Micropropagated Strawberry Plants Research Articles

Possibilities of using RGB-based image analysis to estimate the chlorophyll content of micropropagated strawberry plants

An image analysis method based on RGB features for rapid estimation of the chlorophyll content of leaves of micropropagated strawberry plants (2 cultivars) was presented in the study. An algorithm describing the relationship between the absolute values of chlorophyll content and the colour components of a leaf image captured with a conventional scanner was developed and tested. The accuracy of the proposed method was compared with that of an optical greenness meters designed for assessing leaf chlorophyll content. The chlorophyll content in the strawberry leaves was correlated with the results of measurements recorded by the two optical meters (SPAD-502, CCM-200) and with RGB values of scans of these leaves. The highest values of correlation coefficients were obtained between the chemical analysis results and mean values of the red colour (R) of the scans. However, varietal differences were evident here, which indicates the need for individual calibrations. In the case of the green colour (G), the accuracy was slightly lower; however, no varietal differences were found, thus one calibration can be used for both cultivars. Three formulas: (R-G)/(R+G), (R-G)/(R+G+B), and R/(R+G+B) were selected and their relationship with the changes in chlorophyll content was tested. These variables did not explain the changes in chlorophyll content better than the variable R. The study confirmed the possibility of using the image capture method for the detection of chlorophyll status in strawberry plantlets cultured in vitro.

Endophytic bacteria Arthrobacter agilis UMCV2 and Bacillus methylotrophicus M4-96 stimulate achene germination, in vitro growth, and greenhouse yield of strawberry (Fragaria × ananassa)

Strawberries are one of the most popular fruits in the world. The strawberry production process includes generation of plants through in vitro germination of achenes, micropropagation, and production under greenhouse conditions. In this study, we tested the effect of the plant biostimulant bacteria, Arthrobacter agilis UMCV2 and Bacillus methylotrophicus M4-96, on those processes. Volatile compounds produced by A. agilis UMCV2 stimulated achene germination while B. methylotrophicus M4-96 induced germination through production of diffusible compounds. Growth of in vitro strawberry plants inoculated with A. agilis UMCV2 or exposed to their volatile compounds was also stimulated. Plants inoculated in vitro with A. agilis UMCV2 and B. methylotrophicus M4-96, and then cultured in a greenhouse for 10 months had a 42 % higher fruit yield than un-inoculated controls. Confocal microscopy, used to assess fluorescent in situ hybridization, showed that both bacteria were present in the leaf mesophyll of inoculated plants, but not in control plants. Our results showed that endophytic bacteria, such as A. agilis UMCV2 and B. methylotrophicus M4-96, can be inoculated into micropropagated strawberry plants and colonize the plant tissues, thereby increasing the yield and fruit quality under greenhouse conditions through different mechanisms of plant biostimulation.

MICRORNA EXPRESSION PROFILES IN STRAWBERRY PLANTS

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs in the eukaryotic genome and play important regulatory roles in plant growth and development. To detect strawberry miRNAs and discover the expression difference in different organs and plants, we carried out the detection and quantification of strawberry miRNAs by microarray and real time RT-PCR. 74 miRNAs were checked in strawberry plants by gene chip. Among them, 4 miRNA families (miR159, miR166, miR167 and miR169) are deeply conserved in many plants, including eudicots, monocots and gymnosperms. At the same time, 4 miRNA genes displayed clear expression differences between conventional and micropropagated strawberry plants by microarray, including two up-regulated genes (miR535 and miR390) and two down-regulated genes (miR169a and miR169d). The ratios of conventionally propagated strawberry plants/micropropagated strawberry plants for miR535, miR390, miR169a and miR169d were 2.6884, 2.2673, 0.2496 and 0.3814, respectively. RT-PCR with TaqMan probe was used to validate the microarray result and analyze the expression patterns in organs. The expression of miR164 in the receptacles was higher than that in roots, shoot tips and leaves, while the expression of miR172 was very low in roots.

Tissue Culture Responsive MicroRNAs in Strawberry

MicroRNAs (miRNAs) are 20–24 nucleotide (nt) non-coding regulatory RNAs which play critical roles in plant growth and development. miRNA-encoding genes, which are transcribed by RNA polymerase II, are involved in a variety of processes, including developmental and morphogenesis systems, and hormone and stress responses. To investigate miRNA responses to tissue culture conditions, quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was used to detect differences in miRNA expression between in vitro micropropagated strawberry plants and transplanted micropropagated strawberry plants. Four miRNAs were differentially expressed between them, including one up-regulated gene (miR156) and three down-regulated genes (miR164, miR172 and miR390). The ratios of miRNA expression levels in in vitro micropropagated strawberry plants to micropropagated plants transplanted into soil in greenhouse for 4 months for miR156, miR164, miR172 and miR390 were 6.757, 0.046, 0.035 and 0.050, respectively. The ratio of miR156 expression levels in micropropagated plants transplanted into soil for 5 months to levels in the conventionally propagating runner plants was 3.785. miR156 was expressed highly and was strikingly inversely proportional to the expressions of its target gene SPL9 and miR172 in in vitro micropropagated strawberry plants. We speculate that high expression of miR156 is the main reason for rejuvenation in micropropagated plants.

MicroRNA expression profiles in conventional and micropropagated strawberry (Fragaria × ananassa Duch.) plants

MicroRNAs (miRNAs) are a class of small non-coding RNAs which play a critical role in plant growth and development. To detect strawberry miRNAs and discover the expression difference between conventional and micropropagated strawberry plants, we carried out the detection and quantification of strawberry miRNAs by microarray. The main findings were that 74 miRNAs were checked in strawberry plants and four miRNA genes displayed clear expression difference between conventional and micropropagated strawberry plants, including two up-regulated genes (miR535 and miR390) and two down-regulated genes (miR169a and miR169d). The ratios of conventionally propagated strawberry plant/micropropagated strawberry plant for miR535, miR390, miR169a and miR169d were 2.6884, 2.2673, 0.2496 and 0.3814, respectively. Quantitative reverse transcription polymerase chain reaction applied to the two up-regulated genes (miR535 and miR390) validated the microarray result. This is the first report on differential expression of miRNAs in conventional and micropropagated plants.

Isolation and characterization of genomic retrotransposon sequences from octoploid strawberry (Fragaria × ananassa Duch.)

Strawberry (Fragaria spp.) is a kind of herbaceous perennial plant that propagates vegetatively. The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of LTR retrotransposons were amplified from the cultivated strawberry (Fragaria x ananassa Duch.). Sequence analysis of clones demonstrated that 5 of 19 Ty1-copia group unique sequences and 2 of 10 Ty3-gypsy unique sequences in F. x ananassa genome possessed either stop codon or frameshift. Ty1-copia group sequences are highly heterogeneous (divergence ranged from 1 to 69.8%), but the Ty3-gypsy group sequences are less (divergence ranged from 1 to 10%). Southern dot blot hybridization result suggested that both of the LTR retrotransposons are present in the genome of cultivated strawberry with high copy number (Ty1-copia group 2,875 Ty3-gypsy group 348). RT-PCR amplification from total RNA, which was extracted from leaves of micropropagated strawberry plants, did not yield either of the RT fragments. This is the first report on the presence of RT sequences of Ty1-copia and Ty3-gypsy group retrotransposons in F. x ananassa genome.

EFFECT OF MICRO-PROPAGATION ON THE HEALTH STATUS OF STRAWBERRY PLANTING MATERIAL FOR COMMERCIAL PRODUCTION OF STRAWBERRY RUNNERS FOR QUEENSLAND

Plant tissue culture has been used for a number of years to produce micropropagated strawberry plants for planting into runner growing beds in the Stanthorpe (Queensland) and Bothwell (Tasmania) regions. This process has allowed the rapid release of new cultivars from the LAWS (Late Autumn, Winter, Spring) breeding program into the current runner production system. Micro-propagation in vitro allows plants to be produced during the autumn and winter months, when mother plants would normally be in a fruit production phase in the field in Queensland. The plants produced are of a high health status when they are planted. The subsequent arrival and build up of various diseases in the runner fields are closely monitored. Using tissue culture for the first generation reduces the time the plants spend in the field by twelve months, reducing disease incidence. To date, any disease outbreak has been successfully managed using early detection and rapid response methods.

Growth and photosynthetic activity of micropropagated strawberry plants inoculated with endomycorrhizal fungi (AMF) and growing under drought stress

The plants produced by in vitro methods are free of any microflora contrary to natural systems where plants are colonized by symbiotic fungi. The present paper reports the experiments carried out to evaluate the role of arbuscular endomycorrhizal fungi in development of micropropagated strawberries and their photosynthetic activity (measured by chlorophyll fluorescence) under drought conditions. Mycorrhization strongly affected growth and tolerance to water deficiency of the plants cultivated in greenhouse. Wilting of not-mycorrhized plants was accompanied by drastic increase of Fo and Tfm and decrease of Fm. At the same time, the value of these parameters for mycorrhized plants did not change. Drastic decrease in the value of parameters Fv/Fm, Fv/Fo and Fo/Fm for plants without AMF appeared at the end of dry period. Rise of Fs and decrease Rfd was noted only for not-mycorrhized plants. The plants colonized by fungi, fully recovered their photosynthetic activity when watering was restored.

Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro

In the classical method of strawberry micropropagation, the rooting phase is done in vitro. The trials were undertaken to replace in vitro rhizogenesis by a direct ex vitro rooting. The aim of the present study was to evaluate the morphological and physiological status of strawberry plants rooted by both methods. The micropropagated shoots of strawberry, cultivars Senga Sengana, Kent and Kama were rooted by the standard method at the in vitro stage or they were treated as soft cuttings and rooted ex vitro (in non-sterile conditions). After a 4-week rooting period the plantlets rooted ex vitro had larger root systems than in vitro-rooted ones, as evidenced by a significantly lower ratio of shoot to root dry weights (2.95, 3.33 and 4.24, respectively, for ex vitro rooted Senga Sengana, Kent and Kama plants versus 5.09, 6.95 and 5.04 for in vitro rooted plants of the same cultivars). During subsequent growth, differences in development increased and were most pronounced in runner formation, more than twice as many runners were formed by ex vitro, than by in vitro-rooted plants. Chlorophyll fluorescence parameters showed that photochemical activity was similar in the leaves from plants rooted in vitro and ex vitro. Values of chlorophyll fluorescence parameters indicated that persistent leaves play the key photosynthetic role at the beginning of the growth period. With the formation of new leaves, the photosynthetic activity of the persistent leaves decreased and their function was taken over by the newly formed ones.

DRIFT IN GENETIC STABILITY IN MICROPROPAGATION: THE CASE OF STRAWBERRY

Genetic stability during micropropagation is controlled by numerous factors. Among the internal plant factors, the genotypes, the presence of chimeral tissue and mainly the explant origin, play an enormous role, Moreover, some external factors as the tissue culture protocol, media sequences and the growth substance concentration, have also an important influence on genetic stability. In micropropagated strawberry plants, sporadic occurrences of abnormal fruit setting, with a hyper-flowering habit, were observed in some cultivars, always after numerous subcultures. Therefore, it was arbitrarily decided in 1980 to limit the number of subcultures to ten. In 1990, it was hypothesised that the hyper-flowering habit was related to and connected with the development of special buds, called stipular-buds, These always arise from a specific position on the leaf petiole and show an abnormal phyllotaxy, a very high propagation rate in vitro and a large number of flowers per inflorescence ex vitro. The hyper-flowering habit is unlikely to be due to true mutations, but rather to a reproducible event, possibly DNA methylation. The occurrence of these stipular-buds is reduced by decreasing the benzylaminopurine (BAP) concentration from 1 to 0.5 or 0.2 mg 1 -1 depending on the genotype. It is also necessary to limit the propagation to ten subcultures.

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 µM and 15 µM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 µM and 15 µM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N6-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 µM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred.

Action of Cyclodextrins on Germinating Seeds and on Micropropagated Plants

AbstractEffects of cyclodextrins on germinating barley seeds and on the growth of micropropagated strawberry plants were investigated. Treatment of barley seeds with α‐ or β‐cyclodextrins resulted in a similar delay of germination as reported earlier [1]. The delay of germination occurred with agarose‐immobilized as well as with free cyclodextrins. Since penetration of immobilized cyclodextrins into the seeds is limited, it was concluded that the delay resulted from complexation of growth factors. Soluble cyclodextrins caused a growth retardation with micropropagated strawberry plants and certain morphological changes resembling the ones observed when the action of auxins is enhanced in the respect to cytokinins.

Enhanced Vigor in Vegetative Progeny of Micropropagated Strawberry Plants

Abstract Micropropagated mother plants of strawberry (Fragaria × ananassa Duchesne) often produce more flowers and runners than their runner-propagated counterparts (4, 5), but it is not clear how long this effect lasts. Swartz et al. (5) reported that the increase in runnering of tissue culture plants did not continue after the first flush of runner production in the field, and Marcotrigiano et al. (4) showed that, within the planting year, micropropagation increased runner production in mother plants and their primary runner plants, but not in attached secondary and tertiary runner plants. However, these studies examined runner plants still attached to plants directly out of culture, rather than detached, cold-hardened plants as they are delivered to the grower. In a study where detached runner plants of unknown age were compared, planting stock from micropropagated plants of 4 of 6 cultivars had significantly higher matted row densities ( = 63%) than those derived from conventionally propagated stock (2). The goal of this study was to compare the performance of detached primary, secondary, and tertiary runner plants of micropropagated (MP) and standard (ST) mother plants grown under controlled conditions.

Micro-propagation of strawberry plants in vitro — Effect of growth regulators on incidence of multi-apex abnormality

A morphological abnormality occurring in micro-propagated strawberry plants is described and illustrated. The condition is recognised in established micro-propagated plants by an abnormal phyllotaxy which results typically in 2 or more young leaves emerging together rather than singly and by an excessive number of small branch crowns being produced. Dissection shows that the abnormality originates at the apex and is manifest as a linear (or less often as a circular) arrangement of multiple apices. Such multi-apexing plants “bud-off” normal single-apexed crowns from time to time but the abnormality can persist in the main shoot for 2 years (the limit of our observations). The multi-apexing differs fundamentally from clusters of single apices which, when removed from culture and separated, give rise to normal single-crowned plants. Similarities between the multi-apexing condition and fasciation in other plant species are discussed. Changes in growth regulator constituents of the culture media are shown to control the incidence of multi-apexing. In the presence of indole-3-butyric acid (IBA), higher 6-benzyl-aminopurine (BA) concentration increases the frequency, while adding gibberellin (GA 3) decreases the incidence of affected plantlets. The frequency of abnormality varies greatly between different cultivars. It is suggested that conditions favouring greater proliferation rates in culture may also favour multi-apexing. Results indicate that multi-apexing can be avoided by using appropriate concentrations of growth regulators in culture media which will concomitantly maintain a good proliferation rate.