DNA Synthesis in Microspores in Anther Culture of Wheat (Triticum aestivum L.)

Anthers with mid-unlnucleate microspores were cultured on W5 medium supplemented with 0.5 mg/l kinetin, 2 mg/l 2,4-D and 9% or 3% sucrose. At a series of interval (0, 1, 1.5, 2, 14 days) after cultured, the anthers were labelled with 3H-thymidine (4 MCi/mi) for 24 h, fixed, and then performed autoradiography according to conventional method. Results show that after cultured for 24 h, 3H-thymidine was incorporated into some late-uninucleate microspores (see Plate I, 3), and after for 2.5 days, vegetative nuclei in pollen grains were la- belled (see Plate I, 4). Usually, vegetative nuclei were labelled frequently and generative ones were labelled rarely. Sometimes generative cell which could synthesis DNA might develop suspensor-like structure individually (see Plate I, 13). During early stage of development of a multicellular pollen grain, the DNA synthesis in the cells were synchronized. With pollen development, the synchronism of DNA synthesis was destroyed. When anthers cultured on medium with 3% sucrose, DNA in microspores could be synthesized normally, and the number of labelled microspores was more than that of anthers cultured on medium with 9% sucrose. However, on medium with 3% sucrose, the nuclei in microspores stopped dividing after one or two divisions and the cell wall of them could not be formed and multicellular pollen was not observed. It seems that the absence of multicellular pollen on medium with 3% sucrose was primarily due to the block of cell division and cell wall formation, not due to the interruption of DNA synthesis.     

Effects of Physical and Chemical Factors on Induction Frequency of the Pollen Plantlet and Changes in Starch Formation in Anthers

Physical and chemical facttors affected distinctly induction frequency of the pollen plantlets from anther culture in vitro of Lycium. Experimental results showed that the anthers with their pollen grains at the uninucleate stage when the nuclei were centrally situated, were the best material for the anther culture. The different proportions of various hormones have affected the embryoid formation. When the MS medium was supplemented with 6-BAP (1 ppm) and NAA (0.1 ppm), the induction frequency was increased ( 16.9% ). When 3–15 per cent of sucrose was added in medium, the embryoids were induced and 15 per cent was the optimum. The callus of the filament was inhibited by the increase of the sucrose concentration. Before inoculation the anthers were pretreated at 3–5℃ for 4 days, the frequency of embryoid formation was efficiently increased in comparision with those of untreated anthers. The induction frequency of normal anthers was only 2.8 per cent, but that of the anthers pretreated was 3–9 per cent, the highest was 8.9 per cent .The changes of substances in anther pretreated and in normal anther was compared by means of histochemistry. Under normal conditions, there were a lot of starch accumulated in the inner wall Of the anthers and the distribution of the cytoplasm and the staining of the protein were even: In the anther pretreated, the starch grains have disappeared and the cytoplasm has condensed and the staining of the protein wasn't even. The differences may be related to induction, frequency of the anther culture.     

Pollen Dimorphism and Androgenesis of Paeonia in Vivo

It was observed that anthers of Paeonia lactiflora cultivar “Da-Fu-Gui” produced dimorphic pollen grains, i.e. the normal pollen garins and anomalous pollen grains, frequencies of which were 75.8% and 24.2% respectively. Some of the anomalous pollen grains carried out extra divisions (in vivo) and so the multinueleat or multicellular pollen grains were formed. It seem to indicate that there are several different pathways including asymmetric and symmetric divisions and fusion of the nuclei in the androgenesis.     

籼型光敏雄性不育水稻杂种花粉植株游离小孢子培养成植株

禾本科植物游离小孢子的培养已在水稻、小麦、玉米、大麦等主要农作物上获得成功,且在大麦、玉米上成功地从未经预处理及预培养的游离小孢子培养获得了再生植株。籼稻花药培养能力远远低于粳稻,对其游离小孢子的离体培养研究甚少。本文简要报道这方面的研究结果。     

Plant Development from Isolated Microspores of Oryza sativa L. ssp. indica Derived from the Anther Culture of F2 Hybrids of Photoperiod-Sensitive Male-Sterile Rice

Microspores from a highly anther culturable rice line (Oryza sativa L. spp. indica) derived from the anther culture of F2 hybrids of photoperiod-sensitive male-sterile rice, after 7 days of low temperature treatment and another 7 days of preculture within anthers, were isolated mmechanically. They were cultured in Ne medium containing 3% manitol, 6% sucrose, 5 g/L inositol, 100 mg/L serine, 800 mg/L glutamine, 1 000 mg/L L-proline, 10% (V/V) coconut milk and 2 mg/L, 2,4-D, and 1 mg/L kinetin. After 5 days, microspores initiated first division and subsequently developed into multicellular pollens and calli. Green plant could be recovered when compact calli were transferred onto agar-solidified MS medium containing 3% sucrose, 0.5 mg/L kinetin, 2 mg/L 6-BA and 1 mg/L IAA.     

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     

小麦不同基因型材料花药培养中雄核发育的研究

对小麦离体花药中花粉发育的调查表明：难诱导材料花药在培养初期,花粉退化快,大量小孢子败育,多细胞(核)花粉出现晚、频率低;而易诱导材料花药培养初期,花粉退化相对延缓,多细胞(核)花粉出现早且频率高,这些内在的优势使得更多的小孢子有可能转向孢子体发育,从而获得较高的出愈率。     

小麦不同基因型材料花药培养中雄核发育的研究

对小麦离体花药中花粉发育的调查表明 :难诱导材料花药在培养初期 ,花粉退化快 ,大量小孢子败育 ,多细胞 (核 )花粉出现晚、频率低 ;而易诱导材料花药培养初期 ,花粉退化相对延缓 ,多细胞 (核 )花粉出现早且频率高 ,这些内在的优势使得更多的小孢子有可能转向孢子体发育 ,从而获得较高的出愈率。     

甘蓝花药培养胚状体诱导形成影响因子研究

用甘蓝F1、F2和自交系S33个世代6种基因型材料进行甘蓝花药培养诱导胚状体形成影响因子研究。结果显示:(1)高浓度蔗糖对甘蓝胚状体形成具有显著的诱导作用,6%蔗糖浓度是甘蓝花药培养的最适浓度,其胚状体的诱导率最高达12.2%;(2)材料基因型是影响花药培养的主要因素,F2和F1代材料胚状体诱导效果好,且胚状体诱导率F2代(F2P192和F2P194)18.9%比F1代(F1S17和F1S13)17.1%较高,但差异不显著,自交系S3代材料很难诱导出胚状体;(3)B5培养基比MS培养基更适合甘蓝花药胚状体的诱导培养。结果表明,甘蓝F2代是其花药诱导培养胚状体的最佳基因型材料,B5 2.0 mg/L 2,4-D 2.0 mg/L KT 6%Suc是甘蓝花药诱导培养胚状体的最适培养基。     

Origin of Multicellular Pollen and Pollen Embryos in Cultured Anthers of Pepper (Capsicum Annuum)

Anthers of Capsicum annuum L. were cultured on Murashige and Skoog (MS) medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ kinetin. Inoculated anthers were subjected to 31 °C and development of microspores in anthers of varying stages was observed cytologically using 4′-6-diamidino-2-phenylindol-2HCl (DAPI). Pepper was characterized by a strong asynchrony of pollen development within a single anther. Percentage of pollen at different stages changed with the culture period, and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. In early-bicellular pollen, sporophytic pollen was produced through division of the vegetative nucleus. In mid-bicellular pollen, the generative nucleus may undergo division to produce two or more sperm-like nuclei. However, division of the generative nucleus alone to form the embryo was never observed. The anther stage optimal for embryo production contained a large proportion (>75%) of early-binucleate pollen. Associations were found among the percentage of early-binucleate pollen, the frequency of embryogenic multinucleate pollen, and the yield of pollen embryos.     

To Repeat the Effects of Hormones on the Early Microspore Developments of Wheat in Vitro in Anther Culture

1. The normal development of pollen cells can be transformed by the exoision itself of anther culture: The second mitotic division of pollen grains has been prevented; The frequency of anomalous division of pollen grains was higher than that present in anthers in vive; The generative nuclei after the first mitosis were more or less globular in form and in their subsequent developments most of them do not become spindly-shape which is particular to the generative cells in vive. In the meantime, they show a weak staining reaction with Feulgen reagent. 2. The higher concentrations of hormones were found to enhance the frequency of abnormal division obviously. Of anthers cultured on the four N6 media added with various concentration ratios of IAA to Kinetin 2:10, 10:2, 2:12, and 12:2 mg/l. The mean percentages of abnormal pollen grains were 34.02%, 35.28%, 34.27% and 36.65% respectively. 3. The higher hormone level may promote the formation of multicellular pollen grains obviously. When the IAA concentration was raised up to 12 mg/l, the mean multieellular pollen grain yields per anther increased to 13.3 unit, while the control without hormone was only 4 unit.     

Anther Factor(s) in Barley Anther Culture

The effects of anther tissues were studied systematically on microspores forming multicellular units and furthermore on pollen callus formation in the anther culture of Hordeum vulgare (cv. Sabarlis). Anther productivity was found to be greatly enhanced by use of medium previously conditioned by anthers. In 15 experiments observed, anthers produced 26 times on average more calli in the conditioned medium than in control, in a few cases, even more than 80 times more calli formed. According to this, the authors supposed that cultured anthers released some components, anther factor (s) (AF), which is important to androgenesis in the culture. To achieve high yields of callus, culture was restricted to anthers which had been subpected to cold pretreatment. The temperature stress could not be replaced by the AF. However, for conditioning medium, anthers at binuclear stage were found to be more effective than the test anthers either with or without the pretreatment. Anthers from other 8 barley varieties were also effective for conditioning, as the difference of anther productivity still existed in the culture with conditioned medium between various genotypes tested. Anther response and callus yield were increased in both the culture of anthers at mid and late-uninuclear stage by use of conditioned medium. AF interacted synergistically with m-inositol. Cytological observation showed that AF increased apparently the formation of MPGs, while m-inositol mainly stimulated callus formation from MPGs. To some extent, the effect of exogenous hormone(s) could be replaced by AF. The anther response and pollen callus yield could be much enhanced by increasing anther inoculation density, which also raised the AF level in the culture. Thus, by use of the temperature stress prior to anther culture and culture of test anthers in conditioned medium with m-inositol, or at higher inoculation density, a very high production of pollen callus could be obtained in barley anther culture. For meeting the more specialized requirements of less responsive species or genopypes, the principles given here may be provide some basic information.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pollen callus culture in Triticum aestivum

Summary Pollen shed between 4–8 d from anthers of Triticum aestivum cultured in liquid medium gave rise to calluses. Tillers were harvested at the mid-to late-unicellular pollen stages and chilled for 8 d at 4–5 °C before the anthers were dissected out. Pollen cultures gave about 6 times as many calluses on a per anther basis as anthers cultured on solid medium. With the most productive of 5 cultivars tested, pollen culture results in roughly one callus for each anther used, though the calluses formed by pollen culture were less productive for the regeneration of shoots than calluses derived from anthers cultured on solid medium. The ratio of green to albino shoots is roughly 1 1 for anther cultures but considerably less for pollen cultures.     

Experimental Researches on the Two Pathways of Pollen Development in Oryza sativa L.

The present paper deals with the experimental researches on the gametophytic and sporophytic pathways of pollen development in Oryza sativa L. Subsp. Keng, Cultivar Jinghong No. 2. Three methods of culture were used: (1) The lemma, palea and pistil of excised spikelets were removed and the pedicel was inserted vertically into the medium with the intact stamens standing freely above the medium surface (vertical culture). (2) The spikelets were manipulated similarly but placed horizontally on the medium so that their anthers were directly contacted with the latter ('horizontal culture'). (3) The anthers were excised and inoculated separately (anther culture). In all cases the pollen stage at inoculation was in late uninucleate. N6 basic medium supplemented with or without MCPA (2 ppm) was used. After inoculation the samples were collected periodically for cytological observation. In all cases the pollen passed a short stage of gametophytic development, forming a vegetative and a generative cell, then various pathways commenced in different cultures. In vertical culture, most of the pollen went on .along. the gametophytic pathway up to normal 3-celled stage, but some showed anomalous divisions of vegetative or/and generative nuclei, indicating an initiation of sporophytic development. In horizontal culture, the sporophytic deve]opment went on further, producing some calluses, though the main pollen population remained as gametophyte. In anther culture, the gametophytic pathway to a mature 3-celled pollen was blocked, the unique pathway being sporophytic. In rice, the pollen developed along sporophytic path- way mainly via A route. These comparative investigations indicate that there are two chief factors concerning the switch of pollen development from one pathway to another: first, to be freed from the in vivo restrictions, which, as suggested by Sunderland and as sup- ported by the results of vertical culture in our experiments, is sufficient to trigger the first sporophytic division, and second, 'direct contact with the medium, which is necessary to support the successive growth of multicellular grains and calluses. As to the exogenous hormone, rather than functioning as an agent triggering sporophytic development, it plays an important role in increasing eventual induction frequency, growth rates and differentiating ability of calluses.     

Doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 2. In vitro isolated microspore culture

Previously reported attempts to routinely produce certified doubled haploid sunflower plants were unsuccessful. Isolated microspore culture was assayed in order to overcome the high reactivity of somatic tissues such as anther wall, multicellular hair-type structures, anther connective and parenchymatous vascular bundle. Using filtration or density gradients in liquid medium (N6 supplemented with NAA 1 mg l^-1, BA 0.2 mg l^-1 and maltose 0.44 M), asymmetrical and symmetrical microspore divisions were obtained. However, contaminating cells or groups of cells coming from hairy type structures were very reactive, and able to develop into calluses. A second purification step was therefore necessary after two days in culture, to isolate a population consisting of highly viable swollen microspores. Using this procedure, besides avoiding somatic contaminants, the microspore response in terms of viability and initial division rate was increased, and sustained division and microcallus formation were achieved after addition of aminocyclopropane carboxylic acid, an ethylene precursor.Abbreviations ACC aminocyclopropane carboxylic acid - AOA aminooxyacetic acid - DAPI 4,6 diamidino-2-phenyl indole     

"Real" and Feed Pollen of Lagerstroemia indica: Ecophysiological Differences

Abstract: Lagerstroemia indica is heterantheric, its flowers bearing two kinds of stamens: six peripheral stamens with long, curved filaments and large anthers producing blue-green pollen, capable of emitting pollen tubes and fertilizing ovules; or a central tuft of 35 - 40 smaller anthers producing yellow feed pollen that does not germinate and is collected by insects, mainly bees. The two types of pollen differ in volume, number of pores, pore intine protrusion, wall structure, pollenkitt quality, hydration state, viability at anthesis, longevity and mono- and disaccharide composition. Although total sugar concentrations (glucose, fructose and sucrose) are the same in both types, relative concentrations are different, "real" pollen being sucrose-rich (S / [G + F] = 1.2) and feed pollen glucose-fructose-rich (S / [G + F] = 0.3). A lower degree of dehydration, pore intine protrusion and higher monosaccharide content could make feed pollen more digestible for pollinators. On the other hand, the cytological features of "real" pollen (high dehydration, higher sucrose content) points to longer viability and reproductive function.     

Production of microspore-derived plants by anther culture of an interspecific F1 hybrid between Cyclamen persicum and C. purpurascens

Anthers of a F1 hybrid (2n=41) between Cyclamen persicum (2n=2x=48) and C. purpurascens (2n=2x=34) were cultured to produce microspore-derived plants. Embryoids were produced when anthers, containing microspores at the early uninucleate stage of pollen development, were cultured in B5 medium containing sucrose (90 g l-1) and NAA (0.1, 1 mg l-1) or 2,4-D (0.1 mg l-1) in the dark at 5 °C for 4 days, then at 25 °C for 60 days. The embryoids usually developed into plantlets when cultured in B5 medium containing sucrose (30 g l-1) in the dark at 25 °C. At meiosis, the F1 hybrid, used as source for anther culture, formed some cells with restitution nuclei at telophase and dyads at the tetrad stage, which resulted in the production of viable pollen grains as unreduced gametes. Plants produced by anther culture were grouped into sterile plants with 2n=41 chromosomes and fertile plants with 2n=82 chromosomes. The present findings suggested that the sterile plants were polyhaploids originating from unreduced microspores (n=41) of the F1 hybrid and that the fertile plants were amphidiploids induced by a spontaneous doubling during culture of chromosomes of such unreduced microspores. This revised version was published online in June 2006 with corrections to the Cover Date.     

石刁柏花粉植株诱导及其起源鉴定的研究

采用高渗蔗糖溶液预处理石刁柏花药可以显著抑制花药体细胞分裂和提高花粉愈伤组织诱导率。愈伤组织在转入含低浓度激素的培养基中分化得到了花粉植株。其中单倍体、二倍体、四倍体和非整洁体分别占４．３％、６４．５％、１７．２％和１４．０％。单倍体的频率随愈伤组织培养时间延长而下降,石刁柏幼茎中莽草酸脱氢酶同工酶的多态性表现稳定,用其作为遗传标记结合细胞学方法可以鉴定花粉植株的起源。     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).