Plant donor tissue and isolation procedure effect on early formation of embryoids from protoplasts of Helianthus annuus L.

The effects of factors influencing sunflower protoplast isolation yield, plating efficiency (PE) and the early differentiation into embryoids (embryogenic capacity, EC) have been studied. Only hypocotyl-derived protoplasts divided. The variations of PE and EC in the various treatments did not seem to be linked to the protoplast yields. From statistical analysis of the data, we concluded that, the sunflower genotype, the age and height of seedlings, the part of hypocotyl used, the incubation time (from 6 to 16 hours) in enzymes of explants or of protoplasts alone, influenced PE but large variations were detected for EC. A comparison of the factors effecting EC suggested an origin, inside the hypocotyl, of cells able to give rise, after induction, to embryogenic protoplasts.     

Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annum L.)

A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified V-KM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A₃ and the ethylene inhibitor AgNO₃. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in vivo germinated seedlings. Eighty percent of grafted shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to 10 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - MES morpholinoethanesulfonic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls - 2,4D 2,4-dichlorophenoxyacetic acid     

A system for routine plantlet regeneration of sunflower (Helianthus annuus L.) from immature embryo-derived callus

Immature embryos from inbred commercial cultivars of sunflower (Helianthus annuus L.) were used as donor material for induction of regenerable tissue in vitro. Optimum regeneration frequencies were obtained by transferring the tissue through a sequence of defined media using a specified timetable. The first medium was characterized by a high sucrose content (12%), 10 M 2,4-dichlorophenoxyacetic acid and 5 M abscisic acid. Within 7 days, proliferation of smooth, white, dedifferentiated tissue from the cotyledons was evident. After 3 weeks, the tissues were transferred through a series of 3 media, designed to promote shoot formation, shoot elongation and rooting. Regenerates were obtained with all 7 genotypes tested. From 1983 to 1984, approximately 500 primary R₀ plantlets were regenerated, grown to maturity in a greenhouse and self-pollinated. The resultant R₁ seeds were subsequently field-grown and the plants were evaluated for variation.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Callus and embryoid formation from protoplasts of Helianthus annuus

Sunflower hypocotyl protoplasts have been isolated and cultured. Optimum plating density for cell division and colony formation was in the range of 5 to 7×10⁴ cells/mi in an agarose medium supplemented with BAP (1 mg/l) and NAA (1 mg/l). Plating efficiency was 60% after 21 days of culture. In the resultant culture a mixed population of calli and embryoids was observed. Thirty seven percent of the cell clusters exhibited a developmental pattern similar to an embryoid. Many stages of embryogenesis were observed in the same cultures.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - IAA Indole-3-acetic - BAP 6-benzylamino purine - GA₃ Gibberellic acid     

A shoot regeneration protocol effective on diverse genotypes of sunflower (Helianthus annuus L.)

Summary We describe a protocol, and several experiments that helped lead to its development, for sunflower regeneration. Important factors for sunflower regeneration were explant age, cytokinin type and concentration, basal medium, and explant source. We could not induce shoot regeneration from the explants derived from mature tissues including leaf, petiole, and stem. However, use of juvenile explants such as embryo meristem and primordial leaf tissues allowed routine regeneration of 17 different sunflower genotypes. High frequency of shoot regeneration was achieved with these explants taken from seedlings up to 5 d after germination. Explant age was less critical for embryo meristem explants than for primordial leaf tissues. Of the four basal media tested, MS and B5 media produced higher shoot-regeneration frequencies than did Anderson and woody plant media. The highest shoot-regeneration frequency was obtained with MS medium supplemented with 2 μM BA and without auxin. Addition of 1 μM naphthalene-acetic acid to the medium significantly reduced both the percentage of explants producing shoots and average number of shoots per explant. Regenerated shoots were grown to maturity in a greenhouse.     

Somatic Hybrids of Sunflower (Helianthus annuus L.) Identified at the Callus Stage by Isoenzyme Analysis

Dark grown hypocotyl protoplasts from Helianthus annuus L. cvs. Cerflor and Euroflor were electrically fused to produce somatic hybrids. Following fusion, the protoplasts were cultivated in agarose droplets for four weeks. Macroscopic visible calli (0.1-0.3 mm) were transferred onto solid medium and calli reaching a size of 3 mm were collected. Their isoenzyme patterns were analysed based on two different isoenzymes that allow discrimination between the two cultivars used for fusion. From the examined calli, about 26 % showed an isoenzyme pattern of putative binary heterokaryocytes. The isoenzyme pattern of the non-fused control revealed less than 5% of possibly chimeric colonies.     

Fungal populations on sunflower ( Helianthus annuus ) anthosphere and their relation to susceptibility or tolerance to Sclerotinia sclerotiorum attack

Analysis of the fungal flora from different floret parts of various sunflower (Helianthus annuus) varieties showed that there are differences in both fungal species and frequency, depending on whether the sunflower variety is susceptible (SV) or tolerant (TV) to attack of the flower heads by the ascomycete pathogen Sclerotinia sclerotiorum. The sunflower varieties analyzed were SV: HA 300 and Z 20028, and TV: HA 302, Z AV 8410 and Z 30629. The isolates showed different “in vitro” behavior as biocontrol agents. The most common types of interaction with Sclerotinia sclerotiorum were D2 and D2+ (hyphal contact) for isolates from SV and TV, while some of the isolates from TV displayed antibiosis. The microorganisms that colonize TV florets play a part in an indirect mechanism that protects flowers from ascospore germination and pathogen growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings

Removal of the main root system of sunflower ( Helianthus annuus ) initiates adventitious root development on the lower portion of the hypocotyl. The first cytological changes (enlarged nuclei in the interfascicular parenchymatous cells adjacent to the phloem and some cell divisions) are observed 24 h after root excision. On the basis of experiments in which (a) roots, apical buds and various amounts of cotyledonary tissue were removed, (b) cuttings were subjected to various light regimes, (c) benzyladenine oas applied to cotyledons to create an artificial sink, it was concluded that the roots normally produce factors inhibiting to adventitious rooting and might be a sink for stimulatory substances produced in the shoots. The cotyledons seem to be the major source of these stimulators. Application of aqueous and ethanolic extracts of cotyledons and hypocotyls to cuttings promoted adventitious rooting.     

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.     

The influence of ancymidol on morphology,anatomy, and chlorophyll levels in developing and mature Helianthus annuus leaves

Ancymidol foliar spray at 132 mg·liter^–1 a.i. modified leaf anatomy of developing Helianthus annuus L. Mammoth Russian leaves, but not of mature leaves. Ancymidol was effective in retarding plant growth when applied at the young seedling or at a more mature stage of growth. Ancymidol increased leaf weight per unit area and chlorophyll content on an area and unit weight basis, regardless of stage of leaf development. Total chlorophyll per leaf was also increased in mature leaves. Thus, darker green foliage due to increased chlorophyll content and modified leaf anatomy responses were determined to be independent effects.Texas Agricultural Experiment Station paper no. 22927.     

不同氮素形态对向日葵生长和光合功能的影响

利用砂培试验研究了不同氮素形态对向日葵生长和光合功能的影响。结果表明：NO^3-N(NN)处理的株高、叶面积、干物质重显著高于NH4^ -N(AA)处理,施用NO3^--N的植株有较高的净光合速率(Pn)和较低的气孔导度(C8)、细胞间隙CO2浓度(Ci)。不同处理之间Fv／Fm没有显著差异,NN处理的植株PSⅡ线性电子传递量子效率ФPSⅡ、光化学荧光猝灭系数qP和表观光合电子传递效率ETR都显著高于AA处理的植株;而非光化学荧光猝灭系数qN则明显低于AA处理的植株。碳同化受到抑制是NH4^ -N条件下净光合速率下降的重要原因。     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. III. The role of ethylene

While ethylene is suspected to be one of the many factors that play a role in rooting, some studies have found that ethylene can promote rooting, while others show it to inhibit this process or to have no effect. Using seedlings of sunflower ( Helianthus annuus L. cv. Dahlgren 131) we carried out observations on the rates of ethylene production and the levels of the ethylene precursor, 1-aminocyclopropane-l-car-boxylic acid (ACC), and ACC conjugate, l-(malonylamino)cyclopropane-l-car-boxylic acid (MACC), during the process of root initiation. The changes in these substances in the base of the hypocotyls (the portion that produces roots) were compared to the changes that occurred in the top of the hypocotyls (non-rooting portion). We also supplied a number of presumptive inhibitors of ethylene biosynthesis and inhibitors of ethylene action for short periods during the early and critical stages of root formation. Their effects on ethylene action, synthesis and rooting were examined. We conclude that the wound-induced increase in ethylene, seen within 3 h of production of the cuttings, is a key stimulatory factor in the formation of root primordia. When this increase in ethylene is localized in the lower portion of the hypocotyl, there is a promotion of rooting. On the other hand, higher concentrations in the top of the hypocotyls (as compared to the bottom) may inhibit rooting.     

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Summary Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, gametoplasts, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.     

Leaf expansion of four sunflower (Helianthus annuus L) cultivars in relation to water deficits. I. Patterns during plant development

Abstract. The influence of a slow stress and recovery cycle on the pattern of leaf expansion in four diverse sunflower cultivars ( Helianthus annuus L. cvs. Hysun 31, Havasupai, Hopi and Seneca) was studied in a glasshouse. Stress had no significant effect on the time of flower bud emergence and anthesis, or on final leaf number, but delayed the appearance of leaves at high insertions in all cultivars except Hysun 31.
Leaf expansion was markedly reduced as the predawn leaf water potential decreased from −0.35 to −0.60 MPa, and the predawn turgor pressure decreased from 0.3 to 0.2 MPa, and expansion ceased at a predawn leaf water potential of about −1.0 MPa, i.e. when the predawn turgor pressure reached zero.
The leaves most reduced in final size when water was withheld were those at the insertions which grew the most rapidly in unstressed plants. The maximum reduction in final leaf size of 25–35% was similar in all cultivars and was due to retardation of the rate of leaf expansion: the duration of leaf expansion was actually increased by stress. However, leaves that were initiated during stress, but emerged after rewatering, had final leaf areas at least equal to those in the unstressed plants: in the cultivar Seneca, the final size of leaves of high insertion was significantly greater in stressed than unstressed plants, whereas in the three other cultivars the final leaf sizes were similar in both treatments. All four cultivars examined adjusted osmotically to the same degree, but leaf water potentials in one, Seneca, increased more rapidly after rewatering than in the other three, and this may have contributed to the greater relative leaf size in the leaves of high insertion in this cultivar.     

Characteristics of action potentials in Helianthus annuus

The action potentials induced by nondamaging electrical stimuli in 16- to 22-day-old plants of Helianthus annuus were examined. Typical recordings are presented. Mean values of their amplitudes and conduction velocities in the stem, the strength-duration relation, the 'all-or-none' law and the refractory periods have been determined. The amplitude and velocity of propagation were essentially identical in the upward and downward direction, but greater in the upper than in the lower half. In 'electrically active' plants, the rheobase value is 2 V, the minimum period for stimulation is 1.8 s. and the chronaxie 2.3 s. It is noted that the excitability level between similar plants on the same day and in the same plant on different days is highly variable and undergoes periodic changes.     

Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l^-1 NAA, 0.1 mg l^-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l^-1 TDZ in combination with 0.1 mg l^-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.Abbreviations BA benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - NAA -naphthaleneacetic acid - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.