中国生物工程杂志

Populus tomentosa Carr., LM50, as an elite with characteristics of fast growth, strong stress-tolerance and no flying fluff, is usually recommended as an optimal material for genetic transformation in woody plants. The long-term asexual propagation in practice would cause the decline of excellent traits. During tissue culture, problems such as adventitious bud differentiation and rooting difficulties of explants often occur. Through anther induction approach, the negative impacts caused by the aging of the explants will be eliminated or alleviated, and P. tomentosa can be rejuvenated in a short time, providing more optimal materials for genetic transformation. Meanwhile, haploid individuals are expected to be obtained, which will be used in genomics research and ploidy breeding. Using the ‘LM50’ of the P.tomentosa gene bank in Guanxian County, Shandong Province as the test material, by comparing morphological characteristics and microspore stage, the mononuclear side-stage anthers were selected for regeneration in vitro. The effects of auxins and cytokinins in induction of callus, adventitious bud differentiation and rooting were assessed, respectively. The ploidy levels of the plants generated from anther induction were identified by the flow cytometry and chromosome counting. Furthermore, the leaves of plantlets induced by anthers as explants were used to establish a tissue culture system with high leaf differentiation rate and high rooting rate. The comparison of the microspore development period and the external morphological characteristics of the flower bud shows that most of the microspores were in the mononuclear side stage when the 1/4 inflorescence emerged from floral bud whose size was (1.98 ± 0.06) cm; the anthers in this period were selected to induce callus formation. The medium with the highest callus induction rate was H + 1.00 mg/L NAA + 1.00 mg/L BA, and the induction rate was about 28.89%. The callus was further differentiated into adventitious buds.The optimal shoot induction medium was MS + 0.05 mg/L NAA + 0.50 mg/L BA, and the induction rate was approximately 22.23%; adventitious shoots were inoculated to rooting medium. The optimal rooting medium was 1/2 MS + 0.30 mg/L IBA with 93.30% rooting rate; ploidy identification of 27 regenerated plants cultured in anthers was conducted by flow cytometry and the chromosome compression method, and the plants were all diploid. The optimal medium of in vitro regenerating for leaves and stems of plants originated from anthers was MS+TDZ 0.10mg/L + NAA 0.10 mg/L + BA 0.50 mg/L, and the regeneration rate was as high as 92.23%. The rooting medium for adventitious buds produced by leaf differentiation was the same as that for adventitious buds induced by callus, and the rooting rate was the same. The study obtained ‘LM50’ anther-induced regeneration plants, and established a leaf culture system for the anther regeneration plants, with high differentiation rate and rooting rate, which can be used for the rapid propagation of this excellent clone and genetic transformation of P.tomentosa, and laid the foundation for the molecular design and breeding of P. tomentosa.