The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l^-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Regeneration of tepals,stamens and ovules in explants from perianth of Hyacinthus orientalis L. importance of explant age and exogenous hormones

Regeneration of tepals, stamens and ovules from perianth explants of Hyacinthus orientalis L. in different developmental stages could be controlled by means of exogenous hormones. Perianth explants in a relatively early stage of development were competent for differentiation of tepals on Murashige and Skoog (MS) medium supplemented with 2 mg·1^-1 N⁶-benzylaminopurine (BAP) or zeatin and 0.1 mg·1^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Perianth explants in a later stage of development regenerated stamens and ovules, and marked difference was observed in the activity of BAP and zeatin in this regard. Zeatin stimulated more strongly stamen formation, while BAP enhanced ovule formation. Thus, stamens were formed when the explants were cultured for four months on medium with 2 mg·1^-1 BAP and 0.1 mg·1^-1 2,4-D and then transferred to medium with 0.2 mg·1^-1 zeatin and 0.005 mg·1^-1 1-naphthaleneacetic acid. On the other hand, differentiation of ovules occured in explants cultured for two weeks on the former medium and then transferred to medium with 0.1 mg·1^-1 BAP and 0.01 mg·1^-1 2,4-D. Although ovule formation could also be obtained with 2 mg·1^-1 BAP alone, it was substantially enhanced by the presence of 0.1 mg·1^-1 2,4-D in the medium in the early stages of culture. The results demonstrate the importance of both the developmental stage of the source organ from which explants are excised and of the hormone composition of the medium for the regeneration of different floral organs by perianth explants of Hyacinthus.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Effect of kinetin and GA3 on in vitro ovule embryo culture of Cucumis melo L.

The ability of Cucumis melo embryos of different ages to form plants in vitro was studied in order to rescue hybrid embryos between C. melo and Cucumis metuliferus. Plants were grown in a glasshouse at temperatures ranging from 15°CN-28°CD. Best results were obtained with ovule embryos excised 17 days after pollination. At this age, kinetin of 0.5 mg l^–1 was found optimal for culturing embryo development. Similar results were obtained with ovule embryos excised 14 days after pollination which cultured on 0.5 mg l^–1 kinetin with 0.5 mg l^–1 GA₃.     

An improved protocol for carrot haploid and doubled haploid plant production using induced parthenogenesis and ovule excision in vitro

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

In vitro gynogenesis in cotton (<Emphasis Type="Italic">Gossypium</Emphasis> sp.)

The effects of genotype, pollen or growth regulator-pretreatment of pistils, developmental stage of the ovule (embryo sac) and culture media on induction of gynogenesis, and subsequent plantlet regeneration in vitro were assessed in interspecific Gossypium barbadense × G. hirsutum cotton hybrids. Gynogenesis occurred in all genotypes used when the pistils had been pre-treated with pollen from Hibiscus cannabinus and ovaries were harvested 5 or 10 days after anthesis. The use of culture media, SH and MS, showed no significant differences in responding ovules, embryogenic ovules or embryo germination frequency. Recovered progeny were characterized cytogenetically and microscopically to help documenting their reproductive basis. Root tip chromosome counts of 17 plants established from ovule culture revealed that chromosome numbers ranged from 27 to 44. Although the reproductive mechanisms need to be characterized more extensively by cytological and molecular means, the observations suggest that gynogenesis in cotton involves some unusual reproductive events. Aneuploids could be useful for functional genomic characterization of genome shock, deletion mapping, and germplasm introgression.     

Maternal haploids of Petunia axillaris (Lam.) B.S.P. via culture of placenta attached ovules

Summary Hybridization of Petunia axillaris and P. parodii with Nicotiana tabacum was attempted using the method of in vitro pollination and fertilization. Seedlings were produced when the Petunia species and N. tabacum were used as the maternal parents; however, most of these had the identical somatic chromosome complement of the maternal parent. With crosses involving P. axillaris as the maternal parent, a low frequency of haploids was also produced. Due to the potential of haploids in basic and applied genetic research, additional experiments were carried out to determine whether in vitro pollination was necessary to stimulate haploid production and to more closely define the optimal time for ovule excision and culture. Four treatments were applied to accomplish these objectives. They were: placentas cultured prior to the time of anthesis, with and without pollination, and placentas cultured after the time of anthesis, with and without pollination. In vitro pollination had no effect on the frequency of haploids produced. Placenta attached ovules cultured prior to the time of anthesis produced significantly more haploids than those cultured after anthesis. The preanthesis treatment produced a frequency of 6.5 haploids per 100 ovaries cultured. The culture of placenta attached ovules provides an alternative to anther culture as a means for haploid production.The investigations reported herein were supported by USDA/SEA/CRGO Project 59-2213-1-1-613-0 and the paper (No. 84-3-36) is published with the approval of the Director of the Kentucky Agricultural Experiment StationThe authors are Graduate Research Assistant and Professor, respectively, Department of Agronomy, University of Kentucky, Lexington 40546-0091. The research reported in this paper is in partial fulfillment of the PhD requirements for the senior author     

Plant regeneration from undeveloped ovules and embryogenic calli of Citrus: Embryo production,germination, and plant survival

Embryogenic cultures were initiated from undeveloped ovules of several polyembryonic Citrus species on a basal medium supplemented with either malt extract, 2,4-D alone, or 2,4-D in combination with BA or daminozide. Primary embryos of all responsive cultivars were harvested directly from ovule cultures; secondary embryo harvests were made from Handin orange ovule cultures and long-term embryogenic callus. Differences were observed among cultivars and treatments in percentage of responsive ovules and total number of embryos produced. The most effective treatment for embryo production varied among cultivars. Embryo germination and plant establishment frequencies were determined for this plant regeneration system. Differences among cultivars with respect to regenerate survival percentage were minimal. Plant regeneration via secondary or long-term callus-derived embryos was as efficient as from primary embryos. Critical factors influencing plant production and survival were the production of normal viable embryos, balanced germination, and successful acclimatization to the external environment.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Influence of intraovular reserves on ovule fate in apricot (Prunus armeniaca L.)

 In many plant species with multiovulate ovaries, a considerable reduction in the number of ovules takes place. However, the underlying physiological causes are not clear. In Prunus spp., although flowers present two ovules, usually only one seed is produced. We have followed the development and degeneration of the two ovules in apricot (Prunus armeniaca L.) and examined the extent to which carbohydrates within the ovule might be involved in determining the fate of the ovule. While the primary ovule grows in the days following anthesis, growth of the secondary ovule is arrested. Starch distribution along the different ovular tissues exhibits several changes that are different in the two ovules. Primary ovule growth is inversely related to starch content and this growth takes place independently of pollination since it occurs in the same way in pollinated and unpollinated flowers. In the secondary ovule, starch disappears simultaneously from all ovular structures and callose is layered at the chalazal end of the nucellus. The size of the secondary ovule does not change significantly from anthesis to degeneration, and callose starts to accumulate 5 days after anthesis. Likewise, this process occurs independently of pollination. These results are discussed in terms of the implications of the starch content of ovules in fertilization success and ovule fate. Received: 26 August 1997 / Revision accepted: 17 December 1997     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

OVULE ABORTION IN ‘NONPAREIL’ ALMOND (PRUNUS DULCIS [MILL.] D. A. WEBB)

The earliest indication of ovule abortion in almond (Prunus dulcis [Mill.] D. A. Webb ‘Nonpareil‘) is the deposition of callose (as indicated by aniline blue fluorescence) 2 days after pollination which is 2 days before clear histological symptoms of ovule degeneration are evident and 6 days before fertilization of the viable ovule. Callose deposition begins in the chalazal region of the nucellus where the funicular trace enters the ovule and ramifies into the integuments. As ovule abortion progresses, callose deposition in the inner integument extends as a ring around the nucellus. Movement of the fluorescent dye disodium fluorescein (uranin) indicated that translocation from the vascular trace into abortive ovules becomes blocked at the chalazal position. The dye freely penetrates and diffuses into viable ovules but fails to penetrate abortive ovules. Lack of, or delayed and irregular, megagametophyte development was another characteristic of abortive ovules. Biochemical and histochemical analyses of abortive and viable ovules indicated that carbohydrate depletion parallels ovule abortion. These observations lead to the conclusion that ovule abortion is accompanied by blockage in metabolite supply although whether this blockage is the primary cause or a consequence of ovule abortion is uncertain.     

A refined method for ovule culture in sugar beet (Beta vulgaris L.)

Induction of gynogenesis through ovule culture is a valuable tool to produce haploid and doubled haploid plants in sugar beet (Beta vulgaris L.). However, there is still large room for refining the method. In this study we investigated the gynogenic response of cultured ovules of three sugar beet genotypes, the effect of the application to inflorescences of different pretreatments with mannitol at 4ºC and with 5-azacytidine and 2,4-D, and the effect of the use of different basal culture media and sucrose concentrations. The response was evaluated in terms of percentages of induction of gynogenesis, embryogenesis and callogenesis, as well as of regenerated plants. We showed that a pretreatment with 0.5 M mannitol at 4 °C for 4 days, and with 50 µM 5-AzaC for 1 h, notably improved the percentage of embryogenesis and plant regeneration. Besides, the use of MS basal medium and 60 g/L sucrose was also found beneficial. This study provides new ways to improve the efficiency of haploid induction and plant regeneration through ovule culture in sugar beet, and is potentially applicable to ovule culture in other crops.

     

小叶兜兰的果实生长和雌配子体发育

为了解濒危兰科植物小叶兜兰(Paphiopedilum barbigerum Tang et Wang)胚珠和雌配子体的发育过程,采用常规石蜡切片技术对其果实的生长动态进行了研究。结果表明,授粉后60~75 d的蒴果内种子数量迅速增加,到授粉后120 d时种子充满整个蒴果。授粉后40 d的胎座上分化形成多数由1层表皮细胞包被1列细胞的胚珠原基;授粉后60 d时位于胎座指状结构末端处紧靠表皮细胞下方的孢原细胞分化为大孢子母细胞。之后,大孢子母细胞经过减数分裂和有丝分裂最终形成成熟胚囊;授粉后135 d胚囊发育成熟,附着在胎座上的种子个体分化明显。小叶兜兰胚囊的发育类型为双孢子葱型,胚珠为倒生胚珠,薄珠心,单珠被,成熟胚囊为8核。这为小叶兜兰的生殖生物学及繁殖体系的建立提供理论依据。