Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

The elimination of four viruses from carnation and sweet william by meristem-tip culture

Carnation mottle virus (CarMV), carnation ringspot virus (CRSV), carnation vein mottle virus (CarVMV) and carnation latent virus (CarLV) were all eliminated from both carnation (Dianthus caryophyllus) and sweet william (D. barbatus) plants by meristem-tip culture. Carnation mottle virus was more readily eliminated from D. barbatus than from carnation. Carnation vein mottle and carnation latent viruses were more readily eliminated from carnation than from sweet william: they are rarely found in carnation but CarVMV is found frequently in sweet william. Carnation ringspot was eliminated equally readily from both hosts.     

Elimination of virus and rapid propagation of disease-free sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. cultivar NCo376) using apical meristem culture

The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l⁻¹ 6-benzyladenine and 0.015 mg l⁻¹ 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.     

Production and fingerprinting of virus-free clones in a reflowering globe artichoke

Most of the 24 viruses which infect globe artichoke are detrimental to the crop’s performance and hamper the development of a nursery activity in the respect of current EU legislation. We describe a procedure to sanitize globe artichoke “Brindisino” from Artichoke Italian latent virus (AILV) and Artichoke latent virus (ArLV), while preserving its valuable early flowering trait. ArLV was successfully eliminated by meristem-tip culture, while AILV was removed when two rounds of meristem-tip culture were spaced out with in vitro thermotherapy. In vivo thermotherapy, followed by meristem-tip culture, was also successful in producing virus-free material but was less efficient in terms of the number of plants recovered post treatment. Due to the multi-clonal composition of the populations at present in cultivation, the selected and sanitised clones were fingerprinted by applying microsatellite and AFLP (amplified fragment length polymorphism) markers. One AFLP primer combination produced 28 informative fragments used to evaluate genetic relatedness among the clones in study. Our results demonstrates that AFLP-based molecular fingerprinting enables to verify the true to clone correspondence in nurseries, ensure the effective correspondence between the real and the declared identity of a clone, so that to avoid commercial frauds, and might represents a valuable tool for assessing somaclonal variation occuring during ‘in vitro’ propagation.     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium     

In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgaris L.)

Summary Haploid plantlets from male fertile and male sterile sugarbeet plants could be induced at frequencies up to 2.2% using ovule culture. Ovary culture on media without charcoal resulted in a similar induction frequency. Plant development was inhibited by callus development originating from the mother tissue. When the callus parts were removed and the ovule transferred to a new medium without 2,4 D, callus formation could be inhibited by adding 0.5% charcoal to the medium. Up to 6.1% haploids were induced. Chromosome counts in leaf tips, chloroplast counts and isozyme patterns revealed that all plants were haploid and originated from the haploid cells of the embryo sac. Root tips showed spontaneous polyploidisation.     

The elimination of viruses from Narcissus tazetta cv. Grand Soleil d''Or, and rapid multiplication of virus-free clones

Arabis mosaic virus and narcissus degeneration virus were eliminated from Narcissus tazetta L. cv. Grand Soleil d'Or by meristem-tip culture. Cultures grew equally well in two liquid nutrient media, but tubes 25times75 mm size and temperature c. 12 ^oC were optimal for growth and survival. Virus-free bulbs grew more vigorously than infected bulbs, and gave more and larger flowers with a better colour. To increase the normal multiplication rate, either sector cuts were made in the basal plate, or bulbs were divided into chips (c. 1 g), before planting. Multiplication was greater by chip-cutting. Five virus-free bulbs were propagated to over 2000 in 8 years; theoretically, 20000000 bulbs should be produced in a further 5–6 years, sufficient to replant the Isles of Scilly with virus-free stock.     

A novel strategy for the induction and maintenance of shoot regeneration from callus derived from established shoots of apple [Malus×Domestica Borkh.] cv. Golden Delicious

A novel strategy for the production and maintenance of morphogenic callus for 1 year from mature leaf explants of apple has been developed using micropropagated primary leaves of cv. Golden Delicious. The technique required second generation adventitious buds produced from cultured primary leaves also produced from established shoot cultures. The age at which buds were capable of producing morphogenic callus was critical and found to be when leaflets were 2–3 mm in length. Medium composition affected the maintenance but not the induction of shoot regeneration from callus and the best combination was found to be high calcium, low ammonium and low hormone levels. Adventitious shoots were rooted in vitro and established glasshouse-grown plants showed no phenotypic differences from the plants derived from shoot proliferation. The great advantage of this technique for an increased efficiency of recovery of transgenic plants from transformed cells is discussed and the acquisition and maintenance of cell competence with respect to the formation of shoots in culture is explained. Received: 13 August 1996 / Revision received: 13 November 1996 / Accepted: 6 December 1996     

Micropropagation of sweet orange, Citrus sinensis Osbeck. for the development of nucellar seedlings

Protocol for micropropagation of elite plants of sweet orange (Citrus sinensis) through nucellar embryo culture has been standardized. Three to four nucellar embryos and a zygotic embryo could be excised from a single mature seed and successfully generated as healthy plants in basal MS medium. MS medium supplemented with NAA (1 mg/L) or 2, 4.D (1 mg/L) promoted callus development in both nucellar and zygotic embryos. GA3 (1 mg/L) enriched medium induced plantlets initiation but their growth was very poor. No significant differences were observed between initial growth patterns of nucellar and zygotic seedlings developing from the same ovule. Five to six shoots were obtained from collar region of both category of embryos in MS medium supplemented with BAP (1 mg/L) within 60 days of inoculation. The number of plantlets were almost doubled after their transfer in the same medium and culture for another 30 days. Higher doses of BAP resulted in initiation of callus directly from the embryos. The regenerated shoots (2-3 cm) could be rooted in MS medium supplemented with either only NAA (0.75 mg/L) or NAA (0.50 mg/L) and IBA (2.0 mg/L). A number of plantlets could be obtained from a nucellar embryo grown shoot within a limited time period.