Regeneration and identification of interspecific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton

Abstract

et al. Regeneration and identification of interspecific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton.Asymmetric hybrids between Gossypium hirsutum (YZ-1) and G. davidsonii were obtained by donor-recipient fusion. YZ-1 was considered the recipient and was pretreated with iodoacetamide (IOA), while G. davidsonii was considered the donor and was irradiated with ultraviolet (UV) before fusion. YZ-1 protoplasts stopped growth when treated with 0.5 mmol/L IOA for 20 min, and G. davidsonii protoplasts stopped growth when irradiated with 38.7 J/cm 2 UV for 30 s. Asymmetric somatic hybrids were obtained by electrofusion between the separately treated protoplasts of the 2 species. The regenerated plants were identified by morphological, cytological, and molecular analysis. Most regenerated plants derived from fused protoplasts displayed new morphology; some were intermediate between the two parents and a few displayed recipient-like morphology. Chromosome numbers in these somatic hybrids mostly ranged from 40 to 73. The hybridity was confirmed by random amplified polymorphic DNA and simple sequence repeat analysis. Organelle DNA inheritance of the YZ-1 and G. davidsonii somatic hybrid was investigated by cleaved amplified polymorphism sequence and chloroplast simple sequence repeat analysis, which indicated that recombination and rearrangements might have occurred in some regions of mitochondrial and chloroplastic DNA. This is the first report of completely asymmetric hybrid production via donor-recipient fusion between G. hirsutum and G. davidsonii, which is a novel case in hybrid production following the symmetric fusion and asymmetric fusion based on UV irradiation in cotton.cotton, donor-recipient fusion, IOA, protoplast, UV Conventional breeding programs have made great progress in cotton improvement, but it has become increasingly difficult to make further advances due to sexual incompatibility between some cultivars and wild cotton, especially if the favorable gene is only present in the wild species. An available method for bypassing sexual-crossing barriers is protoplast fusion, which permits transferring desirable agronomically relevant traits from wild cotton to cultivars. This technique has been exploited to enhance resistance and improve quality [1,2] and has produced novel hybrids for incorporation into plant breeding programs [3,4] . In recent years, symmetric and asymmetric protoplast fusion between wild and cultivated species has been successfully achieved in cotton [1,2,5,6] . However, symmetric protoplast fusion transfers not only the desired genes, but also all of the unfavorable ones [3,7] . In asymmetric protoplast fusion, only a part of the wild genome is transferred to a recipient protoplast, reducing the number of undesirable traits incorporated into the recipient genome, but selecting the regenerants is very difficult. So, the donor-recipient fusion with the do-

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

“…Somatic hybridization with viable protoplasts can break the reproductive barriers in the process of sexual hybridization or distant hybridization, and create new germplasm or new varieties that cannot be obtained by conventional breeding [ 12 ]. At present, cell fusion technology has been successfully applied in citrus [ 12 , 13 ], cotton [ 14 ], oilseed rape [ 15 ] and other plants.…”

GhHmgB3 deficiency deregulates proliferation and differentiation of cells during somatic embryogenesis in cotton

“…, 1996). In MTS assays, the protoplast isolation and culture were performed as described (Fu et al. , 2009), and the protoplast number was counted using the hemacytometer.…”

Isolation of Mesophyll Protoplast and Establishment of Gene Transient Expression System in Cotton

“…Cocking [17] 以及 Nagata 和 Takebe [18] 分别报道用酶解 法从番茄根尖与烟草叶片中分离得到大量有活性、 稳定的原生质体。以拟南芥为代表的大多数植物都 建立了有效的原生质体瞬时表达体系, 可以多角度分 析细胞内外源基因的表达特征及可能行使的功能 [19] 。 通过与根癌农杆菌共培养, 周颖等 [20] 将 z108 基因整 合到烟草原生质体的核基因组中, 建立了普通烟草 的瞬间表达转化体系。Yang 等 [21] 建立了简单、高效 的水稻绿色组织原生质体瞬时表达体系, 并用于蛋 白免疫及蛋白互作研究。 棉花是重要的经济作物, 开展原生质体的分离 与应用研究较早。在原生质体培养、体细胞融合以 及植株再生等方面已取得较好研究进展 [22][23][24] 。前人 已将棉花原生质体从愈伤组织 [25] 、子叶 [26] 、悬浮细 胞 [27] 等组织中成功分离, 但分离棉花原生质体存在 植板率低、不同基因型对棉花原生质体的得率和活 性影响差异较大等问题 [28] 。利用棉花原生质体进行 目标基因瞬时表达, 开展功能研究的报道也较少。 5~7 片真叶提取原生质体, 其产量和活性达到最佳 水平 [15] 。本试验利用棉花叶片作为外植体分离原生 质体, 选用生长 12 d 左右, 生长状态良好的棉花子 叶提取原生质体, 获得了 1.0×10 6 mL -1 以上的原生 质体数量, 能满足多次转化后续实验。 酶的种类和浓度的选择应根据植物材料来源和 生理状态而定, 应针对不同的外植体材料选择合适 酶液组合 [30] 。目前, 分离植物叶片原生质体, 最快、 效果最佳的是酶解法, 多采用纤维素酶和离析酶组 合。叶肉原生质体分离的最佳酶液组合对于水稻为 纤维素酶2.0%和离析酶0.7%; 对于玉米、小麦叶均 为纤维素酶1.5%和离析酶0.5% [9] ; 对于烟草为1.0% 纤维素酶和0.5%离析酶, 配合0.5 mol L -1 甘露醇渗 透压更可获得理想纯净的原生质体 [31] 。以拟南芥子 叶为外植体分离原生质体, 其酶液组合为1.5%纤维 素酶和0.4%离析酶 [32] 。棉花原生质体细胞分离已有 较多研究。李仁敬等 [26] 利用4.0%纤维素酶, 0.4%果 胶酶, 酶解6 h 分离出棉花子叶叶肉原生质体。Sun 等 [33] 利用3.0%纤维素酶, 0.5%半纤维素酶, 1.5%果 胶酶, 酶解20 h, 获得棉花高质量的原生质体, 其活 力达到90%。 近年来, 棉花原生质体提取的方法进一 步得到改良。汪静儿等 [28] 运用纤维素酶3.0%、离析 酶1.0%, 酶解20~24 h 获得棉花悬浮细胞高质量原 生质体。付莉莉等 [24] 采用1.5%纤维素酶, 1.0%离析…”