Dr. Ottó Orsós, the forgotten Hungarian pioneer in plant tissue culture

The knowledge of tissue culture deserves attention in respect of understanding the development of universal biology. This study intends to contribute to the past of the plant tissue culture by such data of the history of science which have been unprocessed so far. It seems that the life-work of the Hungarian biologist, Dr. Ottó Orsós is a missing and essential link between those early plant hormone researchers and the representatives of the pioneers of tissue culture schools who have contributed substantially to the development of the modern in vitro plant morphogenesis and plant cell biology. Orsós cultured kohlrabi tuber cubes on White culture medium in a sterile manner. This way, he could efficiently direct the in vitro morphogenesis of the kohlrabi, the regeneration of its shoot and root, and the formation and steps to subculture of pure callus tissues in 1938. He supported the correctness of its statements by means of detailed anatomical examinations. Orsós successfully rooted and aclimatized complete regenerated plants. We may as well call the above system — in remembrance of the creators of the original concept — "Haberlandt-Orsós model". Between the publishing of his main paper in 1938 and 2003, a period of 65 years has lapsed. On the occasion of this anniversary, we bow before this forgotten pioneer.

Model experiments for establishment of in vitro culture by micrografting in apple

Micrografting was used in our experiments for establishment of in vitro culture from one rootstock (`JTE-F') and three scion cultivars (`Remo', 'Rewena' and `Reanda') of apple. Shoot tips of these cultivars were harvested from field and grafted onto in vitro rootstock cultivars. Their survival and development were studied. 42-93% of shoot tips survived and developed further depending on cultivar. Impermanent browning of sticking agar-agar could be observed in 21-25% of the micrografts depending on cultivars but discolouration of agar-agar ceased within one week and did not cause any death of shoot tips. We used micrografting successfully for establishment of in vitro culture from cultivars, from which earlier with conventional methods the culture establishment was not possible because of hard tissue browning. However, further studies are necessary to ensure the survival and development of shoots after removing them from micrografts.

Micropropagation of Rudbeckia hirta L. from seedling explants

We conducted experiments for developing an in vitro micropropagation protocol starting from meristems of Rudbeckia hirta L seedlings. We pre-soaked the seeds in sterile ion-exchanged water for 17 hours, and then achieved surface disinfection in two separate steps. First, we used concentrated household sodium-hypochloride solution for 20 minutes and, also for 20 minutes, we applied hydrogen peroxide of 10%, which was followed by washing with sterile ion-exchanged water three times. For the propagation of seedling meristems, the combination of half-strenght solid Murashige and Skoog (1962) culture medium containing 10 mg/1 of kinetin or 2 mg/I of kinetin + 0.1 mg/1 of 2iP proved to be the most suitable. The average number of shoot-buds developed from the seedling axillary meristem in the best culture media varied between 5 and 17. Without separating them, we inoculated the shoot-bud clusters on MS culture medium containing 2 mg/1 of IAA. After four weeks of incubation we obtained elongated shoots which we separated and inoculated into a new culture medium and we obtained elongated roots. The rooted plants were gradually acclimatised in the cultivation room, potted and carried to a greenhouse, and then planted in open field for subsequent observation. By adopting this method, our laboratory started the micropropagation of the superior and/or elite genotypes of the Rudbeckia hirta L. being of special value in respect of breeding.

In vitro plant regeneration from immature embryo axis and cotyledons of common bean (Phaseolus vulgaris L.)

Phaseolus vulgaris L. is the most important economic species within the genus Phaseolus. It is grown in all parts of the world. Genetic improvement by conventional breeding has met considerable success, although production of hybrids between species within the genus has been limited due to sexual incompatibility. Recent advances in tissue culture have offered the opportunity to produce cultivars, which could not be obtained by conventional breeding methods. The use of tissue culture and genetic engineering is viewed as a logical approach to improve bean production. Gene transfer techniques will have a great impact on legumes. Although the concept of cell totipotency is widely proved, in vitro morphogenesis has not yet been achieved for a large number of cultivated beans. Regeneration protocols are strongly influenced by the genotype. In tissue and cell culture of beans, the factors controlling shoot morphogenesis and somatic embryogenesis are still unknown. The reported data suggest a possible way for future research.

Bean tissue culture and genetic transformation with Agrobacterium

In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS gene as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.

Biotechnologies for ornamental plants: some insights to the Brazilian productive chain

The world industry of ornamental plants is under wide transformations regarding the changes in the cultivation, management, post­harvest technologies and the use of new (bio)technologies to assist the improvement and development of new varieties. This industry worth more than US$ 20 billion, but Brazil shares only 1% of this value. To compete at this market with permanent technological innovation, the country needs to be tuned in with the progresses in the biotechnologies applied to the productive chain of ornamental plants. In this revision, we analyze the Brazilian and world ornamental market emphasizing the role of the biotechnologies in the modernization and increase of the competitiveness of the sector.

The importance of pathogen-free grapevine propagation material in regards to clonal selection and rootstock breeding in Germany

High performing propagation material is essential for a reliable and economical production of quality grapes. Apart from genetic aspects pathogen-freedom is of prime importance in propagation material. In particular virus diseases cause major yield and quality losses and reduced longevity. This is also reflected in the current EU legislation, which focuses on the most common and dangerous viruses: GFLV, ArMV, GLRaV-I and GLRaV-III.Apart from these, locally occurring pathogens, e.g. phytoplasms or agrobacterium, are important as well and should not be present in propagation material. There are several ways to develop pathogen-free clones. Starting with already pathogen-free material is certainly the easiest case, but might not be feasible in local varieties with small acreages and limited vine numbers. In these cases the elimination of pathogens is required, either by heat therapy, tissue culture or somatic embryo genesis.

Susceptibility of sour cherry cultivars to isolates of Monilia laxa (Ehrenbergh) Saccardo et Voglino

In this study, the susceptibility of 7 commercially important sour cherry cultivars to Monilinia laxa was studied. Artificial inoculation was made with M. laxa isolates, which were isolated from different woody plants. Artificial inoculation was prepared in the laboratory and in the field. In laboratory, flowers of sour cherries while in the field, the two-year old twigs were inoculated in 2006 and 2007. According to results of stigmata inoculation, there were infection ability differences among the isolates originated from five different stone fruit host. Cultivars could be sorted into two susceptibility groups. In the field, twig inoculation in 2007 was made at blossom period and in 2007 at harvest. Seven sour cherry cultivars were inoculated with 8-day-old mycelial culture of M. laxa originated from sour cherry and almond. The agressivity and pathogenicity of the two isolates were measured by the degree of floem death: Results showed that year and phenological stage considerably influenced the degree of symptoms caused by the fungus. After artificial inoculation, tissue death progression was studied by fluorescent microscope. According to results, sour cherry cultivars were sorted into disease susceptibility groups. Susceptibility orders were identical to results on stigmata inoculation.

Study of different factors of grapevine regeneration systems and genetic transformation

The most limitating factor for successful transformation is the absence of high-yielding regeneration protocols. However, the anther-derived embryogenic culture is an optimal technique for genetic transformation and it has been widely applied in many important cultivars, but the necessity of further development of regeneration systems has been proved. We attempted to produce somatic embryos on a wide range of genotypes from various tissues; leaves, petioles, stem segments. We started the examination of grapevine regeneration via organogenesis, succeeded in inducing shoot from the meristematic tissue of the base of bud by testing induction medium contained different concentrations of two types of hormones. To optimize the conditions of the Agrobacterium-mediated transformation, we studied the effectiveness of different Agrobacterium-treatments, the use of antioxidants and the sufficient quantity of kanamycin for selection of transformed cells.

Shoot induction and plant regeneration from cotyledon segments of the muskmelon variety "hógolyó"

Cotyledonary segments of the casaba type muskmelon variety "Hógolyó" were used to induce organogenesis. Fifty different hormone combinations were applied to enhance the induction of shoot formation on the edge of the segments. The phases of organogenesis were followed with light- and scanning electron microscope. Shoot induction was achieved with high frequency. The shoots were transferred to hormone free media for root induction. The rooted plantlets were planted out to soil.

NAA was feasible and the method can be applied in transformation experiments.


High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.

Improved clonal approaches to growing black locust (Robinia pseudoacacia L.) in Hungary: a case study

In Hungary black locust (Robinia pseudoacacia L.) is considered as an important exotic stand-forming tree species and due to climate change effects its importance is increasing in many other countries. It has some desirable characteristics from both the practical and research standpoints. As a result of a partly new black locust selection programme new black locust clones were improved and a technology was developed for mass clonal micropropagation of juvenile trees. Clone trials with micropropagated plants were established in the country for evaluating the juvenile growth and the stem form of promising black locust clones under marginal site conditions. Significant differences (P<5%) were found for stem form value which partly verified the genetic gain of the selected clones against the common black locust. It was also proved that tissue culture could offer partly new prospects for the rapid mass cloning of selected genotypes.

Factors affecting rhizogenesis in vitro and acclimatisation of three cherry rootstocks

Factors affecting rhizogenesis in vitro and acclimatisation of three rootstocks of cherry, i.e. Mahaleb, Maxma-14 and Weiroot -10 were investigated.

Rooting was easily achieved within 2-4 weeks on MS-based liquid or agar-gelled media containing auxins IBA at conc. 0.49 or 2.45 pM or NAA at conc. of 0.49 pM. On liquid media with 2.45 pM IBA, a maximum rooting efficiency of 95-100% was obtained. However, high concentrations of auxin delayed the time of root initiation for 3-5 days.

Rooted plantlets were transplanted into pots with a mixture of 3:1 (v/v) peat:perlite and acclimatised gradually to field conditions with efficiency of 60%.

Clonal selection of black locust (Robinia pseudoacacia L.) in Hungary: a review

Black locust (Robinia pseudoacacia L.) is the most important fast growing stand-forming tree species in Hungary. Its importance is increasing in many other countries, too. As a result of a new selection programme 13 black locust clones have been improved for setting up clones trials and seed orchard. In 2003 five of them (R.p. `Bácska', `Homoki', 'Szálas', `Oszlopos' and `Vacsi') were registered as cultivar­candidates. Tissue culture method has proved as a suitable mean of propagating superior individuals. The micropropagated plants have been growing successfully in the clone trials.

Jerusalem artichoke (Helianthus tuberosus L.): A review of in vivo and in vitro propagation

Jerusalem artichoke (Helianthus tuberosus L.) is an old tuber crop with a recently renewed interest in multipurpose improvement. It is a perennial tuberous plant rich in inulin and is a potential energy crop. During food shortages in times of war Jerusalem artichoke received more attention by scientists and farmers because of its multiple uses as a vegetable, medicinal plant, forage plant and source for biofuel. The energy crisis of the 1970s motivated research on Jerusalem artichoke for biofuel as the aboveground plant biomass and the tubers can be used for this purpose. There are different methods to propagate Jerusalem artichoke using tubers, rhizomes, slips (transplants derived from sprouted tubers), stem cuttings, seeds and tissue culture. So, this review was presented to highlight on propagation of Jerusalem artichoke via in vivo and in vitro techniques.