Production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) through culture of unpollinated ovaries

The objective of this work was to produce doubled haploid plants from durum wheat through gynogenesis using unpollinated ovary culture of three local Tunisian genotypes (Jenah Khotifa, Hmira, Azizi) and three improved cultivars (Karim, Khiar, Razzek). A total of 12,000 unpollinated ovaries were cultured in this study. Spikes were either pretreated at 4°C for 14 days or at 4°C in a mannitol solution (0.3 M) for 7 days. Induction was performed using two media. We showed that ovary development, callus and plantlet regeneration was influenced significantly by genotype and growth conditions. The highest regeneration frequency was obtained when the microspore population was in the late mononucleate to binucleate stage. Our results suggested that the cold pretreatment for 14 days was more efficient than the cold treatment in a mannitol solution. Furthermore, the addition of 2,4-D, vitamins and glutamine, and the use of maltose as sugar source in media improved the ovary culture. When the unpollinated ovaries were cultured under the conditions found to be optimal in the present study, a total of 84 plants were produced, all green and haploid. The best levels for regenerated plants were obtained with the cultivars Khiar (3.5%), Hmira (3.1%) and Karim (1.5%). Fertile doubled haploid plants were obtained by colchicine treatment. This result represents a modern tool for breeders to produce durum wheat homozygous lines in a few months.     

Callus Induction and Plant Regeneration from Embryo of Elaeis guineenis Jacq

This paper deals with the preliminary results on callus induction and plant regeration from embryo in vitro of Elaeis guineenis. When mature embryos were cultured on dedifferen- tiation medium they proliferated calli during 30-90 days of culture. Among auxins applied, 2,4-D was more important for callus induction however 2,4-D and NAA combination gave bet- ter result. On the contrary, kinetin inhibited callus formation and growth. These experimental results explain that a higher callus induction frequency depends not only on the constituents of the medium used but also on the genotype of donor plants. After transfering the calli onto kinetin-containing media for a peirad, embryoids, which showed typical configuration of zygotic embryo, could be obtained. The embryoids can further develop into whole plants on a shoot induction medium. Some embryoids have subjected to srveral generations of subculture and still retained the ability to embryoid multification and plant regeneration.     

辣椒花药培养的胚状体分化及其与成苗率的关系

12个辣椒品种的花药培养的结果表明,所有品种均可诱导出胚状体,其中9个品种可获得健壮的再生植株。每一辣椒品种的适宜植物生长调节利配比不同。不同品种的胚状体诱导率和成苗率有差异。成熟的胚状体均能分化成苗,根先分化或停止发育的胚状体很少成苗。     

马铃薯未传粉子房离体培养诱导双单倍体植株

以MS为基本培养基,附加不同水平的生长素,培养未传粉马铃薯子房,经三年试验,从两个品种中获得了双单倍体的绿色小植株,(2n=2x=24);从分化绿苗力很强的球状愈伤组织中,又不断地分化出许多绿色小植株。绝大多数品种,都可以诱导出愈伤组织,一般诱导率为70％左右。品种的基因型和培养基中的生长素种类与水平在愈伤组织分化绿苗中起着重要作用。通过扦插和试管微型薯培养,可以大量繁殖试管苗,这为马铃薯单倍体育种提供了较有利的条件。     

茴香组织培养中体细胞胚胎发生的组织细胞学研究

将茴香幼茎或叶柄的愈伤组织转入附加6-BA和低浓度2,4-D的MS培养基以后,愈伤组织逐步由松软状转变成为颗粒状的胚性愈伤组织,胚状体起源于胚性愈伤组织中的单个细胞或胚性细胞团。在含NAA和6-BA的培养基中,胚状体发育成熟,并再生小植株。茴香的胚状体主要以单细胞内起源方式发生。首先由胚状体单个原始细胞分裂形成2-细胞原胚,2-细胞原胚以三种方式进行分裂:1.T- 形分裂;2.直线形分裂;3.田字形分裂。不同的分裂方式决定了胚柄的有无。茴香胚状体的发育过程与合子胚基本相同。由原胚发育成为球形胚,依次经过心形胚和鱼雷胚阶段,形成成熟的子叶胚。在胚状体发育的每一个阶段,都有其分生组织的活动中心。球形胚期,两团分生组织位于胚体中部对应的两点;心形胚期,位于两侧和中部;鱼雷胚期,分生组织的分布在子叶形成区域呈倒“U”形,在下胚轴部位呈中空的梭形。到子叶期,分生组织从两片子叶伸向胚根,呈“Y”形分布。两子叶间产生茎生长点,由生长点分化出叶原基。胚状体最终发育成为完整植株。     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cytology and Histology in Somatic Embryogenesis of Indica Rice

The somatic embryogenesis was established from mature dehulled seeds. The histological research showed that embryogenic calli were initiated first from absorbed cells of scutellum of mature seed. And then the embryoids derived from the surface of embryogenic callus. Having been the same structure like a zygotic embryo of rice, the embryoids possessed the major parts of scutellum, coleoptile and coleorhiza. In an embryoid, several developmental stages of pro-embryoid, including single embryogenic cells, two, four and multiple cell stage pro-embryeids and some abnormal embryoids were observed. It could be concluded from this experiment that the embryoid from somatic cell culture in Indica rice possessed an original form of a plant in structure like a zygotic did and derived from a single cell.     

The Preliminary Studies on Culture of Unfertilized Ovary of Rice in Vitro

The present paper reports the results of the culture of unfertilized ovaries of rice in vitro. The inducting medium was N6 supplemented with 2 mg/L 2,4-D, 500 mg/L casien hydrolysate and sucrose was 4%. The differentiated medium was N6 supplemented with 2 mg/L Kinetin, 500 mg/L casein hydrolysate and the concentration of the sucrose was 3%. The 4 cultivars and 2 crossed combinations were used as the experimental materials. The experiments were shown the differentiation of the callus occurred amony various cultivars. The induced frequency in the crossed combinations was higher than that in the cultivars. Now 12 green plants and 3 albino plantlets have been obtained. The chromasomes of 11 green plantlets have been examined. Among them, 6 plantlets were haploid (n =12 ) and 5 plantlets were diploid. The embryoids were located in the micropylar end. Some of them possessed the suspensor, similar as zygote embryos. The callus was found from different origin. One of them was originated from haploid tissue derived from the nuclear in the embryo sac. Another was originated from the diploid tissue in the integument or ovary wall. The origin of the callus from the unfertilized ovary was discussed.     

盾叶薯蓣花药培养及单倍体植株的获得

以处于单核期的盾叶薯蓣花药为材料,经愈伤组织途径成功获得单倍体盾叶薯蓣新材料。对盾叶薯蓣花药培养、植株再生过程的研究表明:来自不同居群的外植体对花药愈伤组织诱导有显著影响;W14是适合花药愈伤组织诱导的基本培养基;花药愈伤组织增殖和分化的适宜培养基为:MS基本培养基 BA2.0mg/L IAA0.2mg/L;生根培养基为添加了IBA2.0mg/L、NAA0.4mg/L和0.5g/L活性碳的MS培养基。以流式细胞仪和根尖染色体压片法检查花培植株的结果表明,有8%～12%的个体为单倍体。实验结果为进一步开展单倍体育种及相关理论研究提供了材料和技术基础。     

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.     

The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.)

Microspores were isolated from wheat (Triticum aestivum L.) spikes of various genotypes following an effective pretreatment that induced microspore embryogenesis. The isolated microspores were cultured with or without live ovaries, and with or without medium pre-conditioned by ovaries for varying periods of time. Live ovaries alone increased androgenic embryoid yields up to 4.5-fold over the control for microspores isolated from responsive genotypes. While live ovary co-culture alone was not effective for microspores isolated from recalcitrant genotypes, the addition of medium preconditioned by ovaries to microspore cultures increased embryoid yield by more than 100-fold. Without ovary-conditioned medium, no embryoids could be obtained from some recalcitrant genotypes. Ovary-conditioned medium apparently functions to increase embryoid yields by providing essential substance(s) for elaboration of the embryogenic program already triggered during pretreatment.     

Effect of optimal stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber (Cucumis sativus L.)

Gynogenic plants of pickling cucumber were successfully produced by in vitro culture from unpollinated ovules. Haploids and spontaneous doubled haploids originated directly through embryogenesis. The cucumber ovaries were placed on induction media containing thidiazuron and cultured in the dark for 2-5 days, at 24°C, 28°C or 35°C. After induction, the material was transferred to regeneration media containing !-naphthaleneacetic acid and 6-benzylaminopurine. Results from the regeneration test indicated that the heat treatment increased the efficiency of gynogenesis in the optimal developmental stage of the female gametophyte. The highest number of embryos occurred following the35°C induction treatment. The maximum frequency of gynogenesis was 18.4%, while maximum plant regeneration was 7.1%. The flow cytometry analysis showed that 87.7% of the regenerants were haploid. On the basis of histological studies carried out on the female gametophyte, the best developmental stage for haploid induction seems to be the cellularization stage of embryo-sac formation, when the nuclei are already in position, the membranes have sometimes developed, and the cells are quite uniform in shape and structure.     

红江橙体胚发生及影响因素的研究

红江橙花后４周的幼果,直径约１ｃｍ左右,这时期的胚珠最适于胚性愈伤组织的诱导。以ＭＴ为基本培养基,加上适当浓度的ＩＡＡ、ＢＡ和ＭＥ可显著提高胚性愈伤组织诱导率。体胚发生,以ＭＴ＋ＩＡＡ０．１ｍｇ１－１＋ＢＡ（ＺＴ）１ｍｇ１－１培养基较好,能诱导产生正常体胚,且频率较高。胚性愈伤组织继代次数对体胚发生也有一定的影响,继代６次以内,胚性愈伤组织具有旺盛产生体胚能力;继代至第９次时,产生体胚的能力明显下降;至第１２代时,不能产生胚状体。成熟胚转换成小植株,以ＭＴ＋ＧＡ２ｍｇ１－１＋ＮＡＡ０．１ｍｇ１－１培养基成苗率最高。     

南瓜未授粉子房离体培养及植株再生

以南瓜(Cucurbita moschata)未授粉子房为外植体, 在离体条件下研究了激素种类、基因型、胚囊发育时期、消毒方式和热激处理时间对胚状体诱导效果的影响, 旨在建立完善的南瓜未授粉子房离体诱导再生体系。结果表明, MS+4.0 mg·L^-1 2,4-D+0.5 mg·L^-1 NAA+1.0 mg·L^-1 6-BA和MS+0.04 mg·L^-1 TDZ两种培养基胚状体的诱导率都较高, 分别为19.8%和20.1%, 二者相比, 使用TDZ诱导更加简单; 在6个南瓜品种中, 2个生长势较强的品种(雪峰蜜本和鱷鱼南瓜)胚状体诱导率较高, 适合作为离体雌核诱导的实验材料; 开花当天的子房先切片后消毒可以有效降低愈伤组织的形成, 提高胚状体的诱导率; 在黑暗且35°C条件下热激5天有利于子房的转绿和出胚。将子叶胚转移至无激素的MS培养基上得到再生植株并移栽成活。经鉴定有7个再生植株的染色体数目n=x=22, 气孔保卫细胞叶绿体数的平均值为4.28个, 是单倍体植株。     

几种匍匐翦股颖成熟胚愈伤组织诱导及植株再生

利用成熟种子作为外植体,分析了2,4-D对匍匐翦股颖胚性愈伤组织诱导与植株再生体系的影响,并对体细胞胚的发生过程进行了观察.实验结果表明,在2.0 mg/L 2,4-D 0.1 mg/L 6-BA时胚性愈伤组织的诱导频率最高.随着2,4-D浓度的增加,愈伤组织的诱导和分化能力明显下降.在再生过程中采用1.0 mg/L的6-BA达到了比较好的效果,愈伤组织的再生频率大部分在90%以上.同时发现适当提高肌醇浓度可以使苗长得较为粗壮.在实验中发现匍匐翦股颖的体细胞胚发生过程为球形胚、心形胚、鱼雷胚和子叶胚.     

High Frequency Induction of Somatic Embryoid of Rice in Suspension Culture

On MS and B5 basic media the mature seed of rice (Oryza sativa L.) was induced to produce somatic embryoid. The results showed that the rate of occurrence of somatic embryoid varied with the variety of rice and the concentration of 2,4-D was important to the occurrence of somatic embryoid. For those varieties of rice suitable for induction, the induction factors such as KT, zeatin, BAP, ABA, osmotic pressure and so forth were stringently required at different stages of somatic embryoid development, and they also influenced the formation of somatic embryoid. The results of various layered cultures of embryonic calli proved that the somatic embryoid was derived from the surface layer callus containing embryonic cells. At the induction stage no somatic embryoid was formed in callus. The rate of somatic embryoid formation was over seventy per cent on liquid differentiation medium I.     

烟草未授粉子房胚状体诱导的研究

对烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）未授粉子房胚状体的诱导进行了研究,结果发现,胚状体是单细胞起源,起源于大孢子或卵细胞,接种发育早期的子房,胚状体起源于大孢子;接种发育晚期的子房,胚状体起源于卵细胞;接种发育中期的子种,胚状多起源于大孢子,少数起源于卵细胞,不同的基因型,蔗糖浓度及光照强度等对胚状体诱导率的影响亦有不同。     

Morphogenetic Aspects of Gynogenetic Embryoid and Callus in Ovary Culture of Oryza sativa L.

This paper is a further study on the morphogenetic processes of gynogenetic embryoid and callus formation in rice unpollinated ovary culture. Techniques for culture and for specimen preparation were basieany the same as in previously reported experiments in our lab. During early stage of culture, gynogenetic structures within the embryo sacs were predominantly young pear-shaped proembryos. A few of them developed later along a more or less normal pathway into differentiated embryoids. However, most proembryos grew much larger without embryo differentiation and finally turned to callusing. A discussion is given to the morphogenetic aspects of such kind of callus, which is considered to be a structure somewhat similar to the “protocorm” described by Nostog in barley proembryo culture. In this paper, the process of proembryo abortion and the oecurenee of polyembryony are reported as well.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).