Tissue Culture Studies of Tomato (Lycopersicon esculentum)

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.     

Tomato bushy stunt virus (TBSV) infecting Lycopersicon esculentum

Tomato bushy stunt virus (TBSV) was detected in tomato crop (Lycopersicon esculentum) in Egypt with characteristic mosaic leaf deformation, stunting, and bushy growth symptoms. TBSV infection was confirmed serologically by ELISA and calculated incidence was 25.5%. Basic physicochemical properties of a purified TBSV Egh isolate were identical to known properties of tombusviruses of isometric 30-nm diameter particles, 41-kDa coat protein and the genome of approximately 4800 nt. This is the first TBSV isolate reported in Egypt. Cloning and partial sequencing of the isolate showed that it is more closely related to TBSV-P and TBSV-Ch than TBSV-Nf and TBSV-S strains of the virus. However, it is distinct from the above strains and could be a new strain of the virus which further confirms the genetic diversity of tombusviruses.     

Chromoplast-Targeted Proteins in Tomato (Lycopersicon esculentum Mill.) Fruit

The chloroplast to chromoplast transition during tomato (Lycopersicon esculentum Mill.) fruit ripening is characterized by a dramatic change in plastid structure and function. We have asked whether this process is mediated by an increase in the steady-state level of RNA for plastid targeted proteins. Assays for import of radiolabeled translation products into isolated pea (Pisum sativum L.) chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. We have found striking increases during development in levels of translatable RNA for two such proteins. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increase during fruit ripening. Three of these clones produced in vitro translation products that were imported into pea chloroplasts. This implies that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. Furthermore, our results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis.     

Photoregulation of Phenylalanine Ammonia Lyase is not Correlated with Anthocyanin Induction in Photomorphogenetic Mutants of Tomato(Lycopersicon esculentum)

Photoregulation of phenylalanine ammonia lyase (PAL)(EC 4.3.1.5 [EC] )was analyzed in wild type (WT) and mutants: phytochrome dencient-awrea(au), high pigment exhibiting exaggerated phytochrome response(hp) and the double mutant (au.hp) of tomato (Lycopersicon esculentum(Mill.) cv. Ailsa Craig). Red light, acting via phytochrome,stimulates PAL activity in cotyledons and hypocotyls of tomatoseedlings. The time course of photoinduction of PAL in cotyledonsof the mutants (au and au.hp) and WT seedlings has a peak ofactivity at 4 h, after which the activity falls sharply, exceptin hp seedlings where activity is maintained at a high level.In hypocotyls, photoinduction of PAL also shows an initial rise,reaching a maximum at 3 h, followed by a sharp decline in themutants (au and au.hp) and WT seedlings. However in hp seedlingsphotoinduction of PAL is about 3 fold that in WT. The photoinductionof PAL appears to be dependent on de novo synthesis of proteinand nucleic acids. The use of a PAL specific inhibitor a-aminooxyß-phenylpropionic acid indicated that PAL is an essentialcomponent of the anthocyanin biosyn-thetic pathway in the tomatoseedlings. However, a comparison of anthocyanin biosynthesis[Adamse et al. (1989) Photochem. Photobiol. 50: 107] and PALphotoinduction data revealed that phytochrome mediated inductionof PAL and anthocyanin in the tomato seedlings are not correlated.While au and au.hp mutant seedlings show a similar increasein PAL level as in the WT, there is little formation of anthocyaninin these mutant seedlings. The results indicate that, in contrastto the photoregulation of anthocyanin synthesis which is dependenton the presence of the labile phytochrome (IP) pool in tomatoseedlings, the photoinduction of PAL is mediated via a smallpool of phytochrome in au mutant: stable phytochrome (sP) ora residual /P pool. (Received August 6, 1991; Accepted September 27, 1991)     

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.     

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.     

The Meristem and Quiescent Centre in Cultured Root Apices of the gib-l Mutant of Tomato (Lycopersicon esculentum Mill.)

Cultured root apices of tomato bearing the gib-I mutation, whichreduces the levels of endogenous gibberellins, grew slower andwere thicker than wild-type contols. This was the result ofshorter and broader cells in the menstem of the mutant. Cellsof both cortex and stele were affected, but this did not causeany alteration to the volume fraction occupied by these twotissues in the root meristem. Root caps were longer in the mutantand there were also more layers of rhizodermis. All these effectscould be reproduced in wild-type roots by addition of 0.1µM2S, 3S paclobutrazol (an inhibitor of gibberellin biosynthesis)to the culture medium and could be normalized in mutant rootsby 0.1 µM GA₃. Cell doubling times in the proximal regionof the meristem were similar in mutant and wild-type roots,but were faster in both the quiescent centre (QC) and the capmeristem of the mutant. This latter feature of the mutant rootsis likely to be the cause of their longer caps, while the fasterrate of division in the QC accounts for the additional tiersof cells that were found to build up in the cortical portionof this zone These additional tiers failed to form in mutantroots grown in GA₃, but they could be induced in wild-type rootsby 2S, 3S paclobutrazol. These results suggest that endogenousgibberellins may be partly responsible for the slow rate ofcell growth and proliferation in the QC. Gibberellins, gib-I mutation, Lycopersicon esculentum, meristem, roots, 2S, 3S paclobutrazol, quiescent centre, tomato     

Protein Phosphorylation in Plastids from Ripening Tomato Fruits {Lycopersicon esculentum)

Incubation of plastids isolated from ripening tomato fruits(Lycopersicon esculentum) with [     

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.     

An Apparent Cellulase Complex in Tomato (Lycopersicon esculentum L.) Fruit

Four enzyme-containing fractions were separated by ammonium sulfate fractionation of 2-day, postbreaker tomato (Lycopersicon esculentum L. cv. Manhattan). The pH optima and substrate specificities are reported. The enzymes were identified as a nonspecific β-glucosidase, an exo-β-1,4-glucanase, and two endocellulases. Both endocellulase fractions were able to catalyze the hydrolysis of various insoluble cellulose materials.     

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.     

Ethephon- and Jasmonate-Elicited Pathogenesis-Related Ribonucleases in Cultured Ginseng Cells

Cultured ginseng cells (Panax ginseng C.A. Mey strain R-1) produce two proteins exhibiting RNase activity (Pg1 and Pg2), which, on the basis of their amino acid sequences, have been earlier referred to intracellular pathogenesis-related proteins. An immunoenzyme technique for estimation of these proteins was developed. A close correlation was found between the content of these proteins and the RNase activity of the cultured cells. Ethephon and jasmonic acid activated the RNase activity, ethephon being more efficient. Salicylic acid did not activate Pg1 and Pg2; high concentrations of salicylic acid suppressed the RNase activity of the culture. The protein kinase inhibitor, H-7, reduced the content and activity of RNases both in the presence and absence of ethephon. The results obtained permit a suggestion that ethylene and jasmonic acid signaling pathways, which include protein phosphorylation, are involved in the induction of PR-10 proteins.     

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     

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.     

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

Ripening Physiology of Fruit from Transgenic Tomato (Lycopersicon esculentum) Plants with Reduced Ethylene Synthesis

The physiological effects of reduced ethylene synthesis in a transgenic tomato (Lycopersicon esculentum) line expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme have been examined. Fruit from the transgenic line 5673 ripen significantly slower than control fruit when removed from the vine early in ripening. In contrast, fruit that remain attached to the plants ripen much more rapidly, exhibiting little delay relative to the control. Ethylene determinations on attached fruit revealed that there was significantly more internal ethylene in attached than detached fruit. The higher ethylene content can fully account for the observed faster on-the-vine ripening. All of the data are consistent with a catalytic role for ethylene in promoting many, although not all, aspects of fruit ripening. Biochemical analyses of transgenic fruit indicated no significant differences from controls in the levels of ACC oxidase or polygalacturonase. Because transgenic fruit are significantly firmer than controls, this last result indicates that other enzymes may have a significant role in fruit softening.     

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）。