Soybean Transformation and Regeneration Using “Half-Seed” Soybean Transformation and Regeneration Using “Half-Seed” Explants Explants
Margie M. Paz and Kan WangDepartment of Agronomy, Iowa State University, Ames, IA 50011-1010, USA
Alternative half-seed method
Preparation of explants
and Agro infection
0.3 – 2.8% Transformation Efficiency (coty node method)
AcknowledgementsWe are grateful to Tina Fonger, Juan Carlos Martinez, Joanna Harbaugh, Andrea Ball and Amanda Ehrler for their technical assistance. This work was supported in part by the Iowa Soybean Promotion Board and the North Central Soybean Research Program.
References•Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C,
Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using standard binary vector system. Plant Physiol 129:13-22
•Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167-179
6-day-old seedlings
wounding on coty node
cocultivation, 5 days, dark
regeneration of shoots at (a) 14 days of tissue culture and (b) 28
days
half-seed with embryonic axis
half-seed after embryonic axis
has been removed
seeds soaked overnight in
water
cocultivation, 5 days, light
shoot regeneration at (c) 14 days of tissue culture and (d) 28
days
Rooting
to soil
Shoot elongation
Spraying of R1 plants with Liberty®: green plants are resistant; yellow (dead) plants are sensitive
2.4 – 7.8% Transformation Efficiency (half-seed method)
Southern blot analysis Genomic DNA (10 µg) was digested with Hind III and hybridized with the gus probe. In the positive (+) control, 10 µg of non-transformed soybean DNA was spiked with 1X (63 pg pTF102 DNA) and 5X (315 pg pTF102 DNA) genome equivalents. R0 and R1 refer to first and second generations of transgenic plants, respectively.
Transmission of transgene to the R1 generation was confirmed. Multiple integration was observed in some independent events.
AbstractWe developed an alternative explant system
for transformation via the Agrobacterium method and for organogenesis in soybean. Mature soybean seed was soaked in water and after imbibition the seed was split along the hilum to obtain the cotyledonary tissues that we referred to as “half-seed” explants. The half-seed system does not require precise wounding on explant material that is needed for the cotyledonary node system prior to infection. Transgenic events were confirmed in the R1 generation by herbicide assay and molecular analysis. Transformation efficiency ranged between 2.4%-7.8% with an overall efficiency of 4.4% using half-seed as compared to 0.3%-2.8% using coty node.
Shoot regeneration, 6 mg/L glufosinate
Source of explants
5X p
os c
ontr
ol1X
pos
con
trol
wt s
oybe
an
- 56
-R1-1
- 55
-R1-1
3
- 56
-R1-2
- 56
-R1-3
- 56
-R1-4
- 56
-R1-5
- 56
-R1-6
- 56
-R1-7
- 56
-R1-8
- 56
-R1-9
- 57
-R1-7
- 58
-R1-1
- 58
-R1-2
- 58
-R1-3
- 58
-R1-4
- 58
-R1-5
- 58
-R1-6
- 58
-R1-7
- 58
-R1-8
- 58
-R1-9
pTF102
2.3
4.4
6.6
9.4
Kb
5X p
os c
ontr
ol1X
pos
con
trol
- 58
-R0
Current cotyledonary node method
(a)
(b)
(c)
(d)