Seed development and vivipary in Zea mays L.

Seed development was investigated in kernels of developing wild-type and viviparous (vp-1) Zea mays L. Embryos and endosperm of wild-type kernels began to dehydrate at approx. 35 d after pollination (DAP); viviparous embryos did not desiccate but accumulated fresh weight via coleoptile growth in the caryopses. Concentrations of endogenous abscisic acid (ABA) in the embryo were relatively high early in development, being approx. 150 ng·g^-1 fresh weight at 20 DAP. The ABA content declined thereafter, falling to approx. 50 ng·g^-1 at 30 DAP. Endosperm ABA content was always low, being less than 20 ng·g^-1. There were no differences between wild-type and vp-1 tissues. Immature kernels did not germinate when removed from the ear until late in development. The ability to germinate was correlated with decreasing moisture content in the endosperm at the time of removal; premature drying of immature kernels resulted in greatly increased germination following imbibition. Excised embryos germinated precociously when removed from the endosperm as early as 25 DAP. Such germination could be prevented by treatment with 10^-5 M ABA or by lowering the solute potential (_s) of the medium with 0.3 M mannitol. Treatment of excised embryos with ABA led to internal ABA concentrations comparable to those in embryos in which germination was inhibited in situ. Mannitol treatment did not have this effect, although water-deficit stress of excised embryos resulted in substantial ABA production. Germinated vp-1 embryos were less sensitive to growth inhibition by ABA or mannitol than germinating wild-type embryos. The vp-1 seedlings were not wilty and their transpiration rates were reduced in response to ABA or water shortage.Abbreviations and symbols ABA abscisic acid - DAP days after pollination - FW fresh weight - vp-1 viviparous genotype - _s solute potential     

Interactions of ancymidol with sucrose and α-naphthaleneacetic acid in promoting asparagus (Asparagus officinalis L.) somatic embryogenesis

Interactions of varying ancymidol concentrations with those of α-naphthaleneacetic acid (NAA) or sucrose in embryo induction medium were related to the production and development of asparagus (Asparagus officinalis L.) somatic embryos, and to the ability of these embryos to germinate. A significant sucrose×ancymidol interaction was observed only for the production of bipolar embryos; 4% sucrose with 0.75 mg l^–1 ancymidol gave the best result, 78 g^–1 callus. The frequency of globular embryos decreased as sucrose or ancymidol concentrations increased. Sucrose concentration affected embryo germination; 3% and 4% sucrose were optimal with approximately 60% and 40% of bipolar and globular embryos germinating, respectively. Significant ancymidol×NAA interactions were observed for the production of bipolar and globular embryos and their germination. Varying ancymidol concentrations affected embryo production and germination in combination with 0.1 mg l^–1 NAA, but not with 1.0 mg l^–1 NAA. The treatment combination of 0.1 mg l^–1 NAA with 0.75 mg l^–1 ancymidol produced the most bipolar embryos, 64 g^–1 callus, and the greatest percentages of bipolar and globular embryos germinated, 63% and 42%, respectively. Received: 19 August 1996 / Revision received: 24 April 1997 / Accepted: 22 May 1997     

华重楼离体胚培养及植株再生初探

为探明华重楼离体胚培养及植株再生的基本体系,该文以华重楼离体幼胚为试验材料,以MS培养基为基本培养基,研究不同光照、不同浓度梯度组合的植物生长调节剂对华重楼离体幼胚萌发、成苗的影响。结果表明:培养到60 d时,暗培养条件下华重楼离体幼胚的生长率和萌发率分别比光培养条件下高45.25%、19.17%,故暗培养比光培养更有利于华重楼离体幼胚生长发育。当GA₃浓度相同时,离体幼胚萌发所需时间随IAA浓度增加而延长。不同浓度的GA₃都可促进离体幼胚萌发,促进作用由强到弱依次为5 mg·L^-1GA₃>1 mg·L^-1GA₃> 10 mg·L^-1GA₃。在黑暗条件下,优选得到最适华重楼离体幼胚生长发育配方为1/2 MS+30g·L^-1蔗糖+7 g·L^-1琼脂+0.5 g·L^-1活性炭+5 mg·L^-1GA₃     

Increase in calmodulin level in the early phases of radish seed (Raphanus sativus) germination

Abstract Calmodulin (Cam), the heat-stable, ubiquitous, Ca²⁺-dependent regulator protein, has been purified to apparent homogeneity from germinating radish seeds (Raphanus sativus). The characteristics of radish Cam-molecular weight, absorption spectrum, Ca²⁺-dependent activation of brain phosphodiesterase (PDE)-are very similar to those described for Cam from other plant materials. Radish Cam, like other plant Cam, shows some differences to Cam of calf brain. The total amount of Cam in radish embryos at 24 h of germination is ca. 37 μg g⁻¹ fresh weight. Approximately 95% of the total amount of Cam is present in the soluble fraction (supernatant at 100,000 g). The level in the embryo axis strongly increases in the first 24 h of germination (+540%); this increase is strongly reduced when the germination is inhibited by abscisic acid (ABA). In the presence of Ca²⁺, no ‘free’ Cam (i.e. not bound to other structures) is present in the soluble fraction, suggesting that, during early germination, Cam level is a limiting factor for the activities of Ca²⁺ -Cam-dependent systems. These studies suggest that Cam plays an important role in the early phases of seed germination. An inhibitor of the Ca²⁺-Cam-dependent phosphodiesterase is present in the soluble fraction from radish embryos; this substance decreases during germination. A possible role of this inhibitor during the early germination phases is hypothesized.     

Direct somatic embryogenesis and synthetic seed production from<Emphasis Type="Italic"> Paulownia elongata</Emphasis>

We have developed a reproducible system for efficient direct somatic embryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, -naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to induce somatic embryogenesis. The highest induction frequencies of somatic embryos were obtained on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% Phytagel, 500 mg l^-1 casein hydrolysate and 10 mg l^-1 TDZ (medium MS10). Somatic embryos were induced from leaf (69.8%) and internode (58.5%) explants on MS10 medium after 7 days. Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal media. The maturation frequency of somatic embryos from leaf and internodal explants was 50.8% and 45.8%, respectively. Subculturing of mature embryos led to their germination on the same medium with a germination frequency of 50.1% and 29.8% from leaf and internode explants, respectively. Somatic embryos obtained directly on leaf explants were used for encapsulation in liquid MS medium containing different concentrations of sodium alginate with a 30-min exposure to 50 mM CaCl₂. A 3% sodium alginate concentration provided a uniform encapsulation of the embryos with survival and germination frequencies of 73.7% and 53.3%, respectively. Storage at 4°C for 30 days or 60 days significantly reduced the survival and complete germination frequencies of both encapsulated and non-encapsulated embryos relative to those of non-stored somatic embryos. However, the survival and germination rates of encapsulated embryos increased following storage at 4°C. After 30 days or 60 days of storage, the survival rates of encapsulated embryos were 67.8% and 53.5% and the germination frequencies were 43.2% and 32.4%, respectively. These systems could be useful for the rapid clonal propagation and dissemination of synthetic seed material of Paulownia elongata.Abbreviations BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - TDZ ThidiazuronCommunicated by H. Lörz     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

A practical method for overcoming the dormancy of <Emphasis Type="Italic">Taxus baccata</Emphasis> isolated embryos under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Conditions for obtaining an efficient mass propagation procedure to overcome isolated Taxus baccata embryo dormancy were investigated. The protocol herein described was efficient for overcoming the dormancy of T. baccata isolated embryos under in vitro conditions, enabling the conservation and propagation of this species. T. baccata seeds were unable to germinate directly after collection under in vitro conditions. Very good sterility and germination was achieved by soaking seeds in distilled water at a low temperature (+4°C) at least for 48 h instead of leaching them for 7 d under running water, followed by maintaining isolated embryos on the Murashige and Skoog medium (MS) supplemented with 5 g l⁻¹ activated charcoal. That treatment allowed one to shorten the time of the experiment and gave almost 100% sterility. The best germination was observed in darkness, but to obtain worthy seedlings, it was necessary to place cultures in a 16-h photoperiod after a 2-wk incubation. There was no significant difference in germination between seeds collected from different populations of Southern Poland.     

Enhanced germination of artificial seeds by marine cynobacterial extract

Summary We have developed an improved artificial seed system by using a hot-water extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. Carrot somatic embryos (Daucus carota L.) were divided into two size categories (> 800 m and 425–800 m). High frequency germination (91%) was obtained using the large somatic embryos encapsulated in calcium alginate gel containing 400 mg 1^–1 of extract. This compares to 35% without addition of the extract. A non-dialysate fraction of the extract showed strong germination-promoting activity compared with a dialysate fraction. The germination frequency of artificial seeds containing 100 mg 1^–1 of non-dialysate fraction was more than 90%. Almost all germinating artificial seeds developed into plantlets within 4 days. We also achieved high frequency germination (60%) of artificial seeds encapsulating small somatic embryos (425–800 m) that contained 100 mg 1^–1 of non-dialysate (control 9%). Although the small somatic embryos showed a lower germination frequency than the large embryos, the plantlet development process in these seeds was far more vigorous. Such a high germination frequency has not previously been reported for a carrot artificial seed system.     

Effect of exogenous ABA on somatic embryo maturation and germination in Persian walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l⁻¹ ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.     

The Mechanism of Low Temperature Dormancy in Mature Seeds of Syringa Species

The mechanism of seed dormancy at low temperatures (15-9°C) was investigated in the seeds of Syringa josikaea, S. reflexa and S. vulgaris. Low temperature dormancy in Syringa species was mainly imposed by endosperm embedding the radicle. Different degrees of embryo dormancy may occur in S. reflexa seeds. In most cases the low temperature dormancy was broken completely by removing the endosperm around the radicle. The endosperm did not seem to contain significant quantities of germination inhibitors, and the results indicate that it prevents germination mainly due to its mechanical resistance. The mechanical resistance of endosperm did not change during chilling or during induction of dormancy by high temperature incubation. The strength of the endosperm decreased rapidly in non-dormant seeds before visible germination. Similar changes were not observed in dormant seeds. Generally, the strength of the endosperm was lower in the non- (or less) dormant species S. josikaea and S. vulgaris than in the more dormant S. reflexa seeds. The growth potential of the embryos, measured as their ability to germinate in osmotic solutions (mannitol or polyethylen glycol 4000), was increased by chilling and by GA₃-treatment. The growth potential of untreated S. josikaea and S. vulgaris embryos was generally higher than that of S. reflexa embryos. Acid ethyl acetate fractions of methanol extracts from embryos of all three species contained substances with GA³-like activity in the lettuce hypocotyl test. The activity was found at Rf 0.9–1.0 on paper chromatograms run in distilled water. No significant changes in the activity were detected during chilling or prior to visible germination.     

Carbohydrate status in dormant and afterripened excised wild oat embryos

Germination and carbohydrate concentrations were determined in excised dormant and afterripened wild oat (Avena fatua L. line M73) embryos cultured on N6 medium with and without 88 mM fructose (Fru). Without Fru dormant embryos began to germinate after approximately 2 weeks, and the germination rate was greater at 12 than 16°C. With addition of Fru 80% of dormant embryos germinated in 3 days. More than 80% of afterripened embryos germinated within 1 day on N6 with or without additional sugars. Therefore, relative to afterripened embryos, true embryo dormancy exists in line M73. Concentrations of starch and soluble sugars were initially similar in dormant and afterripened embryos. Culturing dormant and afterripened embryos on medium with Fru resulted in concentrations of glucose (Glu), sucrose (Sue), Fru and maltose (Mal) that were the same or higher than the initial levels. The concentration of starch in embryos initially increased slightly then remained constant or declined, except in dormant embryos on Fru-amended medium, where starch accumulated to 34 μg Glu equivalents (mg fresh weight)^-1 at 52 h. Raffinose (Raf) and stachyose (Stach) concentrations declined over time in all embryos. Carbohydrate concentrations in afterripened embryos on medium without Fru decreased to nearly undetectable levels by 52 h. Soluble sugar concentrations in dormant embryos on medium without Fru also declined by 52 h, but changes were not as extensive as those in afterripened embryos without Fru. In 52 h Raf and Stach were nearly depleted in all afterripened embryos, and in dormant embryos cultured on Fru-containing medium but not in dormant embryos without Fru. The concentration of Stach in dormant embryos without Fru declined 60% at 12 to 18 days coinciding with the potential for germination. The results demonstrate that a decline in Stach concentration is associated with the potential for germination of dormant (D) excised embryos. The mechanism of dormancy-breaking associated with the Raf family oligosaccharides remains to be determined.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^&#x2212;1 sucrose. A sucrose concentration lower or higher than 30 gl^&#x2212;1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^&#x2212;1 tyrosine in the medium. Seedlings germinated in the presence of 0.2&#x2013;0.4 mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and 0.3&#x2013;0.5 mgl^&#x2212;1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60&#x2013;70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Factors influencing production of inflorescence-derived somatic seedlings of sweetgum

Somatic embryos derived from staminate inflorescence tissues of three mature sweetgum (Liquidambar styraciflua) trees were tested for germination and conversion frequencies and early growth variables following pregermination cold treatments. Individual mature embryos were selected from repetitively embryogenic cultures maintained on Woody Plant Medium (WPM) with 1 mg l^–1 naphthaleneacetic acid and cultured on basal WPM at 10 8C for 0, 4 or 8 weeks prior to being transferred to WPM germination medium in the light. After 4 weeks, germinated embryos were planted in potting mix in an acclimatization chamber, grown for an additional 8 weeks and evaluated for conversion and growth. Conversion frequency, which ranged up to 80%, was affected by ortet and clonal line, while the number of first-order lateral roots was affected by ortet, clonal line and cold treatment, with 8 weeks of cold promoting the highest number.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Quercus suber L. Somatic embryo germination and plant conversion: Pretreatments and germination conditions

Summary Osmoticum (high sucrose concentration) and chilling (4°C) treatments were assayed in order to promote germination (radicle elongation) and plant conversion of Quercus suber somatic embryos. Those treatments were applied before embryos were set to germinate on Murashige and Skoog (MS) liquid medium. Chilling for 2 mo. improved germination rate (best results close to 100%). On the other hand, that treatment increased plant conversion, especially when high sucrose concentration was used. The best results were obtained with 150 g l⁻¹ sucrose (75% of cultured embryos converted to plants). Incubation conditions during germination were also studied. Although there were no significant differences on final results, warmer temperatures and darkness increased germination rate. The influence of the different treatments on shoot morphology was also studied. The highest percentages of shoots with normal leaves obtained were close to 25%.     

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.