毛白杨优良无性系‘LM50’花药培养植株再生体系建立*

毛白杨‘LM50’具有速生、抗逆、不飞絮等优良特性,是木本植物进行遗传转化的理想材料。长期无性繁殖会导致优良性状衰退,在进行组织培养时,往往出现外植体不定芽分化和生根困难等问题。通过花药培养可以在较短时间内使毛白杨复幼,从而消除因外植体老化带来的负面影响,为转化研究提供理想的材料。与此同时,期望通过花药诱导创制单倍体植株,为基因组学研究和倍性育种提供材料。以山东冠县毛白杨基因库的‘LM50’为试验材料,对其花药发育时期与花芽形态的关系进行鉴定,选择单核靠边期的花药进行试验,探究了生长素和细胞分裂素在花药愈伤组织形成、不定芽分化及不定芽生根中的作用,建立了毛白杨花药离体再生体系,采用流式细胞仪和染色体压片计数法对诱导获得的再生植株进行了倍性鉴定。进一步利用花药培养再生植株的叶片建立了分化率高、生根率高的植物再生体系。小孢子发育时期与花芽外部形态特征对比表明,花芽长度为(1.98 ± 0.06) cm,1/4花序露出芽鳞的花芽,此时小孢子大部分处于单核靠边期;选择处于此时期的花药诱导形成愈伤组织,愈伤组织诱导率最高的培养基为H + 1.00 mg/L NAA + 1.00 mg/L BA,诱导率约为28.89%;愈伤组织进一步分化为不定芽,最佳分化培养基为MS + 0.05 mg/L NAA + 0.50 mg/L BA,分化率约为22.23%;不定芽接种至生根培养基,最佳生根培养基为1/2 MS + 0.30 mg/L IBA,生根率约为93.30%;利用流式细胞仪和染色体压片法对花药培养的27株再生植株进行倍性鉴定,鉴定植物均为二倍体;再生植株叶片分化成芽的最佳培养基为MS + 0.10 mg/L TDZ + 0.10 mg/L NAA + 0.50 mg/L BA,分化率高达92.23%。该叶片分化产生的不定芽的生根培养基与愈伤组织诱导不定芽生根的培养基相同,生根率一致。研究获得了毛白杨‘LM50’花药诱导再生植株,并建立了再生植株的叶片培养体系,可用于该优良无性系的快速繁育和毛白杨的遗传转化研究,为毛白杨的分子设计育种奠定了基础。

Establishment of Anther-cultured Plant Regeneration System of Populus tomentosa Elite Clone ‘LM50’

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.