I evaluate the proliferation rate when I proliferate the explants in the flow cabinet. If I work with 10 explants and I get 40 explants, the ratio is 4; I think it´sthe only way if the explants are too small and compact

Hallo Saba. I do transfer proliferation clusters to an hormon free medium and after a 2/3 weeks I am able to count the shoots all the best Claudia

I think that the proliferation rate in this case is the number of shoots or buds recovered compared to the total number of explants originally puted in cultivation

I think that all three methods presented above are good. I would focus on the first one.Do you transfer clumps of shoots ? I think, however, that you use media with too high cytokinin content. What species is it and what is the media composition, especially tellus the PGR content of the media. Email: [email protected] , [email protected]

I think it should be better to decrease PGR concentration and use robustshoots one by one.

Fresh and dry weight are criteria used to evaluate proliferation. You may also try to estiolate the proliferated shoots by growing them in the dark, in order to better visualize the shoots and buds.

Hello Saba,

You must be dividing your grown-up/mother clumps into further smaller clumps for subculturing depending upon how the shoots are joined at the basal end. Usually this gives a clear idea of how many sub-clumps you will get from a mother clump. You can think of this like taking/separating out florets from a cauliflower. Also keep a fixed number of explants cultured per vessel/container. If you are generating 10 vessels from one, you get a multiplication rate of 10 and likewise. Don't do it only on just one clump/vessel but take a good number of replicates and, to add more accuracy to it, record the data for at least a few subculture cycles and not just one.

Hope this helps!

Regards,

Sanjeev

Also you can try additional myo-inositol (50-100 mg/L) and dextrose (2g/L) to your media. I tried that with diffirent plant cultures it really increased shoot rate.

Counting initial number is Ok for multiplication rate but you can improve your medium and subculture practice to find out elongated shoots for rooting and finally number of plants produced from single clump in particular numbers of subculture. This will give you efficiency of the system.

Evaluation can be correctly done only after subculturing. Prof Dr Hafsah Jaafar, UniSZA

Do not subculture more than six times ( if multiplication is too good) as this will give poor performance after transplanting.

i believe you can make calculation using PGR and also by transferring the culture in hormone free medium, or else take help of microscope.

Is the compact growth habit is species character or due to your growth regulator effect the culture is showing such a character? Any way subculture your cultured into growth regulator free medium, observe for two weeks for more differentiation and elongation. The next option is determining the proliferation rate on fresh / dry weight basis.

You can use the bud forming capacity. And also you can calculate the total number of shoots induced per explant and the shoot induction rate. Put a range of shoot length, exp.: 1-1.5 cm = 30 shoot/explant. And the remaining shoot less than 1 cm kept in the cluster and subcultured to a fresh medium. The same step repeated many times until all shoots excluded from the cluster.

Try this article:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072594/

Shoot number per explant per cycle of multiplication and shoot fresh and dry weight shall be a better approach to evaluate efficiency of shoot multiplication. Further reducing or eliminating shoot inducing PGR shall also help in shoot elongation and make counting easier.

Good Luck

I agree that assessing number of shoots in a culture with compact growth and small leaves is a difficult exercise. But propagation is considered effective only when proper grown shoots are obtained. So the criteria could be to count such shoots after specified period, the rest may be buds? The second option (more precise) according to me is to take out representative cultures outside the culture vial and count the number using a binocular. This will also mean sacrificing few randomly selected cultures.

I agree to the opinion to subculture the cultures to growth regulator free medium. If it does not work, please shift to ' dark culture ' for some time, AND if this too does not work please supplement GA3 to the culture medium.

My method to evaluate in vitro 'multiplication rate' is that starting with the same size of explants. For example, a 2 cm of two nodal section of shoot from each genotypes are being planted in a specific medium, and after 3 weeks of subculture period, cut out as the same 2 cm of two nodal section from the shoot cultures, then transferred to the same but fresh medium for 3 weeks, and repeat this procedure once more for the stabilization. You can count the final shoot numbers (those are produced from the same size of explant). So that it can be precise measure for the multiplication rate of each genotypes or species cultured in a unique growth medium. If you use different growth media in this way, and you could also measure the nutritional "media effects" on shoot multiplication rates as well. In your case, plants with compact growth habit and small leaves, you can start with one shoot of similar sized explant, transfer as the same way, repeating this for the stabilization, the final shoot counts will be the multiplication rate of your target plant.

You need to an equipment for enlarge and photograph the explants, then you can count and measure the parameters. You can prepare inexpensive portable digital microscope with 10-200X magnification from Dino-Lite company. I think that this can be solve your problem.

The induced GA3 shoot elongation is beneficial for a successful rooting but also to effectively evaluate the shoot proliferation rate since bud formation initials occur in a compact growth habit hard to be quantitative determined. Shoot elongation also has a positive effect in further shoot acclimatization and survival in greenhouse conditions.

Check this paper:

Wochok C.S., Sluir C.J. (1980). Gibberellic acid promotes Atriplex shoot multiplication and elongation. Plant Science Letters. 17:369-369