Dear Rabiul Islam,
The method described in the following article entitled "Development of cormogenic nodules and microcorms by tissue culture, a new tool for the multiplication and genetic improvement of saffron " by Fernández and coworkers may be helpful for your research topic:
Materials and methods
Resting corms of an homogeneous cultivar of saffron (C. sativus L.) were collected from local farmers at Albacete, Spain. Healthy resting corms were rinsed in running tap water for 1-2 h, then washed in distilled water, dipped in 80 % ethanol for 25 s and rinsed three times in sterile distilled water. Afterwards, the corms were surface sterilized for 20 min in a 0.8 % solution of sodium hypochlorite under sonication and then rinsed three times with sterile distilled water. For the initiation of the cultures, the meristematic tissue of corms was used as the initial explant, dissected vertically as a 1-cm-sided cube and inoculated onto a modified MS [14] culture medium supplemented with 0.1 mg·L -1 of 2,4 D (2,4 dichlrophenoxyacetic acid) and
2.0 mg·L -1 of BA (bencylaminopurine), according to Piqueras et al. [19]. The pH of the medium was adjusted to 5.8 with 1.0 N HCl/Na OH and solidified with 0.8 %
agar and autoclaved for 20 min at 121 °C. The explants were cultured in 25 x 100 mm test tubes each with 10 mL of nutrient medium under 16 h cool-white fluorescent light (30 μmol·m -2·s1) at 25 °C. Under these conditions, the meristematic region of the cultured corms produced nodular cormogenic calli able to develop protocorm-like bodies. After the first subculture, the nodular cormogenic
calli were separated from the initial explant and subcultured at 5-week intervals in the same medium (figure 1a). These cultures have been used as the plant material for this work. Only one clone was used for all the experi- ments. For the cultures in liquid medium, 1.5 g of tissue were inoculated in 250-mL Erlehnmeyer flasks containing 100 mL of medium and stirred at 120 rpm. The nodular cormogenic calli obtained as described before were exposed to different treatments to study their
influence on growth and the regulation of morphogenesis in the cultures: several concentrations of MS salts and sucrose (3-7 %) were tested both in solid and liquid cultures as well as adenine sulphate, L-glutamine, casein hydrolisate and sodium di-hydrogen phosphate. To study the effect on the induction of shoots and the development of corms, several growth regulators (BA, Kin (Kinetin), 2ip(2-isopentenyladenine), 2,4-D, paclobutrazol (PAC) and imazalil (IMA) sterilized by filtration) were added to the medium before adjustment of the pH. The experi- ments were repeated twice and each treatment was composed of 24 tubes (1 xplant/tube), three flasks in the case of liquid medium. The morphogenic response was recorded after 6 weeks in culture.
To view the full publication, please use the following link:
https://hal.archives-ouvertes.fr/hal-00885955/document
Hoping this will be helpful,
Rafik
Dear Rabiul Islam,
Nodule formation can be initiated in tissue culture by adding compatible bacteria, but this does not mean being "artificial". There are different protocols availeble, you can find them by performing Scholar Google search.
I am afraid that the procedure mentioned by Rafik is not related to nodulation in a sense of symbiosis formation but rather to nodulation as propagation through node formation in tissue culture.
Thank you very much Professor Rafik Karaman, Dr. Gederts Ievinsh & Mr. Matthew Chidozie Ogwu.
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