Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB

A number of virus elimination protocols have been developed to obtain healthy plants but their success rates have been variable due to the strong virus–host relationship, treatment performances and experimental condition. An in vitro collection of plants of Vitis berlandieri × Vitis riparia Kober 5BB single-infected by Grapevine vitivirus A (GVA), Grapevine fanleaf nepovirus (GFLV), Grapevine fleck maculavirus (GFkV), Grapevine leafroll ampelovirus 1 (GLRaV-1) and Grapevine leafroll ampelovirus 3 (GLRaV-3) was established to carry out thermotherapy treatments. The experimental system allowed to control the effects of host genotype by using the same genetic background, and the environmental condition such as relative humidity, soil and temperature conditions. Trials were conducted in a growth chamber set at 37 ± 0.5 °C for 48 days using in vitro cultures for all infections. ELISA and RT-PCR were used to check sanitary conditions. Results showed complete eradication of GFLV and no effect against GFkV. Different sanitation rates were obtained for other phloematic viruses: 70.2% for GVA, 25.1% for GFLaV-1 and 24.7% for GLRaV-3. The results on phloematic viruses showed a gradient of elimination efficiency which could not be completely explained with the location of viral particles that seems to be only one of the several factors affecting virus susceptibility to heat stress. Moreover, the advantages of this experimental system provide the opportunity to obtain a greater understanding of the thermotherapeutic effects on phloematic viruses.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Asymbiotic seed germination,mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.