In vitro Callus induction of aromatic rice depends on the concentration of 2, 4-D

Abstract Due to growing population, there is an increasing demand of rice production but its productivity is lessened day by day. Aromatic rice has a great demand during festivals in many countries. Kalijira is one of them not only Bangladesh but also all over the world due to its attractive flavor, fine grain and good taste which is generally used to prepare dishes in different special occasions. But there are some limitations to cultivate aromatic rice Such as lack of high yielding variety, fine grain quality, disease or pest resistant, stress and salt tolerance variety and proper cultural management. To overcome this problem tissue culture can be used. However, the lack of a simple and efficient protocol for callus induction in this cereal crop. In this study we tried to find out the potentiality of aromatic rice variety named kalijira for callus induction from mature embryo and to find out the suitable concentration of 2, 4-D for callus induction and proliferation. The highest callus induction were observed when the media was supplemented with 2 mg/L of 2, 4-D and the frequency of callus induction was lowest in 0.5 mg/L concentration of 2, 4-D. This study will be useful for selecting suitable concentration of growth regulator (2, 4-D) for callus induction in future that will be useful for not only national but also international plant breeders.


Description of the experimental site
In this experiment field grown mature seeds of aromatic rice (Oryza sativa L.) variety 'kalijira' were used for callus induction. The explants were collected from Barisal district, Bangladesh.

Preparation of Stock solution
At first stock solution preparation was done for preparing of culture media. Freshly prepared stock solutions were needed which contained different constituents. So, different types of stock solutions (I, II, III, IV, V and VI), organic compounds and growth regulators were prepared separately and stored at refrigerator (Table 1).

Preparation of MS Culture Media
To prepare 200 ml of MS medium 100 ml distilled water was taken in a 500 ml beaker, 4 ml of stock solution I was added in the beaker then 2 ml of stock solution II, III, IV, V & VI was added. 3% sucrose was added. The final volume was adjusted to 200 ml. 0.7 % TC Agar was added after adjusting the PH at 5.7-5.8. Finally media was autoclaved at 1210C for 20 minutes at 15 Psi.

Sterilization of Plant Materials
The dehusked seeds were washed with 10% savlon for 3 min and then washed three times with sterilized distilled water. After washing with sterilized water the explants were dipped at 70% ethanol for one minute and washed three times with sterilized distilled water. After washing with sterilized water the explants were dipped at 50% sodium hypochlorite for 30 minutes and washed three times with sterilized distilled water. All of these works were done in laminar air flow cabinet.

Inoculation of seeds
After surface sterilization the explants were kept onto sterile filter paper placed inside the Petridis. After removal of water from seeds surface, these were inoculated into the culture Petri dish with sterilized forceps and niddle. The explants were inoculated into the MS medium supplemented with 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L of 2, 4-D in each petridish for callus induction. The plates were then incubated in the culture shelves at dark, the temperature and RH were maintained at 25±2o C and 78% respectively.

Callus Induction
Data were collected by counting the calli induced and inoculated seed. Data of callus induction were collected after three weeks of inoculation. All the calli originated from a single seed was considered as one. The frequency of callus induction was calculated as below: Callus induction frequency = No of callus produced No. of seeds inoculated * 100%

Color of callus
After four weeks of incubation the color of callus was observed visually.

Intensity of callus
After four weeks of incubation the intensity of callus was observed visually and graded "+++" for maximum size of calluses, "++" for medium size of calluses and "+" for small size calluses.

Results
In vitro regeneration of plant via calli offers unique facilities of reproducible protocol as well as recovery of somaclonal variants, which can be utilized for the future crop improvement programs. Thus, induction of calli from different explants and subsequent regeneration of complete plants could play an important role.

Treatments used for callus induction
During conducting this research 2, 4-D was used as growth hormone. Different concentration of 2, 4-D were applied for the initiation of callus. In control no hormone was used. MS media was supplemented with different concentration of 2,4-D ranging from 0.5 mg/ L to 3.0 mg/ L for callus induction of rice.

Callus induction frequency
It was noticed that MS medium supplemented with only 2.0 mg/L of 2, 4-D produced highest percentage of callus that is 90% and the MS medium supplemented with only 0.5 mg/L of 2, 4-D produced lowest percentage of callus that is 60%. In those media which were supplemented with 1.0 mg/L, 1.5 mg/L, 2.5 mg/L, 3.0 mg/L showed callus induction frequency of 70%, 70%, 80%, 80% respectively. The callus induction frequency with MS media supplemented with different concentration are shown in Figure 1.

Intensity of callus
In 0.5 mg/L and 1.0 mg/L hormone concentration callus size was same with the seeds that were inoculated. In 1.5 mg/L, 2.0 mg/L and 3.0 mg/L hormone concentration callus size was double than the seeds while In 2.0 mg/L and 2.5 mg/L hormone concentration callus size was three times of the seeds.

Types of callus
The types of callus of different treatment are same and non-embryogenic. The high amount of polyploidization, rough endoplasmic reticulum, polysome, poly-nucleolus, and incomplete cell wall together with abnormal partitioning in non-embryogenic cells, as compared to embryogenic cells. In contrast, vacuolation of cytoplasm, perfect cell wall and partitioning structure, and the high proportion of nucleus/cytoplasm area were recognized in embryogenic cells [16].

Discussion
In any in vitro regeneration technique, callus induction is the primary step. Under present study the callus induction frequency of kalijira, a special aromatic rice variety with different concentration of 2, 4-D was conducted. The callus induction frequency shows significant difference under different concentration of 2, 4-D. The frequency of callus induction was highest when seeds were inoculated in media fortified with 2 mg/L concentration of 2,4-D that support the previous study [1,17] and the frequency of callus induction was lowest in 0.5 mg/L concentration of 2,4-D. All the calli were non-embryogenic, friable and creamy yellowish. 2mg/L of 2, 4-D concentration took less time for callus initiation and 0.5mg/L of 2,4-D concentration took more time.

Conclusion
The present study was conducted to assess the callus induction efficiency of the aromatic rice variety Kalijira on MS medium. The different concentration (mg/L) of 2, 4-D for callus induction were used as follows: 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0. The control replica shows no callus, but other treatments showed callus induction of varying frequency. Among them 2.0 mg/L 2, 4-D has found to induce highest frequency of callus (90%). Other treatments (2.5 and 3.0 mg/L 2, 4-D) showed better result (both 80%). The color of all calli found from different 2, 4-D treatments were creamy yellow and texture of all calli were friable. So, it can be concluded that 2.0 mg/L 2, 4-D was suitable for callus induction and proliferation from mature embryo of Kalijira variety.

Acknowledgement
Thanks to Bangladesh Agricultural University (BAU), Bangladesh for all experimental support.