Establishment Of In Vitro Plantlet Regeneration, Mycorrhiza-Like Structures Of Cathaya Argyrophylla And Mycorrhization Of Abies Chensiensis

Cathaya argyrophylla Chun et Kuang and Abies chensiensis Van Tiegh., tree species belonging to the family Pinaceae, are listed as the first grade and second grade endangered plants by the State Environmental Protection Administration of China, respectively. As natural propagation abilities of these two species are getting lower and lower, they have become critically endangered conifers. This study addresses this serious situation in three important aspects: Firstly, an in vitro regeneration protocol was achieved using plant tissue culture techniques. This plantlet regeneration system provides a step for slowing down the endangered situation of these two species and provides a tool for carrying out related research. Secondly, the mycorrhiza-like structures were induced between roots and calli of C. argyrophylla and ectomycorrhizal fungi, Cenococcum geophilum. This finding can be applied as a useful experimental system to other studies on Cathaya species. Thirdly, an aseptic ectomycorrhizal synthesis between A. chensiensis and C. geophilum was established. The present studies provide useful information to find a new way to restore communities of endangered pine trees.1. Plantlet regeneration of C. argyrophyllaAdventitious buds were formed on stem and root segments excised from sterile young seedlings, when cultured on modified MS media supplemented with 0.5 mg/L BA and 2.0 mg/L 2,4-D (MMS). Stems excised from 8-week-old seedlings showed the highest differentiation capability, i.e., 35.3%. Adventitious buds were also formed directly from stems of 3 to 4-day-old seedlings when incubated on MMS media supplemented with 0.2 mg/L BA and 0.02 mg/L NAA. Shoots elongated successfully on MMS media modified to be at half strength of the major salts and added with 0.03% activated charcoal and 13.3 g/L sucrose. Rooting frequency of shoots was 37.5% on MMS media supplemented with 0.5 mg/L NAA. Plantlets grew successfully on artificial substrates (perlite: vermiculite: soil, 1:2:3) in a green house.2. Aseptic ectomycorrhiza-like formation/infection between root and callus of C. argyrophylla and an ectomycorrhizal fungus C. geophilumCalli formed on stem segments and adventitious roots differentiated from young seedlings of C. argyrophylla were both co-cultured with C. geophilum on a MMS medium containing 0.1 g/L glucose. In callus, fungal hyphae were visible within intercellular spaces 4 weeks after incubation;however, the definite and well-developed Hartig net structures were not yet formed 8 weeks after inoculation. The primary infection occurred in callus. In case of root, the typical ectomycorrhizal structures, i.e., mantle and intracortical Hartig net, were observed 8 weeks after inoculation. This culture system is useful for investigation of mycorrhiza synthesis ofCathaya trees.3. Ectomycorrhizal synthesis between A. chensiensis and C. geophilum Seeds of A. chensiensis germinated at a rate of 33.3% on an agar medium containing 5 g/L of glucose sterilized with 70% ethanol, 0.2% (w/v) HgCl2 solution and 30% H2O2. Seedlings grew well. Sterile seedlings 8 weeks old of A. chensiensis were inoculated with C. geophilum. Typical ectomycorrhizas were formed on seedlings on MMS medium 10-11 weeks after incubation, showing a thin mantle and intracortical Hartig net in lateral roots. This simple in vitro method will facilitate studies on ectomycorrhizal development of A. chensiensis.