Grazia Marino

Comparative effects of sorbitol and sucrose as main carbon energy sources in micropropagation of apricot

In vitro proliferation and rooting capacity of ‘San Castrese’ and ‘Portici’ apricots (Prunus armeniaca L.) were tested on modified MS medium enriched with varying growth regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon energy sources. The interaction of proliferation and rooting media was also studied.Proliferation of both cultivars was proportional to benzyladenine (BA) concentration and enhanced with sorbitol media. However, 8.8 μM BA was often associated with hyperhydricity, particularly when shoots were grown on sucrose media. Newly proliferated shoots elongated better on sorbitol media. The positive influence of sorbitol on proliferation and shoot growth was not due to osmotic effects. Moreover, sorbitol showed a positive carryover effect in hastening rooting of ‘Portici’. By contrast, when transferred to sorbitol rooting media, the shoots of both cultivars generally showed low rooting, with short, thick roots.Up to 70% of the plantlets that produced roots in sucrose media enriched with indolebutyric acid were successfully acclimatized when they were dipped in a benomyl (0.075% w/v) suspension before being transplanted with care being taken to prevent over-wetting of soil.

Storage of in vitro cultures of Prunus rootstocks

In vitro cultures of three Prunus clones (d. 1869, GF 677 and CAB 11E) were successfully stored at +8°, +4° and-3°C following the proliferation phase.Survival of cultures was dependent upon interactions of storage temperature, light, and age of subculture. Up to 100% of the cultures survived at the end of the trials after 170 (at +4°C) and 200 (at-3°C) days storage. Complete dardness appeared more suitable than 16-h (hour) photoperiod for successful storage at-3°C for up to 10 months. One or two weeks in normal growth room vefore storage at-3°C for up to 10 months. One or two weeks in normal growth room before storage enhanced the survival S-1. The proliferation of the cultures following storage at-3°C in the first subculture appeared similar to those under standard growth room conditions.Part of the results were presented as a poster at the 10th Congress of Eucapia in Wegeningen, The Netherlands, 19–24 June 1983.

Micropropagation of Actinidia deliciosa cvs. ‘Hayward’ and ‘Tomuri’

Abstract Benzyladenine (BA) induced the best proliferation responses, as shoot and node production, in Actinidia deliciosa cultivars ‘Hayward’ and ‘Tomuri’, while callus growth was enhanced on zeatin (Z)-enriched media. ‘Tomuri’ showed a higher proliferation and shoot weight increase than ‘Hayward’ on most of the culture media tested, but shoots of ‘Hayward’ produced bigger calli. Numerous roots were observed after 25 days in all shoots of both cultivars transferred from proliferation to indole-3-butyric acid (IBA)-enriched media, especially in ‘Tomuri’. Rooting percentages on media without auxin were just slightly lower than on IBA-enriched media. Acclimatization was strongly affected by temperature; in summer, plants needed 56 days in the hardening chamber if survival was to remain high (75%) on transfer to the greenhouse. By contrast, ∼90% survival was easily achieved in spring. Leaf chlorophyll a and b content was influenced by the culture media; however, it was higher in ‘Tomuri’, particularly in the rooting and hardening phases. Different proliferation, growth, rooting responses and chlorophyll content of in vitro cultures of ‘Hayward’ and ‘Tomuri’ suggest that an interaction between culture media and different endogenous hormone concentrations can be expected, i.e. a cytokinin/auxin balance which is higher for ‘Tomuri’ than for ‘Hayward’.

The influence of ethylene on in vitro rooting of GF 677 (Prunus persica × Prunus amygdalus) hybrid peach rootstock

SummaryTreatments differing from each other for the type of tube closure (i.e., cotton plug for free gas exchange, airtight rubber cap, and rubber cap with ethysorb) and/or rooting culture medium (i.e., enriched or not by 25 to 100 µM acetylsalicylic acid) were compared for their effects on gaseous composition of the culture atmosphere and microcutting rooting of the GF 677 (Prunus persica × Prunus amygdalus) hybrid. Rubber capping, which leads to rapid ethylene accumulation inside tubes, strongly reduced rooting time and in some cases enhanced final rooting percentage over that of cotton plugs. Ethysorb almost completely absorbed ethylene produced by shoots, which showed lower rooting percentages within 9 d than microcuttings cultured in the absence of ethysorb. In contrast, no significant difference in rooting was found between the two treatments after 14 d. Carbon dioxide concentration was similar in all treatments within 5 to 9 d and seemed to be ineffective for rooting. The influence of acetylsalicylic acid on rooting was unclear. Root number and length were not significantly influenced by the treatments. These results demonstrate that the use of airtight closures, leading to rapid ethylene accumulation, can reduce time of rooting expression for GF 677 microcuttings. However, free gas exchange towards the end of the rooting period (from Day 9 to Day 14) is advisable to prevent leaf yellowing. No significant difference in plantlet survival and growth after transfer ex vitro was found among treatments.

Different sealing materials for petri dishes strongly affect shoot regeneration and development from leaf explants of quince ‘BA 29’

SummaryThe effect of different sealing materials [i.e., polyvinyl chloride (PVC) transparent film, and Parafilm (PARA) for Petri dishes was investigated on shoot regeneration from quince (Cydonia oblonga L.) ‘BA 29’ leaf explants. Leaves were excised from proliferating shoot cultures, transversally scored, and placed with the abaxial side down in 60-mm Petri dishes containing 10 ml of Murashige and Skoog modified medium, with 5.4 μM α-naphthaleneacetic acid, 4.5 μM thidiazuron, 200 mg l−1 cefotaxime, and 0.25% (w/v) Phytagel (IM medium) for shoot bud induction, and cultured in darkness at 22±2°C for 28 d. Then the explants were transferred to standard conditions (16-h photoperiod at 30 μmol m−2 s−1 photosynthetically active radiation) on a medium similar to IM, except for lack of NAA, and with 0.65% (w/v) agar instead of Phytagel, for an additional 15–28 d. The sealing combinations PARA-PARA, PARA-PVC, PVC-PARA, and PVC-PVC (in the induction-expression phases) were compared during regeneration and for their carry-over effect on shoot development after transfer of explants to an elongation medium (0.9 μM 6-benzyladenine). Carbon dioxide accumulated at 27.2 mmol mol−1 at the end of induction, and gradually decreased from 35.4 mmol mol−1 on day 9 to 22.5 mmol mol−1 on day 28 of the expression phase in PARA-sealed Petri dishes, being always much higher than after sealing with PVC (1–2 mmol mol−1). Ethylene concentration was 0.1 and 0.04 μmol mol−1 in the first part of the induction and expression phase, respectively, in PARA-sealed Petri dishes, and slightly decreased with duration of exposure to light during expression; while it was absent in most PVC-sealed dishes. The PARA-PARA and PVC-PVC (induction-expression) combinations gave, respectively, the worst and best results of regeneration and successive shoot development.

In vitro performance at high culture pH and in vivo responses to Fe-deficiency of leaf-derived quince BA 29 (Cydonia oblonga) somaclones regenerated at variable medium pH.

Summary The aims of the work were to obtain quince (Cydonia oblonga) BA 29 somaclones tolerant to high culture pH and lime soils, to test the effectiveness of in vitro selection methods based on the selection pressure exerted by pH during regeneration, and to understand the mechanisms for the improved performance under Fe-deficient conditions of eventually tolerant clones. Leaf-derived somaclones obtained at variable medium pH (5.7, 6.0, 6.5, 7.0 and 8.0) were compared with each other and with the control (i.e. shoots derived from BA 29 mother plants by standard micropropagation) for their in vitro growth, proliferation, rooting performance and pH-reducing ability at pH 7.0, 7.5 and 8.0, and for their Fe-chelate reductase (FCR) activity and soluble sugar and organic acid accumulation in the roots and leaf chlorophyll content when grown in soiless culture under Fe-efficient and -deficient conditions. Preliminary results on SPAD analysis on leaves of the same plants transferred to lime soil are also reported. The effect of pH selection pressure was not very clear; however, while all the clones hardly grew and proliferated in vitro at pH 8, the somaclones regenerated at pH 7 generally tended to have better growth and proliferation than control and other somaclones at pH 7.5, especially 7-A. In contrast, most of those regenerated at low pH, but 5.7-A, tended to have lower proliferation rates and shorter axillary shoots. Little differences were found in the rooting performances of the clones, although most somaclones seemed more able to reduce the pH than the control. After 10 d in hydroponic culture under Fe-deficient conditions, almost all the somaclones tended to have some FCR induction in the roots; however, it was detected only in 5.7-A and 7-A at day 30. Leaf chlorophyll determinations in soiless culture and lime soil indicated quite good tolerance to Fe-chlorosis for 6-A, 7-A and especially 5.7-A. When the in vitro and in vivo results are considered together, the somaclones 5.7-A, 7-A and 6-A seem the most interesting, their success in growing in Fe-deficient conditions being probably due partially to sorbitol accumulation in the roots, in particular for 6-A and 5.7-A, and also to inositol and citric acid, respectively for 5.7-A and 7-A. However, further plant evaluation on lime soil is needed to verify these preliminary results and to understand whether some of these somaclones can replace the widespread pear rootstock quince BA 29, as more tolerant to lime-induced Fe-chlorosis.

The effect of the type of closure on the gas composition of the headspace and the growth of GF 677 peach × almond rootstock cell suspension cultures

SummaryThe influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO2 and C2H4 levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO2 increased up to 18%, with a net production of about 0.8 mmol CO2, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 µl liter−1 after 96 h. When aluminum foil closures were used, C2H4 and CO2 increased over the first 72 h, reaching concentrations of about 6 µl liter−1 and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 µl−1 and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs, slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most cells occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

SummaryShoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m−2·s−1 photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.

Adventitious shoot formation in cultured leaf explants of quince and pear is accompanied by different patterns of ethylene and polyamine production, and responses to aminoethoxyvinylglycine

Summary The gaseous environment inside culture vessels is an important factor affecting the growth and differentiation of plant cell and tissue cultures. Carbon dioxide (CO2) and ethylene (C2H4), in particular, play important roles. The effect of reduced gas exchange in sealed Petri dishes has previously been shown to strongly inhibit adventitious shoot formation in cultured leaf explants of quince ‘BA29’, but has not been investigated in pear. In the present work, the accumulation of CO2 and C2H4 in Petri dishes was quantified during the shoot induction and development phases in quince (Cydonia oblonga Mill.) ‘BA29’ and in ‘Conference’ pear (Pyrus communis L.) that have higher and lower regeneration capacities, respectively, on medium supplemented with 4.5 µM thidiazuron (TDZ) and 5.4 µM naphthaleneacetic acid (NAA). In order to evaluate the specific role of C2H4 on the shoot-forming process, the effect of treatment with an inhibitor of C2H4 biosynthesis, aminoethoxyvinylglycine (AVG), was also evaluated during the shoot induction phase. The results showed that AVG strongly reduced both C2H4 accumulation and the rate of production of C2H4, without affecting CO2 accumulation, in both species; whereas it differentially affected the regeneration capacity, enhancing it in quince and reducing it in pear. A time-course analysis of free and soluble conjugated polyamine (PA) titres was also performed in control vs. AVG-treated quince and pear explants during the induction phase. Quince explants exhibited a marked peak in free and conjugated spermidine on day-7 in culture, while pear explants did not. AVG did not alter PA levels, or the PA profile in either species. The addition of AVG to the induction medium is recommended to improve shoot regeneration in quince ‘BA 29’ leaf explants in culture systems that prevent optimal gas exchange. In contrast, the low genotype-specific regeneration capacity of pear prevailed over the effect of AVG treatment in our experimental conditions.

Use of lysozyme for treatment of bacterial contamination in in vitro shoot cultures of fruit plants

SummaryUse of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml−1 egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml−1; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml−1 had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml−1 in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.