In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

Muscle-like Scaffolds for Biomechanical Stimulation in a Custom-Built Bioreactor.

Tissue engineering aims to develop in-vitro substitutes of native tissues. One approach of tissue engineering relies on using bioreactors combined with biomimetic scaffolds to produce study models or in-vitro substitutes. Bioreactors provide control over environmental parameters, place and hold a scaffold under desired characteristics, and apply mechanical stimulation to scaffolds. Polymers are often used for fabricating tissue-engineering scaffolds. In this study, polycaprolactone (PCL) collagen-coated microfilament scaffolds were cell-seeded with C2C12 myoblasts; then, these were grown inside a custom-built bioreactor. Cell attachment and proliferation on the scaffolds were investigated. A loading pattern was used for mechanical stimulation of the cell-seeded scaffolds. Results showed that the microfilaments provided a suitable scaffold for myoblast anchorage and that the custom-built bioreactor provided a qualified environment for the survival of the myoblasts on the polymeric scaffold. This PCL-based microfilament scaffold located inside the bioreactor proved to be a promising structure for the study of skeletal muscle models and can be used for mechanical stimulation studies in tissue engineering applications.

Evaluation of Biomechanical and Chemical Properties of Gamma-Irradiated Polycaprolactone Microfilaments for Musculoskeletal Tissue Engineering Applications.

An appropriate and reliable sterilization technique is crucial for tissue engineering scaffolds. Skeletal muscle scaffolds are often fabricated using microfilaments of a wide variety of polymers. One method for sterilization is 25 kGy of gamma irradiation. In addition, sterilization through irradiation should administer a dose within a specific range. Radiation directly affects the chemical and mechanical properties of scaffolds. The accuracy and effects of irradiation are often not considered during sterilization procedures; however, these are important since they provide insight on whether the sterilization procedure is reliable and reproducible. This study focused on the chemical and mechanical characterization of 25 kGy gamma-irradiated scaffold. The accuracy and uncertainty of the irradiation procedure were also obtained. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analyses were performed to determine whether the crystallinity of the polymer changed after irradiation and whether gamma rays influenced its thermal properties. The tensile parameters of the microfilaments were analyzed by comparing irradiated and nonirradiated scaffolds to determine whether gamma radiation changed their elastic behavior. Dose distribution and uncertainty were recorded with several dosimeters. The results showed that the irradiation process slightly affected the mechanical parameters of the scaffold; however, it did not modify its crystallinity or thermal properties. The irradiation was uniform, since the measured uncertainty was low. The scaffold was pathogen-free after 7 days; this meant sterilization was achieved. These results indicated that gamma-sterilized scaffolds were a promising material for use as a skeletal muscle analog material for tissue-engineering applications because they can be sterilized with gamma rays without changing their chemical structure and mechanical properties. This study provided the dose distribution measurement and uncertainty calculations for the sterilization procedure.

A Temporary Immersion System Improves Regeneration of In Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.) Embryogenic Calli.

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (60Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L-1 2,4-D, and regeneration was achieved with MS medium with 3 mg L-1 BA and 0.5 mg L-1 NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA®, Saint-Mathieu-de-Tréviers, France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials.