Incorporation of Label into Pollen Tube Walls from Myoinositol-labeled Lilium longiflorum Pistils

Compatible and incompatible pollen tubes growing on detached Lilium longiflorum pistils which had been prelabeled with myoinositol-U-(14)C take up a portion of the label and utilize it for biosynthesis of tube wall substance. The label is transferred from pistil to pollen tubes apparently via the secretion products (exudate) of the pistil. The exudate thus appears to have a major nutritional role in pollen tube growth in vivo.     

Inhibition of the Activity of NAD(P)H-Dependent Oxidase in Pistils of Lilium longiflorum by cAMP

The effects of exogenous cAMP on the activities of the stressenzymes were studied using the extracts from stigmas and stylesof Lilium longiflorum cv. Hinomoto without pollination in relationto self-incompatibility. The activity of NADH- and NADPH-dependentoxidases (     

铁炮百合组培中不定芽形成的细胞学观察

以铁炮百合鳞片诱导的无菌苗叶段作外植体,在MS+6-BA2.0mg/L+NAA0.2mg/L培养基上诱导不定芽,效果较好。诱导过程中定期取样进行石蜡制片,观察不定芽分化过程中的细胞组织学变化。结果表明,脱分化始于切口周围的细胞;形态发生的主要方式是由愈伤组织分化出不定芽,不定芽通常起源于愈伤组织团从外到内第2~7层细胞的分生细胞团。     

Further Studies on myo-Inositol-1-phosphatase from the Pollen of Lilium longiflorum Thunb

myo-Inositol-1-phosphatase has been purified to homogeneity from Lilium longiflorum pollen using an alternative procedure which includes pH change and phenyl Sepharose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows that the enzyme is a dimer (subunit molecular weight, 29,000 daltons). The enzyme is stable at low pH values and is inactivated only below pH 3.0. In addition to 1l-and 1d-myo-inositol-1-phosphate, it shows high specificity for 1l-chiro-inositol-3-phosphate. As observed earlier with other primary phosphate esters, d-glucitol-6-phosphate and d-mannitol-6-phosphate are hydrolyzed very slowly. No activity is observed with inorganic pyrophosphate or myo-inositol pentaphosphate as substrate. The enzyme is inhibited by fluoride, sulfate, molybdate, and thiol-directed reagents. Partial protection against N-ethylmaleimide inhibition by substrate and Mg²⁺ together suggests sulfhydryl involvement at the active site.     

根癌农杆菌介导麝香百合遗传转化体系的建立

以麝香百合"white elegance"叶片为外植体,通过根癌农杆菌介导法,研究了影响其转化的因素,建立了稳定而高效的麝香百合遗传转化体系。结果表明,以叶片为受体材料,外植体预培养有利于农杆菌的侵染;3 d的共培养,根癌农杆菌OD600值为0.6左右、侵染10 min,能获得较高的转化率;50 mg.L-1的卡那霉素对叶片有很好的筛选效果。抗性植株经PCR检测,初步证明Mn-SOD基因已转入到麝香百合植株中。     

Microprojectile bombardment-mediated transformation of Lilium longiflorum

We have obtained transgenic lily (Lilium longiflorum) plants after microprojectile bombardment, using the Biolistics PDS 1000/He system, of morphogenic calli derived from bulblet scales, followed by bialaphos selection. Parameters which gave the highest transient uidA expression were used: a bombardment pressure of 1100 psi, a target distance of 6 cm and a 48-h preculture on medium with 3% sucrose. A total of 1800 morphogenic calli were co-bombarded with plasmids containing either the uidA reporter or PAT selectable marker genes. After bombardment, the calli were exposed to 2 mg/l bialaphos. Only 72 of the shoot-forming calli (4%) survived. The 72 shoot clusters produced 342 shoots on elongation medium containing 0.5 mg/l bialaphos. Only 55 plantlets survived subsequent exposure to 2.0 mg/l bialaphos. PCR analysis indicated that 19 of these plantlets contained the PAT transgene. Southern analysis of 3 of the plants indicated that all contained the PAT gene. Received: 21 March 1997 / Revision received: 8 July 1997 / Accepted: 7 August 1997     

Growth of anthers in Lilium longiflorum

The post-initiation growth of 64 anthers (1.1–17.4 mm long) in Lilium longiflorum Thumb. was examined by time-lapse marking experiments in combination with serial sections and the scanning electron microscope. Each anther was characterized by spatial and temporal variation in growth rate. Larger anthers had two, and occasionally three, series of peaks and troughs in local growth rate. Regions of negative growth rate were frequently encountered. When observed over several days, the growth maxima and minima were found to move basipetally as a waveform down the length of the anther. The wavelength was longer in taller anthers; amplitude and frequency were variable, and anthers of the same size were not always synchronous. Distribution patterns of cell division (and elongation, once division has ceased) recapitulate the growth data. Anther growth is a non-steady system, therefore, with growth centers constantly shifting. Implications for future studies in organ growth patterns are discussed.Abbreviation SEM scanning electron microscope     

Temperature effects on self incompatibility in Lilium longiflorum

Summary Detached pistils of the clonal variety, Lilium longiflorum Aral No. 5, were submerged before pollination in 50°C water for 0, 1, 2, 3, 4, 5, 6 or 7 min and then immediately compatibly and incompatibly pollinated. Incompatibility, as indicated by pollen tube length after 48 h at 23.5°C, was eliminated by a 1–2 min submersion while compatibility was removed by a 4–5 min one. The window of incubation temperatures at which incompatible and compatible pollen tubes are clearly differentiated occurred between 15 and 30°C.     

两步外植体法高频再生麝香百合

采用两步外植体法,即以百合鳞片叶为初始外植体,以从初始外植体上长出的芽为次级外植体,成功建立了麝香百合的高频离体再生系统。对不同的BA浓度及次级外植体的不同部位对再生效果的影响,以及组培苗移栽前低温处理的影响进行了研究。结果表明:不同部位的次级外植体中,以短缩茎切片出芽快、整齐、芽数多且粗壮;以MS附加1.0 mg L-1 BA和0.1 mg L-1 NAA的培养基最适于麝香百合的分化。一个中等大小已脱春化的鳞茎通过两步外植体法能扩繁出54 000株左右的新植株,从鳞片叶开始至开花仅需8个月,而且4!低温处理对开花期的影响不大。     

Metabolism of myo-Inositol by Germinating Lilium longiflorum Pollen

Lilium Iongiflorum pollen tubes absorbed myo-[2-³H]inositol produced labeled metabolites which were separated into acid-soluble and -insoluble fractions. The soluble fraction contained labeled myo-inositol, d-glucuronic acid, myo-inositol 1-phosphate, and at least three other unidentified compounds. The acid-insoluble fraction contained considerable chloroformsoluble radioactivity and a labeled residue. Labeled myo-inositol was also absorbed by germinating pollen prior to the time of pollen tube initiation; however, there was a marked reduction in amounts of myo-inositol 1-phosphate and glucuronic acid produced by this pollen in comparison with growing pollen tubes.     

A Calcium-Activated Phytase from Pollen of Lilium longiflorum

A phytase was isolated and partially purified from the pollen of Lilium longiflorum Thumb. Optimum activity was at pH 8.0. The phytase was activated by Ca²⁺ and Sr²⁺ but not by the other divalent cations tested. Activity was inhibited by ethylenediaminetetraacetate. The phytase had a temperature optimum of 55 to 60°C and an activation energy of about 12,700 calories/mole. Extraction of L. longiflorum pollen with 0.1% Triton X-100 increased recovery of the phytase by nearly 4-fold. The phytase had a molecular weight of about 88,000 as determined by gel filtration chromatography and a K_m value of 7.2 micromolar for phytic acid in the presence of Ca²⁺.     

Characterization of an anther-specific glycoprotein in Lilium longiflorum

Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther-specific glycoprotein (LLA-75). LLA-75 protein bound to concanavalin A, suggesting that it was a glycoprotein. Monospecific anti-LLA-75 antibodies were prepared to investigate its distribution during anther development. Immunoblot analyses of total protein from floral and vegetative organs show that LLA-75 glycoproteins accumulated to detectable levels primarily in one discrete stage of anther development, during the microspore and early pollen phase when the tapetum is actively secreting. A cross-reactive protein with molecular mass of 43.0 kD was also observed. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the four isoforms of LLA-75 glycoprotein ranged from isoelectric point 5.6 to 6.1. These affinity purified antibodies to LLA-75 cross-reacted with anther proteins in two dicot species of the Bignoniaceae. In situ localization data using antirabbit immunoglobulin G (IgG) conjugated with gold particles was not definitive but demonstrated that LLA-75 glycoprotein in lily anthers occurred prominently in tapetal tissue and all other tissues to a lesser degree.     

A kinematic analysis of tepal growth in Lilium longiflorum

Time-lapse marking experiments indicate that the growth of tepals in Lilium longiforum Thunb. from 3.7 mm to maturity is triphasic. Phase I (tepal lengths 3.7–10 mm) is characterized by spatial and temporal variation in growth rate and, in the epidermis, a random distribution of mitoses with an acropetal increase in cell area. During phase II (10–90 mm) cell elongation and (later) cell division is restricted largely to basal regions. Cell division ceases when tepals are less than one-third of their mature length of 155 mm. Phase III (90–155 mm) is characterized by the gradual transition from basal to apical growth, and a modification of epidermal cell shape. A sharp peak in growth at the extreme tip of the tepal coincides with anthesis.Abbreviations LRGR local relative growth rate - RER relative elemental rate of growth     

Evidence for a Functional myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen

Addition of myo-inositol to pentaerythritol-based germination media repressed the conversion of d-[1-¹⁴C]glucose to labeled uronosyl and pentosyl units of tube wall pectic substance in lily pollen (Lilium longiflorum Thunb.). Conversion of d-[1-¹⁴C]glucose to labeled glucosyl, galactosyl, and rhamnosyl units was unaffected. The reverse experiment, addition of d-glucose to pentaerythritol-based media, failed to affect the conversion of myo-[2-³H]inositol to uronosyl and pentosyl units although the flow of label into products of myo-inositol-linked glucogenesis was blocked. Results of these experiments are discussed in terms of a functional myo-inositol oxidation pathway.     

DNA loss during C-banding of chromosomes of Lilium longiflorum

Root tip chromosomes were uniformly labelled with ³H-thymidine and replicate squashes were made. One set was untreated, one incubated in Ba(OH)₂ solution, and a further set treated sequentially in Ba(OH)₂ and hot saline-citrate (2 × SSC) to reveal C-bands. All replicates were autoradiographed and comparative grain counts made. Differences in grain numbers per metaphase cell showed that Ba(OH)₂ extracted 40% of label, and that a further 23% was lost in the subsequent SSC incubation. The distribution of grains was mapped along a sample of each of five individually-recognisable chromosomes at the three treatment stages. Within each chromosome, the number of grains per segment did not differ significantly from a random distribution. This was true for all five chromosomes at all three stages of treatment, whether or not the regions were C-banded. — We conclude that DNA extraction occurs progressively during C-banding in Lilium, but that C-bands are not dark because of their relatively high retention of DNA.     

Regeneration and production of transgenic Lilium longiflorum via Agrobacterium tumefaciens

Efficient transformation of lilies is required for their genetic improvement in ornamental and marketable qualities. Although Lilium longiflorum can be transformed by particle bombardment and Agrobacterium, the transformation frequency is low. In this study, we tested new Agrobacterium-mediated transformation methods using shoot segments combined with two different regeneration systems. Shoot segments were co-cultivated for 2?d with Agrobacterium tumefaciens strain AGL1/pCAS04 harboring a binary vector carrying the neomycin phosphotransferase II driven by a promoter from the maize ubiquitin gene. The effect of different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on regeneration was investigated. The results indicated that Murashige and Skoog (MS) medium with 4.4???M BA and 0.5???M ??-naphthalene acetic acid was optimal for shoot formation, and the nodal stem was the best explant for shoot induction. MS medium with 9.0???M 2,4-D and 0.4???M BA was optimal for callus induction. The direct shoot formation method regenerated 187 plantlets per 100 explants, and 74.4% of the regenerants were positive in transgene PCR. The callus regeneration method regenerated 20 plantlets per 100 explants, and 31.5% of them were PCR positive. Southern blotting confirmed the insertion of transgene in the plant host genome. The direct shoot formation method is more than 20-fold more efficient than previously reported transformation method in this species.     

Regulation of self-incompatibility by acetylcholine and cAMP in Lilium longiflorum

Elongation of pollen tubes in pistils of Lilium longiflorum cv. Hinomoto after self-incompatible pollination was here found to be promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, l-alpha-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. Moreover, the elongation was promoted by neostigmine, a potent inhibitor of acetylcholinesterase (AChE; acetylcholine-decomposing enzyme) (EC 3.1.1.7.) and activities of this and choline acetyltransferase (ChAT; acetylcholine-forming enzyme) (EC 2.3.1.6.) in pistils were associated with self-incompatibility. The activity of ChAT was lower after self-incompatible as compared with cross-compatible pollination. Application of cAMP promoted ChAT activities in both cases, whereas activity of AChE in pistils after self-pollination was higher than that after cross-compatible pollination and was suppressed by cAMP in both cases. Furthermore, AChE activity was inhibited by treatment with neostigmine or heating. Our results indicate that the self-incompatibility with self-pollination is due to decrease of ACh and cAMP, causing reduction of ChAT and AC (adenylate cyclase) and concise elevation of AChE and PDE (cAMP phosphodiesterase), and therefore suppressed growth of pollen tubes.     

Two classes of pollen-specific, heat-stable proteins in Lilium longiflorum

Several floral organ-specific proteins in lily anthers do not accumulate to detectable levels until just before anthesis. Antisera were raised against three of these proteins. designated LLA, in pollen of Lilium longiflorum Thunb. cv. Snow Queen. Monospecific antibodies were further prepared from antisera to investigate the specificity and distribution of these proteins during development. In an effort to study the function of these gene products, pollen protein was heat-treated at 90°C for 10 min. Monospecific anti-LLA-32 and -23 antibodies recognized two of these heat-stable proteins with molecular masses of 32 and 23 kDa. Accumulation of the two proteins in anther development was correlated with desiccation that occurred naturally in the pollen. Immunob-lot analyses of total protein from floral and vegetative organs confirmed that both LLA-32 and -23 proteins were pollen-specific. The proteins showed consistent patterns of expression during development and their levels decreased when pollen germinated. The properties of the two proteins differed in responsiveness to both polyethylene glycol 8000 and abscisic acid, and in solubility characteristics. Analysis of amino acid composition indicates that both LLA-32 and -23 proteins are rich in glutamic acid/glutamine and glycine, a characteristic of heat-stable proteins. However, LLA-23 has more polar amino acid residues with a polarity of 57%, two-fold higher than that of the LLA-32. Immunoblot analyses showed that LLA-32 and -23 proteins were immunologically unrelated to the dehydrin-like protein in lily seeds. It concluded that the two classes of pollen-specific proteins have some features in common with each other and with dehydrins. but that each is distinct.