Recovery of haploid and diploid plantlets from anther culture of Citrus clementina Hort,ex Tan. and Citrus reticulata Blanco

Summary

Research on androgenesis in two clementine cultivars (Nules and S.R.A. 63) and two mandarin cultivars (Avana and Tardivo di Ciaculli) was carried out with the aim of obtaining haploids. The anthers, collected at the uninucleated stage were cultured first on 11 media, differing either in the basic medium, the growth regulators, the carbon source and concentration, or the presence of activated charcoal. Calli, shoots, embryos and then plantlets were obtained. Significant differences were observed between the different cultural conditions and genotypes. Cytological observations on calli and plantlets from ‘Nules’ anthers revealed the haploid chromosome number, while mandarin calli and plantlets, and S.R.A. 63 calli had the diploid chromosome number. Electrophoretic analyses on calli and leaf tissues showed that ‘Nules’ had a homozygous genotype and confirmed that these tissues had developed from microspores.     

黄瓜未授粉子房培养获得同源四倍体

为获得稳定纯合的同源四倍体黄瓜材料,采用多种分化培养基对黄瓜(Cucumis sativus L.)未授粉子房培养形成的胚进行培养。结果共获得33个再生植株,经染色体计数后发现7株为同源四倍体植株(2n=4x=28)。利用SSR手段对获得的同源四倍体植株进行了同质性鉴定,发现均为纯合子。利用形态学观察和染色体计数法对同源四倍体单株自交后代的遗传稳定性进行了研究,结果表明自交后代倍性没有发生变异。对此类型同源四倍体植株和经秋水仙素诱导获得的同源四倍体植株分别进行了花粉可染率、花粉萌发率及自交结籽数的观察,发现前者具有高的育性,自交结籽数为15～34粒。上述结果表明,黄瓜未授粉子房培养是获得同源四倍体的一条途径,获得的稳定纯合且育性好的同源四倍体材料可作为特异资源用于黄瓜倍性育种。     

Inheritance of fertility restoration of male-sterility conferred by cytotype Y and identification of instability of male fertility phenotypes in onion (Allium cepa L.)

A novel onion (Allium cepa L.) cytoplasm, cytotype Y, was found in a previous study. Cytotype Y contained unique stoichiometry of coxI and orf725, a candidate gene responsible for male-sterility induction in onions. A S₁ segregating population was produced from a single plant selected from PI273626. Although male-fertility segregated in this population, the ratio significantly deviated from single-gene inheritance. However, genotypes of RF31446 marker perfectly linked to Ms locus-controlling fertility restoration completely matched with male-fertility phenotypes, indicating that male-fertility restoration of male-sterility conferred by cytotype Y might be determined by the Ms locus. One plant derived from the S₁ population showed discrepancy between male-fertility phenotype and RF31446 genotype. Although the RF31446 genotype was homozygous recessive, reduced amount of pollen grains were observed in anthers. Many pollen grains of the unstable male-sterile plant were deformed. Analysis of 13 molecular markers flanking the Ms locus showed no crossover between the Ms locus and the RF31446 marker. Ten more unstable male-sterile plants were identified from open-pollinated progenies of the unstable male-sterile plant. Viable seeds were successfully produced from unstable male-sterile plants, indicating that pollen grains of the unstable male-sterile plants were partially viable. In addition, an umbel containing unstable male-fertile flowers was identified from one of maintainer lines, although both male and female organs might be sterile in these flowers.     

Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.)

Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l⁻¹ from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F₁ showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F₁ was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l⁻¹) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l⁻¹ produced the best result. MS medium containing 90 g l⁻¹ did not produce gynogenic ovules.     

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。     

羽衣甘蓝游离小孢子培养技术研究及应用

以10个羽衣甘蓝杂交种为试材进行游离小孢子培养,研究胚状体发生和小孢子胚成苗的影响因素,以及小孢子植株倍性鉴定和单倍体加倍方法。结果表明,盛花期为最佳取样时期;培养基中13%的蔗糖较为适宜;继代培养以MS+6-BA 2 mg·L^-1+NAA 0.1 mg·L^-1为宜,平均增殖系数为5.06;小孢子再生植株成活率74.6%;小孢子植株自然加倍率为23.33%~37.50%;单倍体试管苗的加倍处理以秋水仙素浓度70 mg·L^-1,处理时间9~11d为宜,加倍率达54.55%。     

Production of virus-free plantlets by anther culture of LiliumבEnchantment’

Anthers of the LiliumבEnchantment’, excised at the uninucleate microspore stage, were cultured on MS media containing 6% sucrose with auxin and cytokinin. When anthers were cultured on the medium with 2 mg l⁻¹ picloram and zeatin, 12–86% of them formed nodular calli. Anthers excised from greenhouse- and field-grown plants showed different responses: anthers of greenhouse-grown plants had a significantly higher capability to form callus and regenerate bulblets than those of field-grown plants. In anthers from greenhouse-grown plants, bulblet formation was dependent on the time at which anthers were excised from donor plants: anthers collected from early forced mother-plants had a higher capability of forming bulblet than others. All regenerated plantlets were diploid, which was substantiated by histological observation showing that the anther-derived calli originated from anther wall tissues. Virus tests by ELISA were made for 49 plantlets selected randomly at transplanting: 20 plantlets (41%) were virus-free, and the rest showed positive reactions for lily symptomless virus, cucumber mosaic virus and/or tulip breaking virus.     

Evaluation of crucial factors for implementing shed-microspore culture of Indonesian hot pepper (Capsicum annuum L.) cultivars

A shed-microspore culture protocol was developed in Wageningen for producing doubled haploid plants in several genotypes of Indonesian hot pepper (Capsicum annuum L.). For transfer of technology to Indonesia, three factors were studied that appeared crucial for successful implementation in practice. First, application in the culture medium of a combination of the antibiotics timentin and rifampicin at the concentrations of 200 and 10 mg/l, respectively, prevented bacterial contamination from the donor explants. Second, in vitro application of colchicine (100 μM) during the first week of culture was highly effective in increasing the percentage of doubled haploid plants. Third, a comparative analysis of the ploidy level of plants regenerated from shed-microspore-derived embryos using chloroplast counts in guard cells of leaf stomata and flow cytometric measurement of leaf nuclear DNA content, revealed that the first procedure is a reliable and an easy to use method for ploidy determination with hot pepper.     

Haploid plant production in Zantedeschia aethiopica ‘Hong Gan’ using anther culture

This report describes advances in the anther culture of Zantedeschia aethiopica. Important factors for improvement as compared to the earlier procedure were: (1) using flowers from inflorescences developed at relatively low temperature during winter, (2) high temperature stress treatment at 32 °C for 2 days in the beginning of the culture, (3) use of Gamborg B5 as anther culture medium, and (4) addition of sucrose at high concentration of 8% in the culture medium. Plants were obtained via a callus phase. Frequency of anthers producing calli was around 4–5%. About 87% of the calli gave regenerants, of which 52% were haploid, 36% were diploid and the rest had other ploidy levels. In addition to chromosome counting, cytological examination of the microspore development and amplified fragment length polymorphism (AFLP) analysis of the regenerants showed that haploid as well as diploid plants originated from the microspores. Finally, 12 doubled haploid (DH) plants could be produced from each inflorescence. One quarter of the DHs equaled the original cultivar in growth vigor, while more than one third showed good fertility, indicating that inbreeding depression was not so severe in this heterozygous species. The improved protocol now enables production of sufficient number of DHs for application of haploid technology in genetic improvement and breeding of Z. aethiopica.     

银杏幼胚离体培养再生植株的研究

 以佛手银杏幼胚为外植体, 研究了不同大小幼胚在不同培养条件下, 愈伤组织和胚状体的诱导发育情况。结果表明: 大于3 mm的幼胚, 在MK +NAA 1.0 mg·L ^{- 1} +BA 1.0 mg·L ^{- 1}培养基上胚状体诱导率最高, 达到53.6% , 最多的一块愈伤组织形成多达38个胚状体。暗培养不利于胚状体的发生。在MK培养基上添加10%椰汁对胚状体的生长发育有很好的促进作用, 有34.5%生长成苗。     

Somatic embryogenesis and plant regeneration in black pepper (Piper nigrum L.): I. Direct somatic embryogenesis from tissues of germinating seeds and ontogeny of somatic embryos

Summary

A protocol was developed for induction, maturation and germination of somatic embryos from the tissues of germinating seeds of black pepper (Piper nigrum L.). Explants were cultured on growth regulator – free solid SH medium maintained in the dark. The first somatic embryos developing directly from the explant tissue were noticed after 60 d of culture. Somatic embryos originated from a ring-like tissue on the micropylar region of the seeds. Sucrose concentration of the medium was found to be crucial for the induction of somatic embryos, and 30 g l^–1 was found to be the optimum. Maturation and germination of somatic embryos were achieved on the same medium. Suspension culture enhanced the process of maturation and germination. Regenerated plants were established in soil. Histology confirmed the ontogeny and each stage of development. Growth regulators were found to inhibit the induction of somatic embryogenesis. Cytological analysis of the regenerated plants revealed the normal chromosome number of 2n=52.     

结球甘蓝—大白菜异源三倍体小孢子培养的研究

 为创建结球甘蓝—大白菜异附加系, 以大白菜二倍体(AA, 2n=2x=20) 与结球甘蓝四倍体(CCCC, 2n=4x=36) 杂交获得的8个异源三倍体(ACC, 2n=3x=28) 单株系为试材, 进行游离小孢子培养研究, 获得再生植株, 并对其进行了染色体数目鉴定和性状调查。结果表明, 异源三倍体(ACC) 的小孢子诱导成胚的能力低于双亲; 在NLN中添加15%的蔗糖和0.1 mg·L^-1 6-BA、0.2 mg·L^-1 2, 4-D、0.05 mg·L^-1 KT的培养基上小孢子胚胎发生最高为0.0250个·蕾^-1, 8个株系间有差异, 平均为0.0100个·蕾^-1; 胚胎再生率只有33.3%。小孢子再生植株染色体数在14～27条之间, 田间性状表现多样。     

Direct somatic embryogenesis in Epipactis veratrifolia,a temperate terrestrial orchid

Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA₃) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L^?1) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA₃. FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.     

Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper (Capsicum annuum L.)

A low percentage of normal-looking embryos (20%) is still a problem in an efficient shed-microspore culture of hot pepper (Capsicum annuum L.) and it is more a serious problem in other androgenesic culture systems of pepper. Therefore, several factors were investigated to refine the protocol in order to increase the percentage of normal-looking embryos. The most important factors which improved the protocol and resulted in a significantly higher percentage of normal-looking embryos produces (>50%), were delayed enrichment of the liquid upper layer medium with 2.5 μM Zeatin and 5 μM IAA, and reduced incubation temperature from 28 °C to 21 °C, both after 3 weeks of culture. Addition of 1% activated charcoal in the solid lower layer of the medium enhanced the total embryo yield only, while an application of abscisic acid and increased osmolality medium had a detrimental effect on embryo production. The use of doubled haploid lines as anther donor plants has clearly decreased the variability and improved the statistical analysis of treatment effects. A higher percentage of normal embryos are produced with this refined protocol, and is therefore more suitable for implementation in the breeding programs of hot pepper.     

Embryo induction via anther culture in papaya and sex analysis of the derived plantlets

We investigated the embryo induction of papaya by anther culture, and identified the sex of plantlets derived from embryos using a sex-diagnostic PCR. Anthers, containing approximately 80% uninucleate pollen, were collected from 10 to 14 mm long male flower buds. They were pre-treated on agar (0.8%) or in liquid medium for 1–5 days at 25 or 35 °C, then transferred to agar medium with 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Agar and liquid media used for the pre-treatment contained water only or MS nutrients with or without sucrose (2.0%). On the agar medium, no embryos were induced at any pre-treatment temperature. In the liquid medium at 25 °C, embryos were induced at about 1.0% (rate of anthers forming embryos) in MS medium with sucrose for 3 or 5 days. At 35 °C, embryo induction rate tended to increase up to about 4.0% when anthers were treated in water for 1 day or MS medium with sucrose for 3 or 5 days. The sex of plantlets established through anther culture was analyzed using a sex-diagnostic PCR. All plantlets were determined as female. From these results, we suggest that all plantlets established through anther culture were of microspore origin, and that the anther culture technique is useful for the breeding of female papaya.     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

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.     

Effects of media,carbon sources and cytokinins on shoot organogenesis in the Christmas tree Scots pine (Pinus sylvestris L.)

Summary

Significant effects of seven basal media and three carbon sources (sucrose, glucose and fructose) on the induction of adventitious buds from embryos of Pinus sylvestris L. were observed. Moreover, hyperhydricity of expiants and shoot regenerants was observed on basal media containing fructose, especially with half-strength Murashige and Skoog (MS), MS, and woody plant medium (WPM). Expiants grown on a Gresshoff and Doy (GD) medium with sucrose produced the highest frequency of regeneration (81%) and with no hyperhydricity observed of developing adventitious shoots. Among three cytokinins tested including BA, BPA, and TDZ (at four concentrations each), 5 μM BA resulted in the highest regeneration frequency and mean number of adventitious shoots per embryo. Shoot régénérants were elongated after transfer to a GD medium containing 2 g-l_–1 activated charcoal and no growth regulators. After one month, rooting was induced on 10% of expiants.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

Temporary immersion and stationary bioreactors for mass propagation of true-to-type highbush,half-high,and hybrid blueberries (Vaccinium spp.)

True-to-type propagules in half-high, highbush, and hybrid blueberries (Vaccinium L. spp.) were produced using stationary (SB) and temporary immersion bioreactor (TIB) systems containing a liquid medium. Multiple shoots were produced in vitro from nodal segments of blueberry cultivars ‘St. Cloud’ and ‘Polaris’, and of six blueberry hybrids obtained from crossing between half-high/highbush and lowbush blueberries. Shoot proliferation was best in a liquid medium containing 4.6 µM zeatin in both TIB and SB systems, but the performance was genotype dependent. Shoot proliferation was better in hybrids than in cultivars. Although SB produced longer shoots with more leaves per shoot in most of the genotypes, TIB-derived shoots were more vigorous and rooted better under ex vitro condition. Liquid culture-derived elongated shoots were rooted ex vitro by treating with indole-3-butyric acid (39.4 mM) before planting on a 3 peat:2 perlite (v/v) medium. Micropropagules were acclimatized and maintained in a greenhouse with 80?90% survival rate of rooted plantlets. Expressed sequence tag (EST)-polymerase chain reaction (PCR) and EST- and genomic-simple sequence repeat (SSR) marker assay formed a homogenous monomorphic banding pattern in the in vitro-derived and donor control plants proving the clonal fidelity of liquid-culture-derived micropropagated plants.