Hi Sujit.

Callus cells can be induced to develop into shoots, roots or both by providing auxins and cytokinins in a defined ratio (see below the attached picture for an example). Double check your auxin/cytokinin ratio in your medium.

The legend of the attached picture:

"Tobacco leaf explants cultured on media with varying concentrations of an auxin (α-naphthaleneacetic acid; NAA) and a cytokinin (6-benzylaminopurine; BAP). Concentrations of NAA are from left to right, 0, 0.01 μM, 0.1 μM, 1.0 μM; concentrations of BAP are from top to bottom 0, 0.01 μM, 0.1 μM, 1.0 μM. At low auxin to cytokinin ratios shoot development predominates, whereas at high ratios profuse root initiation occurs. At intermediate ratios, callus is often the result. Not that type of response also depends on the explant source: tobacco stem pith under the same culture conditions usually shows no development whatsoever unless some auxin is supplied."

http://plantsinaction.science.uq.edu.au/edition1/?q=content/10-2-2-types-differentiation

Sujit, can you please contact me at [email protected]

Check the quality of the plant hormones that are being used to ascertain that they are ok

Thanks everyone. @Edward George Mamati. all the product from Sigma Alrich so no doubt they are good. I have followed all the instruction properly in storage as well. 

Yuan-Yeu and Younes, thanks for the suggestion, i am going to try it and will update in two weeks time. 

Apply some 2,4-D around 2.5 ppm to the medium and this will stop root formation. After this you provide mixture of BAP and Cyatokinin in the ratio of 1 ppm and 0.1 ppm and if you get shooting report it quoting my suggestion 

Although 2iP is a very efficient cytokinin, maybe you could try another cytokinin like BA. And I agree with Younes that your culture could produce high amounts of endogenous auxin so you could omit completely auxin from the medium. If it still does not produce shoots, it is possible that anther cultures from your cabbage - due to the genotype of cultivar- are not suitable for shoot regeneration and you should try another genotype (unless this genotype is particularly important for you, in this case you could try other hormone combinations). Regeneration is strongly genotype-dependent even within a single species.

Hi, Sujit, the organogenisis program are dependent from internal hormone level, but does not from hormones you apply externally.  For the shoots induction you have to induce first "stem cells" , ea. group of the cells which potentially able to give shoots. The idea about 2,-4D treatment is very good, so, try it!

Thanks Tasiu and Csaba, all the hormones are newly bought so should n't have problem with expiry date. BAP i have used last year and early this year. in callus induction 2ip shows better result than BAP therefore, i was continuing with 2ip. but after all the suggestion i got and few more literature review recently done, i am going to try NAA + BAP (1:100), NAA+2ip(1:00) which is 0.001mg/L and and 1 mg/L (NAA and BAP), while another one will be NAA- 0mg/l and BAP / 2ip-2mg/L. Fingers Cross. 

Probably the anther-derived callus has a high level of endogenous IAA. In any case, the best advise to solve this problema is to test new variables susch as different nutrient média, changes in media consistence, culture without growth regulators, other growth regulators....

Hi,Sujit,

I reccomend you to try also 2,4 D/BAP- (!)riboside form /GA3 (1:50:1).

Good luck!

Ivan

Based on your figure it is look like somatic embryo. 

Just go through following literature

(Carloni et al., 2014; Cha-um et al., 2009; Escobar-Guzmán et al., 2008; Germanà, 2010; Górecka et al., 2009; Irikova et al., 2011; Jia et al., 2014; Kou et al., 2012; Kozak et al., 2011; Li et al., 2013; Perera et al., 2008; Prado et al., 2010; Redha and Suleman, 2010; Silva, 2009; Winarto et al., 2011, 2010a, 2010b; Zamani et al., 2000)

Carloni, E., Ribotta, A., López Colomba, E., Griffa, S., Quiroga, M., Tommasino, E., Grunberg, K., 2014. Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry. Plant Cell, Tissue Organ Cult. 117, 311–322. doi:10.1007/s11240-014-0441-4

Cha-um, S., Srianan, B., Pichakum, A., Kirdmanee, C., 2009. An efficient procedure for embryogenic callus induction and double haploid plant regeneration through anther culture of Thai aromatic rice (Oryza sativa L. subsp. indica). In Vitro. Cell. Dev. Biol. - Plant 45, 171–179. doi:10.1007/s11627-009-9203-0

Escobar-Guzmán, R.E., Hernández-Godínez, F., Martínez de la Vega, O., Ochoa-Alejo, N., 2008. In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (Physalis ixocarpa Brot.). Plant Cell. Tissue Organ Cult. 96, 181–189. doi:10.1007/s11240-008-9474-x

Germanà, M.A., 2010. Anther culture for haploid and doubled haploid production. Plant Cell, Tissue Organ Cult. 104, 283–300. doi:10.1007/s11240-010-9852-z

Górecka, K., Krzyżanowska, D., Kiszczak, W., Kowalska, U., 2009. Plant regeneration from carrot (Daucus carota L.) anther culture derived embryos. Acta Physiol. Plant. 31, 1139–1145. doi:10.1007/s11738-009-0332-1

Irikova, T., Grozeva, S., Rodeva, V., 2011. Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol. Plant. 33, 1559–1570. doi:10.1007/s11738-011-0736-6

Jia, Y., Zhang, Q.-X., Pan, H.-T., Wang, S.-Q., Liu, Q.-L., Sun, L.-X., 2014. Callus induction and haploid plant regeneration from baby primrose (Primula forbesii Franch.) anther culture. Sci. Hortic. 176, 273–281. doi:10.1016/j.scienta.2014.07.018

Kou, Y., Ma, G., Teixeira da Silva, J.A., Liu, N., 2012. Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall. Plant Cell. Tissue Organ Cult. 112, 1–7. doi:10.1007/s11240-012-0205-y

Kozak, K., Galek, R., Waheed, M.T., Sawicka-Sienkiewicz, E., 2011. Anther culture of Lupinus angustifolius: callus formation and the development of multicellular and embryo-like structures. Plant Growth Regul. 66, 145–153. doi:10.1007/s10725-011-9638-2

Li, Y., Li, H., Chen, Z., Ji, L.-X., Ye, M.-X., Wang, J., Wang, L., An, X.-M., 2013. Haploid plants from anther cultures of poplar (Populus × beijingensis). Plant Cell, Tissue Organ Cult. 114, 39–48. doi:10.1007/s11240-013-0303-5

Perera, P.I.P., Perera, L., Hocher, V., Verdeil, J.-L., Yakandawala, D.M.D., Weerakoon, L.K., 2008. Use of SSR markers to determine the anther-derived homozygous lines in coconut. Plant Cell Rep. 27, 1697–703. doi:10.1007/s00299-008-0592-z

Prado, M.J., Grueiro, M.P., González, M.V., Testillano, P.S., Domínguez, C., López, M., Rey, M., 2010. Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain). Sci. Hortic. 125, 342–352. doi:10.1016/j.scienta.2010.04.019

Redha, A., Suleman, P., 2010. Effects of exogenous application of polyamines on wheat anther cultures. Plant Cell. Tissue Organ Cult. 105, 345–353. doi:10.1007/s11240-010-9873-7

Silva, T.D., 2009. Indica rice anther culture: can the impasse be surpassed? Plant Cell. Tissue Organ Cult. 100, 1–11. doi:10.1007/s11240-009-9616-9

Winarto, B., Mattjik, N.A., Teixeira da Silva, J.A., Purwito, A., Marwoto, B., 2010a. Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture. Sci. Hortic. 127, 86–90. doi:10.1016/j.scienta.2010.09.004

Winarto, B., Rachmawati, F., Pramanik, D., Teixeira da Silva, J.A., 2010b. Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium. Plant Cell. Tissue Organ Cult. 105, 363–374. doi:10.1007/s11240-010-9876-4

Winarto, B., Rachmawati, F., Teixeira da Silva, J.A., 2011. New basal media for half-anther culture of Anthurium andreanum Linden ex André cv. Tropical. Plant Growth Regul. 65, 513–529. doi:10.1007/s10725-011-9622-x

Zamani, I., Kovacs, G., Gouli-Vavdinoudi, E., Roupakias, D.G., Barnabas, B., 2000. Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture. Plant Breed. 119, 461–465. doi:10.1046/j.1439-0523.2000.00538.x

Thank you Mr. Mafatat lal. will try the previsous suggestion first and see what happen in two weeks time.

My Suggestion is transfer your culture into hormone free  medium this will be germinated as plants within 10-15 days.

resultant plants were haploid. I don't know weather you are looking for double haploid production or not. If you need any assistance send me email: [email protected] 

Thank you Mr. Mafatlal. I will transfer into hormone free media as well. Will keep in touch. thanks

Dear Sujit! 

I would like to offer you to use more effective cytokinin - Thidiazuron for induction of direct shoot organogenesis. I send you references and articles devoted to using new synthetic plant growth regulator - Thidiazuron in plant biotechnology. I hope that my recommendation will bring you success in your researches.

Sincerely yours, SciDoc Victoria Tsygankova

Victor J.M., Murch S.J., Krishna S. Somatic embryogenesis and organogenesis in peanut: the role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis. Plant Growth Regul. 1999. V.28. P. 9–5

Guo, B., B.H. Abbasi, A. Zeb, L.L. Xu and Y.H. Wei. 2011. Thidiazuron: A multidimensional plant growth regulator. African J. Biotech, 10: 8984-9000

Thank you so much Dr. Tsygankova. at the moment, i am using the suggestion i got last week. all the callus sub-cultured on friday last week started showing slight green colour. therefore, i will wait for them for next 10 days. at the same time I will order some Thidiazuron. I am sure you suggestion will give me good result. will give you all update in 10 days time. thank you every one for your valuable suggestions. 

You should place your rooted calli on the media free of growth regulators and after 1 months to the media with cytokinins (BAP).

Thank you Lidia Sarmarina. I was wondering what to do with rooted calli now. 

@Sujit Actually these are not rooted calli. It seems as Somatic Embryo transfer onto hormone free medium soon youwill get green embryo. As I suggested in previous comment. I am waiting from hearing from you about results :)

you should check your CK/auxin balance.  My suggestion is to diminish the concentration of auxin and try applying BAP to the growth media

Mafatlal M. Kher, Thanks for your suggestions. i am getting green calli from Hormone free media. :D. Thank you everyone. I will update more soon. Thanks 

You can try media with 0.5 mg/L IAA, 3 mg/L Zeatin riboside, 0.02 mg/L GA3 and 1mg/L BAP.

Good luck!

@Lidia and Isabel Thanks both of you. we are following you suggestions as well. 

finally, got the plants regenerated :D.

Good job, Sujit, and thank you for updating us.

Dear Sujit Good work and happy to seed that finally somatic embryos germinated. I have suspected that these are somatic embryos. Now I am really very happy that you have obtain similar results.

Best Wishes

Regards

Mafat

Thanks Dr. Mafatlal. your suggestion worked :D.

@Sujit Thanks for appriciation I am not a Doctor I am still a PhD student. About To complete by PhD.

Best Regards

Mafat

well, at least you are doing PhD. :D.