Regeneration of different Cyclamen species via somatic embryogenesis from callus,suspension cultures and protoplasts

The present study is the first report of the establishment of embryogenic callus cultures from seedling tissue, the regeneration of plants via somatic embryogenesis and the development of a regeneration system from protoplast to plant, using three wild species of Cyclamen, Cyclamen graecum Link, Cyclamen mirabile Hildebrand, Cyclamen trochopteranthum Schwarz (syn. Cyclamen alpinum hort. Dammann ex Sprenger). The ability to form embryogenic callus and to regenerate via somatic embryogenesis was strongly genotype-dependent for each species. From 0.5 g callus, up to 1461 somatic embryos were formed in the case of C. mirabile. Culture media with different concentrations of plant growth regulators, CaCl₂ and activated charcoal significantly influenced embryo formation in this species. Up to 1.4 × 10⁶ protoplasts were isolated from 1 g of C. graecum cell suspension. Diverse growth responses of the protoplasts in two embedding agents, agarose and alginate, were observed for the different Cyclamen species. These specific growth characteristics could be used as a selection marker for future fusion experiments. From both protoplast culture systems, somatic embryos were regenerated, grown to plantlets and acclimatised to greenhouse conditions.     

柑橘体细胞杂种超低温保存后的植株再生

 对9年生印度酸橘(Citrus reticaulata)和飞龙枳(Poncirus trifoliata)的体细胞杂种的茎段进行离体培养得到再生植株，用改良的玻璃化法对再生植株茎尖进行超低温保存后获得92％的再生率。这是柑橘体细胞杂种茎尖超低温保存的首例报道。     

葡萄愈伤组织制备原生质体的影响因素研究

【目的】为了获得高质量的葡萄原生质体,【方法】以"鄞红"葡萄茎段愈伤组织为材料,对葡萄原生质体制备过程中的若干影响因子,如酶解液成分、甘露醇浓度、酶解时间、离心条件等方面进行了研究。【结果】0.5 mol.L-1的甘露醇+1%纤维素酶+0.025%～0.05%果胶酶为最佳的酶解液组成;酶解14 h、120×g离心3 min条件下原生质体数量和质量最佳。【结论】研究获得的高质量葡萄原生质体,为下一步的植株再生及葡萄品种遗传改良提供了良好的材料。     

马铃薯栽培种与野生种叶肉细胞融合及体细胞杂种鉴定

 以马铃薯栽培种Solanum tuberosum ‘中薯二号’的无性系3 # 、8 # (2n = 48) 和二倍体野生种Solanum chacoense (2n = 24) 的无菌苗为原生质体来源, 比较了PEG (聚乙二醇) 融合和电融合两种方式马铃薯原生质体融合效果的影响。结果显示, 两种融合方式对细胞的融合率没有显著差异, 但电融合的细胞植板效率和愈伤组织分化能力均显著高于PEG融合法。在早期挑选的生长旺盛的愈伤组织再生的100个株系中, 经RAPD 标记检测有97 个系为体细胞杂种。杂种植株经流式细胞仪倍性分析表明, 52.6 %为六倍体, 14.4 %为八倍体, 14.4 %为非整倍体, 18.6 %为混倍体。叶绿体SSR 引物NTCP29 用于细胞质重组检测, 结果显示, 体细胞杂种中叶绿体具有偏亲现象, 大多数杂种只含有单一亲本的叶绿体类型, 只有16.5 %株系为叶绿体重组的杂种植株。     

百合分子育种研究进展

从百合基因克隆（包括减数分裂基因、花发育基因、花色基因、花粉发育基因以及抗性相关基因）,再生体系建立（包括不定芽再生系统、原生质体再生系统、体细胞胚再生系统、愈伤组织再生系统）,外源基因转化方法（包括农杆菌介导的遗传转化、DNA直接导入系统的基因转化、花粉管通道法）及转基因应用等方面,介绍近年来国内外百合分子育种的研究进展,并讨论了百合分子育种研究中存在的问题和应用前景。     

Plant regeneration from protoplasts of Nicotiana alata ‘Crimson Bedder’ for somatic hybridisation studies

Protoplasts could be isolated readily from mesophyll and suspension cells of Nicotiana alata cultivar ‘Crimson Bedder’ and could be cultured in liquid media to yield small colonies. Suspension culture protoplasts exhibited a higher plating efficiency, and colonies regenerated from mesophyll protoplasts possessed enhanced plant regeneration capacity. These protoplast systems could be used for somatic hybridisation assessment with Petunia using a simple procedure for manual isolation and culture of heterokaryons.     

‘过山香’香蕉原生质体培养及植株再生

 以'过山香'香蕉的胚性悬浮细胞（Embryogenic cell suspensions,ECS）为起始材料分离原生质体,酶解液的组成为：3.5%纤维素酶R-10、1%离析酶R-10、0.15%果胶酶Y-23、204 mmol/L KCl、67 mmol/L CaCl2和0.41 mol/L甘露醇,原生质体产量为3.1×107个/mL PCV ECS （PCV：packed cell volume,细胞密实体积）。分别以培养基‘A’和‘B’为培养成分在液体浅层培养和看护培养两种培养系统中进行原生质体培养。结果表明：在液体浅层培养系统中,采用培养基‘B’比采用培养基‘A’效果好,原生质体的细胞分裂频率和细胞团形成频率分别约是采用培养基‘A'的3倍和10倍;所获得的培养物为只能增殖而不能进一步分化的非胚性细胞团。在看护培养系统中,采用培养基‘A’与采用培养基‘B’时,细胞分裂频率和细胞团形成频率没有显著地差异;所获得的细胞团具有典型的胚性细胞特征。将从看护培养中获得的细胞团转移到体胚诱导培养基上,培养45 d后,从105个原生质体获得1550个体胚。继续在体胚诱导培养基上培养30 d,7.8%的体胚能萌发。萌发的体胚在MS+0.1%活性炭的培养基中发育成健壮植株,移栽后成活良好。     

Somaclonal variation with early juice-sac granulation obtained by inter-specific protoplast fusion in citrus

Summary

Six plants with an early juice–sac granulation trait derived from inter-specific protoplast fusions between embryogenic calli of ‘Bonanza’ navel orange (Citrus sinensis [L.] Osbeck) and mesophyll protoplasts of ‘Dahongpao Red’ tangerine (C. reticulata Blanco) were analysed by flow cytometry and by using molecular markers, including simple sequence repeats (SSR) and restriction fragment length polymorphism (RFLP). The results indicated that all six plants were diploids and had inherited their nuclear DNA from the embryogenic callus parent ‘Bonanza’ navel orange. However, an analysis of morphological and fruit characteristics, and measurements of components of the cell walls in the juice-sacs, showed that they were not true-to-type for ‘Bonanza’ navel orange, especially for fruit traits such as juice-sac granulation and navel structure. These results confirmed that these plants were not hybrids, and were more likely to be somaclonal variants that arose during the regeneration of the navel orange protoplasts. These plants will provide material for studying the mechanism of granulation in juice-sacs, a common phenomenon during the storage of pummelo and other citrus fruits.     

Molecular analysis revealed autotetraploid,diploid and tetraploid cybrid plants regenerated from an interspecific somatic fusion in Citrus

More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo (Citrus reticulata Blanco × C. paradisi Macf) and mesophyll protoplasts of rough lemon (C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed.     

双孢蘑菇同核原生质体育种技术研究：Ⅱ.同核原生质体的杂交

为建立双孢蘑菇同核原生质体杂交育种体系,本文对同核原生质体亲合性反应及杂交异核体F_1代的农艺性状和F_2代的分离与变异规律进行了研究。结果表明:双孢蘑菇同核原生质体的杂交是一个较复杂的性亲合与核迁移过程,其杂交率较低。在按气生型×气生型、气生型×匍匐型、匍匐型×匍匐型组合的520个杂交配对中,共获得113个杂交异核体,平均杂交率为22%。本研究同时建立了以菌落形态、菌丝生长速度、羧甲基纤维素酶相对活性以及酯酶同功酶电泳为手段的杂交异核体鉴定体系。     

植物原生质体全能性表达及其在甘蓝类蔬菜育种上的应用

植物原生质体指植物除去细胞壁后被质膜包被的裸露细胞,在适当培养条件下具有再生成完整植株的全能性。植物原生质体分离、培养及植株再生技术是进行植物育种、基因工程、遗传理论及细胞生理特性研究的平台,具有重要的研究意义。本文概述了植物原生质体全能性表达过程中原生质体的获得、培养和再生等关键技术环节及其在甘蓝类蔬菜育种上的应用,同时讨论了原生质体培养技术中存在的问题,并且对完善植物原生质体培养体系及今后的工作方向提出了建议。     

Novel organized growth and somatic variation of Nicotiana bonariensis protoplast-derived colonies: Transmission to regenerated plants

Mesophyll protoplasts isolated from leaves of axenic shoot cultures of Nicotiana bonariensis underwent sustained cell division and concomitant novel cellular organizational patterns when cultured in liquid Murashige and Skoog medium (1962) containing 2.0 mg 1^?1 naphthaleneacetic acid (NAA), 0.5 mg 1^?1 6-benzylaminopurine (BA) and 9% mannitol. The novel patterns were leaf-like and embryo-like structures which resumed unorganized callus growth prior to shoot regeneration. In addition, the presence or absence of anthocyanin and of chlorophyll pigmentation in the protoplast-derived colonies was also observed. The variation in either presence or absence of anthocyanin in protoplast-derived colonies is restored as anthocyanin synthesis in the regenerated plants. However, the variation for chlorophyll pigmentation observed at the colony stage was transmitted as differences in morphology in the regenerated plants. The mode of shoot regeneration of N. bonariensis protoplasts and the potential value of somatic variants are discussed.     

High-efficiency colony formation and whole plant regeneration from mesophyll protoplasts of Pelargonium × hortorum ‘Panaché Sud’

Summary

This study aimed to establish a plant regeneration system from protoplasts of Pelargonium hortorum, efficient enough to be used for further direct gene transfer experiments. A rapid and efficient system that allowed high efficiency colony formation (40%) and whole plant regeneration (83%), as well as rooting within 4 months, was established using mesophyll protoplasts of the cultivar ‘Panaché Sud’. Protoplast culture in liquid medium was found to be better than culture on solid medium both for cell division and colony formation. The optimum density for high colony formation (31-40%) from viable cultivated protoplasts was 3 – 5 10⁴ protoplasts ml^–1. Reducing the osmotic pressure and increasing the macronutrient and sucrose contents of the culture medium after the first week of culture facilitated the rapid development of colonies. The transfer of microcalli to mannitol-free callus-induction medium produced green calli in all cases. The highest frequency of bud and shoot regeneration from protoplast-derived calli (83%; 6.6 per callus) was obtained at a density of 3 10⁴, on medium containing 0.2 mg l^–1 indole-3-acetic acid (IAA), 1.0 mg l^–1 zeatin and 0.1 mg l^–1 thidiazuron (TDZ). The best results were obtained when the medium was gelled with Gelrite^® and cultures were maintained under low light (12 µmol s^–1 m^–2). Sixty-five percent of protoplast-derived calli underwent bud and shoot regeneration and 2.6 rootable plantlets were obtained per callus after 3 weeks on elongation medium. All acclimatised plants grew normally and gave fertile flowers. However, flow cytometry on 42 plants showed that 40 of these were tetraploids, and only two were diploids, like the mother plant. This protocol can now be used in transformation experiments and applied to other genotypes to improve regeneration.     

影响灵芝原生质体再生的几个因素

本文比较系统地研究了培养基、酶解时间及单双层培养法等因素对灵芝原生质体再生的影响。结果表明,纤维二糖培养基及双层培养法比较适合灵芝原生质体再生。考虑到灵芝原生质体的得率以及不同酶解时间制备的灵芝原生质体的再生率,酶解2.5h的灵芝原生质体用于再生比较合适。     

Plant regeneration from protoplasts of calamondin (Citrus madurensis Lour.)

Diploid callus cells arose from the hypocotyl region of plantlets differentiated by anther culture of Calamondin (Citrus madurensis Lour.). The callus grew vigorously on Murashige and Tucker's medium containing 10 mg l⁻¹ benzyladenine, and differentiated embryoids on a medium with 5% lactose and no plant growth regulators. This embryogenic potential was transferred to protoplasts which regenerated plantlets.

Preculture of callus in liquid medium containing 5% lactose, for 4–7 days prior to protoplast isolation, effected a high yield of protoplasts. Plating efficiency was increased in a medium solidified with agarose and/or layered with liquid medium.     

Somatic hybridization of high yield,cold-hardy and disease resistant parents for citrus rootstock improvement

Summary

Increasing losses of trees to diseases, expansion of plantings into more marginal production areas and the need to control tree size to reduce harvesting costs have elevated the demand for new improved citrus rootstocks in Florida. A major strategy of the CREC rootstock improvement programme has been to use protoplast fusion to produce allotetraploid somatic hybrids that combine complementary rootstock germplasm. Tetraploid citrus rootstocks have been shown to have a built-in tree size control component due to polyploidy. This report focuses on the incorporation of newly identified superior rootstock germplasm into the somatic hybridization programme. Poncirus trifoliata 50–7, a selection of trifoliate orange selected for superior resistance to Phytophthora nicotianae, was hybridized with sour orange, Changsha mandarin, Navel orange, and a seedy white ‘Duncan’ type grapefruit. High-yielding Benton citrange (Citrus sinensis 3 Poncirus trifoliata) was hybridized with Changsha mandarin and sour orange. More than 200 plants of each of these six new somatic hybrids were propagated via tissue culture and rooted cuttings. To assess their horticultural performance, all the somatic hybrids were budded with commercially important scions and planted in replicated trials representing the two most important soil types in Florida. Seed trees of each somatic hybrid have also been planted in the field to determine fruiting potential and level of polyembryony.     

Cybrid/hybrid plants regenerated from somatic fusions between male sterile Satsuma mandarin and seedy tangelos

Somatic hybridization provides an alternative for transferring mitochondria-encoded cytoplasmic male sterility (CMS). Herein, symmetric protoplast electrofusion was conducted between embryogenic callus protoplasts of Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a CMS cultivar, and mesophyll-derived protoplasts of seedy ‘Page’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)] or ‘Nova’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)], to transfer CMS trait. Flow cytometry analysis showed that 14 plants recovered from G1 + ‘Page’ tangelo that displayed typical morphological character of ‘Page’ were diploid, and 6 plants regenerated from G1 + ‘Nova’ tangelo were tetraploid. Genetic compositions of regenerated plants from the two fusions were determined by SSR, CAPS and chloroplast-SSR analysis. Cybrid nature of diploids from G1 + ‘Page’ tangelo with nuclear DNA from ‘Page’, mitochondrial DNA (mtDNA) from the G1 and chloroplast DNA (cpDNA) derived from either parent was confirmed. Tetraploid plants from G1 + ‘Nova’ tangelo were identified as somatic hybrids with random cpDNA inheritance. The regenerated cybrid and hybrid plants hold great potential for Citrus seedless breeding at diploid or triploid levels.     

酿酒葡萄‘神索’体胚发生及再生体系遗传稳定性分析

 以酿酒葡萄‘神索’未成熟合子胚为外植体, 通过植物生长调节剂、光照、温度等因素的控制, 研究了葡萄体胚的产生、保存及植株再生。结果表明, 以NN为基本培养基, 诱导胚性愈伤组织的适宜植物生长调节剂水平是1.0 mg·L ^{- 1} 2,4-D; 诱导体胚的生长调节剂水平是1.0 mg·L ^-1 NAA + 0.5 mg·L ^{- 1} BA, 体胚诱导率为37.5%; 5℃微光条件, 适宜体胚的保存; 体胚成熟及植株再生的植物生长调节剂水平是0.05 mg·L ^{- 1} NAA + 0.5 mg·L ^{- 1} BA, 成苗率为42.1%。利用流式细胞仪并结合染色体计数对体胚及再生植株细胞核DNA含量及染色体鉴定表明, 体胚细胞染色体存在一定的变异, 而体胚再生植株倍性稳定, 细胞核DNA含量及染色体数与供体母株一致。     

草莓悬浮细胞原生质体培养再生愈伤组织

以草毒品种宝交早生的花药愈伤组织建立的悬浮细胞系为试材,对原生质体分离和再生进行研究。结果表明:酶液的渗透压、浓度和配比对悬浮细胞原生质体产量和活力有重要的影响。悬浮细胞在CPW+1.0％ Cellulase R-10+0.5 Macerozyme R-10+0.05％Pectolyase Y-23+0.6mol/L甘露醇+0.5％PVP的酶液中酶解12h,原生质体产量和活力最高,分别达16.35×10~6/g和84.6％。采用液体浅层培养法对原生质体进行培养获得了再生愈伤组织。     

柑桔胚性细胞系的建立

将乙烯作用的专化抑制剂——硝酸银应用于柑桔胚珠培养中,通过交替继代处理和不断选择,成功地建立起能供给原生质体分离并具有良好再生能力的胚性愈伤组织.