Dear Ms. April DeMell,

There is plenty of literature regarding stress studies and metabolic engineering in N.benthamiana, but they do not require optimal growth exclusively.

Optimal growth on soil is under short day ( 8h light/16h dark), light intensity 80–100 μmol/m2s− 1, 21 °C during day and 16 °C during night and around 60% humidity.

Keep in mind that N. benthamiana can also grow under long-day conditions and higher temperatures, but leaf plastids would accumulate more starch.

One can use different developmental stages for infiltration, from seedlings to mature flowering plants, but leaves from young plants (6-8 weeks old) result in higher transformation rates. You could use them for in vitro reproduction inducing a callus.

After transformation increase the humidity to 70%.

I used to cover them with a transparent plastic bag spread with water to prevent contamination and drying.

Here is one possible reference, but there could easily be more recent

ones:

Article Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana)

Here is also one article you might find interesting:

Article Improving agroinfiltration-based transient gene expression i...

I hope it helps

Hi,

I guess when you are saying in the first case is 16 light and 8 dark...

I would suggest you to start with a 12/12 cycle at the beginning (week 1-2) and then switch to the 16/8 (week 3-4).

However, if you can find some previous paper specific for your ATTA experiment that might be the best for your specific case.

Please have a look at the following material:

https://www.sciencedirect.com/science/article/abs/pii/S0981942818304133

https://www.nature.com/articles/366469a0

Article Genome and transcriptome characterization of the glycoengine...

Article Production of santalenes and bergamotene in Nicotiana tabacum plants

Please get the light intensity and spectrum as close as possible to full sunlight as possible during the day

Used to transform Nicotiana tabacum with Agro. And other plants too. The most elegant method to inject it is in leaves or cotyledons even of in vitro growing seedlings.

UPDATE: Based on answers from Nicolò Maria Villa and Francesco Desiderio I tried a 12/12 hr light dark cycle, 25C, and 60% humidity, and 100 umol of light. Within a day the plant leaves were pointing straight upward with unusually long stems, turned yellow-ish, and seem more flexible and more spindle-like (see attached picture). I increased the light to 150 umol, then to 170 umol, both with no change. From other suggestions, I will try to increase the light to 190 umol. Maybe the plants were just too stressed to recover?

This phenotype is not necessarily associated with etiolation, therefore doubling the suggested light intensity might result in worsening their conditions.

As I wrote, it would be optimal to keep a short-day cycle ( 8h light/16h dark), 21 °C during day and 16 °C during night.

Under long-day conditions and higher temperatures N. benthamiana accumulates more starch in the form of amyloplasts (which appear creamy white, thus paler) and the leaves could change axillary angle to reduce the exposure to light.

This is a possible explanation, but more details would be useful:

What kind of illumination are you using?

Why are you keeping the temperature at 25°C?

Are you lowering the temperature at night?

What are the time periods of the previous treatment and the current one?

Have you applied any product that could have caused stress, for instance anything containing growth regulators?

Nicolò Maria Villa

Using LED lights

16hr light/8hr dark day length

25C day/22C night temp

50% humidity

120 umol daylight, 0 umol night

Not adding anything that could cause stress, only 20-20-20 fertilizer (the same frequency and concentration we've been using successfully for years)

Our department just got new growth chambers, and the old ones (which used florescent bulbs) were always fine at a long day cycle (16h light/8hr dark), 25C day, 21C night, 60% humidity, and 150 umol of light. Since we got new growth chambers (that use LED lights), is when we started having the issues.

Based on your suggestions, I think I'll try a short-day cycle ( 8h light/16h dark), 21 °C during day and 16 °C .

I appreciate the help!

April DeMell

Hi,

N. benthamiana can easily grow in 14h light/ 10 hr dark photoperiod with usual fluorescent light intensity, 50-60% humidity and 22-23 degree centigrade temperature. What I suggest is better you can use autoclaved soil rite and wet is and put the N. benthamiana seeds on it (don’t cover seeds with soil) and cover it with cling-wrap till germination. After that just watering them with half strength of Hoagland solution with 2-3 days interval, your plants will be good for sure.

Thanks

SOLVED! Thank you to everyone for all your help!

We recently got new growth chambers, switching from fluorescent to LED light. For plants, you need a white LED light (contains most of the light spectrum). I found out that our growth chamber manager also added a large number of far red light (which, in plants, stimulates a shade avoidance response) which is why my plants looked like they weren't getting enough light. I had these far red lights removed, and the plants look great now!

I wish I had something that could measure the wavelengths of light from the growth chamber! I know tools exist to do this, I just don't have anything nor does anyone I know.

I also changed to a short day cycle, per @Nicolò Maria Villa 's suggestion, which made my plants look greener and much healthier, as suggested in the responses.

The following are the conditions that are now working great:

Using LED lights (full spectrum white lights)

16hr light/8hr dark (short day cycle)

25C day/22C night temp

55% humidity

150 umol daylight, 0 umol night

20-20-20 fertilizer once per week to older plants

Planting in non-autoclaved metro-mix soil, placing seeds on top (not covering them with soil), then leaving them covered with a plastic humidity dome for 2 weeks.

Nicotiana benthamiana (colloquially known as benth or benthi) is a close relative of tobacco and species of Nicotiana indigenous to Australia . The plant was used by people of Australia as a stimulant, containing nicotine and other alkaloids, before the introduction of commercial tobacco (N. tabacum and N. rustica). Indigenous names for it include tjuntiwari and muntju. It was first collected on the north coast of Australia by Benjamin Bynoe on a voyage of HMS Beagle in 1837