We have an exceptional recipe for callus induction and maintenance and get sporadic production of roots and shoots off of this callus with some cultivars when hormone levels drop off in the media, but no definite recipe for media for 'plantlet induction' with consistent results.
Well it had beed observed that if we applied NAA (0.2 mg/l) it become more effective in root induction than IAA. But keep in mind BAP Inhibit root mass accumulation.
As you may know there´re some basic rules but these are just rules because don´t work in the right way all the time. Storage of cultures in the dark in the presence of hormones (both auxins or cytokins) normally stimulate undifferentiated growth. Calli cultured in culture media without hormones or with low levels of cytokins might favoured shoot (or embryos) induction. For mostly of species, as you said, some genotypes have more abilities for morphogenesis than others. Also some hormones are powerful than others however it don´t means that you got what are you seeking. The experience and patience are fundamental with this kind of work. Good luck.
Grape being a woody plant you can refer the article in International Journal of Plant Developmental Biology (2010), 4(1):26-30 Global Science Books
I find that IBA at 0.1-0.5mg/L enhanced root development in rice, tropical woody species.
I applied TDZ (0.5 mg/l) in callus induction than NAA. and supplemented BAP (0.5 mg/l)
hi jessy try BAP alone and in combination of IAA for shooting and for rooting subculture your callus in NAA containing medium.
Hey Jessy first observe that your callus is embryogenic or organogenic. For embryogenic callus you can use combination of high auxins and low cytokinins and for organogenic callus use high cytokinin and low auxin. You can use the auxins and cytokinin alone also. For the formation of root and shoot in your callus you will have to standerdise the perfect media because it varies in every plant.
What media formulation will lead to organogenesis in callus is goverened by the nature and age of the callus. In general callus will lead to shoor organogenesis if you use appropritate concentration of suitable cytokinin and auxin for root induction.
Hi Madhu,
Can you tell me what you mean by observing whether my callus is 'embryogenic or organogenic' in nature?
My advisor has recommended that I wait for one experimental 'media recipe' to see if some of the cells will eventually start to look embryogenic in nature, which would require microscopic investigation for the 'torpedo' stage etc., I think. Is this what you mean?
As for organogenesis, some of the cultures will do this on their own, sporadically. That's when we see some irregular shaped shoots and occasionally roots. Is this what you mean for organogenic callus?
Hello Jessy:
I would like to tell you that I observed similar behavior in callus culture of different Ziziphus species (a woody fruit specie), and just I can induce roots when the medium was supplemented with 1.5 mg/L of BAP and 0.5 mg/L IBA. In addition to the question above; I recommend you to do first the histological studies of your callus and after that you will see the kind of cell are present in the callus that you obtained, because, maybe you are working with callus in which are not possible to develop any organized structure (re-differentiation) from some of your species and maybe you need to change the hormone composition of the medium to induce embryogenic callus line which are the best to produce embryoids and furthermore plantles.
hey Jessy
Generally the embryogenic callus looks friable and creamy and organogenic callus looks compact and greenish. On microscopic study of callus u can easily identify the embryogenic and organogenic callus. After identifying the callus u can easily set your experiments...............Good luck
Thank you for the clarification Madhu. That helps a lot. It turns out I have both types, even occasionally within the same cultivar.
Dear Jessy,
First of all aim to regenerate the shoots by using either TDZ or BAP and then after induction of shoots for enough length, please try to induce roots by using half strength MS with IBA at less conc / lowering N level (NH4NO3) in MS / using basal MS without any hormones will help induce in vitro roots.
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