Effect of dehydration duration of apices on characteristics of in vitro plants of <Emphasis Type="Italic">Fragaria vesca</Emphasis> after cryopreservation

Effect of different duration of dehydration of the apices isolated from in vitro plants on genetic stability was investigated in regenerated plants of wild strawberry (Fragaria vesca L., var. alpine) recovered after cryopreservation according to a precultivation-dehydration protocol. Plant material belongs to a clone (cv. Reine des Vallees) that has been maintained in vitro for more than 25 years in Timiryazev Institute of Plant Physiology. It was shown that duration of desiccation the apices before freezing appreciably affected the rate of postcryogenic recovery of plant growth and coefficient of their subsequent propagation. After 5-h-long desiccation, apices were notable for the highest growth rate. The plants restored from such apices also had the highest coefficient of propagation. For DNA analysis, the samples of leaves were taken separately from each plant after hardening and after cryopreservation. According to the results of RAPD, ISSR, and REMAP analyses, the plants from the chosen clone of strawberry showed some genetic variation prior to cryopreservation (percentage of polymorphic fragments was 9.0%). Plant adaptation to cold did not change the level of genetic variation. Among postcryogenic regenerants, morphologically modified plant forms were not observed, with the level of DNA marker variation decreasing almost two times irrespective of the duration of dehydration. However, in one plant restored after 5-h-long dehydration and cryogenic freezing, a 1200 bp fragment of DNA was lacking, which was detected in all other examined samples (frequency of deviation was 0.9%). Earlier, we did not reveal plant polymorphism of investigated strawberry clone associated with this fragment. Probably, this modification of DNA resulted from the exposure of plant material to dehydration and freezing in liquid nitrogen.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

AFLP analysis of somaclonal variations in Eucalyptus globulus

DNA variations of forty-eight Eucalyptus globulus plants, regenerated by successive culture from seven different explants were assessed by AFLP analysis using 18 primer combinations. At least one variation showed 66.7 % of the analyzed plants, and the numbers of polymorphic bands per plant ranged from 1 to 22. The proportion of polymorphic fragments did not correlate with the numbers of the regenerated plants. However, the more times of successive culture were done the more of polymorphic bands were found within the groups. On average, between 97.39 and 99.88 % of all fragments were shared within the same group. AMOVA analysis showed 39.33 % of the variation was found among the accessions that originated from different calli while 60.67 % was from same calli.     

Oryzacystatin Exogenously Introduced into Protoplasts and Regeneration of Transgenic Rice

Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3′-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration and expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic plants.     

Genetic stability analysis of chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an encapsulation-dehydration cryopreservation protocol

Genetic stability in chrysanthemum (cultivar ‘Pasodoble’) apices was studied at each step of an encapsulation-dehydration cryopreservation protocol: control shoots (A), nodal segments after cold treatment (N), apices after osmotic stress (0.3 M sucrose) and cold treatment (P), encapsulation and culture in 0.8 M sucrose (S), dehydration (D), and cryopreservation (Cr). Two different markers were employed: RAPDs and AFLPs. Throughout the process, the origin of the apices (in vitro shoot from which they were excised) was recorded. Eight complete lines (from which DNA could be amplified after all the steps considered) were studied. Two out of twelve arbitrary primers showed polymorphisms. Three RAPD markers were replaced by three new ones in the Cr sample in one line. Using a different primer, a 700 bp fragment was absent from all samples from the 0.3 M sucrose-culture step (‘P’) onwards, in all the lines studied. The sequences of these fragments were studied to find similarities with known sequences. Polymorphic AFLP fragments were also observed, and most of the differences appeared from step ‘P’ onwards, pointing out the possible effect of this process (preculture on 0.3 M sucrose) in the DNA variation. These results show that genetic variation can appear throughout the cryopreservation process, and the low temperature itself is not the only stress risk of the technique. Therefore, genetic stability of the regenerants obtained after cryopreservation should be monitored.     

Assessment of genetic instabilities induced by tissue culture in alkaligrass

Alkaligrass (Puccinellia chinampoensis Ohwi), one of the important forage grasses in saline-alkalieroded grasslands, has been proved to be invaluable for improving saline-alkali soils. However, little is known of its genetic instabilities during in vitro culture for its artificial breeding. In this paper, a simple and efficient regeneration system of mature seed-induced calli in alkaligrass was established, and the somaclonal variation in the regenerated plants was assessed by inter-simple sequence repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. 18 randomly chosen regenerants were subjected to ISSR and REMAP analysis with the shoot from the same grain of seed as the control. ISSR analysis showed that of the 145 scored bands, 13 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 8.97%. REMAP analysis revealed that 4 out of 127 scored bands were polymorphic, a genetic variation frequency of 3.15% occurred. Cluster analysis indicated that the genetic similarity index calculated on the basis of ISSR data or REMAP data among the 18 regenerated plants and the donor plant was 0.974 and 0.996 respectively. All the results confirmed that somaclonal variation was induced by tissue culture in alkaligrass at a higher frequency, and indicated that the regeneration system could be a viable option for genetic improvement of alkaligrass by biotechniques.     

Comprehensive molecular characterization of tissue-culture-derived Hordeum marinum plants

Summary Scuttelar calli of Hordeum marinum readily and efficiently regenerate functional plants. In order to assess genetic variability among the regenerants we employed multiple analytic tools, which included molecular and biochemical assays. Total DNA extract from regenerated plants was digested with at least two restriction enzymes and hybridized to four nuclear and six mitochondrial coding sequences, in addition to one nuclear and three mitochondrial noncoding probes. SDS-PAGE analyses of hordein extracted from seeds of regenerated plants and activity assays of -amylase were also performed. The nuclear and mitochondrial genomes of 50 regenerated plants demonstrated relative stability when assessed with coding sequences and by biochemical analyses. However, the mitochondrial noncoding probes revealed one qualitative somaclonal variant characterized by a loss of a hybridizing fragment. Moreover, changes in the methylation patterns of the rRNA genes and the nontranscribed spacer were revealed in another regenerated plant. The albino plant regenerated was characterized by a loss of three chloroplast DNA BamHI fragments.     

Freezing of dehydrated calli of spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) in liquid nitrogen and their morphogenetic potential

A cryopreservation method developed earlier was modified for freezing of calli derived from mature embryos of four spring wheat (Triticum aestivum L.) pure lines. The effects of particular stages of cryopreservation protocol on water content, number of calli recovering growth, and rate of morphogenesis were analyzed. Regrowth was observed in 90.5–100% of calli after dehydration and in 93.3–100% after freezing-thawing. Dehydration, but not freeze-thawing significantly decreased the frequency of morphogenetic variation.     

Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild rice Oryza meyeriana Baill

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Somatic hybridization between Lycopersicon esculentum and Lycopersicon pennellii

Summary Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type (¹³⁷Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.     

Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.     

Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild rice Oryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line. Project partially supported by the National Natural Science Foundation of China (Grant No. 392704361, Foundation for Outstanding Young Teachers of China, State Key Program of China and Natural Science Foundation of Hubei Province, China.     

Evaluation of genetic stability in cryopreserved Prunus

Summary The evaluation of the genetic stability of Prunus Ferlenain plants regenerated from cryopreserved apices was investigated. The analysis of plants recovered from frozen material was performed at the phenotypic (developmental competence), cytological (chromosomal preparations) and molecular level [random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)]. No genetic change was detected among the plantlets regenerated from frozen apices in comparison to the non-frozen material, including leaves of the mother tree kept in an orchard and vitroplantlets regenerated from non-frozen apices. This result suggested that the procedure used for Prunus cryopreservation could be used for long-term conservation. The relevance of each strategy for the genetic stability evaluation of the cryopreserved material is discussed.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Cryopreservation of Jiangxi Yanshan Red Bud Taro (Colocasia esculenta var. cormosus cv. Hongyayu) Embryogenic Calli by Encapsulation dehydration

Cryopreservation of Jiangxi Yanshan red bud taro (Colocasia esculenta var. cormosus cv. Hongyayu) embryogenic calli by encapsulation dehydration was studied. The results showed that the optimal preculture condition of cryopreservation by encapsulation dehydration was precultured on MS medium supplemented with 075mol·L-1 sucrose for 3 days. The optimal dehydration method was dehydration by sterile air in a laminar flow hood for 7h or sterile dry silica gel for 11h. the optimal thawing temperature was 37℃ (2min). The optimal culture condition after cryopreservation was first in the dark for 7d and then transferred to the photoperiod of 14h·d-1. The average survival rate of embryogenic calli after cryopreservation by encapsulation dehydration was about 45%. Cryopreservation time and whether the removal of encapsulated calcium alginate had no significant impact on the survival rate. Morphological and cytological study demonstrated that the regenerants were genetically and morphological stable.     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Molecular analysis of plants regenerated from embryogenic cultures of wheat (Triticum aestivum L.)

Total DNAs of plants regenerated from immature embryo-derived 2-month-old embryogenic calli of wheat (cultivars Florida 302, Chris, Pavon, RH770019) were probed with six maize mitochondrial genes (atpA, atp6, apt9, coxI, coxII, rrn18-rrn5), three hypervariable wheat mitochondrial clones (K, K3, X2), five random pearl millet mitochondrial clones (4A9, 4D1, 4D12, 4E1, 4E11) and the often-used wheat Nor locus probe (pTA71), in order to assess the molecular changes induced in vitro. In addition, protoplast-derived plants, and 24-month-old embryogenic and non-embryogenic calli and cell suspension cultures of Florida 302 were also analyzed. No variation was revealed by the wheat or millet mitochondrial clones. Qualitative variation was detected in the nonembryogenic suspension culture by three maize mitochondrial genes (coxI, rrn18-rrn5, atp6). A callus-specific 3.8-kb Hind III fragment was detected in all four cultivars after hybridization with the coxI gene. The organization of the Nor locus of the plants regenerated from Florida 302 and Chris was stable when compared to their respective control plants and calli. The Nor locus in regenerants of Pavon and RH, on the other hand, was found to be variable. However, Nor locus variability was not observed in 14 individual seed-derived control plants from either Pavon or RH sources. In Pavon, a 3.6-kb Taq I or a 5.6-kb Bam HI+ Eco RI fragment was lost after regeneration. In one of the RH regenerants, which lost a fragment, an additional fragment was observed.     

Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]

 Trees were regenerated from six white spruce embryogenic clones after cryopreservation for 3 and 4 years, respectively. Genetic stability was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints. Somaclonal variation was detected in some in vitro embryogenic cultures 2 and 12 months after they were re-established following cryopreservation but not in the corresponding regenerated trees. These results suggest that trees regenerated from cryopreserved cultures in subsequent years are primarily genetically stable in the genomic regions tested and that variation observed due to the in vitro culture process infrequently affects trees regenerated from normally maturing and germinating somatic embryos. However, trees regenerated from somatic embryos that matured or germinated abnormally in in vitro culture exhibited altered RAPD fragment patterns. Received: 20 July 1998 / Revision received: 15 October 1998 / Accepted: 14 December 1998     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.     

Cytogenetic characterization of embryogenic callus and regenerated plants of Pennisetum americanum (L.) K. Schum

Summary Embryogenic calli were derived from cultured segments of immature inflorescences of Pennisetum americanum (pearl millet). The original explants as well as the embryogenic calli and the plants regenerated via somatic embryogenesis were examined cytogenetically. Embryogenic calli were predominantly diploid (2n=14) after one month and six months in culture (92% and 76%, respectively). Tetraploid and aneuploid cells were observed in the original explant (2.5% and 1.2%) as well as in one (4.0% and 4.0%) and six-month-old calli (10.0% and 14.0%). Plants were regenerated from calli that had been in continuous culture for two, four and six months. Of the 101 regenerants, 100 were diploid and 1 was tetraploid. The tetraploid was an albino as were three of the diploid regenerants. Examination of 30 of the regenerants in meiotic diakinesis, anaphase I, anaphase II and quartet stages revealed no cytogenetic differences between control and regenerated plants. Gel electrophoresis for total protein content and alcohol dehydrogenase and malate dehydrogenase activity also did not reveal any differences between the controls and regenerants. The results of this study show that a slight shift toward aneuploidy and polyploidy may occur in embryogenic cultures, but there also is a strong selection in favor of plant regeneration from cytogenetically normal cells.