Method for regenerating viable and fertile citrus plants by tissue culture from explants

A method for regenerating viable and fertile Citrus plants by tissue culture from explants of field-grown mature trees said method comprising the steps of:

• (a) cutting explants selected from fresh shoots of field-grown mature trees of a citrus species selected from the group consisting of C. aurantifolia and C. sinensis;
• (b) decontaminating the said explants by surface sterilization for removing any contaminants which are harmful to the tissue culture process;
• (c) incubating the surface-sterilized explants at a temperature between 27° C. to 30° C. in the presence of about 3 klux white fluorescent light for 15 hrs. a day, in agarified medium-A for the plant explants of C. aurantifolia and medium B for the plant explants of C. siniensis, as defined in Table 1, for a period ranging between 20-30 days, to develop axillary buds and obtain 70-80% infection-free cultures of C. aurantifolia or C. sinensis;
• (d) subculturing the explants of step (c) along with sprouted axillary buds at least 4 times, by sub-culturing the explants of C. aurantifolia in medium-A or C. sinensis in medium-B as defined in Table 1, to produce several aseptic fast-growing shoots;
• (e) excising the meristem domes along with youngest 2-3 leaf primordia of length between 0.2-0.5 mm from the aseptically grown shoots and culturing the meristem domes in medium C as defined in Table 1, at a temperature between 27° C. and 30° C. in the presence of about 3 klux, white fluorescent light for a period of about 15 days to generate shoots of length of about 8 mm;
• (f) subculturing the meristem-regenerated shoots on filter paper bridge employing liquid medium-D as defined in table 1, in order to obtain healthy shoots of an average length of about 1.5 cm without intervening callusing within a period of 20-25 days;
• (g) proliferating the meristem-regenerated shoots of C. aurantifolia or C. sinensis by repeated subculture in medium-A or medium-B, respectively to obtain an average of about 6-10 well-developed shoots, within 4 subcultures of 30 days each;
• (h) inducing roots in excised meristem-regenerated well-developed shoots of C. auranitifolia to the extent of about 100% in 15 days, or C. sinensis to the extent of about 90% in 25 days, in agarified media-E and F respectively;
• (i) transferring the rooted shoots of C. sinenisis to medium-G as defined in Table 1, or allowing the shoots of C. aurantifolia to grow in medium A for a further period of 15 days in order to develop tap roots;
• (j) hardening the rooted shoots ex-vitro in the liquid medium-H as defined in Table 1, for 10 days in a hardening chamber, wherein the relative humidity is lowered gradually from about 90% to about 60% under about 3 klux light intensity from white fluorescent tubes supplemented with light from incandescent lamps at 26° to 28° C. and allowing the rooted shoots to grow in the same medium, for 30 days;
• and
• (k) transferring the hardened rooted shoots to soil containing organic fertilizer for ex vitro growth at relative humidity of 90% to 60% at a temperature of 27° C. to 30° C. in the presence of white fluorescent light for a period of about 7 days to ensure 95% to 100% survival of the rooted shoots of Citrus species namely C. auranitifolia or C. sinensis.