Stefano Morini

Enhanced micropropagation response and biocontrol effect of Azospirillum brasilense Sp245 on Prunus cerasifera L. clone Mr.S 2/5 plants

Inoculation with Azospirillum brasilense Sp245 exerts beneficial effects on micropropagated plants of Prunus cerasifera L. clone Mr.S 2/5, as seen in the results of a comparative analysis of inoculated and non-inoculated explants, during both the rooting and acclimatation phases. The presence of Azospirillum brasilense Sp245 increased root system, root hair biomass production and apical activity. Although the presence of the bacteria had a positive effect on rooting, the addition of indolebutyric acid (IBA) to Murashige and Skoog (MS) medium was seen as indispensable in order to promote the rooting of explants. Aside from the promotion of plant growth, A. brasilense Sp245 protects plants against pathogen attacks, such as Rhizoctonia spp., with a plant survival rate of nearly 100% vs. 0% as seen in the negative control. The biocontrol effect of A. brasilense Sp245 on the fungal rhizospheric community has been confirmed by denaturing gradient gel electrophoresis (DGGE) profiles of the rhizospheric microbial community. This study indicates that A. brasilense Sp245 could be employed as a tool in plant biotechnology.

Effect of 2,4-D and light quality on callus production and differentiation from in vitro cultured quince leaves

The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration, length of induction period and light quality on leaf regeneration of quince clone BA 29 were investigated. After 2, 4 or 6 days of induction with 2.5 mg l−1 or 5.0 mg l−1 2,4-D, leaves were cultured under red, blue, red+blue, far-red+blue, white, far-red light or darkness conditions. Leaves thereby treated showed different responses, with respect to somatic embryogenesis, callus, red-nodular structures or roots. Callus production increased with increasing 2,4-D concentration and induction period, although it was not influenced by light quality; the only exception was far-red+blue light, which reduced callusing response. This result suggested involvement of the blue-absorbing photoreceptor system in the callus formation processes. A high regeneration of red-nodular structures with a meristematic appearance was also observed; from some histological characterizations, we presumed they were adventitious buds that were arrested at an early developmental stage. Red-nodular structures increased with decreasing 2,4-D concentration and induction period. In the regeneration of such structures, the blue-absorbing photoreceptor system appeared to have a negative effect but only at a low photoequilibrium value. In contrast, light quality which activated phytochrome induced an increment in regeneration, but the response did not vary for photoequilibrium values ranging from 0.43 to 0.86. For root regeneration, phytochrome seemed to be the only photoreceptor involved.

Photoregulation of growth and branching of plum shoots: Physiological action of two photosystems

SummaryPlum shoot proliferation was investigated in terms of two distinct processes: axillary bud differentiation and axillary shoot development. Results showed that light quality influenced bud differentiation and interacted with apical dominance in determining shoot outgrowth, resulting in a differentiated structure of shoot clusters and type of branching. Results suggested that blue light, acting through its photoreceptor, increased the number of axillary buds differentiated from apical meristem, but did not remove the apical dominance. Red light removed apical dominance, while reducing the formation of axillary buds; both events appeared to be dependent on the putative amount of phytochrome active form, and independent of light photon fluence rate. On the contrary, blue light action appeared to be dependent on photon fluence rate. In addition, apparent blue-red interactions related to photomorphogenic events fit an antagonistic model for branching regulated by light via cryptochrome and phytochrome photoreceptors. Our results show that the dynamics of shoot cluster development is the product of two events: the formation of new axillary buds and their release from apical dominance.

Effect of photoperiod on some stomatal characteristics of in vitro cultured fruit tree shoots

The effect of the photoperiod on some stomatal characteristics in various leaf lamina zones and in leaves of different age was studied on in vitro grown shoots of Prunus cerasifera clone Mr.S. 2/5, t Malus pumila Mill clone M9, and peach x almond hybrid clone GF 677. Stomatal density was highest in leaves exposed to continuous light and lowest with continuous dark. Photoperiod treatments supplying the same quantity of daily radiation but distributed over different cycles (4 h light and 2 h dark, 16 h light and 8 h dark) led to differing stomatal densities intermediate between those of the above light treatments. The light regime with the shortest light and dark periods was found to be most favorable to stomatal differentiation. Regardless of light treatment, stomatal density was found to be lower in the leaf lamina basal zone as compared to the median and apical zones, and decreasing from the 1st to 3rd to 5th leaf counting down from the apex. The photoperiod effect was also confirmed by a stomatal index. The stomatal axis ratio showed no interaction with the photoperiod but did highlight a tendency to rounder stomatal shape with increasing stomatal age.

Regeneration of somatic embryos and roots from quince leaves cultured on media with different macroelement composition

The effects of different macroelement combinations on somatic embryogenesis of quince (Cydonia oblonga Mill.) were tested. Leaves were excised from shoot cultures of quince clones and cultured on macroelement combinations of 8 different growth media. Callus production varied depending on the medium and the clone combinations. The influence of genotype and macronutrient combination on somatic embryo and root regeneration was also observed. Clone BA 29 showed the highest embryogenic properties and Murashige and Skoog-based medium appeared to be the most favourable for somatic embryo formation. Root regeneration was higher on Woody Plant Medium and Schenck and Hildebrandt-based media. Interactive effects between genotypes and macroelement combinations were also detected both for embryo and root formation. In all treatments, somatic embryos underwent early developmental arrest and failed to convert into plants. Differences in embryo and root regeneration observed among macroelement combinations may be ascribable to different levels of medium nitrogen and probably to the ratio between nitrate and ammonium.

In vitro growth response of Prunus cerasifera shoots as influenced by different light-dark cycles and sucrose concentrations

Trials were carried out to test if the higher growth response shown by shoot clusters of Mr. S. 2/5, a clonal selection of Prunus cerasifera, submitted to short and frequent light-dark regimes could be related to the amount of sucrose added to growth medium.The reduction of sucrose from 30 gl-1 (control) to 22.5 gl-1, 15 gl-1 and 7.5 gl-1 caused a progressive and remarkable inhibition of shoot tip growth. With 15 gl-1 the value of some growth parameters was reduced by more than half. Under 16-h daylength, the best sucrose concentration was 30 gl-1, while with 4-h light-2-h dark no statistical differences appeared between 30 gl-1 and 22.5 gl-1 sucrose. Compared to 16-h light-8-h dark, the 4-h light-2-h dark cycle at the three highest sucrose concentrations gave rise to higher values of fresh and dry weight as well as increasing the number of axillary shoots produced.The increment in growth response induced by the shorter light-dark regime decreased with diminishing growth capacity in the cultures when sucrose concentration was lowered, but it was still appreciable even with 7.5 gl-1. Since the 4-h light-2-h dark cycle induced a favourable effect in culture growth with all sucrose concentrations, we conclude that the greater growth response observed with this light regime was not triggered by carbohydrate availability but by some other unknown factors.

Improving micropropagation: effect of Azospirillum brasilense Sp245 on acclimatization of rootstocks of fruit tree

Abstract The effect of Azospirillum brasilense Sp245 on the micropropagation of three fruit rootstocks: Mr.S 2/5 plum (Prunus cerasifera×P. spinosa), GF 677 hybrid (Prunus persica×P. amigdalus), and MM 106 apple (Northen Spy×M1) was assessed. Rooted shoots were treated with 3×107 of Sp245 cells during transplantation from in vitro cultures to the acclimatization phase. After 60 days, growth parameters were positively affected by Sp245 inoculum. In the case of Mr.S 2/5, an increase in rootstock stem length and node number by 37% and 42%, respectively, compared to the control was noted. In the case of GF 677, the bacterial inoculum increased stem length and node number by up to the 75% and 65%, respectively, compared to the control. The inoculum did not exert on MM 106 for both parameters suggesting that the effects of Sp245 could depend on a specific clone-microbe association. In all cases, however, a higher vigor, consistent with a wider leaf area, was present in the inoculated plantlets demonstrating that the use of Azospirillum can significantly contribute to optimize plant performance during the phase of adaptation of plants to post-vitrum conditions.

Simultaneous Regeneration of Different Morphogenic Structures from Quince Leaves as Affected by Growth Regulator Combination and Treatment Length

Experiments were performed to evaluate the capacity of quince (Cydonia oblonga Mill.) leaves to regenerate somatic embryos and shoots and/or roots simultaneously. Leaves, treated for 2 d in liquid medium containing 2.5 mg dm−3 2,4-dichlorophenoxyacetic acid were cultured for 0, 3, 6, 9, 12, 15, 18, 21 d on a gelled medium supplemented with 1 mg dm−3 kinetin (Kin) and 0.1 mg dm−3 naphthalenacetic acid (NAA) and were transferred to a medium either without growth regulator (GR-) or containing 0.6 mg dm−3 6-benzylaminopurine (BA) + 0.2 mg dm−3 gibberellic acid (GA3) + 0.06 mg dm−3 indole-3-butyric acid (IBA) (GR+). Leaves producing somatic embryos (SEs) only, or adventitious roots (Rs) only, or SEs+Rs simultaneously, were detected on GR- culture medium; on GR+ medium, leaves producing adventitious shoots (Ss) only, SEs+Ss or SEs+Rs+Ss simultaneously, also appeared. Leaves producing both Ss+Rs were never detected. Proportions among the various types of regenerating leaves varied according to both the length of Kin+NAA treatment and the presence or absence of GR in the transfer medium. The greatest variations, both on GR− and on GR+, took place within the first 9 d of culturing on Kin+NAA. After this period, no further substantial differences in the trend of each type of regenerating leaf were observed. The length of the treatment with Kin+NAA also modified the proportions between the different types of morphogenic structures.

Effects of arbuscular mycorrhizal fungi on in vivo root initiation and development of micropropagated plum shoots

SummaryThe influence of arbuscular mycorrhizal fungi on in vivo root initiation and development of Mr.S. 2/5 microcuttings was investigated. Micropropagated shoots of the Prunus cerasifera L. rootstock, clone Mr.S. 2/5, were placed in a sterile rooting mixture or inoculated with arbuscular mycorrhizal (AM) fungi. A part of the uninoculated microcuttings received auxin treatment (IBA 200 mg F1) or were supplied with a non-sterile sievate of mycorrhizal inoculum. Rooting percentage, root system morphology and growth increments of in vivo rooted microcuttings were evaluated. In vivo rooting of Mr.S. 2/5 shoots was obtained successfully but mycorrhizal inoculation did not show a positive influence on rooting ability of microcuttings. First harvest of plantlets was carried out very early, when mycorrhizal and non-mycorrhizal plants showed no significant growth differences. By this time, AM fungi had greatly increased the proportion of the root system present as higher-order laterals and branching intensity of ...

Effect of different photoperiods on in vitro growth of Mr.S.2/5 plum rootstock

Trials were conducted to study the effect of different photoperiods on in vitro growth of Mr.S.2/5 plum rootstock. Three photoperiods-16 h (control), 12 h and 8 h-were applied, with a PAR of 39 μmol m-2 sec-1. Tips collected from in vitro established shoots were used;ggrowth medium was MS with BA at 2.7 μM, GA3 at 0.7 μM and IBA at 0.3 μM.Shoot proliferation after 45 days of growth was not statistically different between 12 h and 16 h of light, while the 8-h photoperiod gave a much lower rate of shoot formation. Shoot quality did not vary noticeably among the three treatments. Chlorophyll content and leaf thickness were not significantly modified even by a 50% reduction in light application. Stomata number and opening increased under 12 h of light.Rooting percentage was reduced by shortening the photoperiod to 8 h while root length was positively influenced with a 12-h photoperiod.