In Vitro Regeneration of Sorghum Plants From Immature Embryos A Potential Tool For Sorghum Transformation. Aniruddha Acharya1, Fabian Strauss1*, Josh Benoit1, Rachel Inoue2, Blacin Doucet1 1 Department of Biology, University of Louisiana at Lafayette, 2 Universidade de Brasília *Poster presenter – frs6493@louisiana.edu Abstract Sorghum is a C4 plant and belongs to grass family. It has high photosynthetic and water efficiency and attains maturity within three months. It can grow on marginal soil with little inputs in terms of water and nutrients and is the fifth most important crop in the world. In Africa and Asia, sorghum serves as an important source for food while in Americas and Australia it is mainly used as animal feed. However, this plant is recalcitrant to genetic manipulation and the first successful transgenic sorghum was reported by Casas et al. in 1993 using particle bombardment, while the first Agrobacterium mediated sorghum transformation was reported by Zhao et al. in 2000. Optimization of tissue culture media like hormones, amino acids, gelling agents along with other parameters like selection of genotypes, explants, phytotoxicity of selection agents, pre-culturing and heat shocking are reported to produce positive outcomes in sorghum tissue culture. In this experiment we regenerated 22 healthy sorghum plants starting with 400 immature embryos thus having a regeneration efficiency of 5.5%. This is an initial report on this technique and we are still collecting data to modify it for higher efficiency. We believe this protocol can be useful in future to generate transgenic sorghum plants. Introduction Sorghum is originated in Africa and is divided into five races, bicolor, guinea, caudatum, kafir and dura (Harlan and De Wet, 1972). Life cycle of sorghum has 3 stages, vegetative, reproductive and mature, each lasting approximately 35 days (Gerik et al. 2003). Though the sorghum genome has been sequenced (Paterson et al. 2009), functional genomics study through transformation is not a routine procedure due to its recalcitrance to genetic manipulation. Through tissue culture and Agrobacterium mediated transformation, useful genes can be incorporated in the sorghum genome, but a robust, efficient, genotype independent and replicable protocol is not available for this process. This study attempted to develop a robust, fast and simple protocol for in vitro regeneration of sorghum. Materials and Methods Germination and Plant maintenance - Sorghum seeds of IS 473 from sorghum mini core collection (Upadhyaya et al. 2009) were used for the study. Seeds were soaked with water for germination. Five day old seedlings were planted in 3ⅹ3ⅹ3.5 inches pots with Metro mix 360. Sorghum field was cleaned of weeds, rowed and fertilized two weeks prior plantation. Two weeks after germination, the plants were transplanted to the field. Plants were watered daily, fertilized every two weeks. Aphid infection was monitored and controlled using neem oil, rosemary oil and peppermint oil based insecticides (Figures 1 and 2). Harvesting immature embryos and surface sterilization - Panicles were harvested between 10-14 days post anthesis to dissect immature embryos. Harvested seeds were surface sterilized with 70% alcohol for 5 minutes, followed by 4% sodium hypochlorite (NaClO) with few drops of Tween 20 shaken for 20 minutes at 200 rpm in a flask. The seeds were then washed five times with sterile water and quickly dried in sterile blotting paper. Callus induction - Immature embryos of the size 1 to 2 millimeters were gently dissected out of the seeds using aseptic techniques. They were placed in Whatman 3 paper soaked with callus induction media in 90ⅹ15 mm petri plates and kept in dark at 27ᵒC for 7 days. This was followed by transferring the embryos to fresh callus induction media solidified with 0.7% agar. Maximum care was taken to prevent embryo injury and the injured embryo were not used. Callus induction media was based on MS media with vitamins from Phytotechnology and supplemented with sucrose, glucose, proline, potassium phosphate, 1mg/L 2,4-D and pH was adjusted to 5.7. Regeneration - Healthy and actively dividing callus were obtained after 14 days (Figure 3A) which was transferred to the first regeneration media in deep petri plates (Figure 3B) . First regeneration media was based on MS media with vitamins and supplemented with sucrose, glucose, proline, asparagine, potassium phosphate, copper sulfate, 1 mg/L 6-Benzylaminopurine, 0.1 mg/L Indole-3-acetic acid, 0.8% agar and pH was adjusted to 5.7. For the first 7days callus in first regeneration media was kept in dark at 27ᵒC followed by low light intensity for next 7 days. Green spots were visible in callus after 14 days which were transferred to second regeneration media (Figure 3C) with higher BAP concentration and high light intensity for the next 14 days. Photoperiod of 14/10 and a temperature of 27ᵒC was maintained. Rooting and hardening - Callus bearing small plantlets were transferred to rooting media in Magenta box (Figure 3D) and kept under high light intensity with 14/10 photoperiod and 27ᵒC temperature for 3 weeks and no sub-culturing was done during this period. Rooting media (Figure 3E) was based on MS media with vitamins and supplemented with sucrose, glucose, proline, asparagine, potassium phosphate, copper sulfate and hormones like 1mg/L NAA, 1mg/L IBA, 1mg/L IAA. Agar (0.7%) was used as solidifying agent and pH was adjusted to 5.7. After 3 weeks magenta box covers were removed and the plants were overlaid with sterile water and were covered in transparent plastic bags with a minute pore for a week for hardening before transferring to soil. A. Callus after 14 days Figure 3. Steps in Sorghum regeneration from immature embryos D. Rooting stage one E. Rooting stage two C. Regeneration stage two B. Regeneration stage one Figure 1. Sorghum seeds germinated in lab and transplanted in field Pretreated with 2% H2SO4 Washed and digested with CTec cellulase Measure glucose content Figure 2. Plants were watered, fertilized, inspected for aphids and pannicles were harvested Results and Discussion In this experiment, 22 healthy sorghum plants were regenerated starting with 400 immature embryos thus having a regeneration efficiency of 5.5%. We were able to complete the process in 10 weeks starting from embryo dissection. We believe the efficiency can be improved with more careful and systematic modification of the media and the techniques. We will also apply this to other genotypes and finally use this method for Agrobacterium mediated transformation of Sorghum. References Casas, Ana M., et al. "Transgenic sorghum plants via microprojectile bombardment." Proceedings of the National Academy of Sciences 90.23 (1993): 11212-11216. Zhao, Zuo-yu, et al. "Agrobacterium-mediated sorghum transformation." Plant Molecular Biology 44.6 (2000): 789-798. Harlan, J. R., and J. M. J. De Wet. "A simplified classification of cultivated sorghum." Crop Science 12.2 (1972): 172-176. Gerik, Thomas J., Brent Bean, and Richard L. Vanderlip. Sorghum growth and development. Texas Cooperative Extension, Texas A & M University System, 2003. Paterson, Andrew H., et al. "The Sorghum bicolor genome and the diversification of grasses." Nature 457.7229 (2009): 551-556. Upadhyaya, H. D., et al. "Developing a mini core collection of sorghum for diversified utilization of germplasm." Crop Science 49.5 (2009): 1769-1780. Carvalho, Carlos Henrique S., et al. "Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency." Genetics and Molecular Biology 27.2 (2004): 259-269. Gurel, Songul, et al. "Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos." Plant cell reports 28.3 (2009): 429-444. Liu, Guoquan, Edward K. Gilding, and Ian D. Godwin. "A robust tissue culture system for sorghum [Sorghum bicolor (L.) Moench]." South African Journal of Botany 98 (2015): 157-160. Acknowledgements We thank Billy Welsh and Kristy Thompson for allowing the use of facilities and tools from UL’s Ira Nelson Horticulture Center. The study is supported by the University of Louisiana at Lafayette.