Hello Brother. Give treatments of 0.5 % Bavistin, 0.5% streptomycin sulfate, and finally 0.1 % Mercuric chloride or sodium hypochloride to avoid contamination's. For propagation of the explant, try MS media without phytohormones till you are getting good number of cultures, then you can use different combinations of the phytohormones in different concentrations. Inform me about your results, I'll help you further, if needed. Adieu.

I used H2O2 at 1m/L it should be filter sterilized and add to the medium after autoclave I think this will overcome the contamination without any damage to the explants. This treatment was very effective for different species.

Please see the follwing manuscript

may decrease the cytokin not more than 1.0 mg/l

Shatnawi, M. A. 2013. Multiplication and cryopreservation of Yarrow (Achillea millefolium L., Astraceae). Journal of Agriculture Science and Technology. 15: 163-173.

Shibli, R. A., Duwayri, M. A., Sawwan J. S., Shatnawi., M. A and Al-Qudah T. S. 2012. Regeneration via somatic embryogenesis of the endangered wild arum (Arum palaestinum). In Vitro Cellular and Developmental Biology-Plant. 48: 335-3340.

May be this help you;

Faisal et al. (2005) investigated thidiazuron (TDZ) effect on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. They found The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5 µM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks.

Faisal, M., Ahmed, N., Anis, M. (2005). Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron. Plant Cell, Tissue and Organ Culture 80: 187–190.

you can use fungicide like bavistin ,mercuric chloride to avoid contamination. you can use different combination of phytohormones with coconut water and adinine sulphate 50mg/L for mulitiple shoot.

I recommend you to use TDZ instead of BAP as Dr. Kaya wrote. In the initiation, I hope that you are putting the medium to rest at least for three days after added the coconut water by filtration, if not do it....... if yes, then try to elongate the shoot from the donor plant to avoid any endogenous contaminants and use 1.5% sodium hypochlorite for 10 minutes; good luck in your future work.

Hi bro. May I know your sterilization method, please? I suggest you to try TDZ or KIN in concentration bêtween 0.5 till 1 mg/l.

Hello friend, you should follow a standard disinfested procedure. In case of vegetative plant part, it better to use Na(OCl) @1.5% chlorine water, soaked for 10 min (three times), three times washing with DDH2O. Try with 2-4 mg/l BAP or 6-10 mg/l Kn with 1-2 mg.l IAA.

First of all you have to check reason for contamination weather it is endophyte or surface sterilization problem if it is endophytic contamination then use some antibiotic agents along with medium.

Here are some useful references which will helps you if you need any of the reference then just inform me

1. Altan F, Bürün B, Sahin N. 2012. Fungal contaminants observed during micropropagation of Lilium candidum L. and the effect of chemotherapeutic substances applied after sterilization. African Journal of Biotechnology, 9: 991-995.

2. Cardoso JC, Teixeira da Silva JA. 2012. Micropropagation of gerbera using chlorine dioxide (ClO 2) to sterilize the culture medium. In Vitro Cellular & Developmental Biology-Plant: 1-7.

3. Cassells AC, Doyle BM. 2006. Pathogen and biological contamination management. Plant Cell Culture Protocols, 318: 35.

4. Fang J-Y, Hsu Y-R. 2012. Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell, Tissue and Organ Culture: 1-10.

5. Grondeau C, Samson R, Sands DD. 1994. A review of thermotherapy to free plant materials from pathogens, especially seeds from bacteria. Critical Reviews in Plant Sciences, 13: 57-75.

6. Langens-Gerrits M, Albers M, De Klerk G-J. 1998. Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant cell, tissue and organ culture, 52: 75-77.

7. Leifert C, Cassells A. 2001. Microbial hazards in plant tissue and cell cultures. In Vitro Cellular & Developmental Biology-Plant, 37: 133-138.

8. Leifert C, Woodward S. 1998. Laboratory contamination management: the requirement for microbiological quality assurance. Plant cell, tissue and organ culture, 52: 83-88.

9. Msogoya T, Kanyagha H, Mutigitu J, Mamiro MKD. Identification and management of microbial contaminants of banana in vitro cultures. Journal of Applied Biosciences, 55: 3987-3994.

10. Reed BM, Buckley PM, DeWilde TN. 1995. Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cellular & Developmental Biology-Plant, 31: 53-57.

11. Reed BM, Mentzer J, Tanprasert P, Yu X. 1998. Internal bacterial contamination of micropropogated hazelnut: identification and antibiotic treatment. Plant cell, tissue and organ culture, 52: 67-70.

12. Thomas P. 2004. A three-step screening procedure for detection of covert and endophytic bacteria in plant. Current Science, 87.

13. Thomas P, Aswath C. Alcohol-Mediated Horizontal Spread of Bacillus Spores and Assessing the Recurrent Sterilization Needs of Culture-Handling Tools Contaminated with Hardy Spores. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences: 1-7.

14. Thomas P, Reddy KM. 2013. Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana. AoB Plants.

15. Thomas P, Sekhar A, Mujawar M. 2012. Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spr...

what we normally use for multiplication of woody crops in our lab is application of 4.4 uM BAP and 0.5um of IBA and it really works. I strongly suggest you to replace NAA with IBA, since NAA is very stable and its efficiency is about 10 time more than IBA. therefor I think when you use NAA, the ratio of CKs/Aux doesn't reach the amount that is required for proper multiplication of the shoots.

with these concentration I mentioned earlier, you will end up with cluster of 3-7 multiplicated shoots per each explant, but if you are interested in having more shoot you can increase the amount of BAP up to 40 uM. this gives you bushy clusters consisting of 20 and more short shoots. about contamination I am agree with Mr Shahzad Pandith.

Good Luck :)

There are already many suggestions forgetting contamination free cultures. My suggestion regarding shoot multiplication is transfer at least 3 subsequent passage on the same high cytokinin medium and wait and watch the cultures. Impatient does not give results as you go on changing the medium frequently.

For Tetrastigma tissue cutlure and your methodology involving coconut water, it is very important to use fresh and green coconuts (from Thailand) for the coconut water as your source of cytokinins and any other useful growth promoting substances. Having coconut water (CW) from Thailand reduced this variability based on my past 10 years of testing. If you wish to have strong growth promoting effects from CW, you have to filter sterilise the CW instead of autoclaving them. The autoclaving process does breakdown the cytokinin nucleotides in the CW, but not impacting greatly the cytokinin bases and ribosides. The second consideration is to use half to one-quarter strength MS or switch entirely to the other mineral nutrient medium formulated for woody plants like WPM or even those used for orchids like Vacin and Went? Adding a "strong" synthetic cytokinin like TDZ should be able to help you to elicit shoot formation, but you may get some potential mutation/somaclonal changes? Meta-topolin, similar to BAP, may be a useful alternative aromatic cytokinin for you to try out. Lastly, it will be useful if you can use Tetrastigma seeds as the starting materials? This will greatly solved the in vivo contamination problems.

https://www.researchgate.net/publication/40768354_The_chemical_composition_and_biological_properties_of_coconut_%28Cocos_nucifera_L.%29_water?ev=prf_pub

https://www.researchgate.net/publication/225926997_A_protocol_for_in_vitro_germination_and_sustainable_growth_of_two_tropical_mistletoes?ev=prf_pub

Article The Chemical Composition and Biological Properties of Coconu...

Article A protocol for in vitro germination and sustainable growth o...

Hello there. I see there are many suggestion here for initiating an in vitro culture of Tetrasigma sp. 1st I suggest you to make sure that the sterilization of the plant material is 100% disinfected, you should try the following method: put the material under tap of water at least for 3 hours, then try to drain that and put in a sterile glass recipient with alcohol (or even 70% alcohol) only and shake it at least for 1-2 minutes, then wash it from the alcohol with sterile bi-distilled water, then add 1:2 1.5% sodium hypochlorite and then 2-3 drops of tween 20, shake it for 8-9 minutes, but be careful when it starts to get whitey in the fragmented parts or yellowish, wash it right away with bi-distilled water (this depend also on the type and variety of the plant), then more 4 times using repeated bath with bidistilled water. As plant material to initiate a culture I suggest you to use very fresh parts from the mother plant, like: buds, nodes. As medium culture, try 1\2 MS using TDZ (try to work on variants to see the differences between their reaction) between 0.5-1.5 mg/l simple or in combination with β-indolilbutiric acid (IBA) 1mg/l for rhisogenesis. It may result callus formation and from callus you could continue this subculturing them on another medium with only 1.5 mg/l β-indolilbutiric acid (IBA) on MS with 30 g sucrose or even without adding any growth regulators, try with active charcoal 3g/l. I did try many variants on Begoniaceae and even other ornamental plants (Sequoia, Aloe vera, Crasulla ovata, Rosa sp, Drosera rotundifolia, and many other ornamental's even on Canabis sativa), but it takes time till you find a right media for each plant you wish to multiply. In case something was effective and gave any positive result please let me know. Thank you and good luck. My best regards

For clonal propagation use nodal explant from 4 to 10th node which gives best response. Design experiment for screening of suitable cytokinin and its concentration , for example in general BAP and Kinetin are frequently used cytokinin, but some times 2-ip, meta topolines and tdz can be used in concentration ranges from 0.50 mg/l to 5.00 mg/l, and TDZ used as lower concentration (0.01mg/l - 0.50 mg/l) for most of dicotyledon plant. Higher concentration of TDZ resulted into vitrification. It is reported that more then 5.00 mg/l other cytokinin cause cellular stress by production of ROS. therefor optimum concentration of cytokinin requirement must be standardize. Other cytkinin such as metatoplins and its isomers are recently used by many researcher for axillary shoot multiplication and there are many review papers are available about metatopolins and tDZ . Some time combination of cytokinin can gives best results. Many researcher reported 1/10 auxin cytokinin ratio helps in axillary shoot proliferation.

Apart from cytokin vessel type glass ware (soda glass, borosilicate, pyrex etc.) or plastic ware, size of glass or plastic ware, inoculum density, closer type explant size, season orientation time of collection etc many factors play significant role for axillary shoot multiplication.

Contamination during culture initiation is basically due to lower concentration of sterilants, short exposure of explants with sterilants, and improper handeling of explants during surface disinfestatin of materials on clean bench. Revise the disinfestation process with respect to concentration of sterilants and treatment time as well as better care for handeling the materials on clean bench. The concentration od NaOCL and treatment time depends on the softness/hardness of explants. For newly developed green shoot explan trequires 0. 5 % to 1.0 % of NaOCl for 10-7 minutes and proper rinsing with strilized water (Sterilized at 121 C for one hour if voume exceeds 200 mlL.

For axillary bud break, better use BAP (0.5- 1.0 mg/l) with IBA or IAA at 0.05 or 0.1 mg/l. Additionslly PPM may be added (1 ml/l) in the initiation medium. Better to use test tube to avoid culture loss for better result.

No if you have research about fruit set in berry .tanks