Infected Plantlets Research Articles

Evaluation of Bacillus amyloliquefaciens as a biocontrol agent against oak decline disease in Quercus trees

Aim of study: This study aimed to investigate the biocontrol potential of Bacillus amyloliquefaciens against Phytophthora cinnamomi infection in Quercus suber (cork oak). Both in vitro and in planta experiments were conducted to assess the effectiveness of B. amyloliquefaciens as a biocontrol agent. Area of study: The microorganism strains, B. amyloliquefaciens and P. cinnamomi, as well as the embryogenic lines of Q. suber used, have a Spanish origin. Material and methods: In vitro experiments involved evaluating the inhibitory effects of B. amyloliquefaciens on P. cinnamomi growth through dual-inoculated agar plates. In planta, dual inoculation tests were performed by co-inoculating plantlets with both P. cinnamomi and B. amyloliquefaciens. Physiological parameters, such as photosynthetic activity, chlorophyll content, and oxidative stress markers, were measured. All experiments were conducted under controlled conditions. Main results: In vitro experiments revealed the inhibitory effects of B. amyloliquefaciens on P. cinnamomi growth. Infected plantlets displayed symptoms of root infection. Dual inoculation tests resulted in plant survival against P. cinnamomi infection. Analysis of physiological parameters indicated variations among treatments and clones, highlighting the distinct response of Q. suber plantlets to the pathogen and underscoring the importance of genetic variability for disease management. Research highlights: This study provides insights into the potential of a strain of B. amyloliquefaciens as a biocontrol agent against P. cinnamomi infection in cork oak. Further investigations are warranted to elucidate the underlying mechanisms of susceptibility and resistance in different clones of Q. suber.

Rhizobium rhizogenes infection in threatened Indian orchid Dendrobium ovatum mobilises ‘Moscatilin’ to enhance plant defensins

The present study illustrates the transformation ability of two wild-type bacterial strains of Rhizobium rhizogenes (MTCC 532 and MTCC 2364) on the embryogenic callus and callus-derived plantlets of a threatened Indian orchid, Dendrobium ovatum. Co-culture of the bacterium with the explants gave marginal hairy root phenotype that failed to multiply in the culture medium. Some primary and secondary metabolites were subdued in infected explants. Moscatilin, the stilbenoid active principle in D. ovatum, was found below the detection limit. The presence of two metabolites viz., Laudanosine, a benzyltetrahydroisoquinoline alkaloid and Lyciumin B, a cyclic peptide, were detected exclusively in the infected explants. The subjugated amino acids and phenolics in the infected plantlets were routed to produce phytoanticipins, and phenanthrenes, strengthening the defence mechanism in infected tissues. This research implies that the plant's defence mechanism activation could have prevented the extensive hairy root formation in the explants, even though nodulations and phenotype transitions were witnessed. Moscatilin has a structural resemblance with Resveratrol, a phytoalexin that combats bacterial and fungal pathogens. The study favours the possibility of Moscatlin being a precursor for phenanthrene compounds, thereby serving as a ‘phytoanticipin’ during the infection phase.

Morphological and molecular characteristics of fungal species associated with crown rot of strawberry in South Korea.

Crown and root rot is the most important and destructive strawberry diseases in Korea as it causes substantial economic loss. In August 2020, a severe outbreak of crown and root rot on strawberries (Fragaria × ananassa Duch.) was observed in the greenhouse at Sangju, South Korea. Infected plantlets displayed browning rot within the crown and root, stunted growth, and poor rooting. Thirty fungal isolates were obtained from the affected plantlet. Isolates were identified based on morphological characteristics and pathogenicity test as well as sequence data obtained from internal transcribed spacer, large subunit ribosomal ribonucleic acid, translation elongation factor, and RNA polymerase II-second largest subunit. Results showed that the crown and root rot of strawberry in Korea was caused by three distinct fungal species: Fusarium oxysporum f. sp. fragariae, F. solani, and Plectosphaerella cucumerina. To the best of our knowledge, F. solani, and P. cucumerina are reported for the first time as the causal agents of the crown and root rot of strawberry in South Korea. Pathogenicity tests confirmed that these isolates are pathogenic to strawberry. Understanding the composition and biology of the pathogen population will be helpful to provide effective control strategies for the disease.

Monochromic Radiations Provided by Light Emitted Diode (LED) Modulate Infection and Defense Response to Fire Blight in Pear Trees.

Pathogenesis-related (PR) proteins are part of the systemic signaling network that perceives pathogens and activates defenses in the plant. Eukaryotic and bacterial species have a 24-h ‘body clock’ known as the circadian rhythm. This rhythm regulates an organism’s life, modulating the activity of the phytochromes (phys) and cryptochromes (crys) and the accumulation of the corresponding mRNAs, which results in the synchronization of the internal clock and works as zeitgeber molecules. Salicylic acid accumulation is also under light control and upregulates the PR genes expression, increasing plants’ resistance to pathogens. Erwinia amylovora causes fire blight disease in pear trees. In this work, four bacterial transcripts (erw1-4), expressed in asymptomatic E. amylovora-infected pear plantlets, were isolated. The research aimed to understand how the circadian clock, light quality, and related photoreceptors regulate PR and erw genes expression using transgenic pear lines overexpressing PHYB and CRY1 as a model system. Plantlets were exposed to different circadian conditions, and continuous monochromic radiations (Blue, Red, and Far-Red) were provided by light-emitting diodes (LED). Results showed a circadian oscillation of PR10 gene expression, while PR1 was expressed without clear evidence of circadian regulation. Bacterial growth was regulated by monochromatic light: the growth of bacteria exposed to Far-Red did not differ from that detected in darkness; instead, it was mildly stimulated under Red, while it was significantly inhibited under Blue. In this regulatory framework, the active form of phytochrome enhances the expression of PR1 five to 15 fold. An ultradian rhythm was observed fitting the zeitgeber role played by CRY1. These results also highlight a regulating role of photoreceptors on the expression of PRs genes in non-infected and infected plantlets, which influenced the expression of erw genes. Data are discussed concerning the regulatory role of photoreceptors during photoperiod and pathogen attacks.

Osmo- and cryotherapy of sugarcane (Saccharum spp. L.) shoot-tips infected with sugarcane mosaic virus (SCMV).

In this work, we investigated the effect of different osmoprotective treatments and of cryopreservation using a droplet-vitrification (D-V) protocol to eliminate sugarcane mosaic virus (SCMV) of shoot-tips excised from in vitro propagated infected plantlets. Shoot-tips of sugarcane (Saccharum spp. L.) were precultured on semisolid MS medium supplemented with 0.3M sucrose for 1day, loaded in solution with 0.4M sucrose and 2M glycerol for 30min and exposed to plant vitrification solution 2 for 15min at room temperature prior to ultra-rapid cooling in liquid nitrogen. Virus indexing was performed by the DAS-ELISA immunoenzymatic test. The presence of SCMV was confirmed in the donor-plantlets derived of infected field material. No virus was detected in the regenerated plantlets from shoot-tips subjected to cryopreservation protocol. The progressive decrease in absorbances occurred from the first preculture treatment and no significant differences (P ≤ 0.05) were found with respect to following steps of D-V protocol. These results indicate that the osmotic dehydration treatments (osmotherapy) and cryopreservation (cryotherapy) may be potentially effective strategies to remove the SCMV from infected plants.

Drought Stress Enhances Up-Regulation of Anthocyanin Biosynthesis in Grapevine leafroll-associated virus 3-Infected in vitro Grapevine (Vitis vinifera) Leaves.

Reddish-purple coloration on the leaf blades and downward rolling of leaf margins are typical symptoms of grapevine leafroll disease (GLD) in red-fruited grapevine cultivars. These typical symptoms are attributed to the expression of genes encoding enzymes for anthocyanins synthesis, and the accumulation of flavonoids in diseased leaves. Drought has been proven to accelerate development of GLD symptoms in virus-infected leaves of grapevine. However, it is not known how drought affects GLD expression nor how anthocyanin biosynthesis in virus-infected leaves is altered. The present study used HPLC to determine the types and levels of anthocyanins, and applied reverse transcription quantitative polymerase chain reaction (RT-qPCR) to analyze the expression of genes encoding enzymes for anthocyanin synthesis. Plantlets of Grapevine leafroll-associated virus 3 (GLRaV-3)-infected Vitis vinifera 'Cabernet Sauvignon' were grown in vitro under PEG-induced drought stress. HPLC found no anthocyanin-related peaks in the healthy plantlets with or without PEG-induced stress, while 11 peaks were detected in the infected plantlets with or without PEG-induced drought stress, but the peaks were significantly higher in infected drought-stressed plantlets. Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress. The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress. Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation. This accumulation involved the up-regulation of two key genes, MYBA1 and UFGT, and their expression levels were further enhanced by drought stress.

Efficacy of Bacillus amyloliquefaciens as biocontrol agent to fight fungal diseases of maize under tropical climates: from lab to field assays in south Kivu.

In the province of South Kivu (Democratic Republic of Congo), warm and humid climatic conditions favor the development and spreading of phytopathogens. The resulting diseases cause important losses in production both in crop and after harvest. In this study, we wanted to evaluate the potential of Bacillus amyloliquefaciens as biocontrol agent to fight some newly isolated endemic fungal pathogens infesting maize. The strain S499 has been selected based on its high in vitro antagonistic activity correlating with a huge potential to secrete fungitoxic lipopeptides upon feeding on maize root exudates. Biocontrol activity of S499 was further tested on infected plantlets in growth chamber and on plants grown under field conditions over an entire cropping period. We observed a strong protective effect of this strain evaluated at two different locations with specific agro-ecological conditions. Interestingly, disease protection was associated with higher yields and our data strongly suggest that, in addition to directly inhibit pathogens, the strain may also act as biofertilizer through the solubilization of phosphorus and/or by producing plant growth hormones in the rhizosphere. This work supports the hope of exploiting such technologically advantageous bacilli for the sake of sustainable local production of this important crop in central Africa.

Potential role of endogeic earthworm Pontoscolex corethrurus in remediating banana blood disease: a preliminary observation

Blood disease is a destructive bacterial infection that causes severe yield loss to the banana industry. Ideally, an environmental friendly yet practical approach is necessitated in the search for effective treatment against the disease. Endogeic earthworms are soil biota that help in improving soil physico-chemical and biological properties and thuspromote plant health. The present study assessed the effect of Pontoscolex corethrurus on banana infected by blood disease. The effect of earthworms was evaluated through observations on stem and root morphology, anatomy and total phenolic contents (TPC). P. corethrurus was inoculated into polybags planted with banana plantlets; inoculation of blood disease bacterium (BDB) into the roots of the plantlets was done using a drenching method. Stem and root sections of the plantlets were preserved in FAA for histological study upon harvest. The remaining sections were freeze-dried for TPC analysis. Root sections of plantlets infected with BDB showed lower root biomass compared to the control and earthworm-inoculated plantlets. Observations under microscope also showed that tissue necrosis at the vascular bundles of infected roots were more severe compared to the earthworm-inoculated and unaffected plantlets. Plantlets inoculated with earthworms had the highest root TPC, followed by the unaffected plantlets and plantlets infected with BDB. Although infected plantlets with earthworm inoculation showed disease symptoms, the disease severity was slightly less. The results suggested the potential role of P. corethrurus in improving plant health for disease management and sustainable agriculture.

New Ascomycetes Associated with Grapevine Dieback in Algeria = أنواع فطر زقي جديدة مرتبطة بالموت التراجعي لكرمة العنب في الجزائر

This study was conducted during spring 2012 to detect the causal organism (s) responsible for a new grapevine dieback disease in Algeria. Samples of grapevine wood were collected from 10 grapevine fields located in two regions (Medea and Tipaza). Several fungi were isolated from the margin between healthy and diseased tissues. Botryosphaeria spp, were identified based on the morphological characteristics of the culture and confirmed by Beta tubulin (s-tubulin) region. The sequences submitted to the GenBank (NCBI) under accession numbers (KC960991)( HQ660477)( AY236931), revealed 99-100% homology. Other fungal species Entoleuca mammata and Rosellinia merrilli. were also isolated at low frequency. Inoculation In vitro of grapevine plantlets, with the two Botryosphaeriaceae species, produced smallest necrosis after five-week incubation; Botryosphaeria obtsusa (Diplodia seriata) were virulent compared with B. dothidea. The species tested were re-isolated from necrosis symptoms on infected plantlets .

Occurrence of Botryosphaeriaceae species associated with grapevine dieback in Algeria

Grapevine dieback is an increasing problem in grape vineyards in Algeria. We conducted field surveys each spring between 2006 and 2012 to determine the fungal pathogens associated with grapevine decline in various grape-growing regions. We isolated and identified 3 species of Botryosphaeriaceae (Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum parvum) from infected wood using a combination of morphological and cultural characters, as well as phylogenetic analyses of ribosomal DNA internal transcribed spacer (ITS) regions and fruiting body. We identified 20 isolates of Diplodia seriata, 13 isolates of Botryosphaeria dothidea, and 7 isolates of Neofusicoccum parvum. Inoculation of grapevine plantlets with the 3 Botryosphaeriaceae species produced necrosis and vascular lesions in the wood after a 6-week incubation; Neofusicoccum parvum isolates were the most virulent, followed by D. seriata and B. dothidea. All 3 species were re-isolated from lesions on infected plantlets. Our results represent the first report of a canker disease of grapevine associated with species of the Botryosphaeriaceae in Algeria.

A Reexamination of the Effectiveness of Ribavirin on Eradication of Viruses in Potato Plantlets in vitro Using ELISA and Quantitative RT-PCR

The potato plantlets singly infected by PVA, PLRV, PVS, PVX and PVY and mix-infected by PVM, PVS and PVY were cultured on MS medium with different concentration of ribavirin. The effects of ribavirin on growth of the plantlets and efficiency of virus elimination were investigated. Results showed that the plant height and fresh weight obviously decreased with increase of ribavirin concentration from 0 mg/L to 150 mg/L, and most of the plantlets could not survive when the concentration reached 200 mg/L. According to the ELISA tests, ribavirin was more efficient for eradicating PVA, PVM, PVS and PVX than PVY and PLRV, and healthy plantlets could be obtained with high frequency (up to 100 %) by culturing with 75 ~ 150 mg/L ribavirin after 2 ~ 3 subcultures. Whereas, only 33 ~ 66 % PVY and PLRV infected plantlets were found to be virus-free after 3 subcultures with 75 ~ 150 mg/L ribavirin. The results of quantitative RT-PCR (qPCR) indicated that ribavirin could obviously reduce virus content in the plantlets. Except PLRV was detected positive after 3 subcultures with ribavirin, the healthy seedlings were obtained from infected stocks at the first or end of propagation and no viruses could be detected at the post-eradication stage. No apparent difference of genetic variation resulted from ribavirin treatment was found by SSR analysis between the control and the treated plantlets. All of these results above proved that ribavirin treatment in vitro was an effective method to eliminate viruses in the propagation of potato.

MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF-MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins.

Gene expression and biochemical changes of carbohydrate metabolism in in vitro micro-propagated apple plantlets infected by ‘Candidatus Phytoplasma mali’

'Candidatus Phytoplasma mali' (Ca. P. mali) is the disease agent causing apple proliferation (AP), which has detrimental effects on production in many apple growing areas of Central and Southern Europe. The present study investigated transcriptional and biochemical changes related to the sugar metabolism as well as expression of pathogenesis-related (PR) protein genes in invitro micro-propagated AP-infected and healthy control plantlets with the aim of shedding light on host plant response to 'Ca. P. mali' infection. Expression changes between infected and control plantlets were detected by quantitative real-time PCR analysis. The most significant transcriptional changes were observed for genes coding for pathogenesis-related proteins and for heat shock protein 70, as well as for some genes related to the sugar metabolism, such as a sorbitol transporter (SOT5), hexokinase, sucrose-phosphate synthase or granule bound starch synthase. Furthermore, biochemical analyses revealed that infected plantlets were characterized by a significant accumulation of starch and sucrose, while hexoses, such as glucose and fructose, and sorbitol were present at lower concentrations. In summary, the present analysis provides an overview of a gene set that is involved in response to phytoplasma infection and, therefore, it may help for a better understanding of the molecular mechanisms involved in phytoplasma-host plant interaction in apple.

Proteome analysis of infection-specific proteins from Japanese birch (Betula platyphylla var. japonica) plantlet No.8 infected with Inonotus obliquus strain IO-U1

This study aimed to identify the proteins produced specifically in Betula platyphylla var. japonica plantlet No. 8 by infection with Inonotus obliquus strain IO-U1. Sterile Japanese birch plantlets were infected with the fungus, and the protein samples obtained at 2 days post-infection were subjected to two-dimensional electrophoresis to detect infectionspecific proteins. The specific proteins were analyzed using MALDI-TOF-MS and identified by peptide mass fingerprinting with MASCOT software. Among the 735 protein spots detected in the infected plantlets, 169 spots were recognized as infection-specific proteins. Of these spots, 91 were analyzed using MALDI-TOF-MS, with two heat shock proteins (Hsp70 and Hsp60) being identified as infection-specific proteins. Hsp70 and Hsp60 may cooperate to refold proteins that were denatured by the infection of I. obliquus strain IO-U1 in the birch plantlets. It is suggested that these proteins are expressed in B. platyphylla plantlet No. 8 by infectious stress with I. obliquus strain IO-U1.

Cationic peroxidase related to basal resistance of Betula platyphylla var. japonica plantlet No.^|^#8201;8 against canker-rot fungus Inonotus obliquus strain IO-U1

This study investigated the time-course changes of the in situ peroxidase (POD) distribution and expression of POD isozymes in Betula platyphylla var. japonica plantlet No. 8 infected with a canker-rot fungus, Inonotus obliquus strain IO-U1. Intact (C1), wounded (C2), and infected (T) plantlets were collected at 2 h up to 30 d. In situ POD activity was detected in the C2 and T plantlets, and the POD activity in the T plantlets was more widely distributed compared to the C2 plantlets. In addition, the area of POD activity localization was almost the same as that of phenolic compounds, although a time lag was found between the appearance of POD activity and phenolic compounds. POD isozymes were clearly detected within the basic range (pI>8.5) in isoelectric focusing electropherograms. The activity of cationic POD isozymes in the C2 and T plantlets was induced strongly compared to the C1 plantlets. In addition, the pattern of time-course changes in the activities of in situ POD and POD isozymes was different between the C2 and T plantlets, suggesting that the responsive mechanisms against fungal infection are different from the responses to wounding. The obtained results suggest that cationic POD isozymes are related to the basal resistance in B. platyphylla var. japonica plantlet No.8 against infection with I. obliquus strain IO-U1.

A gene expression analysis of cell wall biosynthetic genes in Malus x domestica infected by 'Candidatus Phytoplasma mali'.

Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several 'knot'-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.

Comparison of pathogenicity of Clavibacter michiganensis subsp. insidiosus and Pseudomonas viridiflava strains on alfalfa

Hamedan province of Iran is a suitable niche for alfalfa growth but many diseases including alfalfa bacterial wilt, bacterial crown and root rot diseases cause economic crop losses. Bacterial wilt is caused by Clavibacter michiganensis subsp. insidiosus, and bacterial crown and root rot diseases are caused by Pseudomonas viridiflava. In this study, we investigated the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava strains collected from the main alfalfa growing areas of Hamedan province. Pathogenicity of the virulent strains was tested on alfalfa and the bacterial strains caused symptoms, and data were collected about stem length, root length, wet weight and dry weight of the infected plantlets. The data about the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava on alfalfa were compared with each other and were analysed by SAS software and Dunkan's test. Resulted data showed more pathogenicity of C. michiganensis subsp. insidiosus than P. viridiflava on alfalfa. These data also showed that both of these bacteria produced the most losses on wet weight and dry weight of alfalfa plantlets.

HOT WATER TREATMENT – A POSSIBILITY TO REDUCE VERTICILLIUM SP. INFECTION OF HORSERADISH (ARMORACIA RUSTICANA GAERT.)

In the last few years more and more horseradish roots (Amoracia rusticana Gaert.) have been infected by the fungi Verticillium album-atrum or dahliae var. longisporum. Infection can be observed as a dark brown to black internal discoloration of the transport system of horseradish roots. Infected plants bring yield loss and poor quality of processed horseradish roots. Systemic fungicides are neither permitted nor successful. Physical treatments (thermal therapy) are to-date not in use. First experiments with hot water treatment (46°C, 10 min) of horseradish plantlets to reduce Verticillium disease were carried out by the authors in 2005. Hot water dipping of plantlets at 46°C for 10 min had no harmful influence on the development of horseradish plants. Temperature measurements inside the horseradish plantlets (different thickness) showed the desired internal temperature of 46°C after a treatment time of 2 min. Verticillium infected horseradish plantlets were hot water treated (46°C, 10 min) before planting in the field. After eight months of cultivation in the field horseradish roots were assessed for black discoloration caused by the fungus Verticillium sp. Three repetitions, each consisting of 30 horseradish roots of hot water treated and untreated control plantlets were cut horizontally to the roots and determined for discoloration. On average only 7.8% of horseradish roots from hot water treated plantlets showed a weak discoloration in comparison to 45.1% of control roots (no hot water treatment). Additional 6.6% of the control horseradish roots showed a strong development of cavities. These cavities could not be observed in horseradish roots of hot water treated plantlets.

Desempenho in vitro e agronômico de cultivares micropropagadas de morangueiro em vários subcultivos

O morangueiro é uma espécie multiplicada vegetativamente, o que facilita a progressão de viroses a partir de mudas infectadas. A utilização de matrizes provenientes do processo de cultivo in vitro tem sido uma prática na obtenção de mudas isentas de patógenos. Diversos protocolos de micropropagação recomendam a utilização de quatro subcultivos, para preservar a identidade genética dos clones e manter alta taxa de multiplicação. Desta forma, visando a elevar a eficiência do processo de micropropagação, foram utilizadas duas cultivares de morangueiro Oso Grande e Vila Nova, submetidas a nove subcultivos, sendo avaliadas a taxa de multiplicação e a variação somaclonal. Durante o cultivo in vitro, foi avaliada a taxa de multiplicação, a altura e o número de folhas por propágulo. Na aclimatização, determinaram-se a taxa de sobrevivência, a área foliar, a massa fresca e seca da parte aérea e da raiz. As mudas obtidas a partir de matrizes também foram avaliadas quanto ao comportamento agronômico, em ambiente protegido. A maior taxa de multiplicação foi observada durante o 2º e o 3º subcultivos com média de sete a oito propágulos/ explante, respectivamente. Não foram observadas alterações fenotípicas nas duas cultivares, durante as fases de micropropagação, aclimatização e crescimento em ambiente protegido. A cv. Oso Grande apresentou maior desempenho agronômico que a 'Vila Nova'.

Production high class of micro tuber potato seeds (Solanum tuberosum L.). by Using tissue culture technique

The aim of this investigation was produced micro tubers of four potato cultivars Premiere, Bintje, Estima and Escort in vitro. Apical meristems (0.2-0.4 mm) of potato cultivars were excised and cultured on nutrient medium and incubated at 24±2 Cº and 1000 lux light intensity for 16 hrs per day. The developing plantlets were examined serological by using ELISA technique to eliminate the viral infected plantlets. The virus-free plantlets were chopped into pieces with single bud and re cultured on fresh medium for mass propagation. For micro tubers formation in test tubes, the cultures were transferred to another medium containing a high percent of sucrose (60g/L) with different concentrations of kinetin; the cultures were incubated under 16±2 Cº and 8 hrs photoperiod. The plantlets formed micro tubers after 8-10 weeks from culturing. The results showed significant differences among cultivar’s in their response to in vitro culture and micro tubers formation. The results also showed that the kinetin concentration had significant effect on micro tubers, and 1mg/l kinetin concentration was the best. The micro tubers were stored for 10 week at 4Cº to break down the dormancy period, and gave 100% germination under nursery condition. Numbers of tubers derived from micro tubers and normal tubers of these cultivars were compared at the end of season.