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Response to High Frequency Callus Induction Ability from Root Regions of Germinated Embryo in Indica Rice

T.L. Aditya
Plant Breeding Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh.

M.E. Hoque
Biotechnology Division, Bangladesh Rice Research Institute, Gazipur -1701, Bangladesh.

M Khalequzzaman
Genetic Resources and Seed Division, Bangladesh Rice Research Institute, Gazipur-1701 , Bangladesh.

Abstract/ Executive Summary:

High frequency callus induction ability was achieved from well-germinated embryos (on hormone free MS media) of eight indica rice genotypes by culturing on MS medium supplemented with 2 mg l^-12, 4-D and solidified with 0.3% phytagel. When mature seeds were directly cultured, the highest frequencies of callus induction were observed for two genotypes (IR51491-AC5-4 and BR24) out of eight genotypes tested. However, embryogenic callus induction ability of different genotypes significantly differed. Calli from scutellar tissue formed yellow, compact, friable and globular type embryogenic calli designated as embryogenic (E) type I. In contrast when callus produced from root regions of germinated embryo all genotypes showed high callus induction ability and formed soft-friable, pale yellow, shiny and nodular calli designated as embryogenic type II. In both case crystalline, hard, white with suppressed root primordia were formed which were designated as non-embryogenic (NE) calli. The simple procedures described in this paper provide an efficient protocol for reproducible callus induction method of Bangladeshi rice varieties/line, which is pre-requisite for a good regeneration system and a source of fine embryogenic cell suspension cultures for protoplast isolation and genetic transformation method.

Keywords: Induction frequency, Mature embryo, Root, Indica rice

Location: Bangladesh Rice Research Institute

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To establish reproducible callus induction ability derived from root regions of well-germinated mature embryos of eight Bangladeshi indica rice compared with calli derived from scutellar tissue and select the best genotypes for further tissue culture procedures such as regeneration ability, protoplast culture and genetic transformation

Methodology:

Mature dehulled rice seeds (Oryza satriva L.) of eight indica cultivars were used ill this study. Out of eight genotypes four namely BR24, BR26, BRRI dhan29 and BRRI dhan40 were developed by Bangladesh Rice Research Institute (BRRI). One homozygous breeding line IR5I49I-AC5-4 was originated from International Rice Research Institute currently testing in BRRI for salt tolerance. Patnai, Pokkali, Binnatoa were moderately salt tolerant land races cultivated in coastal areas of Bangladesh. Dehulled seeds were immersed in distilled water with one drop of Tween twenty to clean the materials and washed with sterile distilled water. After that seeds were surface sterilized by dipping in 70% (v/v) ethanol for 3 min followed by 25 min in a sodium hypochlorite solution (commercial bleach) by rigorous shaking. Seeds were rinsed for three times by sterile distilled water after each sterilization procedure. In one-step method eight sterilized seeds were aseptically germinated in Petridishes containing 25 ml MS medium supplemented with 3% (w/v) sucrose, 2 mg I^-1 2,4-D at pH 5.8 and semisolidified with 0.3% phytagel designated as MS2 callus induction media. In two-step procedure eight sterilized seeds were germinated in same type of Petri dishes on hormone-free MS medium with 3% sucrose and 0.8% agar at 25±1 °C in the dark. After one week well-germinated seeds were transferred into same MS2 media as used in one-step procedure. Eight Petridishes were represented as eight replications for each genotype. Cultures were incubated at 25±1^oC in the dark. Assessment of callus growth was measured on fresh weight (fr.wt) basis, expressed as a percent weight gain after a certain period of time by using 5-week-old calli. Firstly, the known weight of callus tissue was aseptically transferred onto 10 cm glass tube containing 10 ml of MSI (MS medium+l mg l^-1 2,4-D) callus induction media. After six weeks, the callus pieces from glass tubes were removed with a sterile spatula and re-weighed under same sterile conditions. With regard to one-step procedure of callus initiation, about 2-3 weeks later, the number of seeds forming callus in each treatment (genotype represented as treatment) was counted and converted to percentage. On the other hand for two-step procedure, after 4-5 weeks when primary and some others secondary roots were between 2-5 cm long, callus induction frequency was recorded by counting seeds forming calli in each individual treatment. The percentage of callus initiation was recorded by using the following formula:

Callus initiation (%) = (Number of callus producing seed/ Total number of seed plated) x 100

Weight gain was calculated from initial and final fresh weight by using the following formula:

Weight gain (%) = (Final fresh weight - Initial fresh weight/ Final fresh weight) x 100

Experimental design and statistical analysis: Completely Randomized Design (CRD) was used for conducting experiment and data were analyzed for one way ANOVA by using SAS package as developed by Computer software programme. LSD test was applied for the evaluation of statistical significance of differences.

Source of Information: Pakistan Jaurnalal of Biological Sciences 7 (5): 861-864, 2004; ISSN 1028-8880

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, root regions of germinated embryos was found to be better for high frequency callus induction while seed scutellum axis performed better for embryogenic callus formation. Indentifying genotypes with high callus ability and phenotype of calli were the most important factors to provide an efficient regeneration system and a source of good embryogenic cell suspension cultures of somatic hybridization and genetic transformation methods.

Knowledge Management: Journal