Effect of ectomycorrhizal fungi on survival and growth of micropropagated plants and seedlings of Castanea sativa mill.

 Four ectomycorrhizal fungi (Amanita muscaria, Laccaria laccata, Piloderma croceum and Pisolithus tinctorius) were used to produce mycorrhiza on seedlings and micropropagated plants of Castanea sativa in vitro. Pisolithus tinctorius was most effective in colonizing roots of both micropropagated plants and seedlings. A. muscaria and L. laccata only colonized a few feeder roots of some plants and Piloderma croceum did not form mycorrhizas. Mycorrhization of micropropagated plants increased survival and growth during weaning. Accepted: 27 February 1996     

An Assessment of Genetic Integrity of Micropropagated Plants of Plumbago Zeylanica by RAPD Markers

Clones of Plumbago zeylanica were micropropagated using nodal culture. The application of random amplified polymorphic DNA (RAPD) in assessing the genetic integrity of the micropropagated plants was evaluated by polymerase chain reaction. Twenty arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic fidelity. All RAPD profiles from micro-propagated plants were monomorphic and similar to those of field grown mother plants. No polymorphism was detected within the micropropagated plants.     

Stomatal physiology of a micropropagated CAM plant; Agave tequilana (Weber)

Experiments were designed to assess the capacity of an in vitro cultured CAM plant to control water loss and to examine the response of their stomata to various factors. Detached leaves of micropropagated Agave tequilana plants lost water at similar rates as did field-grown plantlets when dehydrated in air. This was consistent with the fact that stomata from micropropagated plants show similar morphology than field-grown plantlets. In addition, stomata from micropropagated plants responded to various factors in a manner similar to those from field-grown plantlets. It appears that in vitro culture does not affect the capacity of leaves to control water loss nor does it alters the nocturnal stomatal opening of this CAM plant.     

Failure of sexual reproduction found in micropropagated critically endangered plants prior to reintroduction: a cautionary tale

Micropropagation is a useful technique for ex situ multiplication and restoration of critically endangered plant species, but the sexual reproductive behaviour of micropropagated plants is seldom evaluated prior to reintroduction. We examined the critically endangered species Rulingia sp. ‘Trigwell Bridge’, with only three remaining plants known in the wild, as a model case to examine this issue. Abnormalities in micropropagated plants of this species related to four floral traits (lengths of sepals, petals and anthers and width of anthers). The number of pollen grains per flower of abnormal individuals was lower than in plants with apparently normal flowers (wild types), but not significantly so (P = 0.068). Pollen viability for the abnormal plant (0.87 ± 0.26%) was significantly lower than for the plants exhibiting wild‐type floral morphology (45.42 ± 4.47%). Experimental manipulations were used to examine the mating behaviour of normal and abnormal plants. The results showed that both male and female reproductive failure was linked to individuals exhibiting abnormal flowering attributes. Such aberrant reproductive performance in a micropropagated rare species predicates caution when using micropropagated plants in reintroduction programmes, highlighting the importance of screening for reproductive normality prior to release of micropropagated plants (especially for critically endangered species where reliance on in vitro propagation methods is often a necessity). © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 278–284.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh

RAPD markers were used to assess genetic fidelity of 23 micropropagated plants of a single clone (L34) of Populus deltoides. Eleven arbitrary 10-base primers were successfully used to amplify DNA from in vivo and in vitro material. Of these, 5 distinguished a total of 13 polymorphisms common across 6 micropropagated plants. Apart from these 6 plants, the amplification products were monomorphic across all the micropropagated plants, the mother plant and 4 additional field-grown control plants. Our results show that RAPD markers can be used to gain rapid and precise information about genetic similarities or dissimilarities in micropropagation systems that might not be so easily evident from other commonly used techniques.     

Variation in leaf structures of micropropagated rhubarb (Rheum rhaponticum L.) PC49

Alterations in leaf trichomes, stomatal characteristics and epidermal cellular features were investigated in micropropagated rhubarb (Rheum rhaponticum L.) PC49. The results showed that micropropagated regenerants had produced significantly lower stomatal index, but larger epidermal cell size than conventional plants. In addition, altered trichome morphology and abnormal stomata, e.g. twin-stomata were constantly noted only in micropropagated plants. The microscopic observation demonstrated a substantially larger intercellular space in palisade and mesophyll only in leaves of micropropagated plants. But the results showed no difference in chloroplast number and chlorophyll content between micropropagated and conventional plants. All the abnormalities suggest somaclonal variation may have occurred in micropropagated rhubarb PC49.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.     

Growth and biochemical profiling of artificially associated micropropagated oil palm plantlets with Herbaspirillum seropedicae

The challenges of various biotic and abiotic stresses can imperil the growth of micropropagated plantlets either direct or indirectly. Hence, in this study, a mutual relationship was established between diazotrophs and micropropagated plantlets to enhance plant growth and development. Artificial symbiosis was created for different inoculums of Herbaspirillum seropedicae (Z78), namely sonicated cells, broth culture, and pellet cells with micropropagated oil palm plantlets Elaeis guineensis Jacq. Results reveal significant differences on root volume, total protein content, and Brix value for Z78 broth culture treatment compared with plantlets treated with 25% N. High nitrogenase enzyme activities (6.7?×?10^?4?µmol?C₂H₄ g^?1?h^?1) and indole-3-acetic acid production (205.21?µmol (g?FW)^?1) were also detected on roots of plantlets treated with Z78 broth culture. These beneficial traits reviewed that the application of diazotrophs (Z78) in associative manner for micropropagated plantlets hold vast potential for promoting plant growth and plant’s healthiness.     

Influence of mycorrhization on physiological parameters of micropropagated Castanea sativa Mill. plants

 Mycorrhizal micropropagated Castanea sativa plants were studied in terms of growth and physiological parameters following in vitro mycorrhization with Pisolithus tinctorius. Mycorrhization enhanced growth of micropropagated chestnut plants, increased their protein content and photosynthetic rates, decreased the respiratory rates and CO₂ compensation point. RuBisCO activity was not significantly different in mycorrhizal and control plants, although there was an increase in the amount of RuBisCO in the former. Mycorrhization increased plant biomass and improved plants physiological status, thus enhancing the acclimatization process. Accepted: 21 May 1997     

Determination of genetic stability of long-term micropropagated plantlets of Platanus acerifolia using ISSR markers

Inter-simple sequence repeat (ISSR) markers were used to assess the genetic stability of long-term micropropagated plantlets of London plane tree (Platanus acerifolia Willd.). Twenty micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 8 years, as achieved by axillary branch multiplication. Out of 38 ISSR primers screened, 16 primers were found to produce clear reproducible bands resulting in a total of 103 distinct bands with an average of 6.44 scorable bands per primer. Of these 103 bands, 86 were monomorphic across all 20 of the plants tested and 17 showed polymorphisms (16.5 % polymorphism). Based on the ISSR band data, similarity indices between the plantlets ranged from 0.92 to 1.00. These similarity indices were used to construct an UPGMA dendrogram and demonstrated that all 20 micropropagated plants grouped together in one major cluster with a similarity level of 91 %. A total of 1771 scorable bands were obtained from the full combination of primers and plantlets and only 51 (2.88 %) were polymorphic across the plantlets which indicates that this micropropagated line of P. acerifolia is genetically stable.     

Nitrogen-fixing potential of micropropagated clones of Acacia mangium inoculated with different Bradyrhizobium spp. strains

Five A. mangium seedlings of different shoot lengths were selected from a 600-seed screening experiment and micropropagated. Two-week-old rooted microcuttings of the 5 micropropagated clones were inoculated with 3 specific Bradyrhizobium spp. strains in 15 combinations. After 5 months of growth, nodule dry weight and shoot dry weight data showed significant effects of clone and Bradyrhizobium spp. strain. Clones RR-G₁ and IR-M₂ and Bradyrhizobium sp. Aust13c resulted in the highest dry-matter production and most efficient nodulation. No interaction was observed between clone and Bradyrhizobium spp. strain, which indicates that the Bradyrhizobium spp. strain and the host plant can be selected separately.     

A comparison of nine species of arbuscular mycorrhizal fungi on the development and nutrition of micropropagated Rubus idaeus L. cv. Glen Prosen (Red Raspberry)

Growth, development and nutrient status of micropropagated Rubus idaeus cv. Glen Prosen in response to inoculation with nine species of arbuscular mycorrhizal (AM) fungi from three different genera was investigated. The nine species of AM fungi included, Glomus clarum, G. etunicatum, G. intraradices, Gigaspora rosea, Gi. gigantea, Gi. margarita, Scutellospora calospora, S. heterogama and S. persica. Plant responses to AM fungi varied from growth enhancement to growth depression. Depressive growth effects were specific to Gigaspora species. Furthermore, particular species of AM fungi had unique effects on the mineral status of the raspberry plants. Importance of isolate selection for inoculation of micropropagated raspberry plants is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Abnormal chromosomes and DNA content in micropropagated rhubarb (<Emphasis Type="Italic">Rheum rhaponticum </Emphasis>L<Emphasis Type="Italic">.</Emphasis>) PC49

Rhubarb (Rheum rhaponticum L.) is a tetraploid (2n = 44) vegetable crop growing in Europe and North America. The results observed 44 chromosomes for both cultivars Timperly Early and PC49, but showing significant differences in DNA contents between them. Abnormal chromosome numbers (2n = 22 and 32) and chromosomal fragments were observed only in micropropagated plants of rhubarb PC49, suggesting somaclonal variation. The results provide further information on understanding morphological variation and increased disease susceptibility of micropropagated rhubarb PC49 in our previous investigation.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Nuclear rDNA instability in in vitro‐generated plants is amplified after sexual reproduction with conspecific wild individuals

Using micropropagation through tissue culture has become the most used approach worldwide for mass production for the conservation of endangered species. However, the screening of somaclonal variations generated using in vitro culture is usually restricted to the first generation of micropropagated plants, when they have not yet been released in the field. Accordingly, the fate of genetically modified regenerants after sexual reproduction is usually not assessed and changes in the genetic structures of species are unknown. In this work, we assess the cytogenetic stability of two rDNA gene families in the offspring of experimental crosses between accessions generated after in vitro culture and wild individuals of Cistus heterophyllus (Cistaceae). The cytogenetic rDNA profiles (45S rDNA, 5S rDNA) of 118 accessions including wild and in vitro micropropagated individuals and bi‐directional artificial crosses between wild and in vitro‐generated plants were assessed by fluorescence in situ hybridization (FISH) and Ag‐NOR staining. Plants regenerated by micropropagation showed a lower size of the FISH signals in a 45S rDNA site, but this condition was not present in the wild accessions. Three new cytogenetic and cytological variants were present in 36% of the experimental progeny, involving the amplification of one additional 45S rDNA site and the presence of heteromorphic nucleoli. rDNA‐based genomic instability was present after sexual reproduction between wild and in vitro‐generated plants. The results of this study discourage the use of micropropagated materials for plant conservation unless comprehensive surveys of the genetic integrity and stability of the regenerants are performed after crossing between wild and micropropagated plants.     

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Comparative GC–FID and GC–MS analysis of the mono and sesquiterpene secondary metabolites produced by the field grown and micropropagated plants of <Emphasis Type="Italic">Artemisia amygdalina</Emphasis> Decne

The essential oil obtained by hydrodistillation from the leaves of micropropagated plants of Artemisia amygdalina was analyzed by capillary GC–FID and GC–MS and compared with that obtained from the leaves of field growing parent plants. The oil yield from the micropropagated plants was lower (0.05% v/w) than the oil yield obtained from field-grown plants (0.2% v/w). The major constituents of the field-grown plants were p-cymene (21.0%), 1,8-cineole (24.9%), α-terpineol (5.9%), β-caryophyllene (4.7%), germacrene D (4.0%), while as the major constituents from the micropropagated plants were p-cymene (11.3%),1,8-cineole (10.2%), borneol (7.9%), α-longipinene (5.5%), α-copaene (5.5%) and β-caryophyllene (17%). The essential oil from field-grown plant was dominated by the presence of oxygenated monoterpenes (41.5%), monoterpene hydrocarbons (35.9%) and sesquiterpene hydrocarbons (16.3%) while as the essential oil of micropropagated plants was characterized by sesquiterpene hydrocarbons (40.0%), oxygenated monoterpenes (25.2%) and monoterpene hydrocarbons (21.6%).