Occurrence Of Somaclonal Variation Research Articles

Field Performance of Disease-Free Plants of Ginger Produced by Tissue Culture and Agronomic, Cytological, and Molecular Characterization of the Morphological Variants

Ginger (Zingiber officinale Rosc.) is an important spice crop valued for its flavored and medical properties. It is susceptible to soil-borne diseases, which can cause considerable economic loss to growers. In vitro culture is feasible for the propagation of disease-free ginger plants, but has several disadvantages when producing seed rhizomes that can be commercially used, such as long cultivation cycles (usually 2–3 years) and occurrence of somaclonal variation. In this study, dynamic changes in the morphological characteristics of in vitro-propagated disease-free plants of ‘Wuling’ ginger were evaluated by continuous observation and measurement at 30-day intervals, and morphological variants were screened and characterized by agronomic, cytological, and molecular analysis at harvest. Results showed that the plants grew rapidly within 120 days after planting, and the most active growth period was from 60 to 120 days. Eight plants with clear and stable morphological differences were screened out from approximately 2000 plants grown in the field, and they could be classified into two groups (VT1 and VT2) based on tiller number, plant height, leaf color, and leaf shape. By flow cytometry analysis and chromosome counting, the VT1 was confirmed to be diploid, with the shortest plant height, the largest number of tillers and rhizome knobs, and the smallest tiller diameter and rhizome size among the three types of plants. The VT2 was mixoploid, consisting of diploid and tetraploid cells, with significantly reduced tiller number and rhizome knobs, significantly larger stomatal guard cells/apertures, and significantly lower stomatal density. SSR analysis detected DNA band profile changes in six out of the eight variants, including one plant of the VT1 and all the VT2 plants. The findings of this study might contribute to the commercial production of disease-free seed rhizomes in ginger, and the characterized somaclonal variants could provide useful germplasm resources for future breeding.

Assessment of somaclonal variation occurrence in micropropagated Mansonia altissimia (A. Chev.) A. Chev. using molecular marker

Plant tissue culture that offers limited space and short time for clonal propagation of plant species can sometimes be discouraging if true to type clones are not generated. This study investigated direct shoot induction and genetic stability of Mansonia altissimia using molecular markers in order to ascertain that the in vitro propagated plantlets were true to type. Nodal cutting was used as explant, and Murashige and Skoog (MS) medium was supplemented with 6-benzyl aminopurine (BAP) (µM) + naphthaleneacetic acid (NAA) (µM), BAP (µM) + Gibberellic acid (GA3) (µM), Kinetin (KIN) (µM) + NAA (µM), KIN (µM) + GA3 (µM), thidiazuron (TDZ) (µM) + NAA (µM), TDZ (µM) + GA3 (µM), for direct micropropagation. PCR amplification of wild plant and direct micropropagation of Mansonia altissimia were done using 15 RAPD and 6 ISSR primers. The highest shoot height (4.77 cm) and number of leaves (5.33) were obtained from 2.5 µM BAP + 3.0 µM GA3. Moreover, the polymerase chain reaction amplification results showed 100% monomorphic and 1.0 level of similarity coefficients, which indicates that there is no occurrence of somaclonal variation.

Abiotic Stress Induces Morphological, Physiological, and Genetic Changes in Orthosiphon stamineus Benth. In Vitro Cultures

Orthosiphon stamineus is a well-known medicinal herb that has long been used as a traditional treatment for a variety of ailments and disorders. In this study, in vitro grown O. stamineus plantlets were subjected to various stress factors to elucidate the effects of these stresses on the occurrence of somaclonal variation in this species. Murashige and Skoog (MS) media supplemented with 2.0 mg/L kinetin plus 0.5 mg/L IAA was identified as the optimum in vitro regeneration media (OM). The data analysis confirmed the clonal nature of the resulting plantlets. The plantlets were also exposed to various stress factors such as high salinity, abscisic acid (ABA), and high cytokinin concentrations, by subculturing the plantlets on OM added with various concentrations of NaCl, ABA, and high kinetin levels. Exposure to the various stress factors was observed to result in the occurrence of somaclonal variation in O. stamineus, where the incidence of hyperhydricity was found to be the most prominent. An ISSR analysis revealed 70.12% polymorphism among the variants. The results showed that the chlorophyll content and photosynthetic ability of plantlets were reduced when the abiotic stress levels increased.

HD2 overexpression induces dwarfism and elevated histone deacetylase activities in transgenic Ananas comosus variety MD2

Plant tissue culture has served as one of the most efficient biotechnological tools to mass produce high quality major crops. Nevertheless, the use of this technique can result in the occurrence of somaclonal variation, which may be brought upon by genetic and/or epigenetic modulations. The epigenetic modulation of plant growth, particularly on the importance of histone deacetylation/acetylation on genome stability, transcriptional regulation, as well as plant development, has been reported in several species such as Arabidopsis, rice, and maize. However, the information on its role in regulating the growth of major crops such as pineapple is still lacking. Previously, we had reported that supplementation of high cytokinin in the tissue culture media and exposure to salinity and abscisic acid (ABA) resulted in the production of somaclonal variants that exhibited dwarf phenotypes, and that these occurrences were associated with epigenetic modulation via the involvement of histone deacetylases (HDACs). Thus, in the present study, we aimed to further elucidate and verify the involvement of histone deacetylation in the occurrence of dwarfism in tissue culture-derived pineapple plants, through HD2 gene overexpression. AtHD2 gene from Arabidopsis thaliana was synthesized and expressed in phenotypically normal Ananas comosus. Transformation efficiency was also studied, and application of 200-µM acetosyringone was observed to be more efficient than 500 µM vanillin by 10%. The transgenic plants were observed to exhibit dwarfed phenotypes and showed significantly high HDAC enzyme activities. Ploidy evaluation of the transgenic plants showed no ploidy change had occurred in the samples and remained as 2n. The relative expression level of AtHD2 was also higher (3.2-fold up-regulation) in the transgenic plants compared to the wild type plants. Collectively, these results indicate that HDACs indeed played an essential role in the response of A. comosus towards abiotic stress, and that this phenomenon is epigenetically regulated.

Banana somaclonal variation assessed by Amplified Fragment Length Polymorphism profiles at early cycles of in vitro culture

Banana micropropagation for obtaining free-virus plants frequently provokes somaclonal variation that could increase useful genetic variability in this asexually propagated crop. Both exploring the cycle of in vitro culture in which somaclonal variation occurs and the amount of generated polymorphism, are necessary. In this work, preliminary results of somaclonal variation during early cycles of banana in vitro culture are reported. Four randomly selected regenerated plants from the fifth cycle and two samples from the mother plant were analyzed. A total of 36 AFLP primer combinations were assayed, and 24 of them produced amplicons varying among 50- 500 bp. The mother plant presented a total of 125 different amplicons while the regenerated plants jointly showed 131 different amplicons with a mean of 119.75 ± 3.97 per individual. High level of DNA polymorphism (24.43 %) was found among micropropagated plants and, additionally, the occurrence of somaclonal variation at earlier cycles was suggested by multivariate analysis of Principal Coordinates. In this study, somaclonal variation at early cycles of banana micropropagation was validated and the adequacy of AFLP technique to assess it at the molecular level was verified. The phenotypic effects of the detected somaclonal variations remain to be evaluated.

Early detection of somaclonal variation in oil palm callus culture through cytological and SDS-PAGE protein analysis

The major problem of oil palm in vitro propagation is the occurrence of somaclonal variation. This study aimed to obtain somaclonal variation analytical method based on chromosome activities and protein molecular weight level using SDS-PAGE technique on callus culture. Generally, three types of callus were observed, i.e. rooty, nodular friable, and nodular aggregate callus. During mitotic analyses, normal and abnormal cell division of each callus was successfully observed in several phases of mitotic. Rooty callus showed the highest rate of abnormal mitotic divisions up to 7.0 %. In contrast, nodular friable resulted in 3.2 % abnormality rate which is even less than normal plant cell division that was 3.7 %. The most mitotic abnormality type was a reduction-grouping mechanism of the chromosome during methaphase and prophase. SDS-PAGE revealed different protein molecular weight between nodular friable, nodular aggregate, and rooty callus. Protein 20 kDa is a specific protein found in nodular friable callus; this protein was known as a biochemical marker to distinguish embryogenic and non-embryogenic callus.

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

In vitro shoot production, morphological alterations and genetic instability of Melocactus glaucescens (Cactaceae), an endangered species endemic to eastern Brazil

Melocactus glaucescens is a cactus endemic to Brazil. In vitro shoot production represents a viable alternative to conventional propagation, since M. glaucescens grows slowly and requires about ten years to reach its reproductive phase. M. glaucescens can only reproduce by seed and does not ramify or emit lateral shoots. The present work evaluated the morphogenic potential of M. glaucescens by (i) using variations in explant fragmentation and orientation; (ii) the use of plant growth regulators (PGRs); (iii) and the occurrence of somaclonal variation. The results suggest that number of shoots decreased with greater explant fragmentation. Shoot induction can be achieved in culture media with or without PGRs. The evaluation of the morphogenic responses of M. glaucescens explants to PGRs revealed that, when combining naphthalene acetic acid (NAA) with benzylaminopurine (BAP) or kinetin (KIN), BAP yielded a greater number of shoots than KIN. However, with BAP concentrations of 4.44, 8.88, and 17.76μM, shoots were morphologically different from the control plants. The ISSRs analysis showed polymorphisms in shoots produced with or without PGRs and suggests that other factors might affect the genetic stability of M. glaucescens. Our results indicate that M. glaucescens can be propagated using in vitro techniques with or without PGRs. Apical cladode segments can grow in PGR-free medium and can be used as a source of explants. The technique established in this work reduces the removal of seeds from natural populations, and therefore, does not interfere in the reproduction dynamics of this species in its natural habitat.

In vitro micropropagation of the aquatic fern Marsilea quadrifolia L. and genetic stability assessment by RAPD markers

In order to conserve and multiply the aquatic fern Marsilea quadrifolia L., in a long-term in vitro procedure, the effects of different cytokinins, i.e., 6-benzylaminopurine, zeatine riboside, and N6-(2-isopentenyl)adenine, were investigated, varying their concentration and period of supplementation. No clear stimulatory effect on the de novo nodes produced per explant was detected when compared with hormone-free (HF) condition. On the contrary, the rhizome explant micropropagation was inhibited, the inhibition decreasing with the decreasing strength of cytokinins, though without reaching any significant enhancement. Since, as a consequence of the tissue culture procedure, the occurrence of somaclonal variation may introduce genomic alterations, genetic stability was assessed by random amplified polymorphic DNA (RAPD) analysis by comparing eight randomly selected micropropagated plants derived from repeated subcultures, with donor plant. Eighteen different primers generated 189 bands ranging from 100 to 3250 bp, and the same banding profiles were exhibited. No genomic alterations were evidenced in any of the micropropagated plants. Well-developed micropropagated plants were also successfully acclimatized under greenhouse condition. These positive results suggest that the in vitro HF micropropagation could be useful in the development of ex situ conservation programs of M. quadrifolia, even in order to possibly reintroduce the plants in their natural environment.

Nuclear DNA, DNA finger printing and essential oil content variation in callus derived regenerants of Curcuma longa L.

Callus cultures were initiated from shoot base explants of turmeric (Curcuma longa L.) on Murashige and skoog (MS) media supplemented with 2 mg/l 2, 4-dichlorophenoxy acetic acid. Plantlets were regenerated from 90 days old calli on MS media supplemented with 3 mg/l benzyl adenine. Regenerated plants were subsequently transferred to the field. 24 regenerants were analyzed for possible occurrence of somaclonal variation through cytophotometric estimation of 4C nuclear DNA content, analysis of essential oil content and molecular characterization. Significant variation was observed in 4C nuclear DNA content with mean value 8.82 pg and 8.33 pg in 2 regenerants. Analysis of essential oil of 3 regenerants showed significant variation with mean value (0.93 %, 0.73 %, 0.8 %) respectively when compared with the mean oil content 0.48 % of source plant. Inter simple sequence repeat (ISSR) based DNA fingerprinting could also detect variation in regenerants with polymorphism in banding pattern as compared to control. These somaclonal variations detected in callus derived regenerants have enough significance for producing improved varieties of turmeric.

Plant Regeneration and Cellular Behaviour Studies inCelosia cristataGrownIn VivoandIn Vitro

Tissue culture studies of Celosia cristata were established from various explants and the effects of various hormones on morphogenesis of this species were examined. It was found that complete plant regeneration occurred at highest percentage on MS medium supplemented with 2.0 mg/L NAA and 1.5 mg/L BAP, with the best response showed by shoot explants. In vitro flowering was observed on MS basal medium after six weeks. The occurrence of somaclonal variation and changes in cellular behavior from in vivo and in vitro grown plants were investigated through cytological studies and image analysis. It was observed that Mitotic Index (MI), mean chromosome numbers, and mean nuclear to cell area ratio of in vitro root meristem cells were slightly higher compared to in vivo values. However, in vitro plants produced lower mean cell areas but higher nuclear areas when compared to in vivo plants. Thus, no occurrence of somaclonal variation was detected, and this was supported by morphological features of the in vitro plants.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

BackgroundThe recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.).ResultsA modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation.ConclusionsA new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event.

Molecular, C-value and morphological analyses of somaclonal variation in three olive cultivars

The occurrence of somaclonal variation was studied in three olive cultivars ( Olea europaea L.) by using Random amplified polymorphic DNA (RAPD), C-value and morphological analyses. The tissue culture of the cultivars was performed using internode cuttings. The experiment was carried out up to the fifth and seventh subculture in different cultivars. Significant difference was observed in morphological characters among the regenerated plants of different subcultures in all cultivars. Grouping of the genotypes based on morphological data separated some of the regenerated plants from the others due to their morphological differences, however this was not correlated with the time period of subculturing. RAPD analysis produced polymorphic and specific bands in the genotypes studied. Grouping of the genotypes based on RAPD data revealed that in all cultivars the parental genotype and regenerated plants of the first and second subcultures are placed close to each other while the regenerated plants of the latter subcultures stand far from the other genotypes due to their genetic difference. No significant difference occurred in C-value in mother plants and the regenerated plants, indicating that genetic differences are mainly of nucleotide polymorphism/mutations and not due to quantitative change in DNA amount during tissue culture process.

Desempenho in vitro e agronômico de cultivares micropropagadas de morangueiro em vários subcultivos

O morangueiro é uma espécie multiplicada vegetativamente, o que facilita a progressão de viroses a partir de mudas infectadas. A utilização de matrizes provenientes do processo de cultivo in vitro tem sido uma prática na obtenção de mudas isentas de patógenos. Diversos protocolos de micropropagação recomendam a utilização de quatro subcultivos, para preservar a identidade genética dos clones e manter alta taxa de multiplicação. Desta forma, visando a elevar a eficiência do processo de micropropagação, foram utilizadas duas cultivares de morangueiro Oso Grande e Vila Nova, submetidas a nove subcultivos, sendo avaliadas a taxa de multiplicação e a variação somaclonal. Durante o cultivo in vitro, foi avaliada a taxa de multiplicação, a altura e o número de folhas por propágulo. Na aclimatização, determinaram-se a taxa de sobrevivência, a área foliar, a massa fresca e seca da parte aérea e da raiz. As mudas obtidas a partir de matrizes também foram avaliadas quanto ao comportamento agronômico, em ambiente protegido. A maior taxa de multiplicação foi observada durante o 2º e o 3º subcultivos com média de sete a oito propágulos/ explante, respectivamente. Não foram observadas alterações fenotípicas nas duas cultivares, durante as fases de micropropagação, aclimatização e crescimento em ambiente protegido. A cv. Oso Grande apresentou maior desempenho agronômico que a 'Vila Nova'.

REGENERATION AND FIELD EVALUATION OF BANANA (MUSA ACUMINATA, AAA GROUP) PLANTS FROM PROLIFERATING INFLORESCENCE-DERIVED EMBRYOGENIC SUSPENSION CULTURES

Somatic embryogenesis in banana is considered of great promise for the mass propagation of this crop. However, the limited availability of embryogenic competent tissues, together with the possible occurrence of somaclonal variation in suspension-derived plants, has restricted its commercial application. Recently, in vitro proliferating inflorescences have been proposed as a new source of competent explants for the establishment of regenerable embryogenic cell suspensions. The present work focuses on the behavior of the first plants produced with this novel methodology. 'Dwarf Cavendish' banana plants of the local selection 'Gruesa' were regenerated, acclimatized and established in the field. Phenological, morphological and productive parameters were evaluated during the first production cycle under commercial greenhouse conditions in the Canary Islands.

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l −1 NAA + 0.1 mg l −1 BA, and MS + 2.0 mg l −1 IAA + 0.5 mg l −1 Kinetin), and two proliferation media (MS + 0.1 mg l −1 NAA + 0.2 mg l −1 BA, and MS + 4.0 mg l −1 IAA + 2.0 mg l −1 Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.

Somaclonal variation in micropropagated Heliconia bihai cv. Lobster Claw I plantlets (Heliconiaceae)

The occurrence of somaclonal variation is described in various cultures of agronomic interest. Such variation can be of benefit in the development of new flower varieties. In this study, the occurrence of somaclonal variation in micropropagated changes of Heliconia bihai cv. Lobster Claw I was investigated. Stem apexes were introduced in MS culture media with the addition of 2.5 mg L-1 of benzylaminopure (BAP) and 500 mg L-1 of sodium cefotaxime. After selecting the apex stem, it was sub-cultivated in MS media and supplemented with 4.0 mg L-1 of BAP to induce side buds. To conduct the trial, 2,000 plants were selected and compared with plants originated from rhizomes. To calculate the percentage of the variants, the plant stature, the form and color of leaves and pseudostem were evaluated. The plants with buds presenting the same type of variation were considered as variants. The occurrence of three types of somaclonal variants was observed: Variation of the Chlorophyll in the Leaf, Low Stature Variant and Pseudostem and Petiole Color Variant, the latter with ornamental potential. The somaclonal variation rate for Heliconia bihai cv Lobster Claw I, under the proposed conditions, was 61.40%.

Assessment of somaclonal variation in Dieffenbachia plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.

Growth of tissue culture–regenerated salt marsh monocots in a simulated marsh field plot: Implication for wetland creation and restoration

Phenotypically and genetically variable salt marsh plants are needed for wetland creation and restoration efforts. Selected tissue culture regenerants of five salt marsh monocots, Spartina patens, Spartina alterniflora, Juncus gerardi, Juncus roemerianus, and Scirpus robustus, were planted in a simulated marsh field plot that was flood-irrigated with 10 ppt salt water to compare their phenotypic variation for potential use in wetland projects. Plant growth was evaluated after one growing season. Phenotypic variation among regenerants was found in S. alterniflora, S. patens and J. gerardi, indicating the occurrence of somaclonal variation. In S. alterniflora, significant differences occurred among regenerants in stem density. In J. gerardi, significant differences occurred in height and clone circumference. In S. patens, two of the nine regenerants exhibited higher biomass and stem density than some of the other regenerants. By using the random amplified polymorphic DNA (RAPD) technique, genome DNA variation in S. patens regenerants was detected. Genetic variation not only occurred among phenotypically different regenerants, but also among those phenotypically similar for the characteristics measured. Tissue culture–regenerated plants often have desirable genetic characteristics and adaptability as a result of somaclonal variation and may enable a species to perform its ecological functions in created or restored wetlands where ideal environments cannot be achieved. Thus, some previously unrestorable sites may be restorable or marginal marshes made more productive.

Large-scale production of enhancer trapping lines for rice functional genomics

Using a rapid and efficient Agrobacterium tumefaciens mediated rice transformation protocol, we had got about 100,000 independent enhancer trapping lines of japonica rice variety Nipponbare. T 1 seeds had already been obtained from 60,000 lines. Regeneration of transgenic plants could be as short as 3 months starting from callus induction, thus the occurrence of somaclonal variation was reduced to a minimum level. The frequencies of hygromycin-resistant callus induction and plant regeneration were over 90 and 80%, respectively. PCR and Southern blot analysis showed that about 95% hygromycin resistant plants were transgenic. Southern blot and segregation analysis of T 1 seeds showed that about 40% transgenic plants contained single copy insertion, and the average copy number was 1.7. Histochemical assay of T 0 plants revealed expression patterns in root, leaf, embryo and endosperm of the reporter gene β-glucurodinase (GUS). This large-scale enhancer trapping pool provides a valuable tool for functional analysis of rice genome.