Fellow Plant Scientists,
Many plant labs use sucrose in sterile media to ensure optimum conditions for the plant. Adding sucrose helps to normalize and somewhat synchronize the plants' growth, enabling germination, small deviations in measurements (root length, growth rate, among others), and repeatable results. However, sucrose is itself a signal molecule and so may affect the severity or hide specific phenotypes. However, in 1/2 MS salt, 0.5% phytagel, pH 5.8, without sucrose, the germination of WT (Col-0) seems to be random, as well as the growth rate. For example, for the WT seeds that germinate at the same time, some plants grow as though sucrose is in the media, while others are stuck at a late cotyledons stage. Other adjacent labs observe the same phenomenon. All of this seedlings access to the same amount of nutrients, light, and space, so why the disparity in growth rate and development? Is there a minimal amount of sucrose needed in the media to decrease this behavior, while also having a minimum sucrose signaling effect? Better yet, is there a minimum amount of sucrose in the media that is accepted in the literature as "no sucrose" or "low interference"?
Thanks for "listening."
–Thomas Payne
Extended Question:
Thank you all for your answers. To reiterate the question: 0% Sucrose to the media causes germination and growth rate fluctuations and even delay in development, i.e., stuck in the cotyledon stage for a long time, while adjacent plants grow just fine. These growth disparities seemingly are randomly distributed across experiment and allele. Since plants do not have a sucrose supply in the soil, why are these disparities absent in seedlings when grown in soil? Furthermore, sucrose is a signaling molecule, and thus using sucrose in the media may alter growth, physiological response, biochemical pathways, gene expression patterns, etc. Let us consider a plant with a chloroplast deficiency of some kind, in a way where the plant does produce WT levels of sucrose. Thus, adding sucrose to the media may mask a phenotype, is there an amount of sucrose in the media that is accepted in the literature that helps with germination but also does not mask phenotypes helped by sucrose or influence sucrose like effects such as a change in gene expression?
Another way of putting it, is there a media that best mimics soil conditions i.e, similar growth development, transcriptome, proteome, etc? Obviously, transparent media, allows the roots to be exposed to light, thus changing many of the phenotypes listed. Nevertheless, wouldn't a media that closely mimics soil conditions be beneficial to studying plants in the lab? This may have been a bit of a digression...
Melanie Schulz, I only used seeds from the same harvest period, and all my seeds are > 300 um sieve pore size, which ensures healthy seeds and normalizes the seeds across alleles (for the most part).
Hi Thomas,
for the standard MS-medium in our lab we use 10 g sucrose per liter and our Col-0 seeds germinate just fine. Were the seeds you used all from the same batch? Germination rate is also linked to the seed quality, which can be influenced by age and storage conditions.
Cheers and good luck,
Melanie
For us, it depends a lot on the photoperiod.
1.5% sucrose m/v (15 g/Liter) is ideal for continuous light (i.e. without night) and 0.5% is ideal for long days 16h day / 8h night.
In long days, 1.5% sucrose results in pretty high anthocyanin in Col-0 (plants are very purple and growth is affected).
In continuous light Col-0 grows faster with 1.5% than 0.5% sucrose.
We use ES media, but we did not see much difference with MS/2.
There is clearly a difference between ecotypes, so if you do not work with Col-0, you might want to have a look to this paper:
Teng et al., Plant Physiology, December 2005, Vol. 139, pp. 1840–1852
Thank you all for your answers. To reiterate the question: 0% Sucrose to the media causes germination and growth rate fluctuations and even delay in development, i.e., stuck in the cotyledon stage for a long time, while adjacent plants grow just fine. These growth disparities seemingly are randomly distributed across experiment and allele. Since plants do not have a sucrose supply in the soil, why are these disparities absent in seedlings when grown in soil? Furthermore, sucrose is a signaling molecule, and thus using sucrose in the media may alter growth, physiological response, biochemical pathways, gene expression patterns, etc. Let us consider a plant with a chloroplast deficiency of some kind, in a way where the plant does produce WT levels of sucrose. Thus, adding sucrose to the media may mask a phenotype, is there an amount of sucrose in the media that is accepted in the literature that helps with germination but also does not mask phenotypes helped by sucrose or influence sucrose like effects such as a change in gene expression?
Melanie Schulz, yeah I only used seeds from the same harvest date, and all my seeds are > 300 um sieve pore size, which ensures healthy seeds and normalizes the seeds across alleles (for the most part).
I use 20g/litre when I work with solid medium. However, I use 30g/ litre when I use liquid medium and they just work fine for me.
I use 20g/litre when I work with solid medium. However, I use 30g/litre when I use liquid medium and they just work fine for me.
You might want to check these papers:
Article An improved agar-plate method for studying root growth and r...
And please find a paper by Wells et al. 2005 attached to this message.
Cheers, Melanie
I found that 0.1% Sucrose is sufficient for 100% germination but also has a lower sucrose response. i.e. 0.1% sucrose has longer hypocotyls than 1% sucrose during etiolation. Thanks for all your answers, especially Melanie Ringer for her detailed post and providing papers.
For us, it depends a lot on the photoperiod.
1.5% sucrose m/v (15 g/Liter) is ideal for continuous light (i.e. without night) and 0.5% is ideal for long days 16h day / 8h night.
In long days, 1.5% sucrose results in pretty high anthocyanin in Col-0 (plants are very purple and growth is affected).
In continuous light Col-0 grows faster with 1.5% than 0.5% sucrose.
We use ES media, but we did not see much difference with MS/2.
There is clearly a difference between ecotypes
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