Production of potato (Solanum tuberosum, L.) microtubers using plastic culture bags.

We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato (Solanum tuberosum L.). The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production, and bioreactors have been used for this purpose. However, bioreactors are expensive and difficult to handle. The plastic culture bags are relatively inexpensive and are easy to store and sterilize because they can be flattened. Microtuber production involves two stages: plant proliferation in one medium, followed by microtuber production in a different medium. Both steps are carried out using the same culture bag. Using this system, we produced 100 to 300 microtubers per 8 l culture bag, depending on the potato cultivar. We varied the nutrient concentrations in the media and found that a lower sucrose concentration in the plant proliferation medium and lower nitrogen concentration in the microtuber production medium both increased the total numbers of microtubers per bag. Notably, a higher concentration of potassium phosphate increased the numbers of larger microtubers. This is beneficial because larger microtubers are much more tolerant to field conditions. We produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system. These microtubers have been planted directly in the field and utilized for seed potato production.

Highly efficient method of Lithospermum erythrorhizon transformation using domestic Rhizobium rhizogenes strain A13.

Lithospermum erythrorhizon, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact L. erythrorhizon plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in L. erythrorhizon cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for L. erythrorhizon transformation using hairy roots by Rhizobium rhizogenes strain A13, present domestically in Japan. Hairy roots of L. erythrorhizon were generated from explants of the axenic shoots that were infected with R. rhizogenes strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50-70%. Although methods for the systematic stable transformation of L. erythrorhizon plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of L. erythrorhizon genes.