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.     

柑桔体细胞融合再生9个组合的二倍体叶肉亲本类型植株

电场诱导９个柑桔种间及体内体细胞融合,各组合均再生叶肉原生质体亲本类型植株。这些植株经形态学和细胞学检查证明为二倍体（２ｎ＝２ｘ＝１８）。对其中５个组合进行ＲＡＰＤ分析表明,４个组合植株的谱带在所分析的引物上均表现为与叶肉细胞亲本谱带一致,另１个组合,即Ｐａｇｅ柑柚＋粗柠檬,个别植株除含有叶肉亲本的所有谱带外,还扩增出了悬浮系亲本的部分特征带,为杂种类型。探讨了这类植株的产生原因及潜在价值。     

In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus

This paper describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Astragalus membranaceus and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 h prior to culture induced as high as 35.3% tetraploid plants. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploids and tetraploids in A. membranaceus were compared. It was determined that leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in A. membranaceus. As well as generating tetraploids, this technique generated mixoploids in A. membranaceus. Calli derived from mixoploid leaves, were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid and tetraploid by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but no tetraploid.     

甲基胺草磷诱导大蒜多倍体的研究

以通过生理休眠的紫皮大蒜Z-006-10鳞茎茎尖为试材,利用固体培养基中添加甲基胺草磷的离体诱导方法,研究甲基胺草磷不同处理浓度和时间对紫皮大蒜体细胞染色体的诱导效果,并利用形态观察、根尖细胞染色体计数及流式细胞仪分析等方法对得到的再生植株进行倍性鉴定。试验结果表明：10 μmol?L-1甲基胺草磷处理6 d,四倍体诱导率最高,达32.87%。形态观察结果表明,得到的四倍体植株多倍体形态特征明显,与二倍体植株差异明显。根尖染色体压片观察表明,四倍体染色体数为2n=4x=32,对照为2n=2x=16。流式细胞仪倍性分析表明,四倍体体细胞DNA的相对含量是二倍体的2倍,其结果与染色体计数法鉴定结果一致。     

不同倍性蝴蝶兰杂交后代的染色体倍性研究

 对不同倍性蝴蝶兰小孢子母细胞减数分裂及其杂交后代的根尖染色体数进行了检测。结果表 明,除三倍体和三倍体杂交未能产生杂交后代外,不同倍性蝴蝶兰杂交后代的倍性均出现分离。二倍体和 二倍体、二倍体和四倍体杂交均产生二倍体、三倍体和四倍体;二倍体和三倍体杂交产生二倍体、三倍体、 五倍体和非整倍体;三倍体和四倍体随正反交不同而异,三倍体@四倍体的后代倍性有三倍体、四倍体、 八倍体和非整倍体,而四倍体@三倍体的后代倍性有三倍体、四倍体、五倍体和非整倍体;四倍体和四倍 体杂交产生二倍体、三倍体、四倍体、五倍体、六倍体和非整倍体。不同倍性蝴蝶兰的小孢子母细胞减数 分裂普遍存在微核、落后染色体和三分体等异常现象,导致小孢子和杂种后代的倍性出现多样性。     

柑橘种间体细胞杂种的核质遗传研究

为了解柑橘体细胞杂种的核质遗传组成,采用流式细胞仪(FCM)、简单序列重复(SSR)、相关序列扩增多态性(SRAP)以及酶切扩增多态性序列(CAPS)对此前通过对称融合获得的2株种间体细胞杂种进行了分析。结果表明,在丹西红橘+红江橙种间组合中,分析的2株植株中1株为二倍体非对称体细胞杂种,其核DNA具有双亲的部分DNA,并有重组发生,胞质DNA则来源于红江橙(叶肉亲本);而另1株则是异源四倍体体细胞杂种,其核DNA来源于双亲,并有重组发生,胞质DNA则来源于红江橙。     

Stoloniferous shoot protoplast,an efficient cell system in potato for somatic cell genetic manipulations

The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10⁵ protoplasts g⁻¹ fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2n = 2x = 24) ‘C-13’ and diploid (2n = 2x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L₁–L₃). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci (STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.     

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P. parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were ‘Caipira’ sweet orange (Citrus sinensis L. Osbeck) + ‘Rangpur’ lime (C. limonia Osbeck), ‘Caipira’ sweet orange + ‘Cleopatra’ mandarin (C. reshni hort. ex Tanaka), ‘Caipira’ sweet orange + ‘Volkamer’ lemon (C. volkameriana V. Ten. & Pasq.), ‘Caipira’ sweet orange + rough lemon (C. jambhiri Lush.), ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange (C. aurantium L.), ‘Rangpur’ lime + ‘Sunki’ mandarin (C. sunki (Hayata) hort. ex Tanaka), ‘Ruby Blood’ sweet orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, ‘Rohde Red’ sweet orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, and ‘Valencia’ sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. ‘Cleopatra’ mandarin + sour orange, ‘Rangpur’ lime + ‘Sunki’ mandarin, ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Ruby Blood’ sweet orange + ‘Volkamer’ lemon, ‘Rohde Red’ sweet orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Volkamer’ lemon had less trunk rot occurrence, whereas the somatic hybrids ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange, ‘Caipira’ sweet orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Rangpur’ lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy ‘Valencia’ sweet orange budwood. Differences in average scion shoot length indicated that the hybrids ‘Cleopatra’ mandarin + sour orange and ‘Valencia’ sweet orange + Fortunella obovata were intolerant to CTV.     

番木瓜四倍体的诱导及形态学分析

在离体培养条件下,将二倍体穗中红番木瓜无菌试管苗茎尖浸入0.05%、0.1%、0.2%、0.4%秋水仙素水溶液中处理1、2、3、4、5d,诱导多倍体,对照用无菌水处理2d。结果表明0.2%的秋水仙素溶液浸泡处理3d效果最好,诱导率为36.7%。通过体细胞染色体数目检测,变异芽为四倍体或嵌合体,四倍体染色体数目为36(2n=4x=36)。对获得的四倍体和二倍体进行了形态及气孔特征的比较,结果表明四倍体与二倍体植株在叶长、叶宽、叶厚、气孔大小、气孔密度、气孔保卫细胞大小及叶绿体数目等方面存在明显的差异,其中叶片厚度、气孔密度和保卫细胞内叶绿体数目可作为鉴别四倍体与二倍体的重要性状。     

依据多倍体形态特征快速高效发掘柑橘四倍体

基于柑橘多胚性品种存在珠心细胞自然加倍的特点，建立了基于种子催芽、生长室实生播种、幼苗“观根辨叶看油胞”形态初选和流式细胞仪倍性鉴定的发掘柑橘四倍体的快速方法。采用该方法，分别从无酸甜橙、红江橙、贡柑、年橘、新会柑、滑皮金柑的432、506、1 356、1 870、2 749和685株实生苗中，发掘出疑似四倍体5、6、5、17、45、7株；通过倍性检测，从上述6个品种分别获得四倍体4、5、5、14、39、5株，形态初选准确率分别为80.0%、83.3%、100.0%、82.4%、86.7%和71.4%。与之前的四倍体发掘方法相比，本方法四倍体初选准确率高，并且从种子播种到获得四倍体植株耗时不超过40 d。SSR分子鉴定表明，所鉴定的四倍体均来自其二倍体品种珠心细胞自然加倍形成的双二倍体，为柑橘三倍体无核育种提供了珍贵的四倍体亲本资源。     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

Acquisition of triploid germplasms by controlled hybridisation between diploid and tetraploid in Chinese jujube

‘Chenguang’ is the first released tetraploid cultivar (2n = 4x = 48) of Chinese jujube (Ziziphus jujuba Mill.) induced by colchicine. The purpose of this study was to create triploid germplasms by using ‘Chenguang’ as a parent. The controlled crosses between autotetraploid ‘Chenguang’ and diploid ‘Dongzao’ were carried out from 2013 to 2015 by maintaining the 2 parents in a nylon net and employing bees as pollinator. A total of 606 fruits were produced, yet only 17 seeds were obtained from ‘Dongzao’ and none from ‘Chenguang’. Direct sowing of the seeds in greenhouse showed a higher seedling rate than embryo culture. Four progeny plants were eventually obtained in the greenhouse and all of them were confirmed as authentic hybrids by SSR marker technique. At last, 3 triploids and 1 diploid were identified by flow cytometric analysis. Consequently, tetraploid ‘Chenguang’ with high pollen vitality and pollen number per anther could be used as a reliable 2n pollen provider, and the 2x × 4x cross combination type is more suitable for triploid creation in Chinese jujube. This is the first successful production of triploid hybrids by controlled cross between diploids and tetraploids in jujube.     

澳洲指橘与粗柠檬体细胞杂种倍性FCM分析及其花粉活力检测

利用流式细胞仪对5株澳洲指橘(Microcitrus papuana Swingle,2n=2x=18)与粗柠檬(Citrus jambhiri Osbeck,2n=2x=18)属间体细胞杂种进行倍性分析,以已知倍性的二倍体粗柠檬植株的峰对应荧光值(FL)50作为对照,5棵再生植株的FL值分别为51、51、47、52、48,表明这些体细胞杂种植株均为二倍体。同时,实验结果还表明,HR(HighResolution)试剂盒中的核提取液HR-A和染色液HR-B的最佳处理时间分别是5min和2min。体细胞杂种于2002年3月首次开花,对其花粉活力进行分析,结果表明体细胞杂种的花粉活力为50％～90％。     

Colchicine- and trifluralin-mediated polyploidization of Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis

Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis are two important diploid species for rose breeding. Interspecific crosses between diploid species and tetraploid rose cultivars result in triploids with generally decreased fertility. A most promising gene transfer strategy is the production of fertile tetraploid plants before mating with tetraploids. In our study, germinating seeds of R. multiflora and R. roxburghii were treated with colchicine, and cotyledon-stage seedlings of R. multiflora were treated with colchicine or trifluralin in order to obtain tetraploid plants. Also, various concentrations of antimitotic agents and different treatment durations were tested. The results showed the antimitotic agents affected the plant morphological characteristics (i.e. plant height, stem diameter) of R. multiflora and R. roxburghii. Twenty-three tetraploid plants were obtained from the germinating seed treatments (21 from R. multiflora and two from R. roxburghii), but no tetraploid plants were obtained from the seedling treatments. The highest rate of chromosome doubling (25.0%) was achieved when germinating seeds of R. multiflora were treated with 0.2% colchicine for 12 h. At the same time, marked morphological variation was observed and analyzed among tetraploid plants and their corresponding diploids of R. multiflora.     

Tetraploid muskmelon alters morphological characteristics and improves fruit quality

Tetraploid muskmelon plants were induced successfully from diploid Cucumis melo inbred M01-3 (2n = 24) by colchicine. The morphological characteristics and fruit qualities of diploid and tetraploid muskmelons were investigated. The results showed that the leaves and flowers of the tetraploid plants were markedly larger, the plants were obviously higher, and the stems were thicker than those of the diploid plants. Transmission electron microscope observation revealed that the numbers of chloroplast, granule and grana, and the length of chloroplast and granule of the tetraploid plant leaves were significantly more or longer than those of the diploid plants. The soluble solid, soluble sugar and vitamin C contents in the tetraploid fruit were distinctly higher than those in the diploid fruit. The weight of the tetraploid fruit was 30% heavier than that of the diploid fruit. And the seeds from the tetraploid plants were broader and thicker than those from the diploid plants. Together, our results suggest that the tetraploid muskmelon exhibited better agronomical characteristics than the diploid muskmelon and the tetraploid muskmelon could be used as the improved variety and a potential germplasm for the development of triploid fruit.     

Development of seedless and Mal Secco tolerant mutant lemons through budwood irradiation

Mal secco (caused by Phoma tracheiphila (Petri) Kantsch. and Gik.) is the most destructive fungal disease of lemon plantations worldwide and seedless lemons would be preferred by most consumers. Five dosage levels, 0, 3, 5, 7, and 9 kiloradian (krad), of cobalt (⁶⁰Co) gamma irradiation were applied to budstick of ‘Kutdiken’ lemon (Citrus limon (L.) Burm. f.) clone KT-2A. Mutations were stabilized in three vegetative generations. Three hundred fifty-eight and 478 M1V3 (mutation one and vegetation three) plants were evaluated for seed number and mal secco tolerance in the field and the greenhouse, respectively. LD₅₀ was around 5 krad gamma irradiation for ‘Kutdiken’ lemon. The seed number varied from 0 to 34 per fruit. The level of mal secco tolerance also varied significantly among the plants from 1.0 (no symptom) to 4.3 (high level of disease occurance). The stable seedless and mal secco tolerant plants were obtained from 5 and 7 krad irradiation: the three mutants from 5 krad irradiation gave more lemon-like fruits, while 7 krad irradiation caused altered tree morphology and early maturation of fruits. This study shows considerable potential for lemon cultivar improvement aiming to obtain seedless and mal secco tolerant lemons.     

以柑橘多胚性二倍体母本倍性杂交培育三倍体

2009-2012年,以柑橘8个多胚性二倍体品种为母本与近年始花的5个异源四倍体体细胞杂种和1个同源四倍体分别杂交,培育三倍体新种质。连续4年共配置15个杂交组合,授粉4 442朵花,坐果1 484个,平均坐果率33.4%;用于幼胚挽救的果实1 075个,共培养幼嫩种子12 578颗,经生芽、生根诱导获得再生植株2 832个;以流式细胞仪对所有再生植株倍性检测,15个组合共获得三倍体401个,四倍体121个;移栽成活三倍体349个,四倍体98个。对W. 默科特 × NH组合89个株系的三倍体的SSR分子鉴定表明,所有三倍体后代均为双亲的有性杂种。     

脐橙与粗柠檬体细胞杂种果实类胡萝卜素、糖酸遗传的亲本偏向性

 采用高效液相色谱测定柑橘种间体细胞杂种[‘朋娜’脐橙（Citrus sinensis Osbeck）+ 粗柠檬（C. jambhiri Lush）]和其两个融合亲本果实的类胡萝卜素含量，气相色谱测定可溶性糖及有机酸含量，并检测代谢过程中关键基因的表达，比较分析体细胞杂种果实品质遗传及基因表达特点。结果表明，体细胞杂种类胡萝卜素成分及含量均偏向粗柠檬亲本，柠檬酸、苹果酸积累量也偏向粗柠檬；而蔗糖含量处在中亲值；实时定量PCR 技术检测类胡萝卜素代谢途径中的7 个基因，其中4 个基因在‘朋娜’脐橙中的表达量高于粗柠檬，环化途径中番茄红素ε 环化酶基因（CitLcy-e）、玉米黄质环氧酶基因（CitZep）在体细胞杂种中表达量偏向粗柠檬亲本，显著低于‘朋娜’脐橙。可见粗柠檬遗传物质的表达在体细胞杂种中占主导地位。     

Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using SSR markers

Several factors, i.e. the duration of thermal shock pretreatment at 35 °C, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in a variety of cucumber (Cucumis sativus L) ovary culture. The results showed that a thermal shock for 3 days at 35 °C at the start of the culture resulted in higher frequency of embryo formation than 2 or 4 days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO₃ to induction medium had no significant effect on frequency of embryo formation but shortened embryo sprouting period and improved number of embryos formed in each ovary slice. All the experiment materials responded well to ovary culture, and there were no difference among genotypes used in this study. Among the forty regenerated plants obtained, two were identified as haploid plants (2n = x = 7), five were tetraploid plants (2n = 4x = 28), and the rest were diploid plants. Microsatellite markers (SSR) were used to analyze the homozygosity of the diploid plants, the putative chromosome-doubled haploids. Of the 33 diploid plants, 17 (51.5%) were identified as double haploids. Based on the above results, we have established a useful protocol for production of cucumber doubled haploids with ovary culture.     

‘红江橙’天然多倍体的45S rDNA荧光原位杂交分析

 以实生筛选获得的红江橙三倍体和四倍体植株及其二倍体对照为试验材料,利用荧光原位杂交（Fluorescence In situ hybridization,FISH）技术分析了不同倍性红江橙体细胞染色体上45S rDNA的分布。结果表明,红江橙二倍体体细胞中期染色体中存在3个45S rDNA杂交位点,分别位于第1对染色体的核仁组成区和第8号染色体的整个随体上;三倍体体细胞中期染色体中存在4个杂交位点,其中有三个与二倍体基本相同,另外的一个位于第3号染色体的核仁组成区;四倍体体细胞中期染色体中存在6个杂交位点,分布的位置及区域和信号的强弱均与二倍体基本相同。供试材料体细胞间期核与中期染色体中的杂交信号数及强弱程度基本相似。通过分析,提出红江橙三倍体的形成与2n雌配子的形成有关,伴随有外源基因组的重组;四倍体可能产生于珠心系细胞的直接加倍,并且伴随有染色体的断裂和易位。