Mahanta Manisha, Gantait Saikat, Sarkar Sutanu, Sadhukhan Raghunath, Bhattacharyya Somnath
Crop Research Unit (Genetics and Plant Breeding), Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252 India.
All India Coordinated Research Project on Floriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252 India.
3 Biotech. 2023 Mar;13(3):74. doi: 10.1007/s13205-022-03457-z. Epub 2023 Feb 3.
An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (/) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l -Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.
本研究首次建立了一种高效的体外多倍体诱导体系，用于非洲菊杂交种（BGC - 2019 - 01）的高频多倍体诱导。将两周龄的体外发育芽（芽尖）分别用0.1%、0.25%和0.5%（/）的秋水仙碱溶液处理4、6、8和12小时。然后将经秋水仙碱处理的芽尖接种在添加1.5mg/l - 激动素的Murashige和Skoog（MS）培养基上进行多次芽增殖，随后转移到添加1.5mg/l吲哚 - 3 - 乙酸的MS培养基上进行芽生根。通过流式细胞术分析和中期染色体计数，确认了经秋水仙碱处理和再生的植株以及未处理植株的倍性水平。当芽尖用0.1%秋水仙碱处理4小时时，获得了最高频率的四倍体植株（50%）。形态学观察表明，诱导的四倍体植株表现出新鲜芽起始延迟、芽数少但更长以及叶数少但更宽。同样，气孔研究表明，与二倍体对应物相比，四倍体植株的气孔频率较低但明显更大，叶绿体数量更多。在光合色素分析中，四倍体的叶绿素、类胡萝卜素和花青素含量显著更高。在体外驯化和田间生长期间，四倍体植株表现出增殖延迟，但活力更高，叶片变宽变厚。使用保守DNA衍生多态性（CDDP）、小卫星区域DNA定向扩增（DAMD）、简单序列重复区间（ISSR）和起始密码子靶向（SCoT）多态性标记系统，确认了二倍体和四倍体植株之间的遗传一致性。就观赏价值而言，本研究中培育的四倍体对于非洲菊的遗传改良具有极其重要的意义。
