Meristem Culture Research Articles

Field Evaluation of Twelve Clones of Oregano Grown in the Main Production Areas of Argentina: Identification of Quantitative Trait with the Highest Discriminant Value

In Argentina, oregano (Origanum spp.) is one of the most important aromatic species. Leaves and flowering tops are used as seasoning, targeting the retail consumer, industrial and less to export. Local production has low-average yields due to the variability of cultivated material, the vegetative propagation methods used, and the lack of knowledge and adaptive experimentation on advanced cultivation practices. Clones of oregano grown in the country were collected in order to characterize the germplasm used in Argentina. Twelve oregano clones, sanitized by meristems culture and micropropagated in vitro, were field evaluated in three different growing locations. Regardless of growing site, the quantitative variables with more discriminating value were essential oils yield, internode length, length of the longest branch, fresh weight, dry weight of leaf and stem, leaf/stem ratio, and leaf area. Based on the quantitative traits, oregano clones can be classified into four groups. From the observations based on botanical characteristics, it was determined that the evaluated clones belong to three different taxa: Origanum vulgare ssp. vulgare, Origanum vulgare ssp. hirtum, and Origanum x majoricum (hybrid). Within each group, the clones belong to the same taxon.

Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa

Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for non-model plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes should be thoroughly investigated for each experimental condition under investigation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-012-9711-1) contains supplementary material, which is available to authorized users.

Detection and elimination of Begomovirus infecting Trichosanthes dioica (pointed gourd) plants in Uttar Pradesh, India

Begomovirus is widely spread on cultivated crops in India. In a survey, infected plants of Trichosanthes dioica (Pointed gourd) with the symptoms of mosaic and leaf curling were collected and checked for Begomovirus infection through PCR analysis. Application of meristem culture technique for regeneration of virus free plants of T. dioica was assessed. Plantlets were regenerated from infected plants of T. dioica through meristem culture. Regenerated plants were found Begomovirus free as evident by PCR analysis suggesting the suitability of meristem culture technique for elimination of Begomovirus from infected plants of T. dioica. This is the first report on elimination of Begomovirus in T. dioica in India.

Potential of aeroponics system in the production of quality potato (Solanum tuberosum l.) seed in developing countries

The production of potato seed under conventional system has not been effective in avoiding or reducing the build up of pathogens and has consequently led to reduced quality potato seed and low crop yields. Plants once cleaned through meristem culture and induction of tuberization under aeroponics system, produce high quality potato seed tubers rapidly that are free from contamination of pathogens. Further multiplication of potato seed tubers under aeroponics also compliments tissue culture (micropropagation), as it clones minitubers in a short time and reduces numerous labour steps associated with direct use of plantlets from tissue culture into the field in the post flask stage. Minitubers from air-rooted plants are planted directly in the field without a need for acclimatization to such environments. This system, as is the case with micropropagation, has potential to increase income and reduce cost of production of quality seed, thereby, making it more accessible to growers in developing countries where potato production is heavily constrained by the use of poor quality seed tubers. This review gives an insight on the potential of aeroponics in compliment to plant tissue culture in revolutionalizing the potato seed production in the agricultural systems of developing countries. Key words: Aeroponics, tissue culture, potato seed.

Molecular Studies on the Transmission of Indian Cassava Mosaic Virus (ICMV) and Sri Lankan Cassava Mosaic Virus (SLCMV) in Cassava by Bemisia tabaci and Cloning of ICMV and SLCMV Replicase Gene from Cassava.

Cassava mosaic disease, caused by cassava mosaic geminiviruses are transmitted by Bemisia tabaci. The B. tabaci adults from colonies reared on virus free cassava plant produced from apical meristem culture was studied to determine their ability to transmit Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) from cassava to cassava. Virus free plants were confirmed by polymerase chain reaction (PCR) using geminivirus degenerate primers. The virus acquisition access period (AAP) of 48h on virus infected cassava leaves and 48h virus inoculation access periods on virus free healthy leaves were investigated. Both ICMV and SLCMV were absolutely transmitted by whiteflies reared on cassava. Virus specific primers were designed in the replicase region and used to detect virus in B. tabaci after different AAP. The PCR amplified replicase genes from virus transmitted cassava leaves were cloned the plasmid DNA was isolated from a recombinant colony of E. coli DH5α after their confirmation by colony PCR and sequenced them. The nucleotide sequences obtained from automated DNA sequencing were confirmed as ICMV and SLCMV replicase gene after homology searching by BLAST and found to be a new isolates. The nucleotide sequences of new isolates were submitted in GenBank (accession number JN652126 and JN595785).

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.

Meristem culture of selected sweet potato (Ipomoea batatas L. Lam.) cultivars to produce virus-free planting material

SummarySweet potato (Ipomoea batatas L. Lam.) is a staple root crop, consumed as a carbohydrate-rich food source in many tropical countries. The production and productivity of sweet potato is limited by several factors, mainly virus infections. This study was conducted to produce virus-free sweet potato planting material using meristem culture. Meristems without leaf primordia were excised from four virus-positive field-grown local sweet potato cultivars ‘Bellela’, ‘Temesgen’, ‘LO-323’, and ‘Zapallo’. Meristems were cultured in Murashige and Skoog (MS) medium supplemented with different combinations and concentrations of 6-benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), and gibberellic acid (GA3) for shoot initiation. ‘Bellela’ and ‘Temesgen’ exhibited 100% shoot initiation on 5 mg l–1 BAP, 0.01 mg l–1 NAA, and 1 mg l–1 GA3, although the shoots were bushy. ‘LO-323’ showed 90% shoot initiation with the best shoot quality on 2 mg l–1 BAP, 0.01 mg l–1 NAA, and 1 mg l–1 GA3. Shoot tips and nodes from in vitro-grown plantlets were cultured on BAP (0, 0.5, 1.0, or 2.0 mg l–1) or on BAP (0, 0.5, 1.0, or 2.0 mg l–1) + kinetin (Kin; 0, 0.1, 0.5 or 1.0 mg l–1) for multiple shoot formation. The best shoot proliferation was obtained on MS + 0.5 mg l–1 BAP + 0.5 mg l–1 Kin. In vitro-grown shoots were cultured on MS medium containing 0.0, 0.01, 0.1, 0.5, or 1.0 mgl–1 indole-3-butyric acid (IBA). The best root formation was obtained on IBA-free medium. The plantlets thus produced were tested for the presence of ten sweet potato viruses [Sweet potato chlorotic stunt virus (SPCSV), Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato mild speckling virus (SPMSV), C-6 (a flexuous rod virus), Sweet potato latent virus (SwPLV), Sweet potato virus G (SPVG), and Cucumber mosaic virus (CMV)] using nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). Ninety-nine percent of plantlets were free from all viruses.

Virus elimination from ornamental plants using in vitro culture techniques

Viruses are responsible for numerous epidemics in different crops in all parts of the world. As a consequence of their presence great economic losses are being incurred. In addition to the development of sensitive techniques for detection, identification and characterization of viruses, substantial attention has also been paid to biotechnological methods for their elimination from plants. In this review article, the following biotechnological in vitro culture techniques for virus elimination from ornamental plants are presented: meristem culture, thermotherapy, chemotherapy, cryotherapy or a combination of these methods. The plant species, as well as the type of virus determine the choice of a most suitable method. The state of the art in investigation of virus elimination from Impatiens sp. in Serbia is summarized.

FIELD PERFORMANCE OF in vitro SWEET POTATO (Ipomoea batatas L. (Lam)) PLANTLETS DERIVED FROM SEEDSTOCKS

A field trial was conducted to compare the performance of in vitro plantlets derived from the meristem cultures of rootstocks through micro-propagation with the performance of the traditional seed roots in 2009 and 2010. There was no significant difference between the in vitro plantlets and the traditional seed root for haulm length (HL), branch number (BN) and stem number (SN). In vitro plantlets had significantly higher means as compared to traditional seed roots for single storage root weight (SSRW) (305.5 g vs 217.6 g), single plant yield (SPY) (4.1 kg vs 3.0 kg) and plot yield (PY) (16.2 kg vs 12.0 kg). Cultivar NC-150 had higher means than those of the other two cultivars in terms of storage root weight (417.6 g), single plant yield (4.6 kg) and plot yield (18.4 kg).

Evaluation of some therapies and meristem culture to eliminate Potato Y potyvirus from infected potato plants

Meristems (length 100, 200, 300 µm) were excised from infected Potato plants of Binella and Burren cultivars an d cultured on solidied MS medium containing 30 g l �1 sucrose, 5 mg l �1 Ascorbic acid, 5 mg l �1 Pyridoxine, 5 mg l �1 Nicotinic acid, 5 mg l �1 �iamine, 200 mg l �1 Inositole, 2 mg l �1 GA3, and 0.2 mg l �1 kinetin. Virus status of in vitro plantlets was determined by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). Results showed that the highest rate of virus-free pl ants was obtained by using explants 100 µm in length. �e rate of PVY elimination was improved aer treatment by thermotherapy at approximately 37±1°C for 40 days, (81%) in Binella and (75%) in Burren. Chemotherapy was undertaken with (10-20-30) mg l �1 of ribavirin (RBV), �e highest percentage of virus free plantlets 87% in Binella and 82% in Burren were obtained from the ribavirin's concentration (20 mg l �1 ) combined with meristem- tips (100 µm) in length. Severe growth abnormalities were observed on medium containing high concentration of RBV (30 mg l �1 ). Finally, the highest percentage virus free plantlets of PVY (93% in Binella and 87% in Burren) were obtained from meristem-tips 100 µm in length excised aer electric treatments (15 mA/10 min).

Can Cryopreservation Eliminate the Potato Virus X (PVX) and Potato Spindle Tuber Viroid (PSTVd) ?

Virus diseases seriously endanger the potato production, which are the major factor causing yield loss and quality decline of potato. The potato virus X (PVX) and potato spindle tuber viroid (PSTVd) are the most important of potato virus against the potato production. This research attempted to remove the PSTVd and PVX in potato plantlets by using to cryopreservation method and conventional methods (apical meristem culture, thermotherapy and thermotherapy combined with apical meristem culture). The results showed that the survival rate and regeneration rate of potato shoot tips were 83.64% and 72.04%, respectively, treated by cryopreservation approach, being higher than that treated by the apical meristem culture (46.67% and 31.49%), thermotherapy (72.22% and 44.44%) and thermotherapy combined with apical meristem culture (50.54% and 30.38%). Using four methods could remove the PVX, the detoxification effect of the cryopreservation method had the highest rate of 59.13%, followed by thermotherapy combined with apical meristem culture with the rate of 56.02%, and then thermotherapy and apical meristem culture with the rates of 42.61% and 35.83%, respectively, whereas the using four methods to remove the PSTVd were in failure, it suggested that the best controlling PSTVd would be effective through strict quarantine.

Elimination Of CVB (Chrysanthemum Virus B) From A Range Of ChrysanthemumTHE Varieties By Meristem Culture Following Antiviral and Heat Treatments

CVB elimination for retaining healthy protocols from infected chrysanthemum plant was investigated trough combined treatment of meristem culture with synthetic antiviral ribavirin and thermotherapy under in vitro conditions. The biological material used on the experimentation was constituted of six commercial varieties: Dewi Sartika, Saraswati, Yellow Fiji, White Puma, Yellow Puma and White Reagent. Tissue culture initiation was conducted through plantlet establishment using MS supplemented with IAA. Ribavirin was added in media treatments with the concentration of 40 mg/l. Parallel with this step, heat treatment with different durations (1, 2, and 3 weeks) was also conducted on the plantlets. Meristem culture was done following the chemo- and thermotherapy. The experiment resumed the failure single treatment of meristem culture in eliminating CVB from the infected chrysanthemum plantlets. Under heat treatment, percentage of virus-free plantlets increased along with the duration of thermotherapy, though the survival rate of plantlets decreased in lengthened heat treatment. The best results regarding virus free plant percentage was obtained when meristem culture was applied following ribavirin or three weeks heat treatment which effectively eliminated virus particles from the plant.

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.

Initiation, proliferation and development of micro- propagation system for mass scale production of banana through meristem culture

Explants were taken from field grown plants in 2010. The shoot apical meristem of different sizes was cultured on Murashige and skoog’s (MS) medium supplemented with different concentrations and combinations of 6-benzylamino-purine (BAP), kinetin (Kin) and α- naphthaleneacetic acid (NAA) either alone or in combination with each other under different temperature conditions ranging from 23 to 27°C. Shoot formation response from shoot apical meristem showed that MS medium containing 1.0 mg/l BAP showed best response for shoot formation. For shoot multiplication, MS medium containing 1.0 mg/l BAP + 0.25 mg/l kin provided the best multiplication response which was 8 shoot per culture vial within 21.6 days after inoculation into shoot multiplication medium. Shoot formation and multiplication response was also affected by temperature variations. The best results were obtained at 27°C ± 1°C. By increase or decrease in temperature, the rate of in vitro response was also decreased. For rooting of well developed in vitro shoots MS medium supplemented with 1.0 mg/l Indole-3- butyric acid (IBA)+ 0.5 mg/l NAA showed 3.6 roots per plant after 6.8 days of inoculation into rooting medium with an average root length of 2.4 cm. 100% hardening response was obtained in Peat moss after 21 days of transplantation in glass house. The experiments were designed in completely randomized pattern. Key words: Murashige and skoog’s medium, proliferation, banana, micro propagation system.

Initiation, proliferation and development of micro-propagation system for mass scale production of banana through meristem culture

Explants were taken from field grown plants in 2010. The shoot apical meristem of different sizes was cultured on Murashige and skoog’s (MS) medium supplemented with different concentrations and combinations of 6-benzylamino-purine (BAP), kinetin (Kin) and α- naphthaleneacetic acid (NAA) either alone or in combination with each other under different temperature conditions ranging from 23 to 27°C. Shoot formation response from shoot apical meristem showed that MS medium containing 1.0 mg/l BAP showed best response for shoot formation. For shoot multiplication, MS medium containing 1.0 mg/l BAP + 0.25 mg/l kin provided the best multiplication response which was 8 shoot per culture vial within 21.6 days after inoculation into shoot multiplication medium. Shoot formation and multiplication response was also affected by temperature variations. The best results were obtained at 27°C ± 1°C. By increase or decrease in temperature, the rate of in vitro response was also decreased. For rooting of well developed in vitro shoots MS medium supplemented with 1.0 mg/l Indole-3- butyric acid (IBA)+ 0.5 mg/l NAA showed 3.6 roots per plant after 6.8 days of inoculation into rooting medium with an average root length of 2.4 cm. 100% hardening response was obtained in Peat moss after 21 days of transplantation in glass house. The experiments were designed in completely randomized pattern. Key words: Murashige and skoog’s medium, proliferation, banana, micro propagation system.

Agrotechnologies to Enhance Sugarcane Productivity in India

Sugarcane productivity is stagnating in India. Demand for sugarcane is increasing. Scope for extending sugarcane area in the country is limited. Under these circumstances, emphasis must be on increasing sugarcane productivity. Technologies such as situation-specific cultivars, newer planting techniques, heat therapy or meristem culture derived quality seed material, site-specific and cultivar-specific nutrient management, drip irrigation, fertigation, integrated weed management, abiotic and biotic stress management, etc. have the potential to increase yields substantially. This paper reviews some of the recent developments in these areas and suggests how these technologies can be applied at field level to enhance productivity.

ATTEMPTS TO ERADICATE TWO PELARGONIUM VIRUSES (PFBV AND PLPV) BY MERISTEM CULTURE AND SHOOT-TIP CRYOTHERAPY

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex droplet-vitrification cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum 'Stellar Artic' plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method.

Structure and regulation of the Asr gene family in banana

Abscisic acid, stress, ripening proteins (ASR) are a family of plant-specific small hydrophilic proteins. Studies in various plant species have highlighted their role in increased resistance to abiotic stress, including drought, but their specific function remains unknown. As a first step toward their potential use in crop improvement, we investigated the structure and regulation of the Asr gene family in Musa species (bananas and plantains). We determined that the Musa Asr gene family contained at least four members, all of which exhibited the typical two exons, one intron structure of Asr genes and the “ABA/WDS” (abscisic acid/water deficit stress) domain characteristic of Asr genes. Phylogenetic analyses determined that the Musa Asr genes were closely related to each other, probably as the product of recent duplication events. For two of the four members, two versions corresponding to the two sub-genomes of Musa, acuminata and balbisiana were identified. Gene expression and protein analyses were performed and Asr expression could be detected in meristem cultures, root, pseudostem, leaf and cormus. In meristem cultures, mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA. mASR3 exhibited the most variation both in terms of amino acid sequence and expression pattern, making it the most promising candidate for further functional study and use in crop improvement.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-011-1421-0) contains supplementary material, which is available to authorized users.

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.

Use of Sodium Hypochlorite as Media Sterilant in Sugarcane Micropropagation at Commercial Scale

Sodium hypochlorite has been successfully utilized as media sterilant (at total active chlorine concentration 0.002% in the medium) in production of sugarcane plantlets by applying apical meristem culture technique. The process has been scaled up to produce 2.5 million sugarcane plantlets per year. Use of sodium hypochlorite in place of autoclaving has resulted in reducing considerable amount of electricity cost and ultimately reducing the cost of production of sugarcane plantlets.