Inhibition of ethylene action prevents root penetration through compressed media in tomato (Lycopersicon esculentum) seedlings

In the genus Prunus , so far, somatic embryogenesis has not been reported either from cell suspensions or from their protoplast-derived cells. Rhizogenic cell suspensions of Prunus avium L., initiated from adventitious roots developed from cotyledon-derived callus of mature zygotic embryos, have been subcultured for more than one year without losing their morphogenic potential. A yield of 8 × 10⁵ protoplasts ml⁻¹ of packed cells with a viability of 98% has been routinely obtained. Optimum cell division frequency (around 2.5% at day 10 and 4–6% at day 15) occurs in agarose lenses, with Murashige and Skoog (1962. Physiol. Plant 15: 476–497)-based medium supplemented with 5 μ M naphthalene acetic acid, 1 μ M benzyladenine and 0.25 μ M zeatin. Colony formation has been achieved after 35 days with a plating efficiency of 3–4%. Cell suspensions have been initiated from protoplast-derived callus. While the older cell cultures express a rhizogenic response, the younger ones contain early stages of somatic embryo development. Ultrastructural examination confirms the polarization of these structures.     

Comparative Performance of Micropropagated and Seed-Grown Tomato Plants

Morphological, physiological, fruit yield and quality related traits were compared between the seed-grown and tissue-cultured plants of tomato (Lycopersicon esculentum Mill.) cv. Red Coat in a greenhouse. No significant differences were observed for any of the traits studied except for the number of leaves and branches, which were higher in the seed-grown plants than in tissue-cultured plants at the later stages of growth. No phenotypic abnormality of the tissue-cultured plants was observed suggesting that genetic fidelity of tissue cultured plants can be maintained if appropriate plant growth regulators are used with fewer member of subcultures in the multiplication medium.     

火炬松组织培养研究和应用进展

从离体再生途径、影响因素、遗传转化、存在问题等方面对火炬松组织培养研究和应用进展作了介绍和讨论,以期对同类树种相关研究的开展提供参考。     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

Genetic Control of Totipotency of Plant Cells in an in vitro Culture

The main approaches have been considered to studying the genetic control of plant cell totipotency in an in vitro culture. The capacity of cultured plants for callusogenesis, organ formation, and somatic embryogenesis depends on the activity of genes that determine and maintain the meristematic state of cells, level of hormones in the cells, and sensitivity to hormones, as well as on the activity other genes that control different stages of plant morphogenesis.     

Studies on two Gibberellin-like Substances in Young Shoots of Tomato (Lycopersicon esculentum Mill.)

Two gibberellin-like substances were found in the acidic fractionof shoot extracts of the tomato (Lycopersicon esculentum Mill.,cultivar Potentate). These were resolved by paper chromotographywith iso-propanol/ammonia/water (10:1:1) as the developing solventbut not with n-butanol/1.5 N ammonia (3:1). Both substanceswere active in the dwarf maize bioassay on mutants d-1, d-2,d-3, and d-5, and appeared to be more active on d-5 than d-1.Neither was active in the Meteor Pea assay. Neutral and basicfractions were inactive. The relative amounts of these two substances varied accordingto the age of the tissues from which they were extracted andthis feature is discussed in relation to future studies on thephysiology of gibberellin-like substances in vivo.     

Phosphate-Regulated Induction of Intracellular Ribonucleases in Cultured Tomato (Lycopersicon esculentum) Cells

Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems.     

Preferential Promotion of Lycopersicon esculentum (Tomato) Growth by Plant Growth Promoting Bacteria Associated with Tomato

A total of 74 morphologically distinct bacterial colonies were selected during isolation of bacteria from different parts of tomato plant (rhizoplane, phylloplane and rhizosphere) as well as nearby bulk soil. The isolates were screened for plant growth promoting (PGP) traits such as production of indole acetic acid, siderophore, chitinase and hydrogen cyanide as well as phosphate solubilization. Seven isolates viz., NR4, NR6, RP3, PP1, RS4, RP6 and NR1 that exhibited multiple PGP traits were identified, based on morphological, biochemical and 16S rRNA gene sequence analysis, as species that belonged to four genera Aeromonas, Pseudomonas,Bacillus and Enterobacter. All the seven isolates were positive for 1-aminocyclopropane-1-carboxylate deaminase. Isolate NR6 was antagonistic to Fusarium solani and Fusarium moniliforme, and both PP1 and RP6 isolates were antagonistic to F. moniliforme. Except RP6, all isolates adhered significantly to glass surface suggestive of biofilm formation. Seed bacterization of tomato, groundnut, sorghum and chickpea with the seven bacterial isolates resulted in varied growth response in laboratory assay on half strength Murashige and Skoog medium. Most of the tomato isolates positively influenced tomato growth. The growth response was either neutral or negative with groundnut, sorghum and chickpea. Overall, the results suggested that bacteria with PGP traits do not positively influence the growth of all plants, and certain PGP bacteria may exhibit host-specificity. Among the isolates that positively influenced growth of tomato (NR1, RP3, PP1, RS4 and RP6) only RS4 was isolated from tomato rhizosphere. Therefore, the best PGP bacteria can also be isolated from zones other than rhizosphere or rhizoplane of a plant.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0470-z) contains supplementary material, which is available to authorized users.     

The Effect of Various Culture Media on the Formation of Embryo-like Structures in Cultures Derived from Explants Taken from Mature Larix decidua

The effect of various collection dates and nine different culture media on the formation of ‘embryo-like structures’ (ELS) in cultures derived from explants taken from a 42-year-old Larix decidua tree was studied. The best medium was AFC, a medium low in NH₄⁻, NO₃ ⁺, Mg²⁺ and SO₄²⁻ but high in PO₄³⁻ compared with the concentration of these elements in the other media. On AFC, ELS production reached a peak with collections made in late summer during the period when needle primordia are being initiated. For the other media, collection date had a lesser effect on ELS initiation.     

Asexual reproduction,regeneration, and somatic embryogenesis in the planarian Dugesia tigrina (Turbellaria)

In reviewing recent research published in Russian on regeneration and asexual reproduction, the following morphogenetic processes in the planarian Dugesia tigrina are considered: 1) regeneration of lost parts of the body; 2) regeneration of the whole worm from fragments of the body, either by normal regeneration when the inital polarity of the fragment is retained or by somatic embryogenesis when one or more new axes of polarity arise; 3) somatic embryogenesis, or development of individuals from somatic cells; 4) hypermorphosis, or the presence of more than the usual number of organs or body parts, a process that can be interpreted in terms of somatic embryogenesis; and 5) asexual reproduction. Some morphological, biochemical, and physiological studies of the division zone in D. tigrina demonstrate peculiarities of a local breakdown of integrative functions, a breakdown which in turn causes division of the individual to take place at this zone; timing of division is controlled by the organism as an integrated whole.     

NADPH Oxidase Genes from Tomato (Lycopersicon esculentum) and Curly-Leaf Pondweed (Potamogeton crispus)

Abstract: The oxidative burst is an integral component of plant resistance to pathogens. There is accumulating evidence that the oxidative burst is catalyzed by an enzyme with similarities to the phagocyte NADPH oxidase. We have cloned a full length homolog of the gp91 ( phox ) subunit of the plasma membrane NADPH oxidase complex from tomato named LeRBOM. The predicted protein contains 989 amino acids. The large N-terminal domain contains two EF hand calcium binding motifs and one conserved glycosylation site. Six putative membrane spans are present in the C-terminal half of the predicted protein. Extensive homology with the human gp91 ( phox ) subunit was found including conservation of amino acid residues important for heme coordination and substrate binding. We have also isolated partial genomic clones from tomato and from the aquatic plant Potamogeton crispus. These species serve as models for studies of signal transduction leading to NADPH oxidase activation. In tomato, LeRBOH1 expression was too low to be detected on Northern blots. RT-PCR indicated that LeRBOH1 was expressed in all tissues tested.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Japanese larch (Larix leptolepis)

Embryogenic tissue was initiated using LM, LP and MS media from open-pollinated immature embryos of Larix leptolepis. The initiation frequency varied with collection dates. The highest frequencies of embryogenic tissue initiation (60, 67 and 59% on LM, LP and MS media, respectively) were observed from cones collected on July 30. At this time, all the excised embryos were at the cotyledonary stage. ABA over a wide concentration and length of exposure range did not promote maturation, but was beneficial in reducing precocious germination. Of over 400 plants regenerated, 72 were transplanted into soil mixtures and to date, 69 of these (95%) have survived. This revised version was published online in June 2006 with corrections to the Cover Date.     

Retention of Photoinduction of Cytosolic Enzymes in aurea Mutant of Tomato (Lycopersicon esculentum)

The tomato (Lycopersicon esculentum Mill.) aurea (au) mutant has been characterized as a phytochrome-deficient mutant lacking spectrally detectable phytochrome A in etiolated seedlings. Seedlings of au grown under red light (RL) lack phytochrome regulation of nuclear genes encoding plastidic proteins, possess ill-developed chloroplasts, and are slow to de-etiolate. In the present study, the effect of phytochrome deficiency on photoinduction of enzymes in etiolated au seedlings was investigated. The photoinduction of the cytosolic enzymes amylase and nitrate reductase (NR) and of the plastidic enzyme nitrite reductase (NiR) in au was compared with that in the isogenic wild-type (WT) tomato and the high-pigment (hp) mutant with exaggerated phytochrome response. In WT and hp, both brief RL pulses and continuous RL induced amylase, NR, and NiR activities, whereas in au no photoinduction of enzymes was observed with brief RL pulses, and continuous RL induced only amylase and NR activities. The time courses of photoinduction of NR and amylase in au under continuous RL followed patterns qualitatively similar to hp and WT. A blue-light pretreatment prior to continuous RL exposure was ineffective in inducing NiR activity in au. Only continuous white light could elicit a photoinduction of NiR in au seedlings. The norflurazon-triggered loss of photoinduction of NiR in WT and hp indicated that NiR photoinduction depended on chloroplast biogenesis. The results indicate that observed photoinduction of NR and amylase in au may be mediated by a residual phytochrome pool.     

Genetic fidelity of organized meristem-derived micropropagated plants: A critical reappraisal

Summary The commercial multiplication of a large number of diverse plant species represents one of the major success stories of urilizing tissue culture technology profitably. Micropropagation has now become a multibillion dollar industry, practised all over the world. Of the various methods used to micropropagate plants, somatic embryogenesis and enhanced axillary branching have become the principal methods of multiplication. Long-term benefits of this enterprise, however, lie in the production of clonally uniform plants. The concept of genetic uniformity among micropropagated plants derived through organized meristems was exploded by several convincing reports of the incidence of somaclonal variation at morphological, cytological (chromosome number and structure), cytochemical (genome size), biochemical (proteins and isozymes), and molecular (nuclear and organellar genomes) levels. Somaclonal variation is not limited to any particular group of plants; it has been reported, for example, in ornamentals, plantation crops, vegetable and food crops, forest species and fruit trees. The upsurge of these reports, facilitated to a large extent by the technical developments made in molecular biology, is a matter of great concern for any micropropagation system. The economic consequences of somaclonal variation can be enormous in forest trees and woody plants, as they have long life cycles. Therefore, somaclonal variation has to be dispensed with if large-scale micropropagation of diverse plant species is to become not only successful but also accepted by end-users. In the light of the various factors (genotype, ploidy level, in vitro culture age, explant and culture type, etc.) that lead to somaclonal variation of divergent genetic changes at the cellular and molecular levels, genetic analysis of micropropagated plants using a multidisciplinary approach, especially at the DNA sequence level, initially and at various cultural stages, is essential. The results obtained at early multiplication stages from these tests could help in modifying the protocol/s for obtaining genetically true-to-type plants, and ultimate usage by entrepneneurs without any ambiguity.     

Plant Regeneration from Cotyledon Protoplasts of Tomato (Lycopersicon esculentum L.)

otyledon protoplasts of tomato (Lycopersicon esculentum L.) isolated from 2–3 week grown seedlings were cultured in MS liquid medium (2,4-D 1, 6-BA 0.1 mg/l) and fresh medium added subsequently. After 6 weeks culture, the cell clusters were transferred to semisolid medium (the additive same as in liquid medium, agar 0.3%). When the calli grew to 0.5 cm in diameter, transfer them to MS medium (6-BA 2, IAA 0.2 mg/l) for differentiation. The regenerated plants were obtained. After comparing different culture methods, tomato protoplasts grew better in double layers than in agar plate and hanging drops.     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

防风组织培养中畸形胚状体的发生和控制

采用组织培养和石蜡切片法,分析多种因素对防风畸形胚状体发生和发育过程的影响及控制。结果表明,诱导正常体细胞胚高频发生的培养基组合是：启动胚性愈伤组织的培养基是MS 0．5mg／L 2．4-D,蔗糖浓度3％;分化培养基是MS 1mg／L 6-BA 0．2mg／L NAA 0．5mg／L ABA,蔗糖浓度4％～5％;成熟培养基是MS培养基,蔗糖浓度3％。结论：一定浓度的生长素是诱导防风胚性愈伤组织的关键因素,细胞分裂素在体细胞胚的分化和发育过程中起协同作用;蔗糖浓度、ABA、接种方法以及适宜的培养条件和培养容器等均可有效降低畸形胚状体的发生率。     

Development of Multi-Component Transplant Mixes for Suppression of Meloidogyne incognita on Tomato (Lycopersicon esculentum)

The effects of combinations of organic amendments, phytochemicals, and plant-growth promoting rhizobacteria on tomato (Lycopersicon esculentum) germination, transplant growth, and infectivity of Meloidogyne incognita were evaluated. Two phytochemicals (citral and benzaldehyde), three organic amendments (pine bark, chitin, and hemicellulose), and three bacteria (Serratia marcescens, Brevibacterium iodinum, and Pseudomonas fluorescens) were assessed. Increasing rates of benzaldehyde and citral reduced nematode egg viability in vitro. Benzaldehyde was 100% efficacious as a nematicide against juveniles, whereas citral reduced juvenile viability to less than 20% at all rates tested. Benzaldehyde increased tomato seed germination and root weight, whereas citral decreased both. High rates of pine bark or chitin reduced plant growth but not seed germination, whereas low rates of chitin increased shoot length, shoot weight, and root weight; improved root condition; and reduced galling. The combination of chitin and benzaldehyde significantly improved tomato transplant growth and reduced galling. While each of the bacterial isolates contributed to increased plant growth in combination treatments, only Brevibacterium iodinum applied alone significantly improved plant growth.     

番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位

GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶。以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP。LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96％,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99％、97％、91％、89％。经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异。利用75个番茄远缘杂交重组系（IL系）将LeGMP定位在番茄第3染色体上的D区段（3-D）。