Method for producing direct in vitro flowering and viable seed from cotyledon, radicle, and leaf explants, and plants produced therefrom

#This invention relates to a new method for reprogramming plant development to produce viable seeds from flowers derived directly from cultured cotyledons, radicle explants, and in vitro shoots derived from cotyledons and radicle explants. In particular, by manipulating the cotyledon or radicle or a leaf on a suitable media containing a novel growth regulator regime that affects plant development, it is possible to recover fully fertile, viable-seed producing flowers and either eliminate or reduce significantly the plant body. This “in vitro flowering” provides a significant reduction in the time required for seed production and also eliminates concerns surrounding genetically modified organisms (GMO) relating to pollen contamination, either as is or when supplemented with chloroplast transformation by using our in vitro flowering technique.

#The national crop germplasm is at risk due to unregulated entry of infected plant materials and to our own mono-culture breeding practices that have lead to the shrinking of our genetic base. The United States Department of Agriculture (USDA) has already identified 2,000 exotic plant pathogens of which 550 have been recognized as posing a threat to US agriculture. One such plant parasite is Phakospora pachirhzi,the causative agent of soybean rust. This fungus is native to Asia and has already spread to Africa and to South America, thus posing a significant new threat to the soybean industry both in the United States and abroad. To date, none of our commercially important soybean cultivars have been bred for rust resistance. Indeed, there have been harvest losses due to sensitivity to this fungus in other countries.

#Thus, there is a need for a breeding program that would assist in the rapid development of resistant lines against pathogens introduced either by nature, accident or bio-terrorism. The present invention provides a solution to this problem as it provides a robust DNA marker-assisted breeding protocol that allows for rapid development of new lines faster than with traditional breeding programs. Specifically, in vitro flowering allows seed harvest in approximately three months from the time of explant implantation in the tissue culture media, thus enabling, for example, four cycles of soybean production annually.

#The patent or application file contains one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee.

#FIGS. 1a-1d are photographs showing direct viable-seed producing flower bud production from cotyledons in soybean.

#FIGS. 2a and 2b are photographs showing direct viable-seed producing flower buds from in vitro shoots in soybean.

#Definitions

#In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided.

#We define “in vitro flowering” as direct fertile flower production and viable seed production from an explant without a plant body. Given cells of an explant are reprogrammed to terminate vegetative meristem and lead directly to a reproductive state leading to flower and seed production. The immediate result of in vitro flowering is the production of fertile flowers that gives raise to viable seed. In vitro flowering encompasses precocious flowering where the flowers are produced directly from explants having a reduced life cycle but nevertheless produce viable seed. The in vitro flowering of the present invention provides methods of eliciting different developmental fates from cells of an explant by application of a unique novel growth regulator regime. These developmental fates include, but are not limited to: (1) direct viable-seed producing flower bud production on cotyledons leading to the formation and recovery of fertile seed; (2) direct viable-seed producing flower bud production from individual in vitro developed shoots from cotyledon explants leading to the formation of fertile flowers and recovery of viable seed; (3) direct viable-seed producing flower bud production from radicle explants leading to the formation of fertile flowers and recovery of viable seed; and (4) direct viable-seed producing flower bud production from in vitro developed shoots from radicle explants leading to the formation of fertile flowers and recovery of viable seed. Additional developmental fates include production of flower buds and viable seed from in vitro developed shoots from leaf explants. These directed cell fate shifts result from a manipulation of growth regulators in defined concentrations and/or combinations that dictate the cell fate shifts of each explanted tissue.