Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

High frequency production of embryos inDatura innoxia from isolated pollen grains by combined cold treatment and serial culture of anthers in liquid medium

Summary This study concerns the development of pollen embryos as affected by various physical conditions of culture in media devoid of hormones. Freshly isolated pollen, from anthers ofDatura, failed to form embryos regardless of whether they were cultured on liquid or solid medium. In contrast, pollen isolated from anthers precultured on solid medium did form embryos and the response could be increased by prior cold treatment of anthers at 4 °C for 4 days. However, the best results were obtained when anthers were cultured from the very beginning in liquid medium and transferred serially to fresh medium. Under such conditions, the anthers dehisced, allowing spontaneous shedding of pollen grains. It was thus possible to have several fractions of shed pollen continuing their development into embryos. When serial culture was started with anthers from cold-treated buds not only were embryos formed in all the fractions of shed pollen but the frequency was also considerably higher than in any mode of culturing.     

Effects of L-proline and post-plating temperature treatment on Maize (Zea mays L.) anther culture

Summary Comparison of different post-plating temperature regimes with a control treatment (27° C) revealed that a short-term cold (8/14°C:2/2 days or 14°C:4 days) as well as a heat treatment (30°C:14 days) increased the production of embryro-like-structures (ELS) from cultured maize anthers. The beneficial effects of short-term cold treatments were magnified 2–3 times when L-proline (PROL) was added to the induction medium (125–500 mg/L). In the best treatment (14°C:4 days, 125 mg/L L-proline) one genotype produced 143.5 ELS/100 anthers. Anthers subjected to high temperature (30°C:4 days, 30°C:7 days, 30°C:14 days) generally showed a lower response than did cold treated anthers, although genotypic differences were observed. Regeneration frequency did not appear to be affected by the presence of L-proline in the induction medium.Abbreviations ELS Embryo-like-structures - PROL L-proline     

Induction of androgenesis and callus formation inin vitro cultured anthers of a myrtaceous fruit tree (Psidium guajava L.)

Anthers ofPsidium guajava cultivated on either Murashige-Skoog's or Nitsch's basal medium (BM) or the BM supplemented with 10⁻⁶ M benzylaminopurine (BAP) were observed to contain microspores undergoing androgenic segmentations as well as a few multicellular microscopic embryoids. However, final morphogenic response from such cultured anthers was the development of calli. These calli had restricted growth accompanied by their early browning. Suspecting the browning to be due to accumulation of polyphenols, the culture medium was fortified with polyvinylpyrrolidone (PVP). PVP, increased sucrose concentration in the medium and cold pretreatment of anthers decreased the proportion of anthers turning brown as well as delayed the browning of calli, but it was not possible to maintain the calli for differentiation. Cold pretreatment significantly increased the percentage of callusing anthers and also resulted in the early emergence of calli.     

Conditioned medium enhances neuritic outgrowth from rat spinal cord explants

Medium conditioned by primary cultures of fetal or neonatal rat skeletal muscle, fibroblasts, or lung cells dramatically increases the neuritic outgrowth from spinal cord explants. After 7 days in vitro, the outgrowth of neurites from 15- to 16-day fetal rat spinal cord slices grown in conditioned medium (CM) covers a 3- to 4-fold greater area than that from slices grown in fresh, nonconditioned (control) medium. Moreover, the pattern of neuritic outgrowth is markedly different in CM-treated slices. In control slices, the neurites form a tangled, dense network of neurites which usually extend only a small distance from the slice edge, while in CM-treated slices, the neurites form a more open network, with the majority of neurites extending radially for long distances (up to several millimeters) from the slice edge. The effect of CM on neuritic outgrowth is not due to a detoxification or modification of the serum in the medium, because increased neuritic outgrowth was observed in slices grown in medium conditioned in the presence or absence of 10% fetal calf serum. The outgrowth-enhancing factor(s) in CM has a high molecular weight, since all outgrowth-enhancing activity is retained by membrane filters with a nominal molecular weight cutoff of 10⁵ daltons. This factor(s) is stable at 58°C for 30 min, and does not appear to be βNGF or fibronectin.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

Effects of the developmental state of the tissue on the competence for flower bud regeneration in pedicel explants of tobacco

The competence of pedicel explants of tobacco (Nicotiana tabacum L. cv Samsun) to regenerate flower buds in response to auxin was manipulated by preincubating excised tissues in the absence of auxin. When exposed to 1 micromolar 1-naphthaleneacetic acid, these tissues formed fewer buds than controls that were not preincubated. The number of buds eventually formed correlated with the 1-naphthaleneacetic acid concentration in the tissue 6 hours after the start of hormone application. The internal concentrations in pretreated explants were lower than in tissues that were not pretreated due to diminished uptake per milligram fresh weight and increased hormone conjugation. The change in the developmental state induced by auxin deprivation had a dual effect on bud regeneration: (a) the pretreatment caused fewer buds to be formed at any 1-naphthaleneacetic acid concentration tested, and (b) a higher auxin concentration in the medium was required to get a maximum bud number on precultured explants. An increase of the 1-naphthaleneacetic acid concentration in the medium led to an elevated hormone level in freshly cut as well as in preincubated tissues. It was concluded that the developmental state of the tissue directly affects the maximum number of buds that can be regenerated. Apart from that there is an indirect effect exerted via modulation of the ratio between external and internal auxin concentration. The change in this ratio can be compensated for by an adjustment of the auxin concentration in the medium.     

Influence of some adjuvants on <Emphasis Type="Italic">in vitro</Emphasis> clonal propagation of male and female jojoba plants

Summary The influence of different adjuvants, activated charcoal (AC), casein hydrolyzate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA), has been assessed on the shoot production potential of the nodal explants derived from in vitro-raised male and female jojoba (Simmondsia chinensis) shoots. Nodal explants of each sex were cultured separately on Murashige and Skoog medium supplemented with different levels of AC, CW, CH, PVP, and TIBA either alone or along with optimum levels of N ⁶-benzyladenine (BA; 10 μM for male, 20 μM for female). Some differences in response of the explants of both the sexes have been observed in terms of (1) percentage of explants developing shoots, (2) average shoot number, and (3) average shoot length. AC alone proved beneficial for elevating morphogenic response in male as well as female explants in comparison to basal medium or media containing AC and the optimum level of BA. When used alone, CH proved inhibitory for shoot differentiation in both sexes, especially in male explants. Addition of PVP to MS enhanced shoot proliferation in female explants only, but along with BA it increased the response of male explants. BA in combination with different levels of TIBA promoted shoot multiplication in female explants. Thus, explants of both male and female shoots exhibited differential morphogenic behavior under the influence of various adjuvants. However, BA alone proved to be the best for differentiation of shoots in both male (10 μM) as well as female (20 μM) explants.     

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.     

Factors affecting variability in anther culture and in conversion of androgenic embryos ofSolanum phureja

The variation for embryo production in anther ofSolanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. Four clones ofS. phuyreja were grown in a greenhouse under a 16-h photoperiod. The temperature was monitored continuously. Buds (60 per day on 10 days) were collected and the anthers cultured in two groups of five flasks (30 anthers per flask). In the first group, each flask contained the 30 anthers from six buds; in the second group, each flask contained one anther from each of 30 buds. Significantly smaller coefficients of variation were observed for the second group, suggesting that variation for embryogenic capcity among buds was greater than that among anthers within a bud. Variation in embryo yield as a function of greenhouse temperature was examined by stepwise regression analysis. Embryogenic capacity of one clone was adversely affected by high temperatures (31–37°C) that occurred two and seven days before bud harvest. However, similarly high temperatures appeared to enhance the androgenic response of another clone. Conversion of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected conversion rate which ranged from 12.5% to 46.0%. Conversion rate declined on each serial subculture.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid, IAA-indole-3-acetic acid     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Callus induction in culture of Oenothera hookeri and Oenothera picensis anthers

In the present work we try to determine optimum conditions for callus induction in anther culture of Oenothera hookeri and O. picensis. The anther callus yield was increased when the anthers were cultured on modified MS medium supplied with 2 mg dm^-3 2,4-D and 2 mg dm^-3 NAA, in both species. In O. hookeri, best results were obtained when anthers were excised from 7.2 - 9 mm buds at the stage of vacuolated microspores, then pretreated at 4 °C for 2 d and grown under 16-h photoperiod. The response to anther culture of O. picensis was generally very poor compared with that of O. hookeri. The higher yield of calli was obtained when anthers were excised from 6.2 - 8 mm buds at the stage of vacuolated microspores and grown under continuous light. The cold pretreatment of buds decreased anther response in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Callus induction and whole plant regeneration in elite Indian maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbreds

Callus induction and regeneration ability of five elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations influenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l⁻¹ showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l⁻¹ Dicamba induced the highest frequency of organogenic callus. Among the five genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l⁻¹ benzyladenine and 0.5 mg l⁻¹ indole acetic acid promoted the highest frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l⁻¹ naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66.     

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal

Summary Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to the availability of glucose and fructose following heat-induced hydrolysis of sucrose and the AC-mediated adsorption of inhibitory compounds produced during autoclaving.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.     

Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell

 Adventitious shoot buds were induced from leaf and stem explants of Bacopa monnieri on Murashige and Skoog medium supplemented with benzyladenine or kinetin. The source of the explants as well as different gelling agents in the medium were found to influence shoot induction and eventual shoot growth. The best response was obtained in leaf explants taken from shoot cultures grown in medium supplemented with 2 μM benzyladenine and gelled with 0.2% gelrite. A transverse section of the leaf explant incubated in this medium showed several shoot primordia emerging from the leaf surface. This system exhibited a potential for repeated harvesting of the shoots from the original leaf explant as the latter continued to expand and regenerate new shoots, upon repeated periodical subculturing onto fresh medium. However, the callusing response of the plant was very low. Qualitative TLC studies of the regenerated shoots revealed a phytochemical profile similar to that of the field grown-plants. Received: 20 March 1998 / Revision received: 1 December 1998 / Accepted: 12 December 1998