L. E. Heszky

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.

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.

A selective auxin and cytokinin bioassay based on root and shoot formation in vitro

Summary A new bioassay for auxins and cytokinins was developed based on de novo organogenesis. Auxin-dependent adventitious roots and cytokinin-dependent adventitious shoots were induced selectively in tobacco leaf disc culture incubated on nutritive-medium for 28 days. All auxins applied alone (indole, naphthalene, and phenoxyacetic acids) initiated roots, and all cytokinins used alone (purine and urea derivatives) initiated shoots at a concentration range of about 10 −9 M—10 −4 M. The bioassay separated the effects of auxins and cytokinins eliminating their synergistic effects. Abscisic acid and gibberellin (GA 3 ). had no morphogenic activity. **

Restoration of the regeneration potential of long-term cell culture in rice (Oryza sativa L.) by salt pretreatment.

Summary Cell suspension, initiated with seed-derived embryogenic callus and maintained for 2 years, produced morphogenically different cell types. No plant was regenerated from fine and friable cells. Low and sporadic regeneration frequency (0.0-0.3 %) was observed in compact cell clusters. Friable cells (20-21 months old) were exposed to salt stress; 0.75 % NaCI mainly inhibited cell growth and induced cells to become compact, whereas 1.5 % NaCI expressed a selective effect of killing all but embryogenic cells of friable cultures. A 3 month pretreatment with 1.5 % NaCl resulted in a reselection of embryogenic cells, restoring the high regeneration capacity of 2-year-old cultures of Oryzella by 60 % and Karolina by 37 % on salt-free medium. NaCl used at a suitable concentration appeared to be an efficient factor for re-establishment of the regeneration capacity of embryogenic calli derived from cell culture in rice.

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 %.

Effect of cooling rate, cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with −30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at − 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and − 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.

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).