Somatic hybridization as a tool for tomato breeding

Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.Abbreviations cpDNA chloroplast DNA - mtDNA mitochondrial DNA     

Shoot Regeneration from Anther Culture of Sunflower (Helianthus annuus) and Some Interspecific Hybrids as Affected by Genotype and Culture Procedure

Anther culture has been demonstrated to be an applicable technique for the development of doubled haploid, i. e. homozygous lines of many crop species. In some species, androgenetic doubled haploids have already been shown to be a useful tool for breeding. However, anther culture results in sunflower have been rather unsatisfactory up to now. As in other species, anther culture response of sunflower (Helianthus sp.) is strongly affected by physical, nutritional, physiological and genetical factors. By testing a number of different culture parameters, i. e. donor plant stages, culture media and conditions, and appropriate schedule could be worked out for the successful regeneration of shoots – at least for a number of sunflower lines and interspecific hybrids.     

基因型和环境条件对小麦花药培养效果的影响

为进一步提高小麦花培育种效率,明确花药培养力的遗传控制基础,以11个小麦品种及其组配的20个F₁杂种为材料,探讨了基因型、培养基和环境条件对愈伤组织诱导率的影响。在W14D、W14gD、W14GD培养基上,Alondra、Verry、石4185、新春9号和百农3217的花药易被诱导产生愈伤组织,诱导率为25.3%~51.9%,其中石4185是目前公认的花培育种优良亲本,新春9号为新发现的优良花培基因型。以宁春4号配制的部分F₁杂种的愈伤组织诱导率较高,大多数组合高于10.0%,表明宁春4号与供试品种间具有较高的花药培养配合力。小麦花培育种技术要求亲本之一具有较高的花药愈伤组织诱导率或较高的花药培养配合力。小麦花药培养力的遗传控制复杂,表现为数量性状遗传,亲本花药培养力很高,其F₁组合花药培养力不一定很高,这与双亲配合力有关。小麦花药培养中,供体植株生长和愈伤组织诱导的适宜条件为较长的营养生长期、适宜的前期(分蘖期)温度和较高的中期(拔节后期)温度。在添加低浓度生长素和葡萄糖的液体培养基中发现小麦花药直接成苗现象,2,4-D诱导花药直接成苗效果优于Dicamba。随着年度间气候升高的影响,相同基因型花药愈伤组织诱导率呈现增加趋势。     

Embryo rescue in plants—a review

Summary The plant breeders usually rescue inherently weak, immature or hybrid embryos to prevent degeneration. The successful production of plants from the cultured embryos largely depends upon the maturation stage and the composition of the medium. Abortion of embryos at one or the other stage of development is a characteristic feature of distant hybridization. For the first time successful embryo culture to obtain an interspecific cross between Linum perenne × L. austriacum was demonstrated by Laibach (1925, 1929). Since then several refinements have been made in embryo culture/embryo rescue techniques which have been a popular approach for raising hybrids from a number of incompatible crosses. Currently embryo rescue holds great promise not only for effecting wide crosses, but also for obtaining plants from inherently weak embryos, obtaining haploid plants as well as for shortening the breeding cycle.     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

高粱骨干恢复系体细胞变异系的筛选与应用

采用高粱体细胞克隆变异育种方法，对高粱几个重要恢复系晋粱5号、三尺三、忻七等进行了改良，获得了一批综合性状好、特点突出、遗传稳定的无性变异系，其中R111已作为恢复系育成一个杂交种。     

Development of mulberry varieties for sustainable growth and leaf yield in temperate and subtropical regions of India

Sericulture is an agro-based industry, which heavily depends on mulberry (Morus spp.) for its sustainability, as mulberry leaf is the only available feed for the silkworm Bombyx mori L. Sericulture, in West Bengal and other subtropical regions in India, suffers from lack of adequate quantity of quality mulberry leaf during the colder months, the best season for rearing the high yielding bivoltine silkworm hybrids. In order to develop mulberry varieties, which can sustain normal growth during these colder months to yield better, efforts have been made in different research institutes in India. The present report is part of such an effort in which accessions in germplasm bank were screened for better growth, leaf retention and leaf yield contributing associated traits. Twenty three selected parents were crossed, 3,500 seeds were sown, 2,700 seedlings were transplanted to progeny row trail, selected 210 hybrids were evaluated under primary yield trial and finally nine hybrids along with a control variety were studied in detail for all leaf yield contributing traits under final yield trial. Stability analysis was adopted to identify hybrids, which can yield stably across seasons. The hybrids CT-44 and CT-11 out yielding the control by 17.17 and 7.11% were selected as these hybrids yield 7.93 and 8.15 mt/ha leaf respectively during the colder months (February) for their direct use as cultivars in West Bengal and other subtropical areas of India to sustain bivoltine sericulture to produce quality and gradable silk fibers.     

陆地棉品种间杂种优势利用研究进展

本文概述了陆地棉品种间杂种优势的研究和利用状况。理论研究和生产实践均表明，陆地棉品种间杂种存在明显的产量和品质优势。杂交棉高产、优质的重要原因是其适应性广、抗逆性强和稳产性好。陆地棉品种间杂种Ｆ１和Ｆ２皮棉产量的竞争优势分别在１５％和１０％左右，产量构成因素中对产量优势作用最大的是单株结铃数。目前，Ｆ１和Ｆ２均有一定的利用面积，其中印度主要利用Ｆ１，我国主要利用Ｆ２。陆地棉品种间杂种优势有广泛的应用前景。     

玉米自交系花培反应配合力的研究

采用完全双列杂交试验，对６个玉米自交系组配成的１５个单交种的单株粒重配合力和花培出愈率配合力进行了研究。结果表明，在６个自交系中京４３８０的单株粒重一般配合力（ｇｃａ）最高，出愈率的ｇｃａ较高，而Ｂ６７０的单株粒重ｇｃａ最低，但出愈率的ｇｃａ却很高；单株粒重的ｇｃａ与出愈率的ｇｃａ两者不呈正相关；京４３８０×Ｂ６７０这一单交种在１５个杂交组合中出愈率最高，花培反应最强。因此在进行玉米花培育种时，如果选用含有出愈率ｇｃａ较高的亲本自交系的组合作材料，会有较高的出愈率，有可能培育出高配合力的花培系。     

Potential for wild species in cool season food legume breeding

Wild species which are crossable to cultivated pea, lentil, and chickpea have been collected and are maintained in major germplasm collections throughout the world. Wild species of Vicia crossable to the cultivated faba bean have not been found. The primary, secondary, and tertiary gene pools of the cool season food legumes represent potential genetic diversity that may eventually be exploited in cultivated types to overcome biotic and abiotic stresses. Technical difficulties in obtaining hybrids beyond those within the primary gene pool is a major obstacle. Reproductive isolation, embryo breakdown, hybrid sterility, and limited genetic recombination are major barriers to greater use of wild germplasm. Conventional crossing has been successful in producing interspecific hybrids in Lens, Cicer and Pisum and those hybrids are being evaluated for desired recombinants. In vitro culture of hybrid embryos has been successful in overcoming barriers to wider crosses in Lens. The successful transfer of genes from wide sources to cultivated types can be assisted by repeated backcrossing and selection designed to leave behind undesired traits while transferring genes of interest. Molecular marker assisted selection may become a valuable tool in the future use of wild species. In general, too little is known about the possible genetic variation available in wild species that could be valuable in developing resistance to biotic and abiotic stresses. Current efforts on the use of wide hybridization in the cool season food legumes are reviewed and discussed.     

Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza saliva L.)

The effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4-D as control. In the two-step culture protocol, replacing 2, 4-D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther-derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly-regenerated plantlets had well-developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one-step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F₁), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F₁). The PAA-based one-step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F₁) via one-step culture. PAA also enhanced the one-step plantlet formation in rice somatic tissue culture.     

利用SSR快速鉴别花生杂交F1真伪

本研究,我们以6个花生品种为亲本配置杂交组合,田间调查发现亲本间形态表型差异大的杂交F1容易鉴别真伪,而形态表型差异小的则难于鉴别,甚至无法鉴别。为此,我们通过改良Thomson一步法制备DNA模板,建立了一套花生SSR-PCR快速检测体系,并在此基础上利用SSR鉴别花生杂交F1真伪。结果表明,采用Thomson一步法和改良后的一步法提取的DNA模版均能扩展出清晰条带,但改良后制备的DNA模板在4℃下可保存约1个月,未改良的仅能保存一天。利用SSR检测上述群体表明,F1真杂种率为38%～56%,不同亲本间杂交成功率存在差异,同一亲本正反交成功率也存在一定差异。可见,SSR标记用于杂种真伪鉴定具有一定的应用潜力。     

马铃薯高效染色体加倍方法建立与抗寒资源创制

In this work, different treatment combinations including colchicine concentrations and treatment methods as well as different time points was used to investigate the survival rate and chromosome doubling efficiency of potato tissue culture plantlets. The potato chromosome doubling by using colchicine had been successfully optimized. The potato plantlet stems treated with 0.1% colchicine for three days shaking at 120 r min^-1 showed the highest doubling efficiency due to its better contact to the colchicine solution. Compared with other potato chromosome doubling techniques, this method has much higher chromosome doubling rate, shorter time treatment and easier to operate, so that it could provide a higher efficient method for potato ploidy operation. In the meantime, compared with the diploid, the tetraploid interspecific hybrids showed differences in the morphological characteristic, which had higher plants, thicker stem, bigger petals and pollen grain. In addition, no significant difference was found between diploid and tetraploid interspecific hybrids in terms of cold resistance, but both significantly enhanced cold resistance compared with the common potato cultivar. Taken together, the doubled interspecific hybrids could sever for improving cold resistance of potato cultivars.     

桃(Prunus persica)组培快繁研究进展

桃是中国主要栽培水果之一,面积和产量均居世界首位,但生产中还没有大面积推广使用无性系抗性砧木,主要原因是桃的无性系砧木的繁殖技术,包括组培快繁技术还不完善。桃的组培快繁技术可用于脱毒、无性系砧木生产、种质资源离体保存及转基因研究等方面,但桃的组织培养相对其它果树较为困难,且不同的品种之间培养条件差异较大。为了总结分析桃组培快繁过程中出现的关键问题,本综述对桃的组培快繁过程中外植体建立、增殖、伸长、生根和炼苗移栽等环节所涉及的关键影响因子,如培养基中的养分、激素种类和浓度、碳源种类和浓度、最佳培养条件、不同的添加物等,进行了全面的分析总结,提出了桃组培快繁基本技术方案和研究思路,为桃的组织培养相关研究提供参考。     

A New Intergeneric Hybrid between Triticum aestivum L. and Agropyron fragile (Roth) Candargy: Variation in A. fragile for Suppression of the Wheat Ph-Locus Activity

New intergeneric hybrids were obtained between Triticum aestivum L. cv. Tukuho’ (2n = 6x = 42, AABBDD) and Agropyron fragile (Roth) Candargy PGR 8097 (2n = 4x = 28, PPPP) at a frequency of 1.06 %, through the use of direct embryo culture and in ovulo embryo culture. Such hybrids could be used to transfer barley yellow dwarf virus (BYDV) resistance and winterhardiness into bread wheat. The somatic chromosome number in all the hybrid plants was 2n = 5x = 35, as expected. Considerable variation in chromosome pairing was observed among the different hybrid plants. Average meiotic chromosome configuration at metaphase I was 17.29 Is + 6.57 rod Us + 1.97 ring Us + 0.18 III + 0.03 IV + 0.002 VI. The high level of chromosome pairing in some F₁ hybrids was attributed to Ph-suppressor gene(s) present in A. fragile. The hybrids could not be backcrossed to wheat, but amphiploid seeds have been obtained by colchicine treatment.     

Erucastrum abyssinicum × Brassica oleracea hybrids obtained by ovary and ovule culture

Pollen germination and pollen tube growth were studied in the cross Erucastrum abyssinicum × Brassica oleracea. 25 per cent ovules showed pollen tube entry into the micropyle 5 days after pollination. Embryos did not grow beyond the globular or heart-shaped stage. Thus post-fertilization barriers prevent hybridization in this cross. Sequential culture of ovaries and ovules 10 days after pollination helped rescue globular embryos. Three hybrids were obtained. All the hybrids flowered in vitro. Pollen fertility was 6.5 per cent. Leaf esterase and peroxidase isozymes confirmed the hybrid nature. Meiotic metaphase in the pollen mother cells showed 25 chromosomes. The presence of up to 9 bivalents and a quadrivalent indicates high homology between the two genera. The intergeneric hybrids have been multiplied using nodal segment culture. They are a source for obtaining cms cole crops and widening the gene pool of B. oleracea. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hybridization of radish (Raphanus sativus L.) and oilseed rape (Brassica napus L.) through a flower-culture method

Summary Hybridization between radish and oilseed rape has been cumbersome, requiring elaborate embryo rescue techniques. With a modified flower culture method, we have achieved successful hybridization between radish and (transgenic) oilseed rape without the laborious and technically demandingin vitro ovule or embryo rescue techniques.The hybrid nature of the intergeneric hybrids was demonstrated using morphological traits, and DNA analyses. The described method will facilitate the generation ofRaphanobrassica hybrids useful for biosafety studies of the potential for transgenes to spread in weedyCruciferae as well as for breeding programs aimed at introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape.     

How wide can a wide cross be?

Summary Wide crosses in wheat are reviewed in relation to various factors that facilitate wide crossing to show that wide crosses can be as wide as one can make them. Included in this review is a particular reference to wheat-wheatgrass (Agropyron complex) crosses and an update on wide crosses of wheat with various genera of Agropyron complex. Hybrid seed set is too variable to predict whether a wide hybrid, where no seed was obtained in one attempt, will not be possible. High crossability genes seem to facilitate not only fertilization but also seed development, enabling embryo rescue. Variability for crossability occurs not only in wheat but also in alien species. Contrary to conventional thinking, several wide hybrids with wheat can be produced when species with lower chromosome numbers are used as female parents. Pre-and post-fertilization barriers to wide crosses do not appear to be equally strong, and can be overcome by the development and application of various technologies. Considering these aspects of wide hybridization, and based on recent successes in producing previously unsuccessful and very wide hybrids, it is concluded that how wide cross between plant species can be made is an open question and that many new and wider hybrids can be produced in future.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids

The effect of the parental genotypes and colchicine treatment on the androgenic response of wheat (Triticum aestivum L.) F1 hybrids was studied. For this, anthers from three F1 hybrids and their parents were cultured on W14 initiation medium and W14 supplemented with 0.03% colchicine. The number of responding anthers, microspore‐derived structures/100 anthers, green plants/embryos cultured, green plants/100 anthers and albino plants/100 anthers were recorded. It was observed that embryo formation and plant regeneration ability were genetically controlled and genotype dependent. In both treatments the variety Kavkaz had a significantly higher percentage of responding anthers, microspore‐derived structures and green plants/100 anthers than the other genotypes. On the other hand, the variety Myconos also demonstrated high microspore‐derived structure production and green plant regeneration when treated with colchicine. The good response observed in these two varieties indicates the importance of colchicine treatment only for certain genotypes. Green plant production capacity of the hybrids was intermediate to that of the parental varieties. As one parent with a high or even an intermediate response to anther culture could lead to the production of sufficient (for breeding purposes) green plants from the F₁ hybrids, it was concluded that screening the inbred lines for the response to anther culture with and without colchicine treatment could contribute to utilization of breeding material with a low response to anther culture via the proper hybrid combinations.