Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins

Microspore embryogenesis is an inducible pathway interesting from basic and applied perspectives. For plant breeding, it is a powerful tool to produce doubled haploids, useful as pure lines. The most efficient way to produce them is through isolated microspore culture. In eggplant, one of the most important vegetable crops, this method is still poorly explored. So far, it is possible to produce doubled haploids, but not directly from embryos, because they are converted into calli early during their development. In this work we evaluated the effect of abscisic acid, epibrassinolide, polyethylene glycol, and arabinogalactans and arabinogalactan proteins, previously described as promoters of embryo induction and development in other species. When added individually to the standard protocol, all of them significantly increased induction of microspore embryogenesis and callus cell proliferation, producing more and larger calli. Particular combinations of them further improved the efficiency of the method. In particular, gum arabic containing arabinogalactans and arabinogalactan proteins allowed embryos to progress beyond the globular stage, constituting a significant improvement in order to achieve the desired direct induction of viable, germinating embryos. We also evaluated the effect of altering the concentration and relative ratio of naphthaleneacetic acid and 6-benzylaminopurine, used in the standard protocol. Significantly better results were obtained by reducing their concentration. Together, our results shed light on the morphogenic and regulatory roles of these substances on microspore embryogenesis, opening ways to further increase the efficiency of production of androgenic doubled haploids through microspore culture in eggplant.     

Development and characterization of an eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis>) doubled haploid population and a doubled haploid line with high androgenic response

We developed an eggplant doubled haploid (DH) population from a commercial hybrid through androgenesis in microspore culture. Morphological variation, reproductive ability and androgenic responsiveness were evaluated. The DH population showed segregation in vegetative traits related to leaf, flower and fruit, and in reproductive traits such as fruit and seed setting or germination rate. The DH population and subsequent generations also presented variation in the androgenic response, with null, low and high response lines. From this population, we were able to identify the first eggplant highly androgenic DH line (DH36), remarkably similar to the donor hybrid in terms of morphology and reproductive ability, but stably producing four times more calli than the hybrid. The segregating DH population is potentially useful for genetic studies and mapping of several traits, whereas the highly androgenic line DH36 may be used as a model line to facilitate the study of eggplant androgenesis and embryogenesis for both basic and applied research.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

Functional genomics of microspore embryogenesis

Isolated plant microspores, when stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards sporophytic development, with the formation of haploid embryos and ultimately doubled-haploid plants. This process is called androgenesis or microspore embryogenesis, and is widely used in plant breeding programmes to generate homozygous lines for breeding purposes. Protocols for the induction of microspore embryogenesis and the subsequent regeneration of doubled haploid (DH) plants have been successfully developed for more than 200 species. These practical advances stand in stark contrast to our knowledge of the underlying molecular genetic mechanism controlling this process. The majority of information regarding the genetic and molecular control of the developmental switch from gametophytic to sporophytic development has been garnered from four intensely studied (crop) plants comprising two dicotyledonous species, rapeseed (Brassica napus) and tobacco (Nicotiana tabacum), and two monocotyledonous species, wheat (Triticum aestivum) and barley (Hordeum vulgare). In these species the efficiency of microspore embryogenesis is very high and reproducible, making them suitable models for molecular studies. In the past, molecular studies on microspore embryogenesis have focussed mainly on the identification of genes that are differentially expressed during this developmental transition and/or early in embryo development, and have identified a number of genes whose expression marks or predicts the developmental fate of stressed microspores. More recently, functional genomics approaches have been used to obtain a broad overview of the molecular processes that take place during the establishment of microspore embryogenesis. In this review we summarise accumulated molecular data obtained in rapeseed, tobacco, wheat and barley on embryogenic induction of microspores and define common aspects involved in the androgenic switch.     

Assessment of different anther culture approaches to produce doubled haploids in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.     

Genetic,quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured <Emphasis Type="Italic">ms10</Emphasis><Superscript><Emphasis Type="Italic">35</Emphasis></Superscript> tomato anthers

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 ³⁵ gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 ³⁵ mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.     

番茄游离小孢子培养研究进展

雄核发育是小孢子或未成熟花粉细胞沿孢子体发育途径形成植株的过程，也是创制育种材料的有效途径之一。为建立重复性好、再生频率高的番茄小孢子诱导方法，文章通过回顾国内外研究进展，总结了番茄雄核番茄雄核发育各阶段（胚胎发生能力的获得、诱导小孢子分化形成胚状体以及胚状体再生形成单倍体植株）的主要影响因素，并简要归纳了现有的游离小孢子分离培养技术。分析表明，基因型是制约番茄小孢子诱导体系的关键因素，且需与发育时期、培养条件等多个影响因素同期发生，才能使雄核发育偏离配子体途径。最后，展望了番茄小孢子培养未来的研究方向与发展趋势，以期为番茄双单倍体技术的深入研究提供参考。     

Evaluation of androgenic competence through anther culture in common eggplant and related species

Anther culture is a convenient technique to obtain androgenic haploid and doubled haploid (DH) plants. In common eggplant (Solanum melongena), this technique has been used to develop DH pure lines for producing uniform F1 hybrid seed of some commercial varieties. However, a comprehensive study of the variation of this useful trait among different materials of common eggplant and related species is still lacking. In this work, we studied the androgenic response of 12 accessions of common eggplant and related materials from the primary (eggplant complex) and secondary genepools. We cultured anthers of all the accessions under the same experimental conditions, and studied their competence to produce calli, embryos and plants, as well as the quality and origin of the embryos produced. In our conditions, anthers of 11 out of the 12 accessions produced somatic calli, whereas only 5 also produced microspore-derived embryos, with variable results in terms of embryo quality and of frequency of embryo induction and plant germination. Embryos of responding accessions were initially haploid, and reached the DH status, verified with SSR markers, after a defined period of culture. In addition to other aspects common to many androgenesis-responsive species, our results allowed us to extract conclusions particular to common eggplant and relatives, including the difficulty for finding sources of androgenic competence out of S. melongena, the reduced impact of calli in the production of non-DH individuals, and the need to avoid the occurrence of severe anatomical and functional problems in the apex of most embryos, which seriously reduces their germinative success.     

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.     

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7–14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4‐dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.     

普洱茶多糖的提取及降血糖的研究

本试验从云南大叶种晒青毛茶经潮水渥堆做成的普洱茶中,提取茶叶多糖,通过分离、纯化得到纯品茶多糖（TPS）,配制成不同浓度的茶多糖溶液,以研究测定其对小鼠降血糖效果的差异;在动物活体试验中共设四组：正常对照组、糖尿病（DM）模型对照组、糖尿病80mg/kg/d剂量注射组、糖尿病160mg/kg/d剂量注射组,正常对照组和模型对照组注入同等体积的蒸馏水。采用腹腔注射。糖尿病试验小鼠选择经四氧嘧啶诱发造模后合格的小鼠,观 测其体重与血糖的变化情况,为期一周。其试验结果表明： 1.普洱茶多糖具有促进糖尿病小鼠体重恢复的作用,但无显著性差异。 2.普洱茶多糖能有效的降低糖尿病小鼠的血糖值,高剂量的普洱茶多糖（160mg/kg）比低剂量的普洱茶多糖（80mg/kg）降血糖效果要好,在统计上降血糖幅度差异性显著（P<0.05）,并且呈现一定剂量－反应关系。     

植物游离小孢子及其培养所获得的组织在遗传转化中的应用

植物游离小孢子及其培养所获得的组织是植物基因工程中优良的受体,它们具有单倍性和较高胚胎发生能力的特性。因此,以它们为受体构建遗传转化体系可以快速获得纯合的转基因植株。通过综述植物游离小孢子及其培养所获得的胚状体、愈伤组织作为转化受体在根癌农杆菌介导法、基因枪轰击法、激光微孔穿刺法、显微注射法、电激法、PEG介导法等转化技术中的应用研究进展,对目前游离小孢子作为转化受体存在的问题及发展前景进行了探讨。     

High frequency of plant regeneration from anther culture in flax, Linum usitatissimum L.

The effects of induction medium compositions on flax anther culture were investigated in order to improve the efficiency of callus induction and plant regeneration. Anthers were inoculated onto the modified MS medium supplemented with five different combinations of plant growth regulators. The medium containing the combination of 2mg/l 2,4- dichlorophenoxy-acetic acid (2,4-D) and 1 mg/1 6-benzylaminopurine (BAP) produced a significantly higher number of calli forming shoots/100 responded anthers and a significant increase in overall efficiency of regeneration than the same basal medium containing 1 mg/1 a-naphthalene-acetic acid (NAA) and 2 mg/1 BAP (CK). Among the five levels of thiamin hydrochloride tested, the modified MS medium containing 10 mg/1 thiamin hydrochloride significantly increased the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration compared with the same basal medium containing 0.1 mg/1 thiamin hydrochloride. Maltose concentration had a significant effect on the percentage of anthers producing call, the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration. The medium containing 6% or 9% maltose produced the highest overall efficiency of regeneration among the five levels of maltose evaluated. Sucrose concentration significantly affected the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration, and dramatically affected the frequency of microspore-derived plants and the frequency of spontaneous chromosome doubling in microspore-derived plants. The efficiency of doubled haploid line production obtained in this study appears adequate for applied breeding programmes.     

Anther culture response of wild Oryza species

Thirteen different wild species of the genus Oryza were investigated for their response to anther culture and plant regeneration. Callus induction from microspores of anthers was found to be strongly dependent on the species. Although large numbers of anthers from wild Oryza species, including O. barthii, O. latifolia, O. australiensis, O. minuta, O. nivara, O. paraguagensis and O. eichingeri, were cultured, no calli could be obtained. However, calli were produced from anthers of O. punctata, O. perennis, O. alta, O. ridleyi and O. rufipogon, although the frequency of callus induction was different. Similar species-dependence was observed in plant regeneration from microspore-derived calli. In total, 62 plants were derived from anther culture, including 47 albino and 15 green plants (of which 26.7% were haploids) from O. perennis; for the first time, six albino plants were obtained from O. ridleyi. Phytohormone combinations in the callus induction medium were found to influence callus induction and different wild Oryza species exhibited their own preferred phytohormone combinations for anther culture. In general, NK medium containing suitable concentrations of auxin and cytokinin may be successfully applied for anther culture of selected wild Oryza species.     

Regeneration through somatic embryogenesis from petal-derived calli of Rosa hybrida L. cv arizona (hybrid tea)

Summary Somatic embryogenesis in callus cultures of petal explants of rose cv arizona is reported here. The calli from petals initiated on dicamba containing medium were friable and gave rise to embryos after several subcultures while those obtained from other explants did not show embryogenesis. Abscisic acid and phloroglucinol were necessary during maturation and plant development, respectively. The individual embryos grew into true-to-type plants.     

Androgenesis in Hexaploid Spring Wheat F2 Populations and Their Parents Using a Multiple-Step Regeneration System

The main goals of the wheat androgenesis experiment were to study the main phenomena of in vitro androgenesis in anther culture of F₂ populations (10) and their parents (6), to compare the usage of P-4 and C-17 media for the formation of embryo/callus and to demonstrate a new plant regeneration system. The P-4 induction medium was found to be significantly better than the C-17 in the number of responsive anthers (RA) and calli induced (CI) at the 1 % and 0.1 % level, respectively. Genotypic effect was evident in both RA and CI. The yields of F₂ populations in RA and CI were significantly higher than those of their parents regarding both media. The data confirmed the existence of heterosis for RA and CI in F₂ populations. The ratio of green/albino plant regeneration was more favourable in the C-17 derived embryo/calli than in the P-4 derived ones. The frequency of plantlet regeneration was enhanced in the group of unresponsive calli by the application of the multiple-step regeneration system. In this system the calli lacking well developed morphogenic structure were transferred to a new regeneration medium, containing a higher concentration of the same cytokinin, other cytokinin or basic medium, before the occurrence of irreversible changes in their physiology.     

Intra-varietal variation for in vitro plant regeneration in the genus Trifolium

Summary Seedlings of Trifolium repens showed considerable variation with regard to the morphology and growth of their calli, and their ability for in vitro differentiation of shoots. One of the lines selected for regeneration in primary callus cultures also showed shoot formation from protoplasts. Somatic embryogenesis in callus cultures of T. pratense and T. arvense occurred only in selected seedling lines. This paper highlights the importance of screening a large number of plants within a cultivar of outbreeding species to achieve reproducible plant regeneration from tissue culture.     

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.     

The effect of colchicine treatment on microspore division and microspore-derived embryo differentiation in wheat (Triticum aestivum L.) anther culture

Summary The relationship between division symmetry andin vitro microspore embryo genesis was studied using two winter wheat varieties of high embryogenic capacity. Anther cultures were treated with colchicine added to the induction media at concentrations of 0.01%, 0.02% and 0.04%. As a result of the colchicine treatment, the rate of symmetrical divisions increased significantly which was followed by a significant increase in the microspore-derived embryo frequency. The effect of colchicine was not dependent on the concentrations used. On the basis of this it can be supposed that there is a clear relationship between symmetric divisions and microspore-derived embryo differentiation.     

两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察

 MSB培养基添加两种常用植物激素组合，Indole-3-butyric acid (IBA) + kinetin (KT)和2, 4-dichlorophenoxyacetic acid (2, 4-D) + KT，分别命名为IK和DK处理用来诱导陆地棉体细胞胚胎发生。陆地棉品系YZ1下胚轴切段作为外植体在诱导培养基上培养4周后，继代到分化培养基上促进体细胞胚胎发生。结果表明，两种处理均有体细胞胚胎发生，其中IK处理上外植体嫁接33 d后就可见体细胞胚出现，分化率高达97.6%，大多数外植体表现为体细胞胚较胚性愈伤早出现。DK处理中体细胞胚胎发生慢，体细胞胚都是经过明显的胚性愈伤发育而来，胚性愈伤分化率明显低于IK处理，仅为28.6%。此外，IK处理中的外植体在培养过程中大部分都出现了须根，而DK处理中则没有。两种处理中出现了两种不同的体细胞胚胎发生过程，组织学观察结果表明DK和IK处理中的体细胞胚分别主要起源于初生形成层和皮层。