Virus-free Plants Research Articles

MOLECULAR EVALUATION OF THE PRINCIPAL RANUNCULUS ASIATICUS L. VIRUSES

The buttercup (Ranunculus asiaticus L.) belongs to the family Ranunculaceae and is a popular ornamental plant cultivated for the sale of cut flowers and for the production of pot or border plants. The hybrids of Ranunculus asiaticus are susceptible to infection by several viruses, very often present in mixed infection, and all associated with economically important diseases. In the present study the main viruses infecting Ranunculus hybrids (Tomato spotted wilt virus (TSWV), Ranunculus white mottle virus (RWMV), Ranunculus leaf distortion virus (RLDV) and others) were evaluated by double step RT-PCR assays and specific primers, using leaf tissues as starting material. Results showed the presence of some viral species in the samples analyzed, either in single or in mixed infection, and confirm that the molecular evaluation, due to versatility of use and reliability of results, can be easily used as a tool for viruses detection on large scale propagation materials in order to prevent the spread of the principal viruses and assure the production of virus-free Ranunculus plants.

Sweet potato cultivar degeneration rate under high and low sweet potato virus disease pressure zones in Uganda

Sweet potato is a vegetatively propagated crop where vine cuttings from previous crops or volunteer plants are used as planting material. This practice can lead to the accumulation of systemic pathogens, especially viruses. However, the contribution of this practice to degeneration of sweet potato cultivars in Uganda has been only speculative, hence the need to document the rate of cultivar degeneration in high and low sweet potato virus disease pressure zones. Four cultivars of sweet potato – ‘Beauregard’, ‘Dimbuka’, ‘Ejumula’ and ‘NASPOT 1’ – were planted in a series of field trials in central (Kabanyolo) and Eastern (Serere) Uganda over five generations (G1, G2, G3, G4 and G5). The trials started with virus-free planting material and each succeeding trial retained planting material from the previous one, as well as receiving fresh clean material. Data were recorded on virus incidence and severity monthly for 4 months, root yield and vine weight at harvest after 6 months. Virus symptoms were observed 1 month after planting in all the plant generations, with Sweet potato feathery virus and Sweet potato chlorotic stunt virus being the most prevalent viruses detected. The cultivars ‘Beauregard’ and ‘Ejumula’ had highest disease incidence and severity, with the latter collapsing after a single season in both locations. Storage root yields and numbers were greatest in G1 but remained similar although less in all subsequent generations (G2, G3, G4 and G5) for each cultivar. Since it is impractical to provide fresh planting material each year for farmers, the focus should be on breeding more resistant varieties of sweet potato.

Production of virus-free garlic (Allium sativum L.) through meristem tip culture after solar or hot air treatment of cloves

SummaryGarlic (Allium sativum L.) is a clonally propagated crop that can be infected by many virus species which tend to accumulate year-after-year, leading to yield reductions and varietal degeneration. Therefore, the production and use of virus-free planting material is vital to sustain productivity. In this study, Onion yellow dwarf virus (OYDV; Potyvirus), Garlic common latent virus (GarCLV; Carlavirus), Shallot latent virus (SLV; Carlavirus), and Garlic virus X (GarV-X; Allexivirus) were detected in cloves of the commercial garlic cultivar, ‘G-1’ (‘Jamuna Safed’) by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). DAC-ELISA was performed using specific antisera against each virus species, and confirmed through reverse transcription-polymerase chain reactions (RT-PCR) using virus or genus-specific oligonucleotide primers. To obtain virus-free plants of ‘G-1’, cloves were subjected to solar heat treatment for 5, 10, or 15 d, or to hot air treatment (at 37°C, 40°C, or 42°C for 7, 14, or 21 d) combined with meristem isolation and in vitro culture, then indexed for all viruses using DAC-ELISA and RT-PCR. Meristems from solar heat-treated or hot air-treated cloves regenerated after 90 d and 45 d in culture, respectively. Solar heat treatment for 15 d resulted in 17 - 18% regeneration, and all were free from GarCLV, SLV, and OYDV. However, when solar heat-treated for 10 d, only 66% of the 17 - 18% regenerated plants were free from all viruses, including GarV-X. Hot air treatment at 37°C or 40°C eliminated GarCLV, SLV, and OYDV in 20 - 38% and 42 - 62% of regenerated plants, respectively, for the different durations of exposure. However, GarV-X could not be eliminated from any of the regenerated plants by hot air treatment, irrespective of the duration of exposure. Meristems from cloves that had been hot air-treated at 42°C resulted in 50 - 66% regeneration, of which 53 - 57% were free from GarCLV, SLV, and OYDV when treated for 7 d or 14 d, and 29% were free from all viruses when treated for 21 d. For most treatments, an increase in the duration of exposure to heat resulted in reduced regeneration, but increased the rate of recovery of virus-free plants. To our knowledge, this is the first study to report the use of solar treatment for virus elimination in garlic and the development of an efficient tissue culture protocol for the production of virus-free garlic plants using different thermotherapy treatments coupled with meristem tip culture.

Influence of Symbiotic Interaction between Fungus, Virus, and Tomato Plant in Combating Drought Stress

The influence of the three-way interaction between the fungus (Curvularia protuberata), virus (CThTV), and tomato (Solanum lycopersicum) in combating drought stress was evaluated in this study. The plants in this greenhouse experiment were grown under conditions of 400 ± 150 μmol·m-2·s-1 photon flux density, 45% to 50% relative humidity (RH), and 30°C ± 2°C. Tomato seeds were germinated and inoculated with the combination of the fungus and virus at the seedling stage. The plants were allowed to grow for two weeks and randomly selected individuals were utilized. The selected plants were grown in one gallon pots containing organic potting soil. The treatments included non-symbiotic (NS), virus-free (VF), and symbiotic (An) plants. Each treatment received twelve samples and each sample was allowed to grow for an additional two weeks under drought stress. At that time, plants were exhibiting drought stress symptoms including visible wilting. Six samples from each treatment were utilized in determining selected physiological responses of tomato at pre-anthesis stage. The remaining six samples from each treatment were re-watered once and allowed to grow until they reached the anthesis stage. When they showed visible signs of wilting, the same physiological responses measured during pre-anthesis were conducted. The samples of each treatment were utilized at the end of each stage in determining photosynthetic rate, stomata conductance, photosynthetic pigments, water potential, and soluble sugar content. Plant growth, chlorophyll a, chlorophyll b, photosynthetic rate, stomata conductance, water potential, and soluble sugar content were similarly affected by the various treatments. However, carotenoids were significantly higher at pre-anthesis in the symbiotic plants in comparison to other treatments. Additionally, photosynthesis appeared to be significantly higher at anthesis compared to pre-anthesis for all treatments.

Eliminating Potato Virus Y (PVY) and Potato Leaf Roll Virus (PLRV) Using Cryotherapy of in vitro-grown Potato Shoot Tips

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

Identification of Potato Virus Y (Pvy) and Its Economic Importance on Potato Crop

This study was conducted to estimate disease incidence of potato virus Y (PVY) in Duhok Province/ Kurdistan Region/ Iraq and to investigate its effects on the growth and morphology of potato plant and its productivity. High rates of occurrence of viral symptoms in the surveyed field were recorded. The mainly included symptoms were mild to severe yellowing, mottling, necrosis, stunting and malformation of potato plants. The effect of the virus on potato crop was studied using Vegetative growth and yield characters of healthy, current season and tuber borne PVY infected plants. There is differentiation between the growth of the current season, tuber borne PVY-infected and the virus free potato plant. Results showed that infection by PVY leads to reduce many physiological functions of above and underground parts of host plant like size of leaf area, chlorophyll percentage, number of tubers, tuber weight and total yield of a plant. Depending on the results, because of reducing physiological functions of above ground part of potato plant (leaf area and chlorophyll percentage), the number and the weight of tuber decreased, so the productivity of the plant decreased.

Biology, etiology, and control of virus diseases of banana and plantain.

Banana and plantain (Musa spp.), produced in 10.3 million ha in the tropics, are among the world's top 10 food crops. They are vegetatively propagated using suckers or tissue culture plants and grown almost as perennial plantations. These are prone to the accumulation of pests and pathogens, especially viruses which contribute to yield reduction and are also barriers to the international exchange of germplasm. The most economically important viruses of banana and plantain are Banana bunchy top virus (BBTV), a complex of banana streak viruses (BSVs) and Banana bract mosaic virus (BBrMV). BBTV is known to cause the most serious economic losses in the "Old World," contributing to a yield reduction of up to 100% and responsible for a dramatic reduction in cropping area. The BSVs exist as episomal and endogenous forms are known to be worldwide in distribution. In India and the Philippines, BBrMV is known to be economically important but recently the virus was discovered in Colombia and Costa Rica, thus signaling its spread into the "New World." Banana and plantain are also known to be susceptible to five other viruses of minor significance, such as Abaca mosaic virus, Abaca bunchy top virus, Banana mild mosaic virus, Banana virus X, and Cucumber mosaic virus. Studies over the past 100 years have contributed to important knowledge on disease biology, distribution, and spread. Research during the last 25 years have led to a better understanding of the virus-vector-host interactions, virus diversity, disease etiology, and epidemiology. In addition, new diagnostic tools were developed which were used for surveillance and the certification of planting material. Due to a lack of durable host resistance in the Musa spp., phytosanitary measures and the use of virus-free planting material are the major methods of virus control. The state of knowledge on BBTV, BBrMV, and BSVs, and other minor viruses, disease spread, and control are summarized in this review.

Comparing the Effects of Benzyladenine and <i>meta</i>-Topolin on Sweet Basil (<i>Ocimum basilicum</i>) Micropropagation


 Micropropagation of aromatic plants reveals an effective way of obtaining high volume, virus-free plant material of uniform quality. The application of meta-Topolin (mT) (N6-(2-hydroxybenzyl) adenine-9-riboside) and aromatic cytokinin as Benzyladenine (BAP) in the micro propagation of sweet basil (Ocimum basilicum L.) was tested for the first time and plant growth parameters assessed to determine the optimum level of these cytokinins. Additionally, the rate of root-growth inhibition due to these two cytokinins was also assessed. Our results show that 1 mg/l (4.43 mM) BAP and 0.5 mg/l (2.07 mM)mT produced the most favourable effects on new shoot developments. Meta-Topolin was shown to increase the quality of the plants and in comparison with BAP fewer distortions were observed. No significant differences in root-growth inhibition between the mT and BAP were detected. 

Bulbilhos aéreos de alho, provenientes de escapes florais, são infectados por vírus

Em campos de produção comercial de alho é comum observar plantas naturalmente infectadas por vírus dos gêneros Allexivirus, Carlavirus e Potyvirus. Os bulbilhos aéreos podem ser uma alternativa para a propagação de plantas de alho livres de vírus. Desta forma, o objetivo deste trabalho foi analisar a taxa de perpetuação dos vírus de plantas infectadas para os bulbilhos aéreos. Os bulbilhos aéreos obtidos de plantas infectadas foram analisados por RT-PCR utilizando oligonucleotídeos universais para os gêneros Allexivirus, Carlavirus e Potyvirus. A taxa de perpetuação foi de 65% para allexivírus, 20% para carlavírus e 82,22% para potyvírus. Os resultados demonstraram que a perpetuação dos diferentes vírus do bulbo para os bulbilhos aéreos é elevada, inviabilizando a utilização direta dos bulbilhos aéreos provenientes de plantas matrizes infectadas por vírus. Esta metodologia deve ser utilizada somente a partir de plantas isentas de vírus.

Growth and Yield Variations among Generations in Field Cultivation of Virus-free Sweet Potato Plants

Growth and Yield Variations among Generations in Field Cultivation of Virus-free Sweet Potato Plants

Efficient elimination of virus complex from garlic (Allium sativum L.) by cryotherapy of shoot tips

Vegetative propagation of plants, such as garlic (Allium sativum L.), is known to facilitate the transmission of several virus species throughout the plant cycles. This process favors the onset of complex diseases by accumulation of different species in the same plant, resulting in decreased productivity and production quality. Studies have reported the use of cryotherapy of shoot tips, or meristematic clusters, as an efficient tool for obtaining virus-free plants. This study aimed to evaluate the ability of cryotherapy to eradicate virus complex in garlic plants. Bulbils naturally infected with Onion Yellow Dwarf Virus (OYDV), Leek Yellow Strip Virus (LYSV) and Garlic Common Latent Virus (GCLV) were employed as explants for different virus-cleaning treatments tested. Dot-ELISA and RT-PCR analysis were used to demonstrate the presence/absence of virus complex, and histological analysis was also performed to confirm these results. Five days after cryotherapy, structural analysis revealed that cooling had caused cell damage, as indicated by the increased vacuolization of cells after cryotherapy, as well as slight plasmolysis after thermotherapy. Immunolocalization analysis indicated the subcellular distribution of OYDV in garlic shoot tips in association with the development of plasmodesmata, while no OYDV was detected in the first cell layers of the meristematic dome. Cryotherapy successfully removed virus complex, resulting in virus-free plants with enhanced efficiency, compared to conventional meristem culture-based techniques. Moreover, the synergistic effects of cryotherapy and thermotherapy resulted in a 40 % survival rate of shoot tips and the regeneration of 90, 100 and 80 % OYDV-, LYSV- and GCLV-free plants, respectively.

Elimination of mixed ‘Odontoglossum ringspot’ and ‘Cymbidium mosaic’ viruses from Phalaenopsis hybrid ‘V3’ through shoot-tip culture and protocorm-like body selection

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants.

English

Banana bunchy top disease (BBTD) was first identified in DR Congo in 1958. Previously, the disease’s distribution throughout the Congo basin had not been studied, so an initial study, to determine the incidence and severity of BBTD in banana and plantain producing regions of Oriental province, was carried out during 2009 to 2010. Three hundred (300) farms were surveyed across 4 districts and 19 territories, with 30 mats assessed per farm. Visible disease symptoms were recorded and serological tests using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) were carried out on collected samples. Additional surveys were conducted during 2010 to 2012 in Maniema, Northern Katanga, Eastern and Western Kasai, Bandundu and Equateur provinces to assess the distribution of the aphid vector (Pentalonia nigronervosa), BBTD incidence and severity. 92% of mats observed across Oriental province manifested BBTD symptoms but severity levels were low. All plantain and banana cultivars grown in farmers’ fields were susceptible to the disease. The vector, P. nigronervosa, was found on 89% of all assessed mats. In Tshopo district, all samples collected on plants showing disease severity scores 2, 4 and 5 tested positive for the presence of the virus. However, only 48% of plants with severity score 1 and 33% of plants with score 3 gave positive TAS-ELISA results. More importantly, 40% of symptomless plants (score 0) tested positive. The average BBTD incidences in Bandundu, Equateur, Eastern and Western Kasai, Katanga and Maniema were lower than levels observed in Oriental province. The lowest incidence levels were observed in Equateur (43%) and Katanga (35%). Although, BBTD is widespread in the surveyed provinces, the generally observed low severity levels result in limited impact on production. The generalized spread of BBTD in surveyed areas nevertheless underlines an urgent need to identify virus-free plants for multiplication and distribution of disease-free materials to small-scale farmers.

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5′ and parCoat3′ was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found.The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.

SUSTAINABLE STRAWBERRY PRODUCTION IN GEORGIA

In Georgia during the last five years there has been an increase of Organic Strawberry farmers’ interest towards strawberry and blueberry crops. These crops have large export potentials in Georgia and cultivation of them through diversification might develop as one of the most important branches of Sustainable Agriculture Development. Insufficient amounts of organic and virus-free plants of these crops is the principal problem in the development of this branch of agriculture. Farmers are interested in producing organically and biologically safe strawberry plant products without using artificial pesticides and fertilizers. Organic biomass mulches are used widely. Organic strawberry producers are struggling to address the challenges that have been intensified by harmful chemicals and oil-based fertilizers in conventional strawberry production. Three of the most pressing challenges facing sustainable organic strawberry growers are soil fertility, soil-borne diseases and pests. Organic fruit is produced without conventional pesticides, synthetic fertilizers or bioengineering. Choosing organic culture provides a tangible way which is beneficial for the environment, local economies, and public health, both on and off the farm. Our research provides sustainable strawberry organic methods. It also covers integrated pest management and weed control techniques that can reduce pesticide use in strawberry production.

Screening Potato Cultivars for new Sources of Resistance to Potato virus Y

Potato virus Y (PVY) strains have been defined based on genetic reactions in potato indicators expressing hypersensitive reaction (HR) response due to the presence of three different N genes, and also based on genomic information. Nine strains are known currently, with five PVY strains defined biologically, PVYO, PVYC, PVYZ, PVYN, and PVYE. The genetic background of the majority of North American potato cultivars has so far been poorly characterized for the presence of N genes inducing HR towards different PVY strains. Here, the HR response was studied in eight potato cultivars, elicited by five strains of PVY circulating in North America. These PVY isolates included representative isolates of PVYN-Wi, PVYNA-N, PVYO, PVYZ, and PVYN strains. Potato cultivars tested included Russet Burbank, Russet Norkotah, Shepody, Ranger Russet, Western Russet, Alturas, Rio Grande Russet, and Yukon Gem, grown in the U.S., and standard indicators Desiree and Maris Bard with the known genetic background. Three additional strains, PVYN:O, PVY-NE11, and PVYE, were tested on Yukon Gem. Virus-free potato plants were mechanically inoculated with PVY inoculum, and local and systemic foliar symptoms were observed for 8 weeks post-inoculation under different climate-controlled conditions. Virus status of the inoculated plants was tested starting at 3 weeks post-inoculation, by serotype-specific ELISA and RT-PCR, in order to monitor successful infections and confirm the identity of the inoculated PVY isolate. This systematic approach allowed us to identify Nytbr and Nztbr genes present in several North American cultivars. Two more new, putative N genes were postulated to be expressed in the cultivar Yukon Gem, and one additional putative N gene was postulated to be expressed in two cultivars, Yukon Gem and Rio Grande Russet. These N genes may represent valuable sources of resistance against multiple strains of PVY.

Stem apex detoxification culture markedly improved several physiological characters of chrysanthemum ‘YUTAI’

Chrysanthemums host many viruses, viroid species, and sequence variants thereof. In the present study, a stem apex detoxification culture system for chrysanthemum ‘YUTAI’, a cultivar used for chrysanthemum tea, was established by apical meristem culture in vitro on basal Murashige and Skoog medium supplemented with 0.5 mg L−1 benzyladenine and 0.1 mg L−1 naphthalene acetic acid (NAA) to stimulate shooting, and on medium containing 0.5 mg L−1 NAA to afford the highest frequency of rooting. Seven viruses [Chrysanthemum stunt viroid (CSVd), Chrysanthemum chlorotic mottle viroid (CChMVd), Chrysanthemum virus B, Cucumber mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, and Tomato aspermy virus] present in seedlings were synchronously detected using a sensitive multiplex reverse transcription-polymerase chain reaction assay. Two of these viruses (CChMVd and CSVd) compromised plant growth and development (affecting both the photosynthetic rate and height attained), and their levels were investigated in annual plants. Compared with seedlings infected by either or both CChMVd and CSVd, the chlorophyll content, the net photosynthetic rate, and the stomatal conductance and transpiration rates were higher, but the concentration of intercellular CO2 slightly lower, in virus-free seedlings. Virus-infected seedlings were stunted, exhibiting a reduction of 27 % of the mean height of virus-free plants. We suggest that the lower net photosynthetic rate and stunted growth observed in virus-infected seedlings were caused by the virus-mediated degradation of leaf chlorophyll, which in turn compromised plant nutrition.

English

Micrografting is an in vitro grafting technique which involves the placement of a meristem or shoot tip explant onto a decapitated rootstock that has been grown aseptically from seed or micropropagated cultures. Following early experiments of micrografting in ivy and chrysanthemum, the technique has been used in woody species, especially fruit trees. Major work was carried out in different Citrus species for the elimination of various viral diseases. In vitro micrografting has been used for improvement and multiplication of fruit trees as the technique has potential to combine the advantages of rapid in vitro multiplication with the increased productivity that results from grafting superior rootstock and scion combinations. Successful micrografting protocols have been developed for various fruit crops including almond, apple, cherry, chestnut, Citrus, grapes, mulberry, olive, peach, pear, pistacio, walnut, etc. Special techniques have been used for increasing the percentage of successful micrografts with the use of growth regulators, etiolation treatments, antioxidants, higher sucrose levels, silicon tubes, etc. The technique has great potential for improvement and large scale multiplication of fruit plants. It has been used on commercial scale for production of virus-free plants in fruit crops and viroid free plants in Citrus. Micrografting has also been used in prediction of incompatibility between the grafting partners, histological studies, disease indexing, production of disease-free plants particularly resistant to soil borne pathogens and multiplication of difficult to root plants.   Key words: Fruit crops, graft incompatibility, crop improvement, micrografting, propagation, shoot tip grafting.

A protocol for in vitro production of microtubers in Chinese yam (Dioscorea opposita).

Chinese yam (Dioscorea opposita) is an important tuberous crop owing to its dual use as a food as well as a medicine. Tissue culture techniques allow the rapid multiplication of virus-free plant materials. The use of microtubers offers an attractive alternative to in vitro-grown plantlets for the micropropagation and exchange of healthy Chinese yam materials. A protocol for the in vitro production of Chinese yam microtubers was developed in this study. Though we tested both one-step and two-step procedures, only the two-step procedure showed favorable results for tuberization. Media with 60 g L(-1) sucrose yielded the highest microtuber index. We demonstrate that table sugar was an efficient and economical alternative to analytical grade sucrose for microtuber production. Using an orthogonal experimental design, we determined the optimal growth regulator combination for microtuber induction and development. The microtubers obtained from our protocol sprouted readily both in vitro and in soil.

Occurrence of Telosma mosaic virus causing passion fruit severe mosaic disease in Thailand and immunostrip test for rapid virus detection

The cause of leaf and fruit severe mosaic disease in purple passion fruit (Passiflora edulis) in Chiang Mai province, Thailand, was identified and virus diagnostic kit was developed. Symptomatic plants collected from diseased mother plants in the nursery at the Royal Pangda research station (RPRS) was used for virus isolation through single lesion transfer in Chenopodium amaranticolor, followed by virus propagation for purification in Nicotiana benthamiana, and back inoculation to purple passion fruit. Electron microscopy of leaves with typical symptoms revealed potyvirus-like flexuous rod particles, ca. 750 nm long and pinwheel inclusion bodies in the cytoplasm of infected cells. The deduced amino acid sequence of the complete coat protein revealed that the isolated virus was a strain of Telosma mosaic virus with which it shared 84% identity. A rabbit polyclonal antiserum was produced against purified virions, and used to develop a gold-labeled immunostrip for rapid virus diagnosis. The test strip could detect the virus in diseased passion fruit sap up to a dilution of 1: 640, and positive test line could be read within 3–5 min. Application of strip test for virus assay in RPRS nursery's mother plants help screen clean stocks. This strip test kit supports RPRS program of producing virus-free planting materials for farmers.