Long-term Micropropagation Research Articles

In Vitro Long-Term Cultures of Papaya (Carica papaya L. cv. Solo)

In this article, we present proliferation data from 10 years of the continuous in vitro incubation of cv. Solo papaya shoots and propose a reliable method for the long-term micropropagation of papaya, using microshoots developed from the axillary buds of papaya shoots as primary explants. Three different media were assayed. The proliferation medium (PPRM) allowed us to maintain papaya shoots under continuous proliferation for 20 years, maintaining consistent behavior. Most of the shoots developed in the PPRM rooted during the incubation and then acclimated easily, maintaining the ploidy and morphological characteristics of the parental plants, and flowering and setting fruits normally. The PPRM medium consisted of MS medium supplemented with naphthalene acetic acid (NAA) (0.1 mg L−1), benzyladenine (BA) (0.5 mg L−1), gibberellic acid (GA3) (0.5 mg L−1), and adenine hemisulphate (40 mg L−1). The average multiplication rate was higher than 20 shoots per explant during the long-term assay. The elongation medium (PELM) was designed to recover shoots with poor growth and allowed the development of high-quality shoots ready for rooting. It consisted of an MS basal medium supplemented with NAA (0.1 mg L−1), kinetin (KIN) (0.5 mg L−1), and GA3 (1 mg L−1). The rooting medium (PROM) was designed to induce high-quality roots from nonrooted shoots and consisted of a half-strength MS medium and indole-3-butiryc acid (IBA) (1 mg L−1). On PROM, agar can be exchanged for expanded vermiculite. Acclimation took place inside an acclimatization tunnel under progressive hydric stress. After 4 weeks, the plant recovery rate was 90% for plants maintained under continuous proliferation for ten years. The main objective of this work was to provide a micropropagation method which would maintain healthy elite genotypes of papaya for long periods of time and produce a high number of good quality plants.

Clonal micropropagation of essential oil rose cultivars and breeding samples at long-term subcultivationin vitro

The development of explants of 12 cultivars and breeding samples of essential oil rose (obtained with the participation ofRosa damascenaMill.,R. gallicaL.,R. albaL.) during long-term micropropagation was investigated. At the second multiplication stage 5 subcultures were carried out. Increase of the studied morphometric parameters of explants to 3-4thsubcultures was established. In some genotypes, the maximum multiplication index was in the third subculture (сultivars ‘Raduga’, ‘Zolushka’, ‘Lada’, ‘Krymskaya Krasnaya’, ‘Lany’, ‘Vesna’ and samples N37-24, M215), while in others (сultivar ‘Kazanlykskaya’ and samples N138, G2168, N37-2) – in the fourth. The largest increase of the multiplication index in 3-4th subcultures compared to the first (3.0-4.8 times) was found in the cultivars ‘Lada’, ‘Lany’, ‘Raduga’, ‘Krymskaya Krasnaya’ and sample N37-24. In the fifth subculture the multiplication index decreased. However, in most cultivars and breeding samples it was higher than in the first subculture. The best morphogenetic potential was noted for сultivar ‘Raduga’ and samples G2168, N37-2, in which the multiplication index reached 14.2, 14.4 and 11.8, respectively. The minimal ability to propagationin vitrowas in samples M215 and N138 – their multiplication index did not exceed 1.1 - 4.0.

A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation

Morphological variation, genotypic stability and genetic distances among micropropagated tea [Camellia sinensis (L.) Kuntze] cv. ‘Kolkhida’ plantlets after 7 years of propagation in vitro and among plantlets regenerated from callus were compared. Fifteen individuals, randomly chosen amongst a set of 100 plantlets regenerated from callus, and ten individuals, randomly chosen from a set of 100 micropropagated plants were used in the study. Morphological descriptors showed higher variability among callus-derived regenerants than in micropropagated plants. Flow cytometry analysis showed no significant variability of nuclear DNA content among micropropagated plants, however changes in DNA indicated aneuploidy in three out of ten callus-derived regenerants. ISSR analysis showed lesser genetic distances among micropropagated plantlets and maternal cultivar ‘Kolkhida’ compared with callus derived plantlets. Yet, there is some degree of genetic instability after long-term micropropagation. Results confirmed the hypothesis of genetic instability of some species during long term in vitro conservation. Tea vegetative explants plants were propagated in vitro for 7 years. A comparison of tea plantlets derived from callus with plantlets from long-term in vitro propagation revealed genetic variability and confirmed the hypothesis of genotype instability of tea plant.

Morphogenetic, Physiological, and Biochemical Features of Lavandula angustifolia at Long-Term Micropropagation In Vitro

Data on morphogenesis and some physiological and biochemical parameters of explants of five cultivars of Lavandula angustifolia Mill. under long-term micropropagation in vitro during nine passages are represented. Introduction of meristems of the Stepnaya, Sineva, Vdala, Rannaya, and Record cultivars into the culture resulted in the formation of multiple shoots. Maximal number of shoots was observed in the Sineva cultivar (5.7), while minimal number was identified in the Rannaya cultivar (3.9). The experiments with micropropagation of lavender in nine subcultivations showed that the number of shoots increased in all cultivars up to the third subculture and decreased thereafter. The highest multiplication index (12.2) in this passage was observed for the Sineva cultivar. Further subculture were characterized by decrease of this parameter to 7.4–8.6. The lowest multiplication index in the fifth to ninth passages was observed for the Record cultivar (3.8–4.5). The analysis of the water regime of the microshoots obtained via in vitro multiplication showed that the total water content was 83–89%, while fractions of bound and colloid-consumed water made up to 40–57%. Record, Rannaya, and Vdala cultivars were shown to possess maximal water-retaining capacity. The obtained data indicate the active function of chlorenchyma. It was found that the Vdala and Sineva cultivars were characterized by maximal photosynthetic activity of their leaves and shoots and showed the highest viability index. The cultivars studied under the conditions of in vitro cultivation were characterized by a high level of proline, ascorbic acid, and phenolic compounds, especially flavanols. The highest level of proline was observed in the Vdala cultivar (372.87 µg/g), while the highest concentrations of ascorbic acid (31.28 mg/100 g) and phenolic compounds (1103 mg/100 g) were found in the Sineva and Rannaya cultivars, respectively. Shoots cultured in vitro were characterized by a high activity of catalase and low activity of superoxide dismutase and polyphenol oxidase. The highest catalase activity was observed in the Vdala and Sineva cultivars. The Sineva cultivar also demonstrated a high activity of polyphenol oxidase. The results obtained demonstrate the good ability of lavender to micropropagation in vitro, the efficiency of which depended on both the cultivar and the number of subcultures.

Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria.

Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."

Highly endoreduplicated floral organs of somaclonal variants in clonally propagated Phalaenopsis ‘Spring Dancer’

A micropropagation system is essential for obtaining elite clones of Phalaenopsis in the floricultural industry; however, somaclonal variation frequently occurs during long-term micropropagation, which reduces the uniformity and quality of clonally propagated plants. To detect the factors influencing this variation and thus obtain high-quality, uniform Phalaenopsis plants, we analyzed somaclonal variants in the Phalaenopsis ‘Spring Dancer’ (SD) variety collected from an orchid farm, which were grouped into three types according to flower morphology (SD-N, SD-V1, SD-V2). Morphological differences were observed between all SD groups; in particular, the diameter of the petal and thickness of each organ were significantly larger in the variants than SD-N (normal) plants. Both SD variant groups showed the same ploidy level as that of SD-N. The degree of endoreduplication of the petals and flower stalks was higher in the variants (4C–16C) than in SD-N (2C–8C). Cell area was increased and the shape of epidermal cells was changed in both variants. Expression levels of MADS box transcription factor (MADS4) of the flowers were higher than those of the leaves in the variants. Among the three flower organs, MADS4 expression was the highest in the petals of SD-V2. These results revealed that MADS4 is involved in the modification of flower morphology in somaclonal SD variants. High ploidy 2 (HPY2) expression was highest in the flowers of SD-V1, whereas its expression in the leaf was similar to that of SD-N. Among the various flower organs, HPY2 expression was highest in the lips of SD-V1.

Organogenesis and long-term micropropagatlon of Polish pea cultivars

The complete protocol for regeneration and long-term micropropagation of several Polish cultivars of pea (<em>Pisum sativum </em>L.) has been elaborated. The shoots were the most likely regenerated via de novo organogenesis. The adventitious buds formed in callus derived from cotyledons tissue adjacent to the axillary meristems of immature embryos. All cultivars' calli regenerated several shoots per explant on the MS medium supplemented with B5 vitamins and 4.5 mgl<sup>-1</sup> of BAP, however some differences in regeneration capacity among cultivars were observed. The plantlets were subsequently micropropagated with slightly higher efficiency and preserving a good viability over the long-term culture on a medium containing 2.0 mgl<sup>-1</sup> than one with 4.5 mgl<sup>-1</sup> of BAP. The additional step of the pre-conditioning culture of multiplicated shoots on a medium with very low BAP concentration i.e. 0.02 mgl<sup>-1</sup> was applied and appeared to be beneficial before rooting in vitro or grafting. The modified MS-derived medium with the half-strength of MS macroelements but with the full original dose of calcium and supplemented with B5 vitamins and 1.0 mgl<sup>-1</sup> of NAA was developed for effective rooting. The shoots were also sufficiently transferred into ex vitro conditions using grafting. The majority of the regenerated plants had adapted to in vivo conditions in a greenhouse and subsequently has set seeds. The presented protocol provides relatively efficient rate of de novo pea regeneration and would be useful for <em>Agrobacterium</em>-mediated transformation purposes.

Stability of transgenes in long-term micropropagation of plants of transgenic birch (Betula platyphylla)

The stability of integration and expression level of transgenes in long-term micropropagation clones of transgenic birch (Betula platyphylla Suk.) was examined. Multiplexed PCR and reverse primer PCR demonstrated stable integration of transgenes into regenerated plants. Expression levels of the bgt and gus genes among shoot plantlets, subcultured 4, 7, 9 and 15 times, were significantly different. The transcriptional expression level of extraneous genes in regenerated plants decreased with increasing subculture number. Transcriptional gene silencing (TGS) occured in regenerated transgenic lines. The silencing rate of GUS in the 5th subculture plants was 22-65%. TGS in regenerated plants could be reactivated with 5-azacytidine (Azac) at 50-200 microM. GUS and BGT protein expression was reactivated in the micropropagated transgenic birch plants when treated with Azac. A decrease in expression level with increasing number of subcultures is thus associated with DNA methylation.

Changes in the chloroplast ultrastructure in potato leaves during long-term micropropagation in vitro

Changes in the chloroplast ultrastructure in potato leaves during long-term micropropagation in vitro

In vitro micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse.

THE EFFECT OF CYTOKININ TYPE AND CONCENTRATION AND THE NUMBER OF SUBCULTURES ON THE MULTIPLICATION RATE OF SOME DECORATIVE PLANTS

Long-term micropropagation of some plants by tissue culture was investigated. The plants studied can be divided into three groups according to their sensitivity to cytokinins benzyladenine (BA) and kinetin (K). (1) Plants (gerbera cultivars) whose multiplication on BA media is not recommended. Permanent high K level must also be avoided. It must be decreased gradually during a long-term culture. After the 8th or 9th subculture the shoots are to be transferred to the resting medium with 0.1 mg/L K and 1.0 mg/L indoleacetic acid. (2) Plants ( Cordyline, Dracaena, Dieffenbachia) that may be multiplied on media containing either BA or K, the latter being preferable as its concentration may be varied within a larger range (0.5-4 mg/L) without negative side effects. (3) Plants ( Philodendron, Spathiphyllum, Musa) that may be multiplied on BA media without even relatively high BA concentrations (5 mg/L) leading to vitrification. However, in order to obtain the best shoots for rooting, the BA content of the multiplication media has to be reduced to 1/2-1/3 of its original value after the 5th or 6th subculture.

Micropropagation of Passion fruit from Subcultured Multiple Shoot Primordia

A technique for long-term micropropagation of Passiflora edulis from subcultured multiple shoot primordia is described. Excised tissues from leaves cultured on Murashige and Skoog medium (MS medium) supplemented with 3 % (w/v) sucrose, 1 μmol L -1 6-benzylaminopurine (BA) and 1 μmol L -1 indole-3-butyric acid (IBA) formed multiple shoot primordia. These shoot primordia were transferred to a medium containing 10 μM BA and subcultured repeatedly at 3-week intervals. When subcultured shoot primordia were transferred to a medium containing lower concentrations (0.1 or 1.0 μmol L -1 ) of BA, shoots were formed. Subcultured shoot primordia have continued to regenerate plants for more than 3 years. Shoots developed and rooted on MS medium lacking growth regulators. Rooted plants were transferred to pots and grown in a greenhouse. According to the morphology of leaves of regenerated plants, rejuvenation was obtained by this method of in vitro culture.

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N(6)-benzylaminopurine, 10 μM, and α-naphthaleneacetic acid, 1μM, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 μM, NAA 0.1 μM, and gibberellic acid 3 μM to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 μM indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R0) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R0 regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.

Differences between Nerine hybrids in micropropagation potential

Micropropagation ability of nine F 1 clones derived from reciprocal crosses between Nerine angustifolia (Bak.) W. Watson and N. sarniensis (L.) Herb. was investigated. Cultures were started using twin scales from almost full-grown bulbs. Newly formed plantlets in these cultures developed bulblets. Using the in vitro bulblets, a long-term micropropagation procedure was established on solid MS medium with 0.3 mg l −1 IBA. Bulblets more than 100 mg in weight were suitable for subculture. Significant differences in multiplication ability between the F 1 clones were observed during four propagation cycles. In the last cycle, the most outstanding clone still reached a 7.9 times multiplication versus 0.3 times in the lowest producing clone. Removal of the upper half of the bulblets as well as addition of 0.1-1 mg l −1 BA to the medium increased multiplication. The results from this study indicate that large genetic variation for propagation potential can be identified in the course of a breeding programme in Nerine.