G. Gyulai

Effect of copper on shoot and root regeneration in wheat, triticale, rape and tobacco tissue cultures

CuSO4 (0.1–100 μM) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO4 also stimulated root formation. When equal concentrations of CuSO4 were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO4 pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO4, AgNO3, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO3 strongly increased morphogenesis, whereas CuSO4 had no significant effect.

Plant regeneration of NaCl-pretreated cells from long-term suspension culture of rice (Oryza sativa L.) in high saline conditions

Cells of a 2-year-old suspension culture of rice (Oryza sativa L.), grown under 1.5% NaCl stress for 3 months, gave rise to plants through embryogenesis in different saline conditions. The high regeneration potential (59.6%) on salt-free medium decreased rapidly with increasing concentration of salt in the regeneration medium. At 1.25% NaCl, healthy shoots were developed in 14.9% of the cultures. Under 1.5% salt stress, embryo formation and embryo germination (6.1%) was observed but further development into plants was inhibited. Cells not pretreated with salt produced plants at a low frequency (2.6–4.2%) both in salt-free and low saline condition (0.75–1% NaCl). Cells pretreated for 3 months with 0.75% salt did not give rise to plants on all tested media. Plants regenerated from the salt-stressed cultures were transferred to soil and grew to maturity in a greenhouse.

High efficiency adventive embryogenesis on somatic embryos of anther, filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 μM increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 μM BA and 5.4 μM NAA.

Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet (Beta vulgaris L.)

An in vitro method was developed for microshoot initiation from thin-layer explants prepared from the elongated epicotyls of sugarbeet (Beta vulgaris L.). Intact epicotyls of 14-day-old seedlings were excised from the hypocotyls above the cotyledons and allowed to elongate on De Greef and Jacobs (1979) medium supplemented with 0.2 mg/l 6-benzyladenine, 0.2 mg/l gibberellic acid and 0.1 mg/l indole-3-acetic acid in darkness. After a 21-day-incubation, the elongated epicotyls were halved to obtain apical and basal segments prior to removing the leaves and lateral buds. Subsequently, 5–8 mm long, 2–3 mm wide and 0.8–1.0 mm thick tangential sections were prepared longitudinally from the exterior parts of the halved epicotyls. These thin-layer explants were incubated on microshoot initiating media containing various growth regulators. The combination of 1.0 mg/l 6-benzyladenine and the antiauxin 2,3,5-triiodobenzoic acid (1.0 mg/l) resulted in maximum microshoot development (6.3±0.2 microshoots/thin-layer explant). The final efficiency of our tissue culture system was significantly increased by the NaCl (100 mg/l) initiated in vitro rooting of microshoot originated plantlets.

Callus initiation and plant regeneration from inflorescence primordia of the intergeneric hybrid Agropyron repens (L.) Beauv.xBromus inermis Leyss. cv. nanus on a modified nutritive medium.

Plant regeneration from callus of intergeneric hybrid Agropyron repens (L.) Beauv. x Bromus inermis Leyss cv. nanus (AGROMUS) was carried out on a new culture medium designated medium-F. Within 21 days of the plating of inflorescence primordia the initiated callus showed globular structures. From the 21st day of culture, one step plant regeneration occurred on the callus without subculture. The new basal medium reported in this work was effective in callus initiation and plant regeneration of the hybrid AGROMUS by (i) the reduction of the total ion strength (2.6 g/l, 22.5 mM) of macroelements compared to MS (4.5 g/l,45.2 mM), (ii) the use of NH4NO3 as the sole N-source, and (iii) the application of KH2PO4 at an 8 times higher concentration (1160 mg/l,8.5 mM) when compared to the Murashige and Skoog medium composition. This medium provided a 2 to 10 fold reduction in the 2,4-dichlorophenoxyacetic acid supplement needed for the callus initiation and one step plant regeneration after a gibberellic acid (2 mg/l, for 5 days) pretreatment of tillers. The regenerated plantlets were subcultured in multi-shoot culture and potted in soil to grow for further analysis.

Cryopreservation of horse-chestnut (Aesculus hippocastanum L. ) somatic embryos using three different freezing methods

Cryopreservation of somatic embryos of Aesculus hippocastanum L. cultured on nutritive media containing abscisic acid (ABA) at concentrations of 0.75 μM, 7.5 μM and 75.0 μM was evaluated for three cooling methods: (i) slow freezing with cryoprotectants, (ii) fast freezing with cryoprotectants, and (iii) fast freezing with desiccation techniques. The ‘cryoprotectant’ freezing techniques included the embryo pretreatment on ABA containing medium for 4 days, followed by cryoprotective treatment in liquid medium containing 0.5 M dimethylsulfoxide, 0.5 M glycerol, 1.0 M sucrose, and cooled at slow, and rapid rates. Embryos pretreated on a medium containing 0.75 μM ABA, and cooled to −35 °C at 1°C /min, held for 30 min at this transfer temperature and then immersed in liquid nitrogen (LN) had the best embryo recovery (43%). The ‘desiccation’ method involved an air drying step of similar ABA-pretreated, non-cryoprotected embryos followed by rapid cooling. Embryos precultured on 0.75 μM ABA, then subjected to a 4 h period of air desiccation (water content reduction to 13%) showed about the same level of survival (46%) as found with the ‘cryoprotectant’ slow freezing technique. The air-dry ‘desiccation’ method is easier to apply than the more complicated ‘cryoprotectant’ method.

Increase of green plant regeneration efficiency by callus selection in Puccinellia limosa (Schur.) Holmbg.

Three main types of callus have been selected from seeds of salt marsh grass(Puccinellia limosa (Schur.) Holmbg.) subcultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and kinetin. Callus type I differentiated only occasionally. Callus type II produced roots but no shoots under all tested culture conditions. Both green (47 %) and albino plants have been obtained from the embryogenic callus type III. Callus type III was divided into two subtypes (greening and non-greening) according to the presence or absence of green spots. Separated greening embryogenic callus gave up to 87 % green plants, whereas non-greening callus produced only 4 %.

Minibeet initiation from derooted sugarbeet (Beta vulgaris L.) seedlings in vitro

Abstract An in vitro method for the initiation of minibeet development from derooted sugarbeet seedlings was developed. The surface sterilized seeds were germinated for 25 days on sugar-free Murashige and Skoog medium containing half-strength nutritive elements, 10 mg l−1 thiamine, 0.1 mg l−1 gibberellic acid and 7.0 g l−1 agar. After germination, seedlings of six different genotypes were derooted at the root neck and incubated for 90–120 days on minibeet initiation medium containing double-strength Murashige and Skoog elements, B5 vitamins, 100 mg l−1 activated charcoal, 30 g l−1 d -glucose, 6-benzylaminopurine (0.2 mg l−1) and abscisic acid (0.2 mg l−1). Without subculturing, minibeets developed on 20–40% of five different genotypes of seedlings. The developed minibeets were characterized structurally and functionally by means of a histological study and relative sucrose accumulation. Minibeets were compared to tuber-like formations which showed neither polycambial structure nor relative sucrose accumulation. Minibeets gave rise to plants after potting in the greenhouse.

Cryopreservation of Some Halophyte Grasses (Puccinellia Species)

The species of the genus Puccinellia Parl. (Atropis) are perennial halophyte grasses native to the Euro-Siberian region and adventive to North America. Besides the three widely popular species in Hungary, (1) P. peisonis (Beck.) Jav., (2) P. distans (L.) Parl., and (3) P. limosa (Schur.) Holmbg., there have also been species like P. pannonica (Hack) which have disappeared from the Hungarian flora by this century (Simon 1992).

Initiation of Monocot Plant Development In vitro For Studying Plant-Microbe Interaction

In vitro plant regeneration cycles from salt tolerant forage grasses of Puccinellia limosa, P.distand, the hybrid of A. repens x B. inermis and the Festuca arundinacea were initiated in vitro. Plant regeneration pathways were found to be somatic embryogenesis. All the crucial steps in plant development were characterized from the scutellar embryo stage to the coleoptilar stage. The somaclonal variation of the hybrid A. repens x B. inermis was characterized by cytological analysis. Although chromosomes paired essentially as 21 bivalents, the frequency of univalents was found to be higher in the hybrid than in the hybrid-somaclones. The polyamine content (spermine, spermidine, putrescine) was measured to compare embryogeneic and non embryogenic cell line capacities.