Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

Hormone independent root organ cultures of rye (Secale cereale)

This work describes the growth of rye root organ cultures which were capable of being repeatedly subcultured in hormone-free medium. They showed morphological characteristics, growth rate, inability to produce shoots, and response to auxins and cytokinins similar to those of the Agrobacterium rhizogenes (Ri plasmid) transformed hairy root cultures of tobacco and red beet which were used for comparison. The root cultures of rye were initiated from callus produced on a medium containing the growth regulators (plant hormones) 2,4-d and kinetin, then transferred to hormone-free medium. However not all rye explants gave rise to callus that would differentiate into stable hairy root cultures and rye seedling root explants did not grow if placed directly on a hormone-free medium. Rice and wheat produced callus and roots on a medium containing hormones but root organ cultures could not be maintained on a hormone-free medium.     

cDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.     

Tissue culture of Alhagi camelorum – a legume of high regenerative capacity

Alhagi camelorum cultures provide a system with a high propensity for plantlet regeneration from root, hypocotyl, stem, and leaf explants. Excluding leaf explants, all explants regenerate to form shoot-buds on a simple basal medium suggesting a differential morphogenic potential of different parts of the same plant. All parts of the plant including leaves, form shoot-buds on cytokinin-containing media which markedly promote shoot-bud differentiation, alone or in combination with indoleacetic acid or naphthaleneacetic acid. Rooting occurs on media containing indoleacetic acid and naphthaleneacetic acid. 2, 4-Dichlorophenoxyacetic acid is inhibitory for differentiation and induces callusing. Callus induced on benzylaminopurine and 2,4-dichlorophenoxyacetic acid differentiates to form shoot-buds on transfer to cytokinin-containing medium. Upon transfer to basal medium shoots produce roots. Plants have been transferred to soil.     

Promotion of Carrot Suspension Cell Transformation and Plant Regeneration byAgrobacterium Harboring Binary Vector Pretreated with Phenolic Compounds

Non-tumorigenic Agrobacterium tumefaciens strain LBA4404 harboring binary vector pretreated with acetosyringone and complex phenolic compounds significantly increased the rate of carrot (Daucus carota) suspension cell transformation and plant regeneration. Kanamycin resistant colones were selected on media containing 50 mg/L kanamycin. The author's experimental data indicated that transformation frequencies in the group activated by acetosyringone and complex phenolic compounds pretreatment were 0.24% and 0. 17% respectively while that in the phenolic compound non-pretreated was only 0.02%. Transgenic carrot plants were obtained from the resistant colones via somatic embryogenesis on hormone-free media containing 50 mg/L kanamycin. Histochemical analysis showed that high levels of GUS activity appeared in the parenchymatous cells of vascular bundles of roots, stems, leaves and petioles as well as stomatic guard cells, cortical cells, mesophyllous cells and trichome of leaves, also epidermal cells also parenchymatous cells under the epiderm of petiols.     

Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoë) marnierianum

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.     

In vitro plant regeneration from seedling-derived explants of ramie [Boehmeria nivea (L.) Gaud]

Ramie [Boehmeria nivea (L.) Gaud] is one of the most important perennial fiber crops in China. In vitro tissue culture of ramie could serve as an important means for its improvement through genetic transformation. To improve the regeneration capacity of ramie, the effects on plant regeneration of donor plant age, basal medium, plant growth regulators, and culture conditions were evaluated using explants derived from the cotyledon, hypocotyl, leaf, petiole, and stem of ramie seedlings. Cotyledons and hypocotyls excised from 4-d-old seedlings and leaves and petioles and stems from 15-d-old seedlings were optimal explants. The highest regeneration efficiency was obtained on Murashige and Skoog salts with Gamborg’s B₅ vitamins basal medium containing 2.27 μM thidiazuron (TDZ) and 0.054 μM naphthaleneacetic acid (NAA) for the five explant types tested. A photoperiod of 16:8 h (light/dark) was found to be superior than continuous darkness for regeneration of ramie using TDZ. The regenerated shoots were transferred to hormone-free medium for shoot elongation and successfully rooted on half-strength Murashige and Skoog supplemented with 0.134 μM NAA. The rooted plantlets with four to five leaves were transplanted to greenhouse for further growth.     

The jasmonate-induced expression of the Nicotiana tabacum leaf lectin

Previous experiments with tobacco (Nicotiana tabacum L. cv Samsun NN) plants revealed that jasmonic acid methyl ester (JAME) induces the expression of a cytoplasmic/nuclear lectin in leaf cells and provided the first evidence that jasmonates affect the expression of carbohydrate-binding proteins in plant cells. To corroborate the induced accumulation of relatively large amounts of a cytoplasmic/nuclear lectin, a detailed study was performed on the induction of the lectin in both intact tobacco plants and excised leaves. Experiments with different stress factors demonstrated that the lectin is exclusively induced by exogeneously applied jasmonic acid and JAME, and to a lesser extent by insect herbivory. The lectin concentration depends on leaf age and the position of the tissue in the leaf. JAME acts systemically in intact plants but very locally in excised leaves. Kinetic analyses indicated that the lectin is synthesized within 12 h exposure time to JAME, reaching a maximum after 60 h. After removal of JAME, the lectin progressively disappears from the leaf tissue. The JAME-induced accumulation of an abundant nuclear/cytoplasmic lectin is discussed in view of the possible role of this lectin in the plant.     

Transformation of Populus tomentosa by Agrobacterium and Regeneration of Transformed Plantlets

The establishment of efficient transformation system of Populus tomentosa by Agrobacterium is reported. The strains of Agrobacterium used in experiments were: 1. A. rhizogenes R1000, which harboured the Ri plasmid pRiA4b. 2. A. rhizogenes R1000 (pTVK85), which carried the plasmids pRiA4b and pTVK85 Containing supervirulent region. 3. A. tumefaciens C58C1 (pBZ693), the plasmid pBZ693 containing genes 1 and 2. After being cocultured with the bacteria on media containing 0.5 ppm kinetin for 2 days, explants of P. tomentosa were transferred to MS medium containing 500 ppm cefotaxime. Roots appeared on the explants in a week. The roots induced by A. tume[aciens were morphologically different from those induced by A. rhizogenes. The frequency of the explants transformed by A. rhizogenes R1000 (pTVK85) was nearly up to 60%. Some Ri plasmid transformed roots could spontaneously produce adventitious shoots or calli. By adding appropriate plant growth regulators in the media, we could have all of the root lines transformed produce adventitious shoots which would develop into intact plantlets on a hormone-free medium. Some phenotypical differences were observed among clones of the transformed plantlets. Some clones had short internodes, large number of leaves, reduced apical dominance, rich root systems with a great quantity of branches and root hairs, whereas in other clones aboveground parts of plantlets were morphologically normal and only their root systems were different from those of untransformed plantlets. None of the plantlets transformed by A. rhizogenes had the phenomenon of wrinkle leaves and shapes these leaves were analogous to normal plantlets. It was often observed that roots were regenerated from stems above the medium surfaces. Southern analysis on three clones of the putative transformed plantlets by A. rhizogenes R1000 (pTVKS5) showed that two of them were hybridized positively with the probe covering the TL-DNA region of the plasmid pRiA4b.     

Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus

The stems and leaves of the Dolichos biflorus plant contain a lectin that cross-reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.

No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.

The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants.

     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.     

植物离体培养产生草珊瑚有效成分

报道了以草珊瑚外植体诱导愈伤组织的适宜培养基和各种植物激素在诱导及其生长培养中的调节作用。应用薄层层析（ＴＬＣ）和紫外光鉴定，证明所获得的愈伤组织具有合成与原植物材料相同的黄酮类、异嗪上以啶类、延胡索酸等有机酸的能力。亦通过愈伤组织无性系筛选过程，得到一个增长率和代谢物产量比愈伤组织母系高４倍左右的优良愈伤组织系。     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.     

Allometry of sexual size dimorphism in dioecious plants: do plants obey Rensch's rule?

Rensch's rule refers to a pattern in sexual size dimorphism (SSD) in which SSD decreases with body size when females are the larger sex and increases with body size when males are the larger sex. Many animal taxa conform to Rensch's rule, but it has yet to be investigated in plants. Using herbarium collections from New Zealand, we characterized the size of leaves and stems of 297 individuals from 38 dioecious plant species belonging to three distantly related phylogenetic lineages. Statistical comparisons of leaf sizes between males and females showed evidence for Rensch's rule in two of the three lineages, indicating SSD decreases with leaf size when females produce larger leaves and increases with leaf size when males produce larger leaves. A similar pattern in SSD was observed for stem sizes. However, in this instance, females of small-stemmed species produced much larger stems than did males, but as stem sizes increased, SSD often disappeared. We hypothesize that sexual dimorphism in stem sizes results from selection for larger stems in females, which must provide mechanical support for seeds, fruits, and dispersal vectors, and that scaling relationships in leaf sizes result from correlated evolution with stem sizes. The overall results suggest that selection for larger female stem sizes to support the weight of offspring can give rise to Rensch's rule in dioecious plants.     

根芹体细胞胚产量与质量的研究

对根芹的不同外植体在附加不同成分的MS培养基上进行离体培养,以诱导适合于制作人工种子用的高质量体细胞胚。下胚轴、子叶和叶片在含0.5mg／LKT,0.5mg／L2,4 一 D的MSO培养基上诱导与继代胚性愈伤组织,然后转移到附加有100mg／L肌醇、2g／L葡萄糖的MSO无激素培养基上悬浮培养产生体细胞胚,获得了比固体培养基及含有KT的液体培养基中产生的体细胞胚形态发育更正常的大量体细胞胚,这为人工种子制作奠定了基础。     

冬凌草离体培养体系的建立及主要次生代谢产物的测定

以冬凌草叶片为外植体,研究不同浓度激素组合对冬凌草愈伤组织诱导及植株再生的影响,并对不同外植体(茎、叶)诱导愈伤、芽的分化能力及再生植株内主要次生代谢产物的含量进行了比较研究。结果表明:在MS 2.0 mg/L 6-BA 1.0 mg/L NAA培养基上诱导愈伤组织效果较好;在MS 2.0 mg/L 6-BA的培养基上诱导芽的效果较好;叶片和茎段在愈伤诱导培养基上均能产生大量的愈伤组织,但其再分化能力以茎段最好;再生苗生根培养基以0.3 mg/L IBA最好;以叶为外植体诱导的再生植株中冬凌草甲素、迷迭香酸的含量均高于以茎为外植体诱导的再生植株。     

不同培育方式对橡胶树实生苗生理指标、养分及长势的影响

橡胶树(Heveabrasiliensis)种子催芽生长一般使用沙床培育,沙子是不可再生资源,为了选择一种适合橡胶树种子培育方式来替代对沙子的依赖,该研究通过水培、悬空培育和传统的沙培比较橡胶树实生苗第1蓬叶稳定时,苗木的生长势、生理指标及养分含量。结果表明,水培实生苗地上部株高、茎粗、叶面积的长势最佳,壮苗指数和生物量的含量最高,但其根太长,根相对较细。水培的叶、茎、根的可溶性糖、丙二醛、游离脯氨酸、超氧化物歧化酶的含量均较低;水培和悬空培育的叶片和茎的叶绿素、类胡萝卜素及根系活力的含量没有显著性差异,均高于沙培。水培的叶、茎、根中的氮和磷含量最低,沙培的最高;而水培实生苗根和茎中钾的含量较高,叶片中含量与悬空培育、沙培均没有显著性差异;悬空培育在叶、茎、根中钾的含量最低。水培促进了苗木的生长,降低干旱胁迫,提高养分利用率,但后续还需调控根系,建设良好根团。悬空培育的苗木长势较弱,还需进一步完善方法。     

Effect of cadmium on the phenolic compounds formation in the callus cultures derived from various organs of the tea plant

The effects of cadmium (6.3 × 10^?5 M or 10.6 × 10^?5 M) on the growth of tea plant (Camellia sinensis L.) callus cultures derived from leaves, stems, and roots and on the formation, in these cultures, of phenolic compounds, including flavans and lignin, which are characteristic of the tea plant, were investigated. In the calli derived from leaves and stems, cadmium treatment decreased the biomass increment, while in the calli derived from roots, growth characteristics remained at the control level. Under the effect of cadmium, the content of phenolic compounds, including flavans, in the leaf calli decreased, while in the stem and root calli, it either increased (at the cadmium concentration of 6.3 × 10^?5 M), or was close to a control one (at the cadmium concentration of 10.6 × 10^?5 M). The lignin content in the root and stem calli increased, but it did not change in the leaf calli. All this data demonstrate that the cadmium-induced changes in phenolic metabolism of the tea plant callus culture depended both on the cadmium concentration in the medium and on the origin of calli.     

Plant regeneration of Sapindus trifoliatus L. (soapnut) through somatic embryogenesis

Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from leaves of mature (over 60years old) Soapnut (Sapindus trifoliatus L.) tree. Callus was induced from leaf explants and grown on Murashige and Skoog's medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin. Reduction of 2,4-D concentration during subsequent subcultures resulted in formation of embryoids. These embryoids developed further when transferred to a medium containing benzylaminopurine and kinetin and then to a hormone-free medium. Unless 5-methyl tryptophan was added and the level of sucrose raised, the embryoids began to recallus and failed to form plantlets.     

花叶芋的组织培养和植株再生

双色花叶芋(Caladium bieolor)和亮白花叶芋(C.hortulanum)的叶及花序外植体在加有2,4-D 和激动素或只加有2,4-D 的培养基上产生了愈伤组织,它们在转移到无激素或含激动素和低浓度生长素的培养基上以后分化出大量胚状体,并进一步长成小植株。本工作为花叶芋的快速繁殖提供了方法。