Micropropagation with LEDs / Tissue culture with LEDs / Micropropagação com LEDs

I have a lot to tell you about LEDs, plant growth and available options.

On my next post i will extensivelly reveal my thoughs after 5 years steadily searching for all LED models, and respective technical features, with wavelenghts capable of estimulating growth or other physiological responses.

I leave you with an image teaser

like they say in Hungary

Sziia!...

Phormium tenax in vitro

 Fase 1 – Establishment. Cultures have been established of Phormium tenax in vitro from seed and a new method for induction of high germination rates has been discovered. For in vitro propagation seeds need to be isolated from desinfected unopened seed pods  and inoculated in Antifungal containing medium, after mild desinfection. Establishment of healthy cultures has been achieved but inicial trials, left only few live growing material , nevertheless   new trials from 2010 summer seeds will bring abundant cultures since media and growth regulator concentrations have recently been adjusted with success.

Fase 2 – Propagation. Like for the establishment fase, significant progress has been achieved with Phormium tenax proliferation. Moreover our cultures show no loss of the varieagated properties of the mother plant, wich has been mentioned as being a bottleneck for in vitro propagation of the variegated forms of phormium. Growth regulator inbalance is still in test for even better growth rates.

Petunias in vitro

Fase 1 – Establishment. Three diferent varietys of Petunias have be established from seed  in Vitro at Peak Plants tissue culture laboratory, namingly, Petunia grandiflora, Petunia pendula and P. pendula f1 hybrid cv “reflexions”.- Seeds were carefully desinfected with a bleach solution and inoculated in a mixture of half strenght MS and WPM medium. Adition of small concentrations of certain growth regulators in the medium allows secondary embryogenisis to occur and several plantlets can be obtain from a single seed.  Besides this some callus masses form in certain cases wich can be recultured in similar medium allowing some organogenisis to occur but vitrification becomes a problem for most of the plantlets formed by this process. 

Fase 2 – Propagation. Propagation of Petunia after establishment is easy nevertheless certain phisiological problems arise from species with such a high growth rates, being vitrification and whitening of explants the most serious ones. In the case of Petunia little whitening has occured so far, but vitrification is in some cases observed. This problem has been minimised so far by adusting agar oncentration and adding activated charcoal, being the last option the most efective to reduce the phenomena. – Having started from seed material a long intensive cataloging of the cultures is necessary to allow separate cultures to be obtained and ultimatelly, even the selection of chimaeral individuals for market inovation.

Ilex aquifolium In vitro

Fase 1 – Initiation. Ilex aquifolium cultures have been  established in vitro at the  Peak Plants laboratory following the method described by Majada et al. Briefely the method consists in isolating and germinating zigotic embrios, from previously desinfected  Ilex seeds and germinating them in vitro.

There is high seasonal variation in embrio response and  it took more than a year of trials to obtain the first plantlets after undreds of inoculations.

By this methods Peak Plants has, currently multiplying cultures of an endemic Ilex and a variegated form  in a total of about 25 genotipes. The foto displayed here refers to the conversion of the first embryos into plantlets. Our Ilex cultures are currently growing at an outstanding rate  using polypropilene boxes from Globalplas  company.

Fase 2 – Propagation. Ilex cultures display good growth in vitro but genotype diferences can be observed concerning medium adaptation. Tipically genotypes displaying secondary embryogenisis from zigotic embryos have a better after development and growth. It is necessary to make individual salt test trials with this species. Also temporary liquid culture imersion medium is in the agenda for testing, Ilex aquifolium produced in vitro by this method, has high potencial for ornamental purposes due to higher branching of young  plantlets.

Dianthus In vitro

Fase 1 and 2 – The establishment fase and culture medium used for Dianthus has been very similar  to the ones used for Petunias, in fact  the first trials with these herbaceous species were taken using Dianthus seeds. The cultures have very similar needs but besides vitrification Dianthus has also a tendency for whitening after the third week of culture. All other phenomena like callus production,and secondary embriogenesis, follows a very similar pattern for both species.
We have currently 3 species of dianthus growing in vitro in our  culture room, D caryophilus , D. barbatus  and D. chinensis cv Chianty. The next step with these cultures will be adjustment of salt concentrations to allow longer periods before medium transfer operations, and  better overall robustness of explants since propagation rates are more than acceptable. 

Rifampicin, Olea europaea and the establishment in vitro of Olea europaea explants

First of all i must appologise for the eventual dificulties in following the way i have been describing the evolution of my work in the past year. As you know this is a blog of cientific caracter but the way the information is displayed follows a strict cronology, based on the posting order. I cannot change that so i sugest that you read prevoius posts if you fell a bit lost.
My goal concerning Rifampicin, was to contextualize my beliefs about this antibiotic, with a series of curious observations in a way that i could make clear that it's use seems to be only interesting, if combined with other antibiotics or in the case where we want to quickly recover chlorotic material.
Some facts:
I have dealted with field collected explants of Cordovil, Gelega and Madural for 2 years and with established but   endogenously contaminated by bacterium, explants of Galega.

Rifampicine used alone was never clearly effective eliminating bacteria from Olea europaea explants.

Absence of Rifampicin in the culture medium of Olea europaea cv galega explants, regularly exposed to it, caused a marked yellowing of the leafs, from wich explants would recover after 4 to 6 weeks even without the antibiotic.

The recover of explants treated with antibiotics such as PPM or the antifungal preparation described on my last post is much quicker and healthyer with the presence of Rifampicin.

The use of PPM and FLU&CYP  preparations (i will use this expression "flu&cyp" for cetone based solution of cyprodinil and fludioxonil) causes yellowing and sometimes leaf fall.

The joint use of Rifampicin with any of these antibiotics reduces or eliminates theyr toxicity.

From the sentences above wich are well based on repeated observations i am forced to believe that Rifampicine has promissing potencial in the establishment of Olea europaea explants. How? Allowing a reduced impact of some very effective antibiotics on the explants health during establishment fase.

Toxicity of the Fludioxonil and Cyprodinil preparation

Like i said, the usage of this preparation follows a protocol wich i have developed after consecutive trials with newly inoculated explants, as well as, after experimenting a little with non contaminated explants to evaluate plant toxicity.
Acute toxicity seems to affect only very small and sensitive portions of tissue or explants, but so far i have only exposed, Ilex , Gardenia, Rose, Petunia and Dianthus to these substances. Asside from that there is the question of the solvent wich is Cetone, and this product by itself might carry some harmfull properties to the culture medium. My conslusion for Olea europaea was that the toxicity afects only uninodal explants in my most comon concentration of 1ml of the concentrate per liter of culture medium. Explants rescued from the treatment 2 or 3 weeks after inoculation, can be recovered clear of fungus except some white moulds, specially filamentous ones ( similar to cotton candy).

Here we see explants tretaed with cyp.& flu. antifungal preparation.
Rifampicin here like with PPM seems to greatly reduce the chlorosis phenomena on the treated explants. 

Chemical war on fungus ( the use of a new antifungal preparation against fungal tissue culture contamination )

The use of PPM in the culture medium against the fungal contaminations was inefective.

Contaminations were coming back after 3 weeks of culture, and the subsequent fungal colonies seemed to be unafected by PPM dosage, wich was about 2ml per liter of culture medium.

I had still my secret weapon to test against this problem.

Like i described before, it consists of extracting Cyprodinil and Fludioxonil from a comercial phytosanitary product. Please be so kind to ask for detailed process of elaboration of the solution....I have briefly described it in a previous post.

So what are these antifungal agents?
Fludioxonil is an analogue synthetic molecule based on the natural compound pyrrolnitrin produced by the soil-borne bacterium Pseudomonas pyrrocinia. Chemists had to find a similar molecule with a more stable behaviougr since pyrrolnitrin was very sensitive to light. Find more here: https://docs.google.com/viewer?a=v&q=cache:CKjtrEL0blYJ:www.farmassist.com/postharvest/images/GraduateTechBroFinal.pdf+fludioxonil+pseudomonas+pyrrocinia&hl=en&pid=bl&srcid=ADGEESiM6VmOAKqhw5soFVwAQTlEfK4E8t2iNTxtVYs1hi0F4SyVQA9zsFMVaLk7Cri-5crbTtqXmTvsDpt9JhzN2kFVO9-ePR1EOOtDK123bEfW1ypP343W_tTMSYwvTO9JOfAAWVE_&sig=AHIEtbSwxwnu6J9lXOOJeWm56ghW_oWj3w Fludioxonil is considered a low human risk compound.

Cyprodinil is a synthetic compound discovered when analising the anti-fungal activity of a range of sulfonylureas. It is a systemic substance with a broad spectrum activity for a number of plant diseases, including Botrytis cinarea. Find more here:http://pubs.acs.org/doi/abs/10.1021/bk-1998-0686.ch024
In terms of safety Cyprodinil is untill further notice a relatively safe substance to humans, and generally safe for nature. Its most acute toxicity is by contact and inalation. Persistence in the soil has been demonstrated but mobility is very low. The most harmfull consequences for cyprodinil are for aquatic ecossystems, having very high impacts even in small dosis, nevertheless its poor mobility makes is reasonably safe as long as simple precautions are taken.