Analyzing somaclonal variation in micropropagated bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Summary In a micropropagation program, where it is of paramount importance to produce true-to-type planting material, somaclonal variation of any kind is undesirable. Variation among plants regenerated from tissue culture is termed ‘somaclonal variation’. In banana, somaclonal variants of different type have been reported with regard to plant morphology. This article discusses various factors due to which somaclonal variations may arise. Somaclonal variation may be detected by visual screening or by using molecular markers such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and by cytological studies. Although somaclonal variation is undesirable in the context of micropropagation, it can be used to advantage for genetic improvement of banana, as has been described.     

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.     

Micropropagation of Daylily (<i>Hemerocallis fulva</i>) from Crown-Tip Explants and Assessment of Somaclonal Variation of <i>in Vitro</i>-Propagated Plants Using SCoT Markers

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriate micropropagation protocol and growth regulators for commercial scale multiplication. In this research, nine multiplication media (MM) augmented with different concentrations of 6-benzyl adenine (BA), Kinetin (Kin), and Thidiazuron (TDZ), Three rooting media (RM) supplemented with three levels of α-naphthalene acetic acid (NAA) and three types of soil mixtures (v/v); Coco peat/Vermiculite/Sand (CVS), Peat moss/Perlite/Sand (PPS) and Peat moss/Perlite (PP) were used in the micropropagation protocol of daylily plants. MM2 showed the maximum shoot length and the number of leaves, while MM9 showed the maximum number of shoots. The RM1 showed the maximum root length and the number of roots. During acclimatization, CVS, PPS, and PP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plant height and diameter. The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted (SCoT) Markers. Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to 1598 bp size. Thirty one out of 51 loci were presented in the mother plants. 40 loci were polymorphic, 11 were monomorphic and 7 were unique. The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with 5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway, thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used during multiplication subcultures. This research can be successfully used for other ornamental and medicinal plants’ bulk multiplication, germplasm conservation, and future genetic improvement.     

植物体细胞无性系变异研究进展

植物体细胞无性系变异是植物组织培养中的普遍现象,泛指在植物细胞、组织和器官培养过程中, 培养细胞和再生植株中产生的遗传变异或表观遗传学变异。植物体细胞无性系变异的发生有其遗传学基础, 可从形态学、细胞学、生物化学和分子生物学等多个方面对其进行综合检测和鉴定。植物体细胞无性系变异是植物育种的有利资源, 但同时也是植物微繁和遗传转化工作中需要克服的一大难题,一直被众多研究者所关注。本文分别从细胞学和分子生物学两个层次综述了植物体细胞无性系变异的遗传学基础及其鉴定方法的研究进展,并就其在植物品质改良中的应用现状、存在的问题和应用前景进行了讨论。     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

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.     

Microsatellite DNA somaclonal variation in micropropagated trembling aspen (Populus tremuloides)

Microsatellite DNA markers of ten simple sequence repeat (SSR) loci were used to examine somaclonal variation in randomly selected micropropagated plantlets derived from three different Populus tremuloides donor trees (genotypes). The plantlets were obtained from tissue cultures of dormant vegetative buds, and those derived from the same donor tree, grown in the greenhouse, did not exhibit any sign of visible morphological variation. No microsatellite DNA variation was observed among 13 somaclones of one tree and 4 somaclones of another tree at eight of the ten SSR loci. However, despite the small number of micropropagated progeny per tree sampled, microsatellite DNA variation was detected among the plantlets derived from the same donor trees at two SSR loci. The primer pair for the SSR locus PTR5 revealed somaclonal variation in 1 out of the 13 plantlets obtained from one genotype, while the primer pair for the PTR2 SSR locus revealed somaclonal variation in one out of the four plantlets obtained from another genotype. The variation at the PTR2 locus resulted in the appearance of a new allele of increased size, possibly due to an addition of the repeat units, while the variation at the PTR5 locus resulted in the appearance of third allele, presumably due to the presence of a single extra chromosome or duplication of a chromosomal segment. These results demonstrate that the genetic fidelity of micropropagated plants of P. tremuloides cannot always be assured and somaclonal variation can occur even when tissues of well organized vegetative buds are used for tissue cultures; that somaclonal variation cannot always be detected at the gross morphological level; and that microsatellite DNA markers provide useful and sensitive markers for determining the clonal fidelity and somaclonal variation in P. tremuloides.     

Genetic analysis of enhanced-axillary-branching-derived Eucalyptus tereticornis Smith and E. camaldulensis Dehn. plants

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. Received: 8 October 1996 / Revision received: 22 July 1997 / Accepted: 30 July 1997     

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

In vitro biotechnological approaches on <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews: advancements and opportunities

In vitro biotechnological advancement of Vanilla plays a major role in germplasm conservation, genetic engineering, accelerated clonal multiplication and production of disease-free plants with enviable aromatic properties. Several attempts have been taken place for the establishment of efficient in vitro protocol for Vanilla in the past few decades. Optimization of various conditions during different phases of micropropagation, for instance development of in vitro aseptic cultures, multiple shoot regeneration, rooting and acclimatization of the plantlets are discussed in this review. In addition to basic micropropagation techniques, various other in vitro biotechnological applications such as clonal fidelity assessment, genetic transformation, synthetic seed technology and cryopreservation are also highlighted. Apart from the existing data, applied aspects like embryo rescue, mutation breeding, genetic engineering, protoplast fusion, somaclonal variation, in vitro enhancement of vanillin production through cell suspension culture, hairy root culture or bioreactors and cryopreservation need to be investigated further. Overall, the current review gives a synopsis on progress and prospect of in vitro culture of Vanilla.     

Progress in the biotechnology of trees

An increasing world population and rise in demand for tree products, especially wood, has increased the need to produce more timber through planting more forest with improved quality stock. Superior trees are likely to arise from several sources. Firstly, forest trees can be selected from wild populations and cloned using macropropagation techniques already being investigated for fruit tree rootstocks. Alternatively, propagation might be brought aboutin vitro through micropropagation or sustained somatic embryogenesis, with encapsulation of the somatic embryos to form artificial seeds. Tree quality could be improved through increased plant breeding and it is likely that experienced gained, to date, in the breeding of fruit species will be useful in devising strategies for forest trees. Since the development of techniques to regenerate woody plants from explant tissues, cells and protoplasts, it is now feasible to test the use of tissue culture methods to bring about improvements in tree quality. Success has already been achieved for tree species in the generation of somaclonal and protoclonal variation, the formation of haploids, triploids and polyploids, somatic hybrids and cybrids and the introduction of foreign DNA through transformation. This review summarizes the advances made so far in tree biotechnology, and suggests some of the directions that it might take in the future.     

Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev)

Two different DNA-based techniques, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers, were used for fingerprinting kiwifruit genotypes and for detecting undesirable genetic variation in micropropagated plants. The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Two cluster analyses were performed to express - in the form of dendrograms - the relationships among the genotypes and the genetic variability detected. Both DNA-based techniques were able to amplify all of the genotypes, but only SSR markers could detect genetic variation induced in micropropagated plants of cv. Tomuri. Two hypotheses were formulated to explain these results, both of them are in agreement with the results obtained using these two types of molecular markers. We conclude that when the tissue culture technique is used, the analysis of somaclonal variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for SSR and RAPD markers.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

In vitro manipulation and propagation of medicinal plants

Well developed techniques are currently available to help growers meet the demand of the pharmaceutical industry in the next century. These protocols are designed to provide optimal levels of carbohydrates, organic compounds (vitamins), mineral nutrients, environmental factors (e.g. light, gaseous environment, temperature, and humidity) and growth regulators required to obtain high regeneration rates of many plant species in vitro and thereby facilitate commercially viable micropropagation. Well-defined cell culture methods have also been developed for the production of several important secondary products. An overview of the regeneration of medicinal plants by direct and indirect organogenesis and by somatic embryogenesis from various types of explants is presented, and the use of these techniques combined with other biotechnological approaches to improve medicinal plants through somaclonal variation and genetic transformation is reviewed.     

植物体细胞无性系变异研究进展

植物体细胞无性系变异是植物组织培养中的普遍现象,泛指在植物细胞、组织和器官培养过程中,培养细胞和再生植株中产生的遗传变异或表观遗传学变异。植物体细胞无性系变异的发生有其遗传学基础,可从形态学、细胞学、生物化学和分子生物学等多个方面对其进行综合检测和鉴定。植物体细胞无性系变异是植物育种的有利资源。但同时也是植物微繁和遗传转化工作中需要克服的一大难题,一直被众多研究者所关注。本文分别从细胞学和分子生物学两个层次综述了植物体细胞无性系变异的遗传学基础及其鉴定方法的研究进展．并就其在植物品质改良中的应用现状、存在的问题和应用前景进行了讨论。     

Gerbera micropropagation

Gerbera jamesonii (gerbera) is an important cut-flower in the global floricultural industry. Micropropagation is the main system used to clonally propagate gerbera in vitro resulting in the production of millions of plantlets each year. Numerous types of explants and protocols for micropropagation have been established and used for gerbera. Shoot tips are the commonly used explant while adventitious shoot induction from the capitulum is also a popular method. Most papers in the literature have focused on testing the influence of different types and combinations of plant growth regulators with the aim of improving the regeneration and multiplication stage of one or few cultivars. Genotype is one of the most influential factors on the response of gerbera in vitro. Despite this, no successful universal protocol has yet been developed for multiple cultivars, limiting the usefulness of current protocols for commercial biotechnology labs. Slow-growing endogenous bacteria are one of the most important problems in gerbera micropropagation but require more studies on control and prevention. Individual shoots are normally easy to root, usually in excess of 90% of plantlets, but the acclimatization stage requires improvements and new technologies to increase the survival of plants. Epigenetic variations in micropropagated gerbera are frequently observed only with high concentrations of cytokinins in the culture medium but somaclonal variation is rare.     

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.     

Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers

Use of high levels of growth regulators during micropropagation results in undesirable clonal variability in important commercial crops such as banana. The present study investigated the effects of high levels of cytokinins on micropropagation in banana (genotype AAB), and the genetic stability of plantlets was assessed using RAPD and ISSR markers. Cytokinins, such as BA and kinetin were added to the routine shoot multiplication medium at concentrations up to 10 mg l⁻¹. After 12 weeks of culture involving three subcultures, the maximum number of shoot buds were produced in cultures receiving either 5 mg l⁻¹ BA (80 shoot buds) or 4 mg l⁻¹ kinetin (62 shoot buds). Certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro. To check the genetic stability, RAPD and ISSR profiles of micropropagated plantlets obtained from different cytokinin-treatments were compared with control microplants maintained on MS medium as well as the field-grown mother plant. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands. Thus a total of 17,400 bands were generated showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation in all the plantlets analysed. Based on these results a protocol for high rate shoot multiplication was worked out leading to uniform shoot production.     

Evaluation of genetic stability using FRAPD markers as novel method along with antioxidant and anti-diabetic properties of micropropagated <Emphasis Type="Italic">Salacia chinensis</Emphasis> L.

Salacia chinensis L., a perennial medicinal plant, is well-known for its well-documented anti-diabetic properties. The daily growing demand in pharmaceutical industry is stimulating the conservation and wide-ranging production of the plant using plant tissue culture techniques (micropropagation). In the present study, the plants generated by direct micropropagation from nodal explants were assessed using fluorescently labeled RAPD (FRAPD) primers. Although standard RAPD primer bands in agarose gel showed genetic stability, using FRAPD analysis in genetic DNA sequencer as a novel strategy showed more accurate and reliable method has indicated by the evidence in 5% genetic variation. Antioxidant and anti-diabetic activities of micropropagated plants versus mother plant were examined using DPPH, FRAP, α-amylase, and α-glucosidase assays. The results showed that the micropropagated plants, which are able to produce higher amount of secondary metabolites than the mother plant, possess higher in vitro antioxidant and anti-diabetic properties.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.