the greenhouse and one in the field
• Each hybrid plant was screened for transgene
  expression via DAS-ELISA

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Methods DAS-ELISA

• Bt endotoxin ELISA kit from Agdia, Inc.
• Leaf samples ( 200 mg) taken at seedling stage
• Presence of protein in sample expressed
  colorimetrically
• OD is read with plate reader at 405 nm and
  converted to Absorption Ratio (AR)
• AR (OD of test plant)/(OD of negative control)
• Examine segregation ratios to verify gene copy
  number as well as transgene expression levels

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96-well ELISA plate
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Mean absorption ratios (AR) of Cry3Aa expressing
plants in four hybrid families of tetraploid
potato first greenhouse trials of 16 plants per
family
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Segregation ratios of Cry3Aa expressing and
non-expressing plants in four hybrid families of
tetraploid potato first greenhouse trials of 16
plants per family

• A segregation ratio significantly different from
  11 was observed in one family

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Mean absorption ratios (AR) of Cry3Aa expressing
plants in five hybrid families of tetraploid
potato second greenhouse trials of 16 plants
per family
Again, expression levels not significant, however
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Segregation ratios of Cry3Aa expressing and
non-expressing plants in five hybrid families of
tetraploid potato second greenhouse trials of
16 plants per family
High expected ratio due to APM-2 having high
frequency of 2n pollen
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Segregation ratios of Cry3Aa expressing and
non-expressing plants in two hybrid families of
tetraploid potato field trial
Results slightly significant at 95 confidence
level
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Pooled Data
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Conclusions

• Levels of Cry3Aa expression do not differ between
  reciprocal hybrids
• The transmission rate of the Cry3Aa gene appears
  to differ in reciprocal hybrids between Atlantic
  and Katahdin
• An unexpectedly greater number of expressers to
  non expressers was observed in three separate
  sets of hybrids

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Possible causes of different frequencies of
Cry3Aa expressing hybrids in reciprocal hybrids

• More than a single copy of the transgene exists
  in Atlantic TC1 accompanied by transgene
  silencing in Katahdin X Atlantic TC1
• Preferential gametic selection

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With more than one copy of the transgene in
Atlantic TC1

• Expect higher expressers to non-expressers in
  both Atlantic TC1 X Katahdin and Katahdin X
  Atlantic TC1
• Expect gene silencing in Katahdin X Atlantic TC1
  non-expressers
• PCR of non-expressers

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Methods

• DNA extraction (Sigma Extract-N-Amp)
• Primers to amplify Cry3Aa
• Forward 5GAGCTGCAAGGCCTTCAAAACAAT
• Reverse 5TCTAGCACGCTAAGGGTCATCTCT
• 1 cycle 4 min 94ºC 40 cycles 1 min 94ºC, 1
  min 58ºC, 1.5 min 72ºC 1 cycle 5 min 72ºC
• 1.0 agarose gel
• Stain with ethidium bromide

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PCR results of amplification of genomic DNA of
expressing and non-expressing progeny of Katahdin
x Atlantic TC1
500bp
Results confirm observed segregation ratio from
ELISA
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Conclusion

• PCR results agree with ELISA results
• If not transgene silencingthen gametic
  selection?
• How does segregation compare in a different
  hybrid using Atlantic TC1 as female parent?

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In order to determine if the phenomenon of
unexpectedly high numbers of transgenic progeny
were obtained from Atlantic TC1 as a female
parent and to verify the single transgene copy,
we examined a cross between Atlantic and the 2n
pollen-producing diploid hybrid, APM-2
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Conclusions

• Single transgene copy in Atlantic TC1 was
  supported by segregation data in four of five
  families
• Absence of the transgene in non-expressers was
  verified by PCR in two families so transgene
  silencing does not appear to be the cause
• The unexpectedly high frequency of Cry3Aa
  expressers in Atlantic TC1 X Katahdin is likely
  due to preferential selection of transgenic
  female gametes in this cross

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Acknowledgements

• Dave Douches
• Richard Veilleux
• Sue Tolin
• Vladamir Shulaev
• Suzanne Piovano
• My fellow graduate students