title / Genetics of transformation and regeneration in horticultural brassicas
/ DEFRA
project code / HH0909SFV
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Genetics of transformation and regeneration in horticultural brassicas
DEFRA project code / HH0909SFV
Contractor organisation and location / John Innes Centre
Colney Lane
Norwich, Norfolk
Total DEFRA project costs / £ 280,561
Project start date / 01/02/99 / Project end date / 31/07/02
Executive summary (maximum 2 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (Rev. 6/02) 3
Projecttitle / Genetics of transformation and regeneration in horticultural brassicas
/ DEFRA
project code / HH0909SFV
This aim of this research programme was to determine the genetic basis of in vitro shoot regeneration and Agrobacterium tumefaciens-mediated transformation in Brassica oleracea. Previous work over many years has resulted in the development of protocols to allow a few selected genotypes to be transformed. This programme was designed to move away from the empirical variations of plant tissue culture methods, to understand the genetic control of three principle factors that affect the transformation process: shoot (and root) regeneration, tissue sensitivity to Agrobacterium tumefaciens infection, and background antibiotic resistance, to determine the nature of the genetic control of these traits and facilitate the selection of amenable breeding material for use in GM breeding programmes. Early in the programme a pilot study of the background resistance to antibiotics found no variation in sensitivity to kanamycin within the DH mapping population. As a result the control of this character was not investigated further.
In vitro shoot and root regeneration was investigated from cotyledonary petioles and hypocotyl segments, two explant types commonly used in Brassica transformation. Detailed analysis of variation for these characters within selected lines of a reference mapping population of B.oleracea, allowed the identification of both additive and dominance components for the genetic control of shoot regeneration from these explant types with additive effects the most important. To demonstrate the heritability of shoot regeneration from cotyledonary petioles, predictions were made on the inheritance of shoot regeneration in selected selfed (F2) and backcrossed (BC) populations. The data from these families show that shoot regeneration potential can be passed on to subsequent generations and that shoot regeneration rates could be increased significantly.
Susceptibility to Agrobacterium tumefaciens was also found to be controlled largely by additive effects. A central region of linkage group O9 of the Brassica oleracea genetic map was identified as being closely associated with this trait. The heritability of this trait from generation to generation was again demonstrated in F2 and BC generations.
Susceptibility to A. tumefaciens and shoot regeneration potential are good phenotypic markers to assist in selecting genotypes for transformation. However, tissue culture blackening and the mode of shoot regeneration were also found to be important factors in the production of transformed shoots. Transgenic plants were not recovered from any genotypes that exhibited tissue culture blackening. Genotypes that regenerated shoots via a callus phase showed the highest level of transformation success, even when the level of susceptibility to Agrobacterium was reduced. The results of an analysis of the genetic control of multiple shoot regeneration suggest the potential to introduce this character into desirable plant breeding material through hybridisation is high. The elimination of tissue culture blackening may also have a positive effect on the frequency of transformed plants produced.
One DH line (1012) has been identified as highly amenable to transformation. The results of transformations with two disarmed strains of Agrobacterium tumefaciens (LBA4404, EHA101) and co-transformation with another (AGLl) illustrates the efficiency of the transformation system in terms of the total number of plants produced, the number of independent transgenic lines produced and the speed of production of transgenic shoots and flowering plants. The rapid production of a high frequency of transgenic lines demonstrates the potential of this DH line for use as a valuable research and development tool for testing gene function in Brassica.
CSG 15 (Rev. 6/02) 3
Projecttitle / Genetics of transformation and regeneration in horticultural brassicas
/ DEFRA
project code / HH0909SFV
Scientific report (maximum 20 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (Rev. 6/02) 3
Projecttitle / Genetics of transformation and regeneration in horticultural brassicas
/ DEFRA
project code / HH0909SFV
Summary
The aim of this programme was to determine the nature of the genetic control of three principle factors that affect the transformation process: shoot and root regeneration, tissue sensitivity to Agrobacterium tumefaciens and background resistance to commonly used antibiotics. This information will explain why some genotypes can be transformed while others cannot, and facilitate the selection of amenable breeding material for use in GM breeding programmes. This work builds on DEFRA funded project (HH1103SFV) to develop efficient transformation in horticultural Brassica species. This study utilises a doubled haploid (DH) mapping population of B.oleracea derived from a cross between B.oleracea ssp alboglabra (A12DHd) and B.oleracea ssp italica (GDDH33) and the associated genetic map 1, 2 to identify quantitative trait loci (QTL’S), associated with in vitro shoot and root regeneration and susceptibility to Agrobacterium tumefaciens. These characters are used to predict transformation success measured in terms of transformation events and regeneration of transgenic plants. These predictions are also applied to a co-transformation system to facilitate the use of clean gene technology. In addition, the development of a rapid, high throughput transformation system for the analysis of gene function in Brassica oleracea is reported. Early in the programme a pilot study of the background resistance to antibiotics found no variation in sensitivity to kanamycin within the DH mapping population. As a result the control of this character was not investigated further.
Shoot and root regeneration
In vitro regeneration of shoots and roots was investigated from two explant types, cotyledonary petioles and hypocotyl segments, commonly used in Brassica transformation. Fifty five DH lines and the two parental genotypes from the A12DHd/GDDH33 mapping population1, 2 were screened for in vitro shoot and root regeneration from the two explant types. Cotyledonary petioles and hypocotyl segments were excised from 4-day-old seedlings and cultured on regeneration media containing 2mg/l BAP. 100 cotyledons and 50 hypocotyls were established per line. The number of explants regenerating shoots (or roots) was scored after 16, 23 and 44 days. Only the results of the 44-day score are discussed here.
Variation in the frequency and mode of shoot regeneration was observed between the two parental genotypes A12DHd and GDDH33 (Figure 1). Significant differences were observed between genotypes (p < 0.001) for shoot regeneration from cotyledon and hypocotyl explants and for root regeneration from cotyledonary explants across the DH population. Shoot and root regeneration from cotyledonary petioles were not antagonistic (r= 0.01). Regeneration frequencies of roots from hypocotyl segments were not consistent across replicates and were not investigated further. Shoot regeneration rates were similar from hypocotyl explants and cotyledonary petioles (r=0.66). However, individual DH lines showed significant difference in regeneration response between the two tissue types. Subsets of the A12DHd/GDDH33 mapping population were screened for in vitro shoot and root regeneration on three separate occasions. Screening the population at different times and under different environmental conditions did not alter the distribution of regeneration frequencies across the population.
Figure 1: Shoot regeneration from cotyledonary petioles (bottom row) and hypocotyl segments (top row) of A12DHd (right) and GDDH33 (left). For both hypocotyl and cotyledonary explants of GDDH33, regeneration is associated with a clearly visible initial callus phase, while shoot regeneration from A12DHd appears to be direct from cells at or near the cut surface.
To identify potential QTL’s associated with shoot and root regeneration from cotyledons and shoot regeneration from hypocotyl explants the regeneration responses of the 55 DH lines were compared against the genetic map of the population using the mapping programme MapQTL3. Putative QTLs for shoot regeneration from cotyledon and hypocotyl explants were located on linkage groups 01 and 08 respectively, and for root regeneration from cotyledons on linkage group 05. Only the profile for shoot regeneration from cotyledonary explants on linkage group O1 was significant, with a LOD score (Log likelihood of a particular marker being associated with the trait) greater than 3.04 (Figure 2). A second peak on linkage group O1 (with a LOD score of 5.2) was not associated with any markers on the genetic map, and was an artefact of the program (due to there being a large barren area with no markers between 64.9 cM and 114.2 cM). Data generated from the two previous screens were also entered into MAPQTL®. For these data sets the QTL profile on linkage group 01 was not significant, however, the activity of the profile was similar for all years with a peak found at the same location. Single marker analysis, performed to identify RFLP markers linked to shoot regeneration from cotyledonary petioles also showed markers on linkage group 01 to be significant.
Figure 2: QTL profiles for in vitro shoot regeneration from cotyledonary petioles for 1997(green), 1999(blue) and 2000 (red) screens.
Quantitative traits are influenced to varying degrees by the combined effects of both environmental and genetic factors. To determine the relative importance of these factors in the control of shoot and root regeneration in Brassica oleracea, a subset of 12 DH lines with contrasting shoot regeneration potentials from the two explant types were selected for use in a diallel crossing programme.
Enough F1 seed was obtained from 128 of the possible 132 F1 hybrids to screen for shoot regeneration after 44 days in culture. 100 cotyledonary explants and 50 hypocotyl explants from each of the 140 genotypes used were screened for (1) presence or absence of shoots and (2) number of shoots produced per explant. Shoot regeneration rates from cotyledonary explants for the 128 F1 and the 12 DH parental lines are presented in Table 1.
Table 1: Diallel table of shoot regeneration from cotyledonary explants, after 44 days in culture. N.B where no F1 seed was available, as indicated in bold, the results from the reciprocal cross was used.
MaleFemale / 5070 / 3070 / 5047 / 5117 / 4052 / 6024 / 4030 / 2072 / 5118 / 2069 / 1027 / 1002
5070 / 1.00 / 1.00 / 1.00 / 1.00 / 0.98 / 0.99 / 0.93 / 0.99 / 0.95 / 0.98 / 0.68 / 0.94
3070 / 1.00 / 1.00 / 0.98 / 1.00 / 1.00 / 1.00 / 0.98 / 1.00 / 1.00 / 1.00 / 0.89 / 0.86
5047 / 1.00 / 0.98 / 0.99 / 0.99 / 0.94 / 0.98 / 0.98 / 0.94 / 0.97 / 0.77 / 0.80 / 0.95
5117 / 1.00 / 1.00 / 0.99 / 0.84 / 0.90 / 0.71 / 0.92 / 0.71 / 0.55 / 0.77 / 0.53 / 0.49
4052 / 0.98 / 1.00 / 0.98 / 0.90 / 0.90 / 0.80 / 0.96 / 0.70 / 0.53 / 0.88 / 0.62 / 0.74
6024 / 0.99 / 1.00 / 1.00 / 0.88 / 0.80 / 0.40 / 0.67 / 0.66 / 0.61 / 0.73 / 0.09 / 0.57
4030 / 0.94 / 0.99 / 0.92 / 0.92 / 0.89 / 0.34 / 0.30 / 0.61 / 0.56 / 0.32 / 0.22 / 0.41
2072 / 0.99 / 1.00 / 0.94 / 0.89 / 0.56 / 0.53 / 0.62 / 0.31 / 0.20 / 0.46 / 0.04 / 0.15
5118 / 0.95 / 1.00 / 0.97 / 0.55 / 0.53 / 0.61 / 0.56 / 0.20 / 0.18 / 0.70 / 0.13 / 0.08
2069 / 0.97 / 1.00 / 0.61 / 0.71 / 0.81 / 0.73 / 0.38 / 0.42 / 0.68 / 0.10 / 0.08 / 0.12
1027 / 0.79 / 0.89 / 0.92 / 0.72 / 0.70 / 0.27 / 0.24 / 0.04 / 0.13 / 0.06 / 0.01 / 0.00
1002 / 0.94 / 0.86 / 0.95 / 0.49 / 0.86 / 0.58 / 0.30 / 0.15 / 0.08 / 0.12 / 0.00 / 0.02
Two-way analysis of variance suggested that 15 % of the variation observed within the diallel table was a result of non-genetic or environmental effects, with the remaining 85 % attributable to genetic effects. The analysis of variance described by Hayman (1954a) 5 was used to describe the genetic variation within the diallel table in terms of additive gene action and/or dominance relationships. This analysis reveals both additive and dominant gene effects were significant for shoot regeneration from cotyledonary explants (p=0.01) No significant maternal effects were observed. The results also indicate directional dominance though the extent of this seems to vary between the parental lines suggesting that the lines carry different numbers of dominant alleles (Figure 3).
Figure 3: Graphical illustration of the variance and covariance of each genotype in relation to the parental mean. Arrows indicate how dominance relates to expression of the trait.
Genetics component analysis6 was also carried out to determine the proportion of the phenotypic variance that can be attributed to additive genetic variance. This is known as the narrow sense heritability or specific combining ability and is a measure of the proportion of the variation that can be passed from one generation to the next. For breeding purposes a strong additive effect is desirable, if this trait is to be introduced into subsequent populations. A high narrow sense heritability of 70.9% was calculated for shoot regeneration from cotyledonary petioles indicating that the potential to introduce this trait into new material is considerable. The genetic components analysis indicates that there is incomplete dominance of this trait (the mean degree of dominance (ÖH1/D) was 0.615).