Plant Regeneration - Science topic

I would suggest you study the following article

Dear @Reza Ghahremani

Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded successfully to the Somatic Embryogenesis induction using different explants, conditions, and concentrations of PGR. The details can be accessed at:

Dear Swati,

Please take a look at these 2 interesting papers attached. I hope it will be useful for the discussion.

Polyethylene glycol and abscisic acid improve maturation and regeneration of Panax ginseng somatic embryos

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Best regards,

Jean

we normally use Ck and auxin,(or 2iP) but it may be very basic

Dear @Amina Ilyas You may find a detailed discussion related to your query at the link given below as to why 70% ethyl alcohol is used for surface sterilization:

I would like to add that I am fully convinced with the explanation by @Yuan-Yeu Yau on that thread.

Tween 80. Maybe Triton-X-100. Especially Tween 80

Michel Branchard you can scan the two papers into electronic files first. Then you upload the files into ResearchGate.

For hairy root transformation after co-cultivation you just go for MS+2,4 D (0.5 mg/ml) in less concentration or u can use IAA in 2-4 mg/ml.

Very good and true remark! Working in biotechnologies I’ve often encountered the same problem, i.e. the impossibility of obtaining reproducible results of my own experiments, carried on by the same people, keeping strictly the same process conditions and quantities of materials taken with high precision. Furthermore, duplicate or triplicate samples in simultaneous experiments give very different results. This has happened to me since I work with biochemical, fermentation processes, involving living matter, bacteria, plant and animal biomass, active sludge. For comparison, when I worked with chemical processes (synthesis), the repetability and reproducibility of the results was very high. My explanation is that the biochemical processes are much more complex, dynamic, rather unpredictable, and the biological factor is responsible for the poor reproducibility. Bacteria and living matter generally work by complex mechanisms that involve very different biochemical pathways, specific enzyme and high instability even for the same conversion process. Often it is not possible to know exactly which product will be obtained, on what biochemical pathway, under what environmental conditions and at what time in two identical experiments involving bacterial metabolism. Although as a whole, even in detail, science has made tremendous progress, it seems that living matter has its strict information that does not allow us to fully control it. This is an explanation in the lack of explanation...

A lot of researchers have used AGL1 for genetic transformation on different plant species. Plants regenerated from calli with erratic phenotype are not necessary due to the Agrobacterium per se. The erratic phenotype or regenerants could be raised from tissue culturing practicing, or due to somaclonal variation.

You may find useful these references:

However, epigenetic remodelling is not something you can manipulate at will, it depends on the cell developmental state, the tissue type, the environmental conditions, and the hormonal stimuli present.

My advice is look for available regeneration protocols for close relatives of the species you want to regenerate and test how these work on your case.

Most trees, including conifers, have extremely large genomes with high duplication and tend to have a large number of transposable elements of various classes. It may be hard to promote plant embryogenesis from callus, and sometimes direct organogenesis is more effective.

An example:

The transformed rice seeds having kanamycin resistant gene as selection marker can produce albino plant in vitro. This is not unusual as kanamycin at particular concentration blocks ribosomal RNA synthesis in plastids resulting in no synthesis of chlorophyll.

Dear Camila

Please refer to the following links and articles about the protoplast culture and plant regeneration in woody trees.

Are those cells dead?? You said you cultured it in MS "basal' medium. Does this MS basal powder you used to make medium contains other important ingredients such as vitamins...etc? There are many types of MS powder mix sold from companies. Some contains only macro-elements, some contains both macro-elements and vitamins,......etc.

Yes, I have done that on tobacco. See attached paper. Pictures of the results from different steps are shown in p.160 of the paper. However, I have not tried that on cotton. You can use the paper as your reference.

Thanks Andrew

plant regeneration efficiency. Furthermore, is there any reliable method to distinguish homozygote regenerants from the heterozygotes in pea.

Not aware of anything from the Ghats (not my region) but these may help:

Balch, J.K., D.C. Nepstad, L.M. Curran, P.M. Brando, O. Portelab, P. Guilhermeg, J.D. Reuning-Scherera, and O. de Carvalho Jr. 2011. Size, species, and fire behaviour predict tree and liana mortality from experimental burns in the Brazilian Amazon. Forest Ecology and Management 261: 68–77.

Cochrane, M.A. 2003. Fire science for rainforests. Nature 421: 913-919.

Cochrane, M.A., and M.D. Schulze. 1999. Fire as a recurrent event in tropical forests of the eastern Amazon: effects on forest structure, biomass, and species composition. Biotropica 31: 2–16.

Kinnaird, M.F., and T.G. O’Brien. 1998. Ecological effects of wildfire on lowland rainforest in Sumatra. Conservation Biology 12: 954-956.

Uhl, C., and J.B. Kauffman. 1990. Deforestation, fire susceptibility, and potential tree responses to fire in the eastern Amazon. Ecology 71: 437–449.

I would be keen to hear if you find anything more relevant.

Dear Dr. Kaushal

You can use Potassium nitrate or Ammonium sulfate for callus regeneration.

Thank You.

To prevent green coloration you may change the photoperiod (increase the dark cycle). Variations in NAA/BA combinations may also provide desired results.

Dear Hawer,

From root explant follow organogenesis protocol in tissue culture then you can get some callus and from there you can able to get plantlets. If you will clear about genus name it will be easy to help you.

Regards,

Aloka

Yes, somatic embryos have less genetic variability compared with the regenerants from calluses. Somaclonal variation depends upon the concentration of auxin particularly 2,4-D. If plants are not produced from a callus after 4-6 months, attempts should not be made to regenerate plants and new cultures should be initiated.

Please read this paper:

Singh, R. J., T. M. Klein, C. J. Mauvais, S. Knowlton, T. Hymowitz, and C. M. Kostow. 1998. Cytological characterization of the transgenic soybean. Theor. Appl. Genet. 96: 319 - 324.

Three good textbooks about plantations, mainly in the tropics, but they do not refer to Populus euphratica:-

Evans, J. 1992. Plantation forestry in the tropics. 2nd edition. Clarendon Press, Oxford, UK. 403 p.

Garforth, M. and Mayers, J. (Editors). 2005. Plantations, privatization, poverty and power. Earthscan Forestry Library, London, UK. 294 p.

Susuki, K. et al. (Editors). 2006. Plantation technology in tropical forest science. Springer, Tokyo, Japan. 292 p.

In the late last century and early 2000s major reports on plantations were produced by FAO, Rome, Italy, especially authored by Jim Ball and Jim Carlyle. These may be available online. The Forest Research institute in Izmit, Turkey (formerly the Poplar research Institute) may have written on P euphratica.

I hope this helps somewhat.

Regards, Jeff Burley

The plant in the picture is undergoing necrosis. Necrosis can sometimes occur spontaneously in vitro as a consequence of stress (sometimes even transferring your plants to fresh medium can cause stress), but it can be also due to medium composition; please check if the composition of your cultivation medium is appropriate, also check its other properties, like pH.

If this is the first time that you're trying to cultivate that plant in vitro, you should look for optimization of the medium composition. If you have done that before and had no necrosis, then just forget about this plant and look for healthy, viable explants.

When studying prescribed fire, you have the added benefit of establishing permanent plots prior to the burn which you can sample both before and repeatedly after the fire. Pre-burn conditions that can be important to vegetation recovery and tree regeneration include the presence of plant species that can recover vegetatively (a strong determinant of what comes back!), depth of the litter layer (if any, which can determine local mortality due to fire intensity or smoldering), and local crown closure or canopy cover (e.g., measured using hemispherical photography or a spherical densiometer). I hadn't thought of sampling the seed bank, as suggested by Mathiventhan, but that is a good idea and could be important in some systems too.

My experience is in the Pinus contorta forests of western Canada, where vegetation responses and forest recovery can be very patchy, especially under a mixed fire regime or the relatively cool conditions that managers are comfortable with for a prescribed burn. So make sure you have lots of sample plots and be prepared to stratify your results by pre-existing vegetation (you mention a forest-grassland mosaic) and local burn severity.

Yes...What do you want? some hairy roots really sprout shoots. I dont care, as I can use the shoots for transformation. But if one is only after those shoots, are they transformed too and perhaps root easier later on?

I would suggest callusing in vitro using cytokinins and auxins with subsequent transfer of obtained calli into the fresh liquid medium in e.g. bioreactor systems such as temporary immersion ones.

There is no report available on micropropagation study on Clinacanthus nutans for more information see link