Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Tissue Culture of Cassava on Chemically Defined Media

Defined media that promote the initiation and undifferentiated growth of callus derived from stem explants of four cultivars of cassava, Manihot esculenta Crantz, are described. Growth rates and yields of cassava callus after 4 weeks of culture are shown to be comparable to those of callus of Nicotiana tabacum L. cv. Wisconsin No. 38. Nitrogen sources of ammonium nitrate or of ammonium chloride plus succinate supported growth of all four cultivars. Sucrose was superior to glucose as a carbon source. The cassava cultivars differed in their response to increasing concentrations of sucrose between 0.5% (w/v) and 3%, two of them increasing in dry matter with increasing sucrose concentrations of up to 3%. When cultured in the light on defined media that contained higher ratios of cytokinin to auxin, callus of the latter two cultivars turned green. Roots but not shoots differentiated from the callus of all cultivars. The influence of hormone concentrations, sucrose level, and nitrogen source on greening and root formation is summarized.     

Callus in Rosmarinus officinalis L. (Lamiaceae): A morphoanatomical,histochemical and volatile analysis

This study aims to evaluate the anatomy of calli and their chemico-morphological characteristics after in vitro culture. Callus was induced from leaf explants of Rosmarinus officinalis derived from in vitro grown plants on media containing 2,4-dichlorophenoxyacetic acid (2,4D) and thidiazuron (TDZ) supplementation. Both capitate and peltate glandular trichomes were recognized in R. officinalis callus by light and electron microscopy. Histochemical tests showed positive reactions to lipophilic and terpenoid compounds for capitate trichomes and parenchyma or meristematic cells. Volatiles were isolated by simultaneous distillation–extraction and analysed by gas chromatography. Specifically, the use of 2,4D was shown to have a positive effect on the capacity of R. officinalis friable callus to produce volatile monoterpene compounds relative to TDZ. The main volatiles produced by callus were α-pinene, β-pinene and camphor produced in media containing 0.5 and 1.0 mg L^? 1 of 2,4D. Significant quantities of α-pinene, β-pinene and camphor were produced with 0.5 mg L^? 1 of 2,4D. On the basis of this accumulated evidence, it can be concluded that R. officinalis callus produces volatiles in developing glandular trichomes and that cellular aggregates and in vitro culture can be used for the production of volatiles under in vitro controlled conditions from selected plant material.     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

An improved system to establish highly embryogenic haploid cell and protoplast cultures from pollen calluses of maize (Zea mays L.)

Regenerable, embryogenic haploid cell suspensions were initiated and established from type II pollen calluses of two selected Chinese maize genotypes (No 592 Y and 592.A2 LY). The induction frequency of friable, embryogenic callus (type II) was highly dependent on three factors: genotype, medium, cold pretreatment, and on their interactions. Repeated callus and cell selection during the culture procedure led to stable haploid suspensions consisting of fine clusters each containing 20–50 cells. The selected cell lines were able to maintain their morphogenic ability during long-term subculture (2 years). Protoplasts were successfully isolated from subcultured, friable, embryogenic pollen calluses and cultured on N₆BM and N₆K media using a feeder layer, obtained from 2-day-old suspension culture. Healthy plants were regenerated from protoplast-derived calluses.     

Development of haploid cell lines from immature barley,Hordeum vulgare,embryos

Callus and suspension cell lines were derived from haploid barley embryos produced by the Bulbosum method. Embryos 1 to 2 mm long callused on medium containing a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast-growing nodular, beige callus (Type 1), slow-growing, light brown, watery callus (Type 2) and a dense, light yellow, nodular callus (Type 3) were recovered. Type 3 callus was embryogenic and was produced on embryos 1 to 2 mm in length. Although callus cultures gradually became polyploid, a small proportion of haploid cells was retained and the majority of regenerated plantlets were haploid. The organogenic potential of long-term (Type 1) callus cultures was generally low and decreased with time. Attempts to inducede novo shoot formation in Type 1 cultures were not successful.     

Growth of the haploid phase of the myxomycete Physarum flavicomum in defined minimal medium

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemically defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, l-arginine and l-methionine. Cell yields of 1.4x10⁷ cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 μliters of oxygen/mg protein·hr in the minimal medium. The morphology of cells maintained in this medium appeared to be “normal”. l-valine replaced either glycine or l-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

<Emphasis Type="Italic">In vitro</Emphasis> studies to produce double haploid in <Emphasis Type="Italic">Indica</Emphasis> hybrid rice

The aim of this investigation was to improve in vitro the technique of production of double haploid in Indica hybrid rice by combining anther culture, hormone shock and doubling chromosome. It was discussed how to avoid somaclonal variation during culturing and to reduce the time of this process. The anthers of KDML 105 × SPR 1 (Indica × Indica) were cultured in Linsmaier and Skoog (LS) medium, which contained nutrients, growth regulators [(2,4,-dichlorophenoxy acetic acid (2,4-D) and naphthalene acetic acid (NAA)] and organic compounds, and then subcultured by inducing embryo-like structure (ELS) LS media. During 4 weeks used LS media supplemented with 10 μM KNO³ + 2 mg/L 2,4-D + 2 mg/L NAA + 20% coconut water + 1 mg/L of activated charcoal had induced high embryogenic frequent callus with length of 4–5 mm. The supplementation of 0.2 g/L colchicine and 100 μM 2,4-D was the most efficient in LS media. Over 70% of viable double haploid ELS were produced in 8 weeks and subcultured only twice compared with conventional anther which takes more than 12 weeks. This new technique can therefore be applied to rice in order in shorten time to produce higher number of double haploid plantlets.     

Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

Efficient tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of haploid poplar derived from anthers

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 629–633. The text was submitted by the authors in English.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgaris L.)

Summary Haploid plantlets from male fertile and male sterile sugarbeet plants could be induced at frequencies up to 2.2% using ovule culture. Ovary culture on media without charcoal resulted in a similar induction frequency. Plant development was inhibited by callus development originating from the mother tissue. When the callus parts were removed and the ovule transferred to a new medium without 2,4 D, callus formation could be inhibited by adding 0.5% charcoal to the medium. Up to 6.1% haploids were induced. Chromosome counts in leaf tips, chloroplast counts and isozyme patterns revealed that all plants were haploid and originated from the haploid cells of the embryo sac. Root tips showed spontaneous polyploidisation.     

Tissue culture and wetland establishment of the freshwater monocots <Emphasis Type="Italic">Carex,Juncus, Scirpus</Emphasis>, and <Emphasis Type="Italic">Typha</Emphasis>

Summary Cell cultures of freshwater wetland monocots were regenerated, plants were grown in the greenhouse, and then established and evaluated in wetlands. Typha (cattail), Juncus (rushes), Scirpus (bulrushes), and Carex (sedges) were studied because they are common, dominant, high biomass wetland-adapted plants, tolerant of chemically diverse ecosystems. The goal was to define micropropagation and wetland establishment protocols. Tissue culture systems defined for numerous monocot crop species can be readily applied to wetland plants, with a few modifications. Issues addressed were selection of explant material, shoot and root regeneration conditions, culture age verses regenerability, greenhouse acclimatization needs, plant uniformity and requirements for wetland establishment. In vitro-germinated seedlings were an excellent source of pathogen-free regenerable tissue. T. latifolia, T. angustifolia, and J. accuminatus were regenerated from callus induced in the dark with picloram, then transferred to medium with benzyladenine in the light to promote shoot organogenesis. J. effusus, S. polyphyllus, and C. lurida could not be regenerated from callus, which turned black. They could be regenerated directly by culturing intact seedlings directly on cytokinin media in the light. Shoots rooted with little or no auxin. J. effusus rooting was promoted by the addition of charcoal to the medium. Covering plants for the first 2 wk with plastic facilitated greenhouse establishment. There were high rates of greenhouse and wetland survival. No abnormal plants were observed. These regeneration systems could be utilized for the production of wetland plants for potential application in habitat restoration and wetland creation, and would provide an alternative to field collection.     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.     

Organogenesis in Callus from Shoot Apices of Pisum sativum

Shoot formation was observed in callus from apical cells of pea (Pisum sativum L. cv. Century). Shoot apices from 4-day-old plants were macerated and the resulting cell masses grown on agar media. The callus formation and shoot production occurred within 4 to 6 weeks in defined media containing 0.2 to 5.0 μM benzyladenine and 1 μM naphthaleneacetic acid. While most callus produced one or more shoots at high frequency, root formation did not occur regularly. Plants obtained by these procedures were grown to maturity producing flowers and pods.