JOHNSTON, IOWA
Pioneer Hi-Bred International, Inc.

Molecular analysis of T0 plants transformed by Agrobacterium and comparison of Agrobacterium-mediated transformation with bombardment transformation in maize
--Zhao, ZY,Gu, W, Cai, T, Tagliani, LA, Hondred, D, Bond, D, Krell, S, Rudert, ML, Bruce, WB, Pierce, DA

Agrobacterium-mediated transformation methods have been used principally in dicotyledonous plants. Monocotyledons are not considered to be natural hosts for Agrobacterium. Recently Agrobacterium-mediated transformation has been reported in a number of agriculturally important cereal crops, such as rice, wheat, barley and maize. Even though DNA analysis has been used in these transformation studies to confirm stable integration of T-DNA into plant genome, more complete information on the molecular analysis of the Agrobacterium-transformed events in monocotyledons is still not available. Those data include T- DNA integration pattern, transgene copy number, the integration of binary vector backbone DNA sequence into plant genomes, transgene expression as well as the direct comparison of Agrobacterium-transformed events to bombardment-generated events etc. In this current study, we use maize Hi-II line as a model system to provide the above information. Hi-II embryos were genetically transformed with LBA4404 harboring the super-binary vector created by Japan Tobacco Inc. The regenerated T0 plants were used for these analyses. Microprojectile bombardment of Hi-II embryos was used for comparison. The results indicated that Agrobacterium has a number of advantages over bombardment for H-II transformation.

Immature embryos of Hi-II and N6 media were used for Agrobacterium (LBA4404 ) transformation. Super binary vector pPHP10525 (Figure 1) was constructed with pSB1and pSB11 vectors obtained from Japan Tobacco Inc. The T-DNA of pPHP10525 contained GUS and bar genes. The second intron of the potato ST-LS1 was inserted into the coding region of the GUS gene to prevent expression of GUS in Agrobacterium. Maize genomic DNA was digested with SpeI for GUS integration, with XbaI for bar, spec, tet and virG integration, and with SphI for PTU (plant transcription unit) of GUS and bar. GUS protein was assayed with GUS-Light Assay Kit (Tropix, Inc. 47 Wiggins Ave., Bedford, MA01730).

1. Agrobacterium-mediated Hi-II immature embryo transformation: Fresh Hi-II immature embryos were transformed with LBA4404(pPHI10525). Stably transformed calli were recovered on bialaphos-containing medium and confirmed with X-gluc staining. T0 plants were regenerated from those calli. With optimized conditions, the embryo-based transformation frequency can be as high as 32.8 to 50.5% (Table 1).

Figure 1. pPHP10525.

Table 1. Agrobacterium Hi-II transformation frequency.
 
Expt. No. No. of immature embryos    Transformation frequency (%)
  Inoculated  GUS+ event   
1 195 64 32.8
2 97  37  38.1
3 65 30 46.2
4 103 52 50.5
Sum 460 183 39.8

2. Southern analysis of Agrobacterium transformed T0 plants: To understand the molecular characteristics of Agrobacterium-mediated maize transformation, T0 plants from 107 embryo-derived events were used in Southern analysis. These T0 plants were divided into two groups as follows: Group-1: Single plants from each of those embryo-based events that were transformed with Agrobacterium at 1x1010, 2x109, 1x109, 5x108, and 1x108 cfu/ml. The purpose of this assay is to determine transgene insertion pattern, copy number and presence or absence of backbone DNA of the binary vector as well as for verifying the effect of Agrobacterium concentration on these parameters. Group-2: 25 plants derived from 5 embryos (5 plants/embryo), where all 5 plants derived from a single embryo showed the same phenotype for the bar and GUS genes; 25 plants derived from another 5 embryos (5 plants/embryo), where the 5 plants showed variable phenotypes for the bar and GUS genes. The purpose of this assay is to identify the number of independent events generated from a single embryo. The way these events are defined is described in the following chart. Southern results are listed in Table 2 and 3 and shown in Figures 2, 3, 4 and 5.
 
Items LowCopy/Simple Insertion Multicopy/Complex Insertion
Bands in integration blots* 1-3 >3
Bands in PTU blots* 1 with proper size >1
Copy No. for both genes 1-3 >3
Rearrangement no yes or no
Backbone DNA  no yes or no
* The blots for both GUS and bar genes.

Table 2. Southern results of the events transformed with different concentrations of Agrobacterium.
 
Agrobac Concen. No. event Low/Simple 

Event %

Multi/Comp 

Event %

Backbone 

Event %

1 x 108 12 8 67 4 33 2 17
5 x 108 24 13 54 11 46 4 17
1 x 109 49 30 61 19 39 3 6
2 x 109 14 6 43 8 57 1 7
1 x 1010 8 5 63 3 37 0 0
Total 107 62 58 45 42 10 9

Table 3. Number of independent events produced from a single embryo.
 
 
Number of independent events
Embryo No. 5 Plants with same phenotype 5 Plants with varied phenotypes
1 1 2
2 2 2
3 3 2
4 3 3
5 5 4
Total 

Average

14 

2.8 events/embryo

13 

2.6 events/embryo

Sum:  27 events/10 embryos Multi-event index = 2.7
Figure 2. A Southern blot showing presence of the GUS gene.

Figure 3. A Southern blot showing integrity of the GUS transcription unit (PTU).

Figure 4. A Southern blot showing presence of vector backbone sequences

Figure 5. A Southern blot showing multiple events from the same embryo.

3. Comparison of Agrobacterium transformation with microprojectile bombardment: A number of comparisons were made for the T0 plants derived from Agrobacterium transformation and from bombardment transformation in Hi-II. The comparisons include transgene copy number, insertion pattern (Table 5), transgene co-expression (Table 6), and GUS gene activity (Table 7 and Figure 6 and 7). Based on these comparisons Agrobacterium-mediated transformation shows a number of advantages over bombardment: (1) a higher proportion of low copy/simple insertion patterns; (2) a significantly higher proportion of events with higher expression levels of GUS. In addition, Agrobacterium gives a significantly higher frequency of transformation, compared to our experience with bombardment (33-51% vs. 7-10% of treated embryos).

Table 4. Comparison of DNA analysis results.
 
Method Event No. Low copy/ Simple Multicopy / Complex
Bomb* 133 8% 92%
Agro. 107 58% 42%
*Bombardment transformation with Bt and pat PTU

Table 5. Comparison of transgene co-expression.
 
Method Events GUS+ / bar+ GUS- / bar+
Bomb* 17 15 88% 2 12%
Agro 50 47 94% 3 6%
* Bombardment transformation with another inbred line.

Table 6. Comparison of GUS activity in T0 plants.
 
    GUS scores
Method Events +++ ++ + or -
Bomb 14 6 (43%) 3 (21%) 5 (36%)
Agro 43 31 (72%) 6 (14% 6 (14%)
GUS score +++: =>100,000 units/ug protein; ++: 10,001 to 99,999 units/ug protein; + or -: 0 to 10,000 units/ug protein

In summary, if transformation events with low copy and simple insertion pattern are considered as desirable events, the efficiency of producing desirable transgenic events with Agrobacterium would be:

39.8% (transformation frequency) x 2.7 (multi-event index) x 58% (low/simple) = 62 desirable events /100 embryos.

while the efficiency of producing desirable transgenic events with gun bombardment would be:

10% (transformation frequency) x 1.15 (multi-event index) x 8% (low/simple) = 1 desirable event /100 embryos. 


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