Storage usually affects virulence of plant pathogenic bacteria. It is better to use fresh culture. Look at different protocols^

Davis, A.M., Hall, A., Millar, A.J. et al. Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods 5, 3 (2009). https://doi.org/10.1186/1746-4811-5-3

Clough, Steven J., and Andrew F. Bent. "Floral dip: a simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana." The plant journal 16.6 (1998): 735-743.

I'd recommend always using fresh culture. Plants take more time to grow, so it's worth not taking unknown risks during the transformation.

For the long term, You can grow them on a plate.

When your plants are ready to be transformed, just a day or two before grow the liquid culture to desired OD.

Here is a working protocol of mine, I can assure you will get better results.

https://www.protocols.io/view/progeny-test-for-generating-single-copy-homozygous-5xrg7m6

Use fresh ones. I use fresh ones all the time for all transformations, including transient assay. You don't want to waste time at the end finding out your transformation efficiency drops or zero. That is not affordable for a researcher, in terms of time.

Hafiz Muhammad Rizwan

Hafiz Muhammad Rizwan

< can we dip the arabidopsis seeds directly to agrobacterium running solution for 3 to 5 days and then dip into antibiotic such as hygromycin 1or 2 hours >

First, if the Agrobacterium can penetrate the hard seed coat or not, I am not sure. Or, Maybe the bacteria is very easy to get into the seed coat, and overwhelm the seed (rotten by the bacteria)? But the most important issue is: once the bacteria go in, they might infect only a part of the *seed*, then the seed develops into a 'chimeric plant' (only some cells are transformed). It is unlike 'floral dip' transformation (see next post).

Hafiz Muhammad Rizwan

I want to add here, *floral dip* works through female reproductive tissues, before they develop into a plant. So, they should grow into a homogeneous transgenic plants (as opposed to "chimera"). See this paper below (from PNAS):

"In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of *ovules and embryo sacs* was observed only if the Agrobacterium were applied 5 d or more prior to anthesis [1]."

• Figure: Close-up of two ovules (partially overlapping in photo) showing staining of *embryo/embryo sac* rather than entire ovules [1].

[1] http://www.plantphysiol.org/content/123/3/895

Hafiz Muhammad Rizwan

Hafiz Muhammad Rizwan

< If luckily got the good results by this way, so will be worthy ...>

So, you are expecting that when you transform a seed, when the seed grows into a plant (see attachment), the plant will be a *homogeneous* transgenic plant (i.e. every cell of the plant carried the foreign gene-of-interest (GOI)? How? (click to view the whole picture)

Right! I know several attempts to employ simplified methods to get GM plants (do not mix with transient expression), which were severely criticized for lack of proof that they have got the target.

Hafiz Muhammad Rizwan

< So if in the case: (expecting) by this way transformed the empty vector (pCAMBIA1301/1302 with GUS/GFP marker) and later confirmed by GUS staining /GFP fluorescent in roots, shoots, leaves, stem, etc, so will it be enough for *homogeneous* confirmation ? or not? >

• No, you cannot. This approach is not scientifically sound. The GUS staining can show that the root looks like blue, but it doesn't provide proof that every single cell contains the GOI. This goes to GFP fluorescence, too.
• After the seed incubates with Agrobacterium, some cells of the seed might be transformed. So, the transgenic plant derived from this seed will be chimeric for GOI. Only the GOI passes down to the next generation through germ line cells (eggs or sperm), the progeny can then be *homogeneous* transgenic plant line. (see next post)

Hafiz Muhammad Rizwan

There is more than expectation (science is behind it). Here is an old paper for you. Arabidopsis seeds were used for genetic transformation. They did transformation in the T1 seeds (they called their start material as T1). Then grew them into T1 plants. Most of T1 putative transgenic plants would be chimeric. Then they collected T2 seeds from the T1 plants which gone through kan selection. Again, they grew T2 in selection medium and grew the survived ones into T2 plants. Then, they collected T3 seeds from the selection-survived individual T2 plant. They used T3 seed population from each T2 plant for 3:1 ratio analysis (kan resistant). Since the GOI has passed through 2 generations (from T1 to T3), some GOI did go through germ line cells and transmitted to next generation. These lines are *homogeneous* transgenic lines.

Hafiz Muhammad Rizwan

Organogenesis vs. (somatic) embryogenesis:

• In "in-planta" transformation case, your bona fide transgenic line is derived from a single transgenic cell (harboring your GOI) and grow into a plantlet through *organogenesis*. Organogenesis is the process by which new organs and even whole plants are usually formed in response to wounds from other parts of the organs. The photo is taken from your another RG discussion (your in planta transformation), an example of organogenesis.
• For transgenic lines regenerated through callus somatic embryos, single transgenic somatic cell (harbor your GOI) will keep dividing and grow into a whole plant. So, each cell of the plant should contain the GOI copy, too. In somatic embryogenesis, first, a structural cell (*somatic embryo*, not go through sexual fertilization ) similar to *zygotic embryos* (go through sexual fertilization) is formed, and then the entire plant is regenerated

Hafiz Muhammad Rizwan

If use the method described in the published paper ("Arabidopsis seed transformation ....."), then you have to. The paper was published in 1987 (only 4 years after the very first transgenic plant -tobacco- was published in Nature (May, 2013) [1]). They used nptII gene as both selectable marker gene (SMG) and GOI. At the time the nptII gene (kanamycin-resistant gene) was quite popular. The gus gene or other "screenable genes" were not available at the time yet, I guess (?). Otherwise, they could have seen GUS blue spots scattering around in the seeds (their transformation starting material).

[1]https://www.nature.com/articles/497040a#:~:text=In%20May%201983%2C%20the%20Van,first%20transgenic%20plants%20became%20fact.

As for when the gus gene was first used in plant transformation (mentioned in my prior answer), I checked:

1987 Creation of the GUS reporter system by Richard Jefferson and Mike Bevan. The paper describing their work is now the most highly-cited plant transformation paper to date [1].

[1] https://bbsrc.ukri.org/documents/plant-transformation-pdf/#:~:text=1987%3A%20Creation%20of%20the%20GUS,included%20the%20GUS%20reporter%20gene.

Hafiz Muhammad Rizwan

1. (Case 1) You transform your Passion Fruit seeds. Then, let the seed grows into a plant (chimeric). Then let it go through a few generations to let the GOI passes through germline. But, this is very impractical for your woody Passion Fruit plant. As a student or postdoc, you just don't have time to wait for that. It is practical for Arabidopsis, because of its short life cycle. For the 'organogenesis' scenario, you can only have transgenic shoots growing out the transformed spots/cells on the seed when organogenesis occurs. But, do you know if organogenesis occurs on seeds? Or, specifically on Passion Fruit seeds?? Organogenesis usually occur at 'wounded' sites of a plant. Will seed transformation cause 'wounds' on seeds and induce organogenesis process?

By the way, what is the seed size of your Passion Fruit variety? Just curious.

2. (Case 2) The seed is just like a piece of 'explant'. You can do transformation on it--> Agrobacterium-mediated in your case; then let the seed can develop into an antibiotic-resistant callus; then regenerate transgenic lines from those callus. But, I don't know why you want to use *seed* as an explant? Why don't you use other tissues as explants? Is Passion Fruit seed easily to induce callus?? Different plant species, they have different preferred tissues for inducing calli. for example, in my experience, carrot roots are easier for inducing callus. But, for cotton, we use hypocotyl for callus induction. Which tissue is easier for Passion Fruit to induce calli?

3. (Case 3) Is using the in planta (tissue-culturing free) transformation method (see picture in my previous answer ). You have already established this method in your lab for rapidly passion fruit genetic transformation. This is the best method, in my opinion. Why do you bother to change another method?

Hafiz Muhammad Rizwan

1.

Is this in general, or for passion fruit?

2. < sow into pot media and after the seedlings were with 4-6 leaves did GUS staining so they show GUS blue color ...>

That is interesting. But, we don't know if it is a homogenous transgenic plant? What is the pattern of GUS blue staining? Are they uniformly stained blue, like for a whole leave, a whole stem...etc? How many plants derived from seeds did you test? Did they all stain blue? Can we see a picture?

Hafiz Muhammad Rizwan

I was taught to make the dipping solution with suspended bacteria fresh each time. The initial Arabidopsis dip protocol found that you can get successful transformation with cultures grown different lengths of time so that would be one way to be able to dip on many days in a row.