HMW-GS variation of somaclones and its variants in Triticum aestivum

Research on somacolonal variation in hexaploid wheat (Triticum aestivum L.) has been conducted a lot and morphological, cytological, and biochemical variations have been observed. Evidence for gene mutations has been obtained through the analysis of gliadin and glutenin in wheat grain, but loci of mutations have not been located. The aims of this study are: firstly, to detect mutation loci of specific gene (Glu-1) to provide exact evi-dence for gene mutation by analyzing changes of high-molecular-weight glutenin

Micropropagation of Daylily(Hemerocallis fulva)from Crown-Tip Explants and Assessment of Somaclonal Variation of in Vitro-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 ofα-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.

Somaclonal variations of Soybeans (<i>Glycine Max.</i>L. Merr) stimulated by drought stress based on random amplified polymorphic DNAs (RAPDs)

In soybeans, drought stress causes 50% yield losses. Breeding for drought tolerance in soybeans has been widely developed using various methods, among which is polyethylene glycol (PEG-6000) induction to simulate drought in vitro. In a previous experiment, three somaclones with different levels of tolerance were generated. The objectives of this research were to determine the RAPD patterns of those somaclones and to investigate the correlation of the RAPD patterns to the drought tolerance characteristics. The results showed eleven RAPD primers capable of amplifying the DNA genome of soybeans, among which four primers were monomorphic and seven were polymorphic. Two of the polymorphic primers, OPK7 and OPK12, are capable of differentiating medium tolerance traits from other traits. Bands that are specific for medium tolerance against drought were 450 bp and 650 bp in size, generated by the OPK7 primer, and the band of 2000 bp, generated by the OPK12 primer. However, there was no band capable of differentiating between sensitive and tolerance varieties/lines, although some changing of the DNA sequence was detected in this research. This indicates that there are other factors responsible for the expression of drought tolerance.

Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.

In vitro Screening of Fusarium Wilt-resistant Germplasm Resources of Red Edible Seed Watermelon( Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao)

[ Objective] This study aimed to establish an appropriate technology system for in vitro screening of Fuzarium wilt-resistant germplasm resources of red edible seed watermelon and obtain variants resistant to fusaric acid, thus providing resistant materials for breeding Fusarium wilt-resistant red edible seed watermel- on. [ Method] Using Zhongxin No. 1 red edible seed watermelon advemitious buds as screening materials and fusaric acid （FA） as a stress agent, in vitro screen- ing of Fusarium wilt-resistant red edible seed watermelon clonal variants and identification of Fusarium wilt-resistance of the germplasm resources of red edible seed watermelon were performed. [ Result] The results showed that the appropriate FA for in vitro screening of Fusarium wilt-resistant red edible seed watermelon vari- ants was 15 mg/L. In vitro screening system for Fusarium wilt-resistant red edible seed watermelon variants was established preliminarily and FA-resistant regenera- ted plants were obtained. Among the 36 germplasm resources of red edible seed watermelon, there were 2 highly resistant materials, 6 moderately resistant materi- als, 11 slightly resistant materials and 17 highly susceptible materials. [ Conclusion] This study confirmed preliminarily that in vitro screening method is effective for obtaining resistant materials of red edible seed watermelon.

Functional Analysis for Rolling Leaf of Somaclonal Mutants in Rice (Oryza sativa L.)

This study was carried out to facilitate the functional analysis of rice genes. Some 297 insertion plants (1.7%) of the entire lines with the endogenous retrotransposon Tos17 were produced. Phenotypes of these plants in the S2 genera-tion were observed in the field according to different leaf types. Rolling leaf mutants showed thinner sclerenchyma-tous cells, defective arrangement of vascular bundles, and well-formed bulliform cells as compared to the parental cultivar. Two new copies of Tos17 were detected in the rolling leaf type. In the new leaf type, the copy number and activation of Tos12, 15 did not appear as ‘Ilpum’. Flanking sequence tag (FST) analysis of Tos17 in the rolling leaf mutant indicated that new copies of Tos17 were transposed on chromosomes 11 and 12. Annotated homologues of the tagging genes on chromosome 11 were arabinoxylan rabinofuranohydrolase isoenzyme AXAH-I and II. The tagging gene in chromosome 12 was highly correlated with 6 kinds of genes including a transcript regulated factor and a rough sheath 2-like protein. This rolling leaf and flanking sequence data will stimulate the functional analysis of rice genes.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

Resistance to Bacterial Leaf Blight in a Somaclonal Rice Mutant HX-3 at Cellular Level

The interaction between rice host and its pathogen Xanthomonas oryzae pv. oryzae (Xoo) at cellular level was studied by using a resistant somaclonal mutant HX-3 and its susceptable donor Minghui 63. After inoculation with Xoo strain Zhe 173 (Chinese pathotype Ⅳ), the activity of superoxide dismutase (SOD) and peroxidase (POD) in the callus of Minghui 63 was increased dramatically, and the active oxygen(O2 ) was produced at a higher rate; Meanwhile, the callus grew slowly with the reduction of protein content Compared to the activity of SOD and POD, the production rate of Oa and the fresh weight in HX-3 callus varied little after the inoculation It could be proposed that there were great differences between the resistance of HX-3 and Mighui 63 at cellular level. There was no difference detected concerning resistance to bacterial leaf blight in HX-3 between the plant and the callus.

Study on Somaclonal Variation of Spring Wheat

Somaclonal variation of calli and regenerated plants of spring wheat were detected by using technique RAPD in the study. Calli at different culture stages and regenerated plants derived from young spikes and immature embryos were used as materials. Molecular variation could be reflected from electrophoresis pattern of RAPD fragments at different culture stage in calli, and in regenerated plants derived from different explants, even no phenotype variations were found. Somaclonal variation in calli and in regenerated plants appeared regularly: A higher frequency of variation in hybrids F2 was detected than that of the cultivar that is stable genetically. High variation frequency of RAPD fragments appeared in calli when cultured 75 days. The identical variations of RAPD fragments were observed in calli and in the regenerated plants induced from different genotype or explants. The variation frequency detected is higher in regenerated plants than that of in calli. RAPD could be applied easily and simply to determine variation in level of DNA at each stage cultured in vitro.

Vital Parameters Assessments of Starvation Tolerance of in vitro Populus alba Culture

Populus alba is a large woody deciduous plant.The plant has been introduced to shooting,then multiplication of rooting on Murashige and Skoog(MS)medium.This work was designed to estimate the effect of two factors(low levels of 1-Naphthaleneacetic acid NAA and sucrose)on P.alba response resulting in 6 treatments compared to the control,with twelve measured responses.There was a significant difference in some measurements in morphology,like plantlets fresh-weight,shoot-,root-length,and leaf number.In the physiological measurements,there were significant differences in all the measured parameters.The low concentrations of sucrose and media composition/power(MS grams/L)led to starvation in plants;however,these conditions led to enhancement in some morphological and physiological parameters to overcome the starvation effect,compared to the control.The RAPD-PCR molecular marker(four decamers)was used to evaluate the new individuals’genetic variation(instability),resulting in a total polymorphism percentage of 50.83%.It was formerly known that the plantlets were identical to each other and to the mother plant.In this study,however,the use of distinct media power,hormonal and sucrose levels resulted in molecular variation reflected in P.alba’s morphological and physiological responses.

Studies on Immature Embryo Culture In vitro in Wheat： Genetic stability analysis of somatic variation in main agronomic chara

The genetic stability of variations of main agronomic characteristics in progenies from the regenerated plants of immature embryo culture in vitro was studied. The results showed that the variations of earliness in maturity, high 1000-grain weight, plant height and grain weight per spike were heritable and tended to be stable in IE3 and completely stable in IE4 The wide variation of main agronomic characteristics induced by embryo culture in vitro provided the probability of selection in wheat improvement. Some somaclonal lines with useful variations could be used directly in wheat production.

Somaclonal variations as a mean for obtaining regenerants with different growth rates in poplar (<i>Populus ×berolinensis</i>Dipp.)

Adventive regenerants of Populus × berolinensis Dipp. were obtained on 1/2 MS salts with sucrose, vitamins, BA, TDZ and NAA using stem, petiole and leaf explants. They differed from each other in plantlet dimensions. More than 50 clones were produced from initial regenerants by excision and rooting of apexes and lateral shoots. Differences in stem length and thickness were observed between 200 field plants produced from in vitro plantlets. Differences in stem length were conditioned mainly due to different number of internodes and not by internodes’ lengths. Plants grown from cuttings excised from highest and smallest plants retained the abilities of mother plants to grow with different rates. It is concluded that somaclonal variability can be used for selection of fast growing poplar plants which are necessary for industrial plantations. These plants can be used for this purpose without the limitations existing for transgenic plants.

The Application of Somaclonal Variation in Early Maturity,High Yield and High Quality Improvement in Wheat

Yield characters, maturity and grain protein content of somaclones derived both from immature embryo of cultivar 77(2)-Spring and single-cell culture of cultivar NE7742 in vitro were studied and the wide variation was found. Somaclones with maturity 8 days earlier than or the same as CK NE 7742 (high yield, early maturity and high quality), combining with high quality (grain protein content 15.5% - 18%) and high yield (the same as 7724 or higher) have been found and selected and now multiplied for 8 generations. The results of cultivar comparison trial in 1995 showed that several somaclones (the yields were significantly higher than CK DN120) could be used directly in wheat production. The studies confirmed that somaclonal variation is one of the effective ways for early maturity, high-yielding and high-quality improvement in wheat.

Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.

Restorable variation of cytoplasmic type of male sterile line in rice by in vitro culture

Rice male sterile (MS) lines, IR66707A and IR69700A, which possess the cytoplasm of Oryza perennis and O. glumaepatula respectively, belong to the cytoplasmic type. Their sterility could be maintained but not be restored. By somatic cell culture of these two MS lines, 47 somaclones with 465 R1 plants were obtained. All of the 465 R1 plants were sterile in the spring season in Guangzhou. According to the expression of the R1 plants and the level of similarity to their donor parents, they could be divided into three types. The plants of type I were male sterile. The sterility of some somaclones of this type could be restored by the test crossing varieties or alternated to fertile by changes of some environmental conditions. The hybrid F1 of test cross from the MS somaclones in type I was fertile while the hybrid F1 from the donor MS lines was still sterile. The R1 plants of type Ⅱ were similar to the donor parents and also male sterile. The hybrid F1 from all of the plants of type Ⅱ crossed to test