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The feasibility of in vitro propagation of intergeneric hybrid Fragaria x Potentilla, named “Serenata" was tested by using six MS and, respectively, six LF-based proliferation media supplemented with different combinations and concentrations of benzylaminopurine (BAP), kinetin (Kin), indolylacetic acid (IAA), 3-indolylbutiric acid (IBA), and giberellic acid (GA3). A high multiplication rate combined with good quality of proliferated shoots and in vitro rooting potential was induced by media containing 1.0 mg/l BAP, 1.0 mg/l IAA and 0.1 mg/l GA3. Ten primers (from 48 previously tested) were selected and used in RAPD analysis to assess the genetic stability of these shoots. The lack of polymorphisms in micropropagated plants screened through molecular markers was proved by identity of the banding patterns.
In order to establish the major factors affecting in vitro micropropagation of intergeneric hybrids Fragaria × Potentilla, respectively ‘Pink Panda’ and ‘Serenata’, basic culture media Murashige-Skoog (MS), Lee-Fossard (LF) and Knop, were supplemented with 6-benzylaminopurine (BAP), kinetine (Kin), indole-3-acetic acid (AIA), indole-3-butyric acid (AIB) and gibberellic acid (GA3), in different combination and concentration. In ornamental strawberry ‘Serenata’, which showed a genetic potential of shoot regeneration significantly higher compared with ‘Pink Panda’, a high multiplication rate associated with a high vigor of shoots was obtained on MS medium supplemented with 0.5 mg/l BAP + 0.1 mg/l AIB + 0.1 mg/l GA3. The same combination of growth regulators, added in MS medium in higher concentrations, namely 1.0 mg/l BAP + 0.2 mg/l AIB + 0.1 mg/l GA3 led to the highest rate of multiplication in ‘Pink Panda’ intergeneric hybrid of Fragaria × Potentilla.
2012 •
In order to establish the major factors affecting in vitro micropropagation of intergeneric hybrids Fragaria × Potentilla, respectively ‘Pink Panda’ and ‘Serenata’, basic culture media Murashige-Skoog (MS), Lee-Fossard (LF) and Knop, were supplemented with 6-benzylaminopurine (BAP), kinetine (Kin), indole-3-acetic acid (AIA), indole-3butyric acid (AIB) and gibberellic acid (GA3), in different combination and concentration. In ornamental strawberry ‘Serenata’, which showed a genetic potential of shoot regeneration significantly higher compared with ‘Pink Panda’, a high multiplication rate associated with a high vigor of shoots was obtained on MS medium supplemented with 0.5 mg/l BAP + 0.1 mg/l AIB + 0.1 mg/l GA3. The same combination of growth regulators, added in MS medium in higher concentrations, namely 1.0 mg/l BAP + 0.2 mg/l AIB + 0.1 mg/l GA3 led to the highest rate of multiplication in ‘Pink Panda’ intergeneric hybrid of Fragaria × Potentilla.
In order to evaluate the genetic stability and uniformity of ornamental strawberry plants micropropagated by using a new and highly efficient protocol we have developed recently, RAPD markers were used with intergeneric hybrids ‘Pink Panda’ and ‘Serenata’. Micropropagated shoots selected at random from four subcultures onto either Murashige & Skoog or Lee & Fossard media, each of them supplemented with 6-benzylaminopurine (BAP) at 1.0 mg l-1, indolylacetic acid (IAA) at 1.0 mg l-1 and gibberellic acid (GA3) at 0.1mg l-1, were subjected to molecular analysis. Ten deca-nucleotide primers (among 48 tested) were chosen for RAPD analysis, all of them indicating genetic stability for micropropagated plants of the investigated varieties of ornamental strawberry.
The effect of growth regulators, explant source and culture age on genetic stability of plants obtained from tissue culture propagation of ornamental strawberry “Serenata” were examined. Genomic DNAs of in vitro-derived shoots and control plant were extracted and compared by RAPD-PCR analyses. Ten primers (from 48 previously tested) were selected and used in RAPD analysis to prove the clonal fidelity (i.e. genetic stability) of the tissue culture-derived ornamental strawberry plants. The lack of polymorphisms in micropropagated plants screened through molecular markers was used to suggest genetic fidelity. Identicaly banding patterns of the RAPD profiles obtaining from vitroplants, regenerated via organogenesis or meristems culture, suggested that in the ornamental strawberry, variety “Serenata”, neither explant source, nor callus age or limited number of subcultures, in basal media supplemented with low concentration of growth regulators, were associated with occurence of somaclonal variation.
Ex vitro experiment was conducted at the greenhouse and nursery facilities of the Plant Tissue Culture Laboratory of Department of Horticulture, Faculty of Agriculture, Suez Canal University during the period 2009-2012. Strawberry ex vitro growth performance of tissue culture-derived plants from different propagation methods (meristem, direct regeneration and callus-derived plants) were examined with standard runner-propagated mother plants based on morphological traits and genetic analysis using RAPD. Ex vitro growth performance examination showed that leaf morphology and serration patterns were almost similar among plants obtained from meristem, direct or callus regeneration. Tissue culture plants have higher number of runners while standard runner plants were higher in flower production compared with in vitro propagated plants. RAPD analysis indicated that plants of cv. 'Tudla' from the different in vitro propagation methods were genetically stable, while in cv. 'Festival', callus-derived plants deviated from normal genotype. The obtained results confirmed the usefulness of RAPD in determining genetic stability of plants clonally propagated in vitro as well as detecting the somaclonal variants, which might be used further in breeding programs.
Experimental Biology
In vitro сlonal propagation of repairing hybrids of wild strawberry Fragaria ananassa Duch2017 •
An efficient protocol was developed for sustainable mass multiplication of strawberry (Fragaria × ananassa Duch cv. ‘Chandler’) through multiple shoot induction. Shoot buds were most successfully induced from runner tips, when these were cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg l-1 α-naphthalene acetic acid and 1 mg l-1 6-benzylaminopurine. Maximum shoot multiplication and proliferation occurred on MS medium with 2 mg l-1 kinetin. MS basal medium with 0.5 mg l-1 indole-3-acetic acid and 2 g l-1 activated charcoal proved to be the best for root induction and elongation from separated shoots. Autoclaved sand and soil with intermittent water spraying could optimize the primary acclimatization of in vitro generated plantlets. A pot mixture combined with sand, soil and farm yard manure (1: 1: 1 v/v) resulted in the acclimatization of 92% of plantlets. To prove the genetic uniformity of propagules, in vitro generated clones were DNA fingerprinted using selected ISSR primers.
As an important stage in micropropagating ornamental strawberry, in vitro rooting of microshoots on media containing different concentrations of auxins was investigated in two intergeneric hybrids Fragaria x Potentilla, respectively “Pink Panda” and “Serenata”. IBA at either 0.25 or 0.5 mg/l, and IAA at 0.5 mg/l concentration, were added to solidified Murashige and Skoog (1962) basal medium containing half strenght macroelements and half Lee-Fossard microelements. In all treatments, 0.1 mg/l of GA3 was also added to the basal medium. IBA was found to be the most effective auxin in promoting rhizogenesis, with the concentration 0.25 mg/l giving the highest rooting rates for both varieties, respectively 100% for “Pink Panda”, and 80% for “Serenata”.
As some of the ornamental varieties of strawberry obtained from Fragaria x Potentilla crosses are lacking the ability to form runners, their in vitro propagation is dependent on either direct or indirect organogenesis. The influence of culture medium composition and explant type were investigated in two genotypes of ornamental strawberry, “Pink Panda” and “Serenata”, respectively, in order to establish an efficient protocol for regeneration by indirect organogenesis. Aiming to a good rate of callogenesis and shoot regeneration, the effect of different combinations and concentration of growth regulators (2,4-D, IBA, and BAP) added in culture media (either MS or LF) were evaluated with leaf and petiole explants. It was found that the highest frequency of explants forming callus have been induced in both varieties investigated on the LF basal medium containing 0.5 mg/l or 1.0 mg/l 2.4-D and, respectively, 3.0 mg/l BAP. A maximum of 100% leaf explants, and 92% petiole explants formed calli having characteristics of those regenerating shoots in “Serenata” variety. Similarly, a maximum of 92% petiole explants formed callus in “Pink Panda” intergeneric variety.
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