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)