Speeding up the Recovery of Stable Transgenic Tomato lines

Improvements in the means and methods of studying transgenic crops are a science in their own right. Testing and analysing different model species for their ability to be reliably and efficiently transformed using a simple method, easily selected for and fast growing cycles has led to the adoption of such species as Arabidopsis thaliana as the plant of choice for this research.

A recent piece in Plant Cell, Tissue and Organ Culture (the article is behind a paywall, but here is a preprint) reports on an alteration to a standard regeneration medium used by the authors when regenerating transformed tomato lines. Noting that tomato plants had become commonly used in theirs and other labs for studies including biotic and abiotic stress resilience and with the successful transformation of the tomato genome by CRISPR/Cas9 likely to increase its importance, the authors performed a literature review looking for growth regulators that may reduce the time for regeneration of transgenic lines when combined with the zeatin growth regulator contained in their medium.

What they found was a number of articles that cited the use of indole-3-acetic acid (IAA) with positive regeneration effects but with no mention of its effect on regeneration time.

Therefore, the researchers tested the time for regeneration of tomato explants transformed with Agrobacterium tumefacians when grown on their standard medium compared to media containing IAA.

The experiment

The researchers took seeds of Solanum lycopersicum cultivar M82 and germinated them on a Murashige and Skoog based medium. Parts of the plants were cultivated a day prior to being transformed with A. tumefaciens that had a kanamycin-resistance plasmid inserted into it.

After transforming the explants they were plated on their standard selective media. After a week they transferred onto either the standard media as a control or on the same media containing either 0.01, 0.05, 0.1 or 0.5mg/L of IAA. After two weeks they were again transferred onto fresh media with the same concentrations of IAA.

The shoots were allowed to grow from the explants until they were 3mm tall and then they were transferred to a rooting media with either no added IAA or with 1mg/L IAA.

By the end of the experiment they had a total of 750 explants per IAA concentration.

The presence of the transferred DNA from the A. tumefaciens in the regenerated plants was confirmed by a β-glucuronidase histochemical assay and by PCR amplification of the selectable marker.

The results

The DNA form the A. tumefaciens was found stably inserted in the transgenic lines while control lines were missing the transgenic DNA.

Of all the tested media, those containing the IAA had a lowered transformation efficiency compared to the roughly 90% efficiency of the standard media described in the body of the article. Instead, the IAA media returned an efficiency of 48% and 54%.

Table 1 from article – Results for recovery of stable transgenic lines of Solanum lycopersicum cv M82 from Agrobacterium tumefaciens-infected cotyledon explants cultured on selective plant regeneration medium supplemented with different indole-3-acetic acid (IAA) concentrations

IAA (mg/l)

Total number explants

Total number rooted plants

Average transformation efficiency (±) SE*

Total time for recovery of transgenic lines (weeks)

0

750

660

88 ± 2.2

17

0.01

750

390

52 ± 1.0

15

0.05

750

375

50 ± 1.5

11

0.1

750

405

54 ± 1.2

11

0.5

750

360

48 ± 2.0

16

Transformation efficiency values shown are the average from 5 experiments ± the standard error (SE) calculated from 3 biological replicates *Average transformation efficiency was calculated as percent of stable transgenic lines recovered from the total number of cotyledon explants infected with Agrobacterium tumefaciens

As can be seen from the table above, the time for recovery of the transgenic lines were reduced by 6 weeks when grown on media containing 0.05 or 0.1 mg/L of IAA.

Given the results, the researchers now implement a new protocol for transforming and regenerating transgenic lines of tomato plants as per the figure below.

tomato-regeneration-standard

Figure 2 from article showing the revised method for trangenic tomato regeneration using media containing IAA.

Discussion

Six weeks saved might not seem like much of a win, but when multiple lines of stably transformed lines of plants are generated in a shorter period of time the following research can be conducted earlier and the knock-on effects of reproducing results, publishing papers or adjusting experiment conditions can be undertaken earlier.

Lots of little wins can lead to big results and hopefully this adjustment helps a number of researchers achieve results in shorter time-frames.

 

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