Plant material: Leaf, nodal and internodal calli of variety Shital were used in present investigation.
Genetic transformation material: Agrobacterium strain, plasmid and gene:
Genetically engineered A. tumefaciens strain LBA4404 was used for infection in the pre-cultured explants. The strain is being maintained at the Biotechnology lab. under Bangladesh Agricultural University. This strain contains plasmid pBl121 of 14 kDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct- i. The uidA gene encoding GUS (β-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. ii. The npt II gene encoding neomycin phosphotransferase II (npt II) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. iii. The CIPK sense gene encoding calcineurin B-like protein conferring abiotic stress tolerance. Calcineurin B -like proteins (CIPK) Calcineurin (Cn) is a unique Ca2+ dependent serine/threonine protein phosphatase (PP2B) of cytosol, which plays an important role in the coupling of Ca2+ signals to stress responses. Using degenerate primers from the conserved domains and by library screening a full-length cDNA (CIPK, 972 bp) was isolated from pea (accession no: AY883569). Plants respond to adverse environments by initiation a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein interacting protein kinases (CIPKs). Putative CIPK genes (OsCIPK01 - OsCIPK30) survived for their transcriptional responses to various abiotic stresses, like drought, salinity, cold, polyethylene glycol and abscisic acid treatment. To prove that some of these stress-responsive CIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (CIPK 03, CIPK 12, CIPK 15) were over expressed in Japonica rice. Transgenic plants over expressing the transgenes CIPK 03, CIPK 12, CIPK 15 showed significantly improved tolerance to cold, drought and salt stress, respectively. Under cold and drought stresses, CIPK 03, CIPK 12, over expressing transgenic plants accumulated significantly higher content of proline and soluble sugars. and putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants, against different stresses.
Methods
Treatments: There were 3 factors in this experiment. Factor A consisted of three types of callus, factor B consisted of two inoculation times and factor C consisted of two co-cultivation periods. A. Explants: Leaf, nodal and Internodal callus B. Infection time: 3 and 5 minutes C. Co-cultivation period: 24 and 48 hours Total no. of treatments were 12 (3x2x2). Each treatment consisted of 4 vials and replicated three times. Design: Factorial in Completely Randomized Design (CRD) Media used Media used in the present study were as follows. A. For callus induction For induction and maintenance of callus, MS medium supplemented with different concentrations and combinations of BAP and NAA were used. B. For Agrobacterium culture Two types of culture media, namely, YMB (Yeast Extract Mannitol Broth) medium and LB (Luria Broth) medium were used with kanamycin as antibiotic to grow the strain of genetically engineered Agrobacterium tumefaciens. YMB medium was used for Agrobacterium maintenance and LB medium was used as Agrobacterium working culture medium for transformation work. C. For co-cultivation MS media without growth hormones were used for co-cultivation. D. For washing explants after co-cultivation Cefotaxime (200 mg/l) was used for washing the explants after co-cultivation. E. For Post-cultivation and regeneration MS media supplemented with 2 mg/l BAP, 1 mg/l NAA and 100 mg/l cefotaxime were used for this purpose. F. For selection and regeneration Low selection medium: MS media supplemented with 2 mg/l BAP, 1 mg/l NAA, 20 mg/l kanamycin and 100 mg/l cefotaxime were used. High selection medium: MS media supplemented with 2 mg/l BAP, 1 mg/l NAA, 30 mg/l kanamycin and 100 mg/l cefotaxime were used.
Preparation of Culture Media
Preparation of MS medium, Agrobacterium culture medium, LB (Luria Broth) medium, GUS assay solution was prepared.
Sterilization Techniques
Sterilization of culture media and glasswares and instruments were sterilized.
Data Recording
To investigate the effects of different treatments and responses of different varieties to callus induction subsequent inoculation and regeneration, data were collected from the different parameters as given: a) Number of survived callus, b) Number of survived callus, c) Number of callus positive for GUS assay, d) Percentage of callus positive for GUS (Percent GUS expression) assay.