Investigation of the effect of explants physiology, as well as of medium and culture conditions on micropropagation of xMalosorbus Florentina

Aim of this study was to develop an effective in vitro protocol for the propagation of rare and endangered native plant of Greek flora xMalosorbus florentina in order to use it as an ornamental landscape plant as well as for its conservation. This species is, so its micropropagation will contribute to its conservation. Main problems that had to be addressed during in vitro culture were contamination and browning of explants during initial culture establishment and difficulty during in vitro rooting of microshoots. MS with 1.0 mg l-1 BA and 0.1 mg l-1 IBA was used as basal in vitro culture medium. Explants excised from adult plants, shoot tip explants and explants collected in March and April showed more browning and had higher content of total phenols than explants excised from juvenile tissue, nodal explants and those collected during any of the other months of the year. Explants content in lignin, which was estimated using FT-IR spectroscopy, was lower in apical than in lower explants, while differences as regards to origin or season of explants collection were not observed. Discriminant analysis of IR spectra in the region of 1450-1700 cm-1, where peaks of phenolic compounds and lignin are found, with TQ analyst grouped spectra of explants as regards their location, their origin and the season of their collection. Microscopic observation of apical explants excised from normal shoots or spurs of adult plants, monthly during one year, showed that there was a vascular connection of buds with shoot and that hairs covered scales and young leaves of buds. In parenchymal cells, starch accumulation from July through September and fill with fibrous structures from October to January, as well as crystals of calcium oxalate throughout the year were observed. Apical explants excised from adult plants growing wild on Mt. Parnitha were more difficult to establish in vitro (14%) compared to explants excised from micropropagated plantlets or sprouts of burned plants during fire of 2007 in Panitha (29-36%). Nodal explants excised from in vitro grown seedlings were established at the highest percentage (83%), giving the most shoots per explants (5.2). Nodal explants from the base of sprouts produced shoots at higher percentage (60%) than explants from upper locations (20-31%), but any differences in proliferation rates of established cultures ceased after the third subculture. Explants form micropropagated plantlets were established at higher percentage when they were cultured in medium with 1.0 mg l-1 TDZ (54%). TDZ and ΒΑ gave more shoots per explants (3.3) than zeatin, kinetin and 2iP or the control without plant growth regulators, but shoots in medium with TDZ were not elongated. After subculture in basal medium, explants from TDZ surpassed those from BA and other media in shoot number (4.0 shoots/ explant) without falling short in shoot length. Generally, in vitro cultures established from adult plants, with the exception of one culture, showed lower multiplication rates compared to cultures from sprouts or micropropagated plantlets, while explants from seedlings had to be treated 0.5 mg l-1 GA3 in order to increase their proliferation potential. Cover of culture vessels with sanitas membrane limited the problem of shoots hyperhydricity that was appearing with the use of plastic film or plastic cap, while it simultaneously increased proliferation rate of explants. ΒΑ was proved more effective for shoot proliferation compared to zeatin and 2iP, while kinetin was considered inappropriate since it led to the formation of a limited number of short shoots unsuitable for rooting. Presence of ΗΒΑ (0.1 mg l-1) on the medium of shoot proliferation increased multiplication rate. Cytokinin levels between 1.0 and 2.0 mg l-1 contributed to the production of high shoot number without significant restriction of their length. Replacement of BA of the basal medium from TDZ in concentrations 0.1-2.0 mg l-1 gave higher shoot proliferation rates, having though a negative effect in shoots elongation. Transfer of shoots and shoot clusters from medium with TDZ to basal medium with full or half concentration of BA and IBA brought their elongation. Use of low concentration of TDZ (0.1 mg l-1) in alternate cultures with BA is proposed, since it increases blastogenesis compared to continuous use of BA. Although WPM increased blastogenesis, MS was preferred to WPM because microshoots produced in WPM showed symptoms of stress and rooted at lower percentage than those produced in MS. Microshoots cultured in solid ½MS medium with auxin for root induction for only one week followed by culture in medium without plant growth regulators rooted at higher percentage and formed less callus than those cultured continuously in medium with auxin. Microshoots produced in culture established from adult plant rooted at higher percentage (32%) in medium with combination of 0.5 mg l-1 IBA and 8.0 mg l-1 IAA, while microshoots produced in culture established from sprouts rooted at higher percentage (65%) in medium containing only ΗΑΑ (4.0 or 8.0 mg l-1). Method of brief dipping of microshoots base in liquid solution 1000 mg l-1 ΗΒΑ was less effective in root induction, while it increased callus formation. Application of darkness during the first week of culture in root induction medium is not recommended because it led to the production of excessive callus at the base of microshoots without contributing significantly to the improvement of rooting percentage. Addition of AC (2 g l-1) on root induction medium completely inhibited microshoots rooting, while when it was added on the medium of root elongation, only during the first week, it increased rooting percentage and reduced callus formation. Microshoots produced in medium with low concentration of cytokinin generally rooted at higher percentages than those coming from higher cytokinin concentration. Microshoots excised from juvenile cultures were more capable to root (51-58%) forming more roots than those excised from cultures of adult plants (16-32%). Higher acclimatization percentages and faster growth rate of plantlets was succeeded by robust plantlets with rich root system, that were transferred ex vitro in mixture of peat-perlite 1:1 (v/v), end of winter-early spring. The 83% of plantlets was acclimatized ex vitro independently of their origin, and plantlets of juvenile origin, although developing the same height as those originating from adult plants, had shorter internodes and thus more compact shape. Addition of AC in the medium of root elongation in vitro favored following acclimatization, increasing percentage of plantlets survivor (up to 100%)