Establishment of a high-efficiency plant regeneration and transformation system for the elite maize inbred lines from three heterotic groups

Abstract

Maize (Zea mays L.) is one of the most important crops in the world and its agronomic traits could be improved by genetic transformation with desirable genes. A successful transformation must depend on a high-efficiency in vitro plant regeneration and genetic transformation system. In our studies, six media compositions were used to induce callus from the immature zygotic embryos of seven maize inbred lines and LM6 was proved the best callus induction media, with high callus induction percentage (CIP) and callus quality. Furthermore, four phytohormones were analyzed on their effects on callus induction, the results indicated that 2,4-dichlorophenoxyacetic acid (2,4-D) played an important role in callus initiation, but, 3.3 mg/l Dicamba could provide higher embryogenic callus induction percentage (EIP) than 2 mg/l 2,4-D; both 6-benzyl aminopurine (6-BA) and kinetin (KT) could decrease CIP, EIP and callus quality at the concentration level of 0.2 mg/l. The experimental result also revealed that media LM6-CI was preferable to LM6 in callus induction. The selected LM6-CI media, together with other two media (LM6-EI and LM6-PR), were used to identify 18 elite maize inbred lines from three heterotic groups on their tissue culture characteristics, as a result, the eight materials, including 3189/4380, 4380/Sanzong5, 8103, Xianzao17, 18-599(red), 501, 178 and Ji53 belonging to group Reid or Compound Germplasm, presented higher CIP, EIP and plant regeneration percentage (PRP) than others; the four materials, including Huangzao4, Huangye4, Jing24 and Ji853 from Tangsipingtou group, were not easy to be differentiated into plants, in spite of high CIP. Maize inbred line 18-599(red) as a representative was further used to establish genetic transformation system, its embryogenic calli, initiated from immature zygotic embryos, were transformed with the plasmid p35SBarNos harboring Bar gene by microprojectile bombardment, after selection and differentiation culture, partial bombarded calli were regenerated into green plantlets and further fertile plants. The results of molecular identification for fertile regenerated plants showed that Bar gene had been integrated into maize genome and the transformation frequency was high up to 66.7%. All these results were beneficial for the studies on in vitro plant regeneration and genetic transformation in plant.