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Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

Qi W, Tinnenbroek-Capel IE, Salentijn EM, Schaart JG, Cheng J, Denneboom C, Zhang Z, Zhang X, Zhao H, Visser RG, Huang B, Van Loo EN, Krens FA - Sci Rep (2015)

Bottom Line: In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged.After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant.The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People's Republic of China.

ABSTRACT
The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

No MeSH data available.


Related in: MedlinePlus

The process in which wild-type vegetation tissues were identified from a chimeric crambe GM shoot.This figure exemplifies the process in which wild-type vegetation tissues were identified from a chimeric shoot of line 2 after the 15 μM DEX treatment and 5-FC selection. There were 3 axillary buds obtained from the shoot in total. As showed in the right corner, the blue arrows mean a round of regeneration and selection with 5-FC; plants in pale represent those killed by the selection, and green plants indicate survivors. The drawn numbers are the actual number of plants handled. All the survivors were tested by Gus-staining at the end of Step 6, ‘O’ means GUS positive, and ‘X’ means negative. So, finally from this specific starting plant, five surviving shoots obtained were GUS-negative, and three were GUS-positive. And this result gives a thorough indication of the chimeric nature in the starting shoots.
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f9: The process in which wild-type vegetation tissues were identified from a chimeric crambe GM shoot.This figure exemplifies the process in which wild-type vegetation tissues were identified from a chimeric shoot of line 2 after the 15 μM DEX treatment and 5-FC selection. There were 3 axillary buds obtained from the shoot in total. As showed in the right corner, the blue arrows mean a round of regeneration and selection with 5-FC; plants in pale represent those killed by the selection, and green plants indicate survivors. The drawn numbers are the actual number of plants handled. All the survivors were tested by Gus-staining at the end of Step 6, ‘O’ means GUS positive, and ‘X’ means negative. So, finally from this specific starting plant, five surviving shoots obtained were GUS-negative, and three were GUS-positive. And this result gives a thorough indication of the chimeric nature in the starting shoots.

Mentions: In Fig. 9,it is a flow-diagram demonstrating how the WT shoots werefinally isolated. The recovery after 5-FC selection, of such a high percentage of WT non-transformed shoot clusters demonstrated that the transformation protocol developed in our lab for crambe using cotyledonary node explants (CNE) with meristematic tissue32 may give rise to chimeras. However, such chimerism was never observed in earlier or later transformation experiments when looking e.g. at segregation ratios in T1 or T2 progenies of transgenic lines. A possible reason can be that here a less stringent (shorter) Km selection was applied and the continued 5-FC selection without Km allowed the scarce WT cells to proliferate and become prominent. Still, it was obvious that in the experiments described here chimerism did occur in the original shoots put on rooting medium and subjected to DEX; line 1 showing a higher percentage of WT cells (2.4% of the original number of axillary buds contained WT cells) than line 2 (0.9%). The two lines used for establishing the protocol were analyzed at the start by GUS staining and molecular analysis by PCR and Southern hybridization and proven to be transgenic. The two lines were selected based on their difference in T-DNA copy number with line 1 having a single insert and line 2 having more than one insert to monitor any differences in efficiency in obtaining recombinant plants. In crambe, transformation usually gives 49% single insert and 35% 2–3 copy inserts32. The PCR and Southern hybridizations apparently cannot exclude the possibility of a few WT cells to be still present. There was no evidence indicating that any axillary-bud explant from the rooting shoots in the beginning was totally non-transgenic (WT). Using the CNE protocol of transformation in crambe, a stringent and prolonged selection period on Km seemed to be required in order to avoid the survival of WT cells32. Final assurance for obtaining fully transformed plants is to go through a seed phase and working with T1 or T2 generations. Moreover in present research, a passage through a seed-phase should also ensure the homogeneous recombinant nature of the subsequent generations.


Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

Qi W, Tinnenbroek-Capel IE, Salentijn EM, Schaart JG, Cheng J, Denneboom C, Zhang Z, Zhang X, Zhao H, Visser RG, Huang B, Van Loo EN, Krens FA - Sci Rep (2015)

The process in which wild-type vegetation tissues were identified from a chimeric crambe GM shoot.This figure exemplifies the process in which wild-type vegetation tissues were identified from a chimeric shoot of line 2 after the 15 μM DEX treatment and 5-FC selection. There were 3 axillary buds obtained from the shoot in total. As showed in the right corner, the blue arrows mean a round of regeneration and selection with 5-FC; plants in pale represent those killed by the selection, and green plants indicate survivors. The drawn numbers are the actual number of plants handled. All the survivors were tested by Gus-staining at the end of Step 6, ‘O’ means GUS positive, and ‘X’ means negative. So, finally from this specific starting plant, five surviving shoots obtained were GUS-negative, and three were GUS-positive. And this result gives a thorough indication of the chimeric nature in the starting shoots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4566089&req=5

f9: The process in which wild-type vegetation tissues were identified from a chimeric crambe GM shoot.This figure exemplifies the process in which wild-type vegetation tissues were identified from a chimeric shoot of line 2 after the 15 μM DEX treatment and 5-FC selection. There were 3 axillary buds obtained from the shoot in total. As showed in the right corner, the blue arrows mean a round of regeneration and selection with 5-FC; plants in pale represent those killed by the selection, and green plants indicate survivors. The drawn numbers are the actual number of plants handled. All the survivors were tested by Gus-staining at the end of Step 6, ‘O’ means GUS positive, and ‘X’ means negative. So, finally from this specific starting plant, five surviving shoots obtained were GUS-negative, and three were GUS-positive. And this result gives a thorough indication of the chimeric nature in the starting shoots.
Mentions: In Fig. 9,it is a flow-diagram demonstrating how the WT shoots werefinally isolated. The recovery after 5-FC selection, of such a high percentage of WT non-transformed shoot clusters demonstrated that the transformation protocol developed in our lab for crambe using cotyledonary node explants (CNE) with meristematic tissue32 may give rise to chimeras. However, such chimerism was never observed in earlier or later transformation experiments when looking e.g. at segregation ratios in T1 or T2 progenies of transgenic lines. A possible reason can be that here a less stringent (shorter) Km selection was applied and the continued 5-FC selection without Km allowed the scarce WT cells to proliferate and become prominent. Still, it was obvious that in the experiments described here chimerism did occur in the original shoots put on rooting medium and subjected to DEX; line 1 showing a higher percentage of WT cells (2.4% of the original number of axillary buds contained WT cells) than line 2 (0.9%). The two lines used for establishing the protocol were analyzed at the start by GUS staining and molecular analysis by PCR and Southern hybridization and proven to be transgenic. The two lines were selected based on their difference in T-DNA copy number with line 1 having a single insert and line 2 having more than one insert to monitor any differences in efficiency in obtaining recombinant plants. In crambe, transformation usually gives 49% single insert and 35% 2–3 copy inserts32. The PCR and Southern hybridizations apparently cannot exclude the possibility of a few WT cells to be still present. There was no evidence indicating that any axillary-bud explant from the rooting shoots in the beginning was totally non-transgenic (WT). Using the CNE protocol of transformation in crambe, a stringent and prolonged selection period on Km seemed to be required in order to avoid the survival of WT cells32. Final assurance for obtaining fully transformed plants is to go through a seed phase and working with T1 or T2 generations. Moreover in present research, a passage through a seed-phase should also ensure the homogeneous recombinant nature of the subsequent generations.

Bottom Line: In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged.After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant.The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Key Laboratory for Bioresources of Saline Soils, Provincial Key Laboratory of Agrobiology, Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People's Republic of China.

ABSTRACT
The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

No MeSH data available.


Related in: MedlinePlus