A. Pellegrineschi,1 L.M. Noguera,1 S. Mclean,1 B. Skovmand,2 R.M. Brito,1 L. Velazquez,1 R. Hernandez,1 M. Warburton,1 and D. Hoisington1
1Applied Biotechnology Center and 2Wheat Program, CIMMYT

Introduction

A group of 129 "Bobwhite" sister lines, generated at CIMMYT in the mid 1970s from the cross CM 33203 (pedigree: Aurora//Kalyan/Bluebird/3/Woodpecker), were used in this study. They are highly responsive materials, reported to be transformable.

The objectives of this study were: 1) to use transformation protocols and genotype data to screen 129 Bobwhite accessions for their transformation ability; and 2) to identify the most transformable and responsive accessions based on their ability to regenerate and adapt to tissue culture, and on their agronomic characteristics.

Materials and methods are described in:

Pellegrineschi A., L. M. Noguera, S. McLean, B. Skovmand, R. M. Brito, L. Velazquez, R. Hernandez, M. Warburton, and D. Hoisington.  Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Submitted to Plant Cell Reports.

 

Results

Somatic embryo induction

Cultures transferred to selective medium were checked for somatic embryo formation. The effect of genotype on scutellum embryogenesis is summarized in Table 1. Most (111) of the 129 genotypes tested produced somatic embryos (Table 1). Eleven accessions showed the highest yield (nearly 100%) of embryos producing embryogenic callus. There were no distinct differences in stage development, with the exception of the number of scutella differentiating somatic embryos. Generally, the first globular stage somatic embryos were observed 4-5 days after transfer, and the globular stage usually formed directly from the scutellum. This was followed by a high frequency of repetitive somatic embryogenesis. Early globular stages were followed by full differentiation of the somatic embryo.

Transformation frequency and selection efficiency

Transformation efficiency was evaluated based on regeneration performance on selective medium. Healthy, fully differentiated embryogenic calli were scored (1 callus per embryo, Table 1) as number of regenerating calli divided by total number of immature embryos bombarded (one regenerating callus was scored as 1). Somatic embryo germination frequency was 0-89%. Accessions responded in four different ways to bombardment: 1) no regeneration, 2) herbicide tolerance and bombardment susceptibility, 3) herbicide sensitivity, bombardment tolerance, and high regeneration, and 4) herbicide sensitivity and bombardment tolerance but low regeneration (Table 1). Transformation efficiency was calculated as the effective number of transgenic plants obtained divided by the total number of immature embryos bombarded.

The most efficient lines were SH-98 26 and SH-98 56 (Table 1). Other accessions (SH-98 15, SH-98 88, and SH-98 121) gave higher regeneration frequency but less overall efficiency due to "escapes" (plants surviving the selection process but not transgenic). Accessions SH-98 26, SH-98 29, SH-98 56, SH-98 96, SH-98 97, SH-98 110, SH-98 128, and SH-98 129, the best lines for transformation, were tested further as described in Materials and Methods; results are shown in Table 2.

Molecular screening of transgenic plants

Shoot tissue harvested from BastaTM resistant plants was screened with PCR to verify the presence of the Bar gene in the plant genome. Results indicated that all plants analyzed from all experiments contained the Bar gene. Amplified DNA fragments (approximately 350 nucleotides) from transgenic plants were identical in size to the controls, and all hybridized with the plasmid probe. Fifty independently transformed plants were analyzed for copy number (Bar gene) by Southern blot analysis in which a gene copy reconstruction lane was included. Where the Southern analyses indicated there were multiple copies of the transgene (Figure 6: lanes 6 to 13 and 19 to 26), all copies appeared to cosegregate yielding progenies with all or no copies. This suggested that all copies of the transgene were inserted at the same genetic locus. The Bar transgene was inherited and expressed in the T1 and T2 generation lines tested. Most of the initial transgenic wheat plants were at least partially fertile. Fertility was usually restored in subsequent generations, indicating that partial sterility observed in the T0 generation was not, in most cases, an inherited trait.

Inheritance of the marker gene

Selected progeny were evaluated again for resistance to BastaTM. Resistant and sensitive seedlings were clearly distinguishable after spraying with 0.3% BastaTM. A segregation ratio of 3:1 was observed for 500 of 600 independent transgenic events tested (randomly taken). The BastaTM resistant (T1) progeny of plants that gave a segregation ratio of 3:1 were analyzed by PCR and Southern hybridization. All BastaTM-resistant progeny contained bands that hybridized with Bar probe; none of the sensitive progeny hybridized and may have been escapes (data not shown).

Statistical analysis

Results of the average, standard deviation, minimum and maximum of embryogenesis, regeneration, and transformation efficiencies are shown in Tables 1 and 2.

 

Discussion


Figure 1. Response of Bobwhite line SH 98 26 to selection medium.1
 1 Bombarded embryos (left); controls (non-bombarded) (right). All explants on regeneration medium with 5 mg/l BastaTM. 

The use in biolistic transformation of a highly responsive wheat genotype can enhance efficiency. To identify highly responsive genotypes, it is necessary to optimize and standardize tissue culture conditions and transformation efficiency, and to identify the physiological conditions of the material to be transformed.

Standardization of the physiological status of the donor plants was a critical factor for comparing transformation abilities of the Bobwhite accessions. After testing under various conditions (data not shown), a uniform non-stressed growth environment was selected for the optimal growth of the donor plants.

The choice of the zygotic embryo development stage was also important. Various development stages were screened for their response to the transformation process. The dimension of the embryo (1 mm on the longest side) was taken as standard in all accessions regardless of "days after pollination" because at this stage scutella are more responsive to tissue culture. In the transformation experiments, accessions SH-98 26 and SH-98 56 were slightly more efficient (overall efficiency: more than 70%), although their ability to differentiate somatic embryos was less than other accessions (Table 1). Their performance could be explained by their high sensitivity to herbicide selection (non-transformed controls were not able to produce plants under selection conditions).

Of the two high performing varieties, the variety SH 98 26 was selected as "super transformable" because it is early maturing, does not have the 1B/1R translocation, and may be a suitable parent in breeding programs. Genetic analyses of T1 and T2 progeny provided conclusive evidence of the incorporation of the Bar transgene into wheat chromosomes. In most cases the Bar gene was inherited with a Mendelian ratio of 3:1. However, in some progeny the phenotype "BastaTM resistance" was expressed in the T1 generation with an unusual pattern of segregation, but the T2 generated from the BastaTM-resistant T1 plants segregated at the expected Mendelian ratio (3:1).


Figure 2. PCR analyses of transformants of line SH 98 26.1
 1 DNA was extracted from leaves. Each lane represents an independent event. Plants 10 and 16 did not survive the BastaTM treatment. 		      
Figure 3. Southern blot analysis of regenerated T0 plants from line SH 98 26 after SmaI restriction digest plasmid (unique site on UbiBar plasmid).1

1 DNA digested with SmaI and probed with UbiBar plasmid Dig-labeled by nick translation.
2 Lanes 1 and 14 contain a 1 Kb ladder; Lanes 2, 3, 4 15, 16, and 17 represent copy number references (10, 5, and 1 copy number, respectively); lanes 5 and 18 are negative controls; and lanes 6 to 13 and 19 to 26 are from BastaTM resistant plants. 

 

Table 1. Regeneration and effective transformation efficiency of 129 Bobwhite lines.1 
Bobwhite  Embryogenesis Regeneration Transformation  Bobwhite  Embryogenesis Regeneration Transformation
line Bombarded Control Bombarded Control Bombarded Tr./Emb. line Bombarded Control Bombarded Control Bombarded Tr./Emb.
SH 98 01 50.35  51.86 49.15 1.20 47.94 1.05 SH 98 66 41.90 42.74 28.26 13.64 14.62 1.00
SH 98 02 66.26 69.57 62.55 3.70 58.85 1.20 SH 98 67 60.59 59.98 36.88 23.71 13.17 1.00
SH 98 03 33.91 34.59 22.31 11.59 10.72 2.10 SH 98 68 29.60 29.89 26.09 3.51 22.58 1.00
SH 98 04 36.99 36.62 29.30 7.69 21.61 1.00 SH 98 69 37.13 41.74 37.13 0.00 37.13 1.00
SH 98 05 33.83 34.17 28.99 4.84 24.16 1.02 SH 98 70 41.35 42.67 41.35 0.00 41.35 1.10
SH 98 06 0.80 0.90 0.80 0.00 0.80 1.00 SH 98 71 66.92 68.92 40.67 26.25 14.42 1.20
SH 98 07 40.98 42.30 40.98 0.00 40.98 1.04 SH 98 72 88.09 92.49 32.20 55.88 0.00 1.00
SH 98 08 7.41 7.63 7.41 0.00 7.41 1.00 SH 98 73 84.11 85.79 51.85 32.26 19.59 1.00
SH 98 09 61.79 64.88 55.77 6.02 49.75 1.00 SH 98 74 61.64 61.02 53.57 8.06 45.51 1.00
SH 98 10 38.12 38.88 38.12 0.00 38.12 1.00 SH 98 75 64.08 64.72 58.90 5.17 53.73 1.10
SH 98 11 39.20 38.80 28.22 10.98 17.25 1.00 SH 98 76 67.77 76.17 59.26 8.51 50.75 1.25
SH 98 12 8.34 8.42 6.45 1.89 4.56 1.00 SH 98 77 49.56 51.15 43.68 5.88 37.80 2.25
SH 98 13 23.25 26.13 20.78 2.47 18.31 1.00 SH 98 78 47.01 48.42 35.90 11.11 24.79 1.00
SH 98 14 97.68 100.00 76.53 21.15 55.38 1.30 SH 98 79 57.95 60.85 55.45 2.50 52.95 1.00
SH 98 15 109.15 100.00 71.65 37.50 34.15 1.00 SH 98 80 32.57 33.22 32.57 0.00 32.57 1.00
SH 98 16 89.59 94.07 71.61 17.98 53.63 3.00 SH 98 81 80.77 79.96 50.00 30.77 19.23 1.00
SH 98 17 24.34 24.83 5.19 19.15 2.05 1.00 SH 98 82  0.00 0.00 0.00 0.00 0.00 0.00
SH 98 18 37.50 37.13 0.00 37.50 0.00 0.00 SH 98 83 79.14 88.95 54.55 24.59 29.96 1.00
SH 98 19 48.52 49.00 39.90 8.62 31.28 1.20 SH 98 84 62.34 64.34 52.97 9.38 43.59 1.30
SH 98 20 3.85 4.32 3.85 0.00 3.85 1.00 SH 98 85 43.72 45.03 35.38 8.33 27.05 1.10
SH 98 21 43.64 45.03 43.64 0.00 43.64 1.00 SH 98 86 35.48 37.26 35.48 37.26 35.48 1.00
SH 98 22 54.85 56.49 46.85 8.00 38.85 1.50 SH 98 87 46.04 46.96 43.82 2.22 41.60 1.00
SH 98 23 47.89 50.28 47.89 0.00 47.89 1.02 SH 98 88 100.00 103.00 54.41 80.00 0.00 0.00
SH 98 24 56.72 57.85 56.72 0.00 56.72 2.02 SH 98 89 95.45 100.00 50.00 45.45 4.55 1.00
SH 98 25  20.00 19.80 20.00 0.00 20.00 1.00 SH 98 90 0.00 0.00 0.00 0.00 0.00 0.00
SH 98 26 73.81 74.55 73.81 0.00 73.81 2.40 SH 98 91 50.00 49.50 50.00 0.00 50.00 1.40
SH 98 27 16.39 18.42 8.70 7.69 1.00 1.00 SH 98 92 94.75 95.70 68.09 26.67 41.42 1.00
SH 98 28 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 93 54.05 60.75 51.72 2.33 49.40 1.00
SH 98 29 62.16 64.03 62.16 0.00 62.16 1.40 SH 98 94 42.29 43.14 35.54 6.76 28.28 1.00
SH 98 30 48.12 50.53 48.12 0.00 48.12 1.00 SH 98 95 74.13 73.39 45.10 29.03 16.07 1.20
SH 98 31 22.41 22.86 22.41 0.00 22.41 1.20 SH 98 96 72.20 72.92 70.54 1.67 68.87 3.25
SH 98 32 61.79 61.18 45.13 16.67 28.46 1.00 SH 98 97 90.92 100.00 77.19 13.73 63.47 1.00
SH 98 33 48.61 49.10 34.88 13.73 21.16 1.00 SH 98 98 68.93 71.14 44.44 24.49 19.95 1.00
SH 98 34 30.62 34.42 24.56 6.06 18.50 2.20 SH 98 99 61.95 63.81 50.36 11.59 38.76 1.00
SH 98 35 14.03 14.48 5.80 8.24 0.00 0.00 SH 98 100 37.95 39.84 28.57 9.38 19.20 1.00
SH 98 36 22.92 23.60 22.92 0.00 22.92 1.00 SH 98 101 55.20 56.31 36.15 19.05 17.11 1.00
SH 98 37 22.48 23.60 22.48 0.00 22.48 1.00 SH 98 102 81.00 80.19 57.60 23.40 34.20 1.00
SH 98 38 57.03 58.17 42.32 14.71 27.62 1.00 SH 98 103 100.00 100.00 80.85 35.21 45.64 1.00
SH 98 39 23.03 22.80 23.03 0.00 23.03 2.00 SH 98 104 79.99 83.99 62.14 17.86 44.28 1.00
SH 98 40 38.88 39.27 11.96 26.92 0.00 0.00 SH 98 105 59.08 60.26 45.56 13.51 32.05 1.00
SH 98 41 0.02 0.03 0.02 0.00 0.02 1.00 SH 98 106 67.12 66.45 55.36 11.76 43.59 1.00
SH 98 42 0.03 0.04 0.03 0.00 0.03 1.00 SH 98 107 96.26 97.22 58.76 37.50 21.26 1.00
SH 98 43 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 108 98.81 100.00 66.46 35.35 34.10 1.00
SH 98 44 0.05 0.05 0.05 0.00 0.05 1.00 SH 98 109 22.10 21.88 13.94 8.16 5.78 1.00
SH 98 45 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 110 82.10 82.92 71.43 10.67 60.76 1.10
SH 98 46 0.26 0.26 0.26 0.00 0.26 1.00 SH 98 111 73.74 82.89 40.41 33.33 7.08 1.00
SH 98 47 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 112 81.87 84.49 51.26 30.61 20.65 1.00
SH 98 48  0.00 0.00 0.00 0.00 0.00 0.00 SH 98 113 92.71 95.49 30.80 61.90 0.00 0.00
SH 98 49 25.00 25.80 25.00 0.00 25.00 1.00 SH 98 114 94.35 99.07 71.28 23.08 48.20 1.20
SH 98 50 0.16 0.17 0.16 0.00 0.16 1.00 SH 98 115  0.00 0.00 0.00 0.00 0.00 0.00
SH 98 51 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 116 100.00 100.00 83.81 50.00 33.81 1.00
SH 98 52 0.09 0.10 0.09 0.00 0.09 1.00 SH 98 117 100.00 100.00 89.47 47.06 42.41 1.00
SH 98 53 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 118 100.00 100.00 81.71 37.04 44.67 1.50
SH 98 54 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 119 100.00 100.00 80.87 40.91 39.96 1.00
SH 98 55 24.91 28.00 24.02 0.89 23.13 1.10 SH 98 120 100.00 100.00 85.07 37.50 47.57 1.00
SH 98 56 73.47 75.82 73.47 0.00 73.47 2.10 SH 98 121 100.00 100.00 87.50 41.67 45.83 1.00
SH 98 57 34.28 35.31 28.66 5.62 23.04 1.00 SH 98 122 0.00 0.00 0.00 0.00 0.00 0.00
SH 98 58 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 123 25.64 25.38 25.64 0.00 25.64 1.00
SH 98 59 39.32 40.10 39.32 0.00 39.32 1.00 SH 98 124 0.00 0.00 0.00 0.00 0.00 0.00
SH 98 60 48.20 47.71 34.20 14.00 20.20 1.10 SH 98 125 0.00 0.00 0.00 0.00 0.00 0.00
SH 98 61 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 126 100.00 100.00 86.62 23.26 63.36  1.10
SH 98 62 71.34 80.19 49.68 21.67 28.01 1.50 SH 98 127 0.00 0.00 0.00 0.00 0.00 0.00
SH 98 63 0.00 0.00 0.00 0.00 0.00 0.00 SH 98 128 93.38 96.18 76.71 16.67 60.05 1.00
SH 98 64 58.28 60.03 47.37 10.91 36.46 1.00 SH 98 129 82.92 87.06 72.92 10.00 62.92 1.00
SH 98 65 62.28 65.39 49.55 12.73 36.82 1.00                     
1 Accessions with high transformation efficiency highlighted. Transformation efficiency calculated by dividing total number of transgenic plants obtained by total number of embryos bombarded. 

 

 

Table 2. Results of statistical analyses of the 8 best Bobwhite lines for embryogenesis, regeneration in selective medium, and transformation efficiency.1
  Embryogenesis Regeneration Transformation
Bobwhite line Bombarded Control Bombarded Control Bombarded Tr./Emb.
SH 98 26 72.09±13.34 75.35±13.39 71.17±14.68 0.00 70.86±14.48 2.42
SH 98 29 59.23±15.56 63.49±18.22 61.90±11.29 0.00 60.92±11.58 1.34
SH 98 56 70.34±9.82 70.14±5.98 70.34±9.83 0.00 69.02±6.94 2.13
SH 98 96 69.17±9.92 69.97±4.73 69.17±9.93 2.43±1.55 66.96±6.14 3.2
SH 98 97 90.96±5.95 93.33±4.92 76.63±9.02 13.98±4.54 66.96±4.53 1.1
SH 98 110 81.07±7.15 81.25±5.23 70.95±6.32 10.77±4.87 60.80±6.28 1
SH 98 128 91.15±4.85 93.54±4.22 78.13±8.21 20.13±5.61 58.27±10.40 1
SH 98 129 80.55±7.01 85.59±6.11 71.82±9.61 10.08±3.58 60.04±9.61 1
1 Data pooled from 3 repetitions with over 2000 embryos per transformation. 

 

Acknowledgments

This research is funded in part by the Australian Cooperative Research Center for Molecular Plant Breeding, in which CIMMYT is a participant. The "Bobwhite" family, developed by Dr. S. Rajaram, is stored in CIMMYT's germplasm bank, as "in trust" material under the FAO agreement.


© CIMMYT July 2001

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