Development of efficient cell and tissue culture regeneration system for genetic transformation of maize towards improved pest resistance

Improvement of maize genotypes via genetic engineering technology requires an efficient plant tissue culture regeneration and transformation system. The production of genetically transformed plants depends both on the ability of target to integrate foreign genes and on the efficiency of genetically transformed cells to regenerate into whole functioning plants. The DA-BAR/GMA Corn project 'Development of Efficient Cell and Tissue Culture Regeneration System for Genetic Transformation of Maize Towards Improved Pest Resistance' specifically aimed to develop an efficient regeneration system from cultured cells, tissues and organs via somatic embryogenesis and/or organogenesis, identify maize genotypes with high regeneration potential, evaluate the field performance of tissue culture derived maize and optimize conditions for transient transformation using the particle inflow gun. Fifty one (51) yellow and white maize inbred lines developed by the Institute of Plant Breeding-UP Los Baños (23 lines) and International Maize and Wheat Improvement Center (CIMMYT, 28 lines) were evaluated for their tissue culture response. Of the 51 lines tested, only 34 inbred lines (66.7%) produced embryogenic callus (E-callus) and the remaining 18 lines produced non embryogenic callus. Based on E-callus formation, the maize inbred lines tested were classified as having 1) poor response C 20% E-callus formation), intermediate response (21-49% E-callus formation) and good response (50% E-callus formation). For yellow inbred lines, 13.8% (4/29) of the lines tested showed good E-callus response (50-63% E-callus), while for white inbred lines, 25% (3/12) of the lines tested showed good response (67-77% E-callus). Two types of E-callus were initiated from immature embryos, type 1 E-callus (compact nodular with many scutellum like-bodies) and type 2 E-callus (creamy to yellow friable callus). For most inbred lines tested, initiation of type 2 E-callus was generally lower compared with type 1 E-callus, 17.4% and 21.3%, respectively. Shoot regeneration was obtained in only 64.7% (22/51) of the inbred lines tested. For inbred lines with good E-callus formation, shoot regeneration ranged from 50% to 100%. Based on E-callus formation and shoot/plant regeneration, five(5) IPB inbred lines Var 4, Var 2 and P 53, Pi 17 and Pi 23 and two (2) CIMMYT yellow inbred lines CL00331 and ARMMP 36 were selected as suitable lines for genetic transformation. Using the optimized callus induction protocol, shoot regeneration from four selected local inbred lines ranged from 50% to 100%. The conversion or regenerated shoot into whole plantlets either multiple shoots or single shoots ranged from 45.2% to 66.2%. The single shoot regenerants, typical of a germinating embryo, accounted to only 16.9% of the total regenerated plantlets. Morphological evaluation of tissue culture regenerated (R0) plants in the field showed both normal (65.5%) and off-type or somaclonal variant (37.5%) plants generated. Vegetative and reproductive growth of tissue cultured generation 1 (R1) plants from both normal and off-type plants were comparable with seed derived control plants. Transient transformation of corn explants was conducted using the reporter Gus gene. The conditions for particle inflow gun (PIG) transformation were optimized. Higher rates of transient transformation, measured through expression of the Gus gene, were obtained using pre-cultured embryos as target explants, distance of 5.5 in from the point of discharge to the target cells and pressure of 900 to 1100 kPA for optimum particle delivery formation. The significant results obtained from this project, including the identification of inbred lines with high regeneration potential, optimized protocols for E-callus formation, shoot/plant regeneration, improved rooting and ex vitro establishment and generation of normal plants, and transient transformation, would hasten the genetic engineering of maize for pest resistance and higher crop productivity. The regeneration and transformation systems developed could now be used for stable transformation of maize. The optimized regeneration system could likewise be used for other in vitro manipulations in maize.