In vitro culture of sugarcane infected with maize streak virus (MSV)

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Selection ofMycosphaerella fijiensis-resistant cell lines from micro-cross sections of banana and plantain

Anin vitro selection system using microcross sections of banana and plantain cultivars belonging to AAA and AAB genomic groups were used to produce plants resistant against the Black Sigatoka disease. The fungus resistant plantlets were obtained in a double selection system. This involved in a first step the use of a fungal crude filtrate and in the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted from the fungusMycosphaerella fijiensis (M. fijiensis), the causal agent of Black Sigatoka disease. Resistant plantlets obtained from the double selection system were inoculated with conidia ofM. fijiensis in a growth chamber to reproduce Black Sigatoka symptoms. Compared to non-treated control plantlets, which were highly susceptible to the fungus, 10.7–19.3% toxin-resistant plantlets which arose from tissues that went through the double selection system were resistant againstM. fijiensis. This technique of using micro-cross sections for selection on fungal toxins seems to be amenable to differentMusa genotypes for the production of fungus-resistant plants.F. A. Schulz died 11. 3. 1995     

Chitosan improves development,and protects<Emphasis Type="Italic"> Vitis vinifera</Emphasis> L. against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.Communicated by S. Gleddie     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Comparison of carbonic anhydrase activity among various species of plantlets

During plant tissue culture, the culture container is small and sealed; the concentration of CO₂ in the microenvironment is relatively low. The plantlet growth is restrained for the shortage of CO₂ in the culture container. Carbonic anhydrase is a zinc-containing metalloenzyme that catalyzes the reversible conversion of bicarbonate to CO₂. The determination of carbonic anhydrase of leaves from Atractylodes lancea (thunb.) DC, Orychophragmus violaceus (L.) O.E. Schulz, Brassica juncea (L.) Czern.et Coss. cv. Luzhousileng, Brassica campestris L. cv. Chuanyou No.8, Brassica napus L cv. Oro, Brassica carinata Braun, Raphanus sativa L. var. raphanistroides Makino and their plantlets indicates that the carbonic anhydrase activity of leaves from both plantlets and fields varies from plant species to plant species, the carbonic anhydrase activity of leaves of Atractylodes lancea (thunb.) DC is the lowest among those plants, and the leaves of all plantlets are lower in carbonic anhydrase activity than the same species of plants from fields. The comparison of the growth rates of those plantlets shows that their relative growth rates are significantly different, plantlets of Atractylodes lancea have the slowest relative growth rate among those plants, and plantlets of Brassica juncea have the greatest relative growth rate. The relationship between RGR of plantlets and their CA activities is a significant linear function. It seems that there was certain correlation between carbonic anhydrase activities of plants and their growth rates. It suggests that in vitro, the greater the carbonic anhydrase activity of plantlet is, the higher its net photosynthetic rate, and the faster its growth rate. Those results offer a foundation to a rational medium choice in plant tissue culture.     

In vitro propagation of mature trees of Alnus incana (L.) Moench.

Mature trees of European grey alder (Alnus incana) were micropropagated on a modified MS medium containing 2.5 M BA, 6.2 mM (500 mg l^-1) NH₄NO₃ and 1.5% glucose. Prior to in vitro culture, mature scions were multiplied through grafting and cutting techniques. Shoot tips from cuttings were established in vitro. After six months of culture, shoots were rooted either in vitro or in vivo and plantlets were transferred to greenhouse conditions.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt and rhizosphere colonization in pot-grown banana plantlets

Streptomyces sp. strain g10 exhibited strong antagonism towards Fusarium oxysporum f.sp. cubense (Foc) races 1, 2 and 4 in plate assays by producing extracellular antifungal metabolites. Treating the planting hole and roots of 4-week-old tissue-culture-derived Novaria banana plantlets with strain g10 suspension (10⁸ cfu/ml), significantly (P<0.05) reduced wilt severity when the plantlets were inoculated with 10⁴ spores/ml Foc race 4. The final disease severity index for leaf symptom (LSI) and rhizome discoloration (RDI) was reduced about 47 and 53%, respectively, in strain g10-treated plantlets compared to untreated plantlets. Reduction in disease incidence was not significant (P<0.05) when plantlets were inoculated with a higher concentration (10⁶ spores/ml) of Foc race 4. Rhizosphere population of strain g10 showed significant (P=0.05) increase of more than 2-fold at the end of the 3rd week compared to the 2nd week after soil amendment with the antagonist. Although the level dropped, the rhizosphere population at the end of the 6th week was still nearly 2-fold higher than the level detected after 2 weeks. In contrast, the root-free population declined significantly (P=0.05), nearly 4-fold after 6 weeks when compared to the level detected after 2 weeks. Neither growth-inhibiting nor growth-stimulating effects were observed in plantlets grown in strain g10-amended soil.     

In vitro propagation of Sandersonia and Gloriosa

The in vitro reactions of two closely related endangered species Sandersonia and Gloriosa were studied. Both species responded similarly in vitro. Callus production was initiated on a basal medium containing 2, 4-dichlorophenoxyacetic acid. Roots were formed on removal of this hormone. Tuber explants from particular parts of the tuber produced plantlets on a range of hormone concentrations. Repeated longitudinal sectioning of the bud also resulted in multiple plantlet formation.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Application of rapd in the determination of genetic fidelity in micropropagated Drosera plantlets

Summary Random amplified polymorphic DNA (RAPD) markers were used to verify the clonal fidelity of two micropropagated Drosera species, D. anglica and D. binata, which were regenerated by adventitious budding from leaf explants and shoot tips, respectively. Twenty arbitrary decamers were used to screen 15 randomly selected plantlets of each species. No genetic variation was detected among D. binata regenerants, whereas a 0.08% polymorphism frequency was estimated for D. anglica plantlets. These results indicate that the regeneration of plants through shoot-tip culture is a low-risk method for generating genetic variability, whereas material regenerated through leaf explants requires further verification.     

Micropropagation of mature wych elm (<Emphasis Type="Italic">Ulmus glabra</Emphasis> Huds.)

Explants of mature vigorous donor trees of wych elm (Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.Abbreviations DED: Dutch elm disease - BAP: 6-Benzylaminopurine - IBA: Indole-3-butyric acid - TDZ: Thidiazuron - WPM: Woody Plant Medium - DM: Dubovský Minimal Medium Communicated by D. Bartels     

Control of leaf-tip necrosis of micropropagated ornamental statice by elimination of endophytic bacteria

Summary Leaf-tip necrosis of micropropagated statice plantlets is a serious problem in commercial laboratories in Taiwan. Endophytic bacteria were detected in plantlets obtained from commercial laboratories with a leaf-tip necrosis problem. Endophytic bacteria were detected in flower stalks collected from four different statice farms at frequencies ranging from 61 to 100%. All plantlets regenerated from flower-stalk explants that tested free of endophytic bacteria did not develop leaf-tip necrosis. The most frequently detected endophytic bacteria were Pasteurella multocida, Stenotrophomonas maltophilia, and Alcaligenes sp. Most endophytic bacteria in statice plantlets were eliminated by the subculture of plantlets on medium with augmentin, cefotaxime, or augmentin plus cefotaxime. Those plantlets freed from endophytic bacteria by subculture on antibiotic-amended medium did not develop leaf-tip necrosis. Our results show that leaf-tip necrosis of micropropagated statice plantlets is associated with endophytic bacteria, and that the disease can be controlled by using explants pre-tested to be free from endophytic bacteria or by the subculture of affected plantlets on antibiotic-amended medium.     

Arbuscular mycorrhizal (AM) technology for the conservation of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn.: an endangered medicinal herb

Curculigo orchioides Gaertn. (family Hypoxidaceae) is an endangered anticarcinogenic and aphrodisiac herb, native of India. This study reports the effect of three arbuscular mycorrhizal (AM) fungal inocula on post-transplanting performance of ‘in vitro’ raised C. orchioides plantlets. The three AM fungal inocula consisted of two monospecific cultures of Glomus geosporum and G. microcarpum and one crude consortium of AM fungal spores isolated from rhizosphere soil of C. orchioides growing in natural habitat. Complete plantlets of C. orchioides were raised by direct organogenesis of leaf explants on half strength Murashige and Skoog’s medium devoid of any growth hormone. C. orchioides plantlets responded significantly different to all three mycorrhizal treatments. Mycorrhization enhanced the survival rate of C. orchioides plantlets to 100%. The inoculated plantlets fared significantly better than the uninoculated ones in terms of biomass production and number of leaves and roots per plant. Mycorrhizal plantlets exhibited higher concentrations of photosynthetic pigments as well as minerals P, Mg, Cu, Zn, Mn and Fe in both shoots and roots. Among the three inocula tested, plantlets inoculated with the mixed consortium of AM fungi consistently performed better in terms of the parameters evaluated. The study suggests use of mixed consortium of AM fungi over monospecific cultures for the sustainable cultivation and conservation of endangered medicinal plant: Curculigo orchioides.     

Transgenic plants of Vitis vinifera cv. Seyval blanc

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.     

Effect of agar concentration on growth and anatomy of adventitious shoots of Picea abies (L.) Karst

The development of adventitious shoots of Picea abies was affected by the agar concentration in the culture medium. Increasing the agar concentration from 0.5 to 2.0% decreased vitrification, but at the same time reduced shoot growth and rooting potential. Slightly vitrified plantlets could become acclimatized to greenhouse conditions. The mesophyll of needles developed in vitro was interspersed with large air spaces; the lower the agar concentration, the larger the air spaces. After transfer to the greenhouse, the new needles from the acclimatized plantlets had an anatomy approaching that of plants growing in field.     

Intergeneric somatic hybrid plantlets between Dianthus barbatus and Gypsophila paniculata obtained by electrofusion

Hypocotyl-derived protoplasts of Dianthus barbatus that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.     

In vitro insect-feeding bioassay to determine the resistance of transgenic rice plants transformed with insect resistance genes against striped stem borer (Chilo suppressalis)

Summary To determine the degree of insect resistance in transgenic plants, different bioassays are used which typically use either whole plant or small pieces of leaves or stems of transgenic plants, following culture under greenhouse conditions. An in vitro insect-feeding bioassay is presented which permits the infestation of transgenic plantlets with newly hatched larvae from the striped stem borer. The bioassay consists of the germination of rice seeds in vitro using Murashige and Skoog medium in test tubes, and then infestation of each 3–4 cm long seedling with one neonate larva obtained from surfacesterilized eggs of Chilo suppressalis. The infested in vitro plantlets are kept in culture rooms at 25°C for several days and then the seedling damage and the growth of the larvae are analyzed. Senia (japonica variety) homozygous transgenic rice plants were used for these experiments. The plants were transformed with either the cry1B or the maize proteinase inhibitor (mpi) genes. Both genes confer resistance to Chilo suppressalis. With non-transformed plants the larvae grew and developed normally, feeding on the small rice plantlets. In contrast, with cry1B plants, the neonate larvae died during the first days of the infestation. These plantlets recovered completely and developed similarly to the non-infested control plants. With transgenic plants transformed with the mpi gene, the neonate larvae did not die but grew more slowly compared with the controls. Thus, this in vitro insect-feeding bioassay is a rapid and easy method to detect the resistance of cry and mpi transgenic plants to stem borers such as Chilo suppressalis.