In vitro regeneration in cole crops

The experiment was conducted to optimize a suitable protocol for in vitro regeneration in cole crops. Callus initiation was excellent in the variety Early Tropical. Highest percentage of callus proliferation was observed in Early Tropical (75.0%) followed by Tangail Special Pauslali (55.0%) and the lowest in Tara (40.0% ). Maximum callus proliferation (68.5%) was observed in MS + 3.0 mgL BAP + 0.1 mgL 2,4-D + 2.0 mgL AgNO3 . Callus proliferation was lowest (40.0%) in MS + 2.5 mgL BAP + 0.1 mgL 2,4-D + 2.0 mgL AgNO3. MS medium supplemented with 3.0 mgL BAP + 1.0 mgL 2,4-D + 2.0 mgL AgNO3 was the best for shoot initiation & plantlet regeneration. The highest number of shoots per vial was 7.20 and the lowest number of shoots per vial was 4.40. Among the concentration MS + 3.0 mgL BAP + 0.1 mgL 2,4-D + 2.0 mgL AgNO3 showed the highest performance of shoots per vial. The variety Tangail Special Pauslali was the best for root initiation.


Introduction
The genus Brassica belongs to the family Brassicaceae under the order Rhoedales (Benson, 1957) has three categories namely the Rape seed, the Mustard and the Cole.(Yarnell, 1956).Cabbage (Brassica oleracea var.capitata) and cauliflower (Brassica oleracea var.botrytis) are the most important members of cole crops in the tropic and temperate regions of the world (Siddiqui, 2004).In Bangladesh, out of 260.32 thousand hectares of vegetables growing area, cabbage and cauliflower covers 9.64% of the total land and contributes 13.97% of total production (BBS, 2004).The curd (white head of cauliflower) is an early stage of inflorescence development as its formation invariably precedes floral initiation (Bose and Som, 1986).Conventional breeding methods are time consuming, extending over seven to eight years involving crossing and wise selection of desirable traits.In addition, genetic incompatibilities in some cases restrict many important gene transfer by interspecies hybridization.More recently, genetic engineering (gene technology) has advanced and opened a new avenue for crop improvement.An efficient tissue culture system is thought to be essential to the success of plant genetic engineering.This technique can be used to add desirable traits from wild to existing cultivars.Therefore, it is essential to develop an efficient transformation system (Henzi et al., 2000) which offers the potential for the introduction of specific genes from any source (related or unrelated plant species or even from animal) into the existing elite plant lines.And to do, a well adapted regeneration protocol for cabbage and cauliflower should be developed using the locally cultivated varieties.Keeping the above facts in mind, the present study was taken to standardize a suitable protocol for in vitro regeneration of different genotypes of Cole crops.

Materials and Methods
The seed materials of two cabbage varieties like Tara, Summer Star and two cauliflower varieties like Early Tropical, Tangail Special Pauslali were collected from the local market of Mymensingh town and the experiment was conducted in the Tissue Culture Laboratory of the Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh.Healthy and uniform seeds were surface sterilized with 70% ethyl alcohol and 0.1% HgCl 2 .Half MS used for in vitro germination.Six days old seedlings were used as source of explants and cultured on MS medium supplemented with different concentrations of BAP (2.0, 2.5, 3.0 mgL -1 ) with a constant concentrations of 2,4-D (0.1 mgL -1 ) and AgNO 3 (2.0 mgL -1 ) for callus induction and subsequent plantlet regeneration.The regenerated plants were transferred to sterile culture vessels containing rooting media (half strength MS medium supplemented with 0.5 and 1.0 mgL -1 NAA).When the plantlets became 5-6 cm in length with sufficient root system, they were transferred in plastic pots containing a 3:1 mixture of autoclaved soil and sand for hardening and incubated at 25±1 °C in the culture room for a week.The plants were finally transferred to the pots to the field condition.

Results and Discussion
Initiation and maintenance of callus: Calli raised from hypocotyls of each of two varieties of cabbage and cauliflower were successfully maintained by subculturing at an interval of 15 days on MS medium supplemented with different concentrations of BAP (2.0, 2.5 and 3.0 mg L -1 ) with constant concentration of 2,4-D (0.1 mg L -1 ) and AgNO 3 (2.0 mg L -1 ).
After two to three subcultures, the calli exhibited variation in color and texture i.e. calli became friable in texture and greenish in color.The subcultured calli then started regeneration by shoot bud initiation.

Effect of hormone × variety interaction:
The results related to hormone × variety interaction for shoot regeneration characters like, number of shoots per explant, number of shoots per callus and number of shoots per vial is presented in Table 3.The interaction between hormone × variety for the concentration and combination of 2.0, 2.5 and 3.0 mg L -1 BAP were found to be non significant.Among the characters the higher number of shoots per vial was 7.20 and the lower number of shoots per vial was 4.40.Among the concentrations, MS + 3.0 mg L -1 BAP + 0.1 mg L -1 2, 4-D + 2.0 mg L -1 AgNO 3 (T 3 ) produced the higher number of shoots per vial.BAP was added to MS medium supplemented with 0.1 mgL -1 2,4-D and 2.0 mgL -1 AgNO 3 .

Fig. 3 .
Fig. 3. Response of different treatments on cabbage and cauliflower varieties towards callus proliferation