不定胚再生植株的染色体数目变异

本实验以甜瓜“小麦瓜”和“青皮绿肉”品种为试材,经不定胚诱导再生植株。分别调查了不同继代培养时间后形成再生植株的染色体数目,并将之与对照染色体数目相比较,发现通过诱导不定胚所得到的再生植株中存在着一定的变异,而且经过不同继代培养时间后,所得到的不定胚再生植株的变异程度不同,随着时间的增加,染色体数目的变异率从3.3%增加到30%,变异幅度也从2n=23~24增加到2n=13~48。从而得出结论：不定胚再生植株染色体数目变异程度随着培养时间的增加而增加;培养时间在1~2个月内所得到的不定胚再生植株的变异较少。此外,不定胚再生植株的染色体数目变异程度也因品种而异。 Abstract:Chromosomal number of different of somatic embryos regenerated plants were investigated in melon variety “xiaomaigua” and “Qingpilurou”.Certain variations of chromosomal number were found among the regenerated plants compared with normal sample,and range of variation covered from 2n=23～24 to 2n=13～48 with the increase of generation,the rate from 3.3% to 30%.The results indicated that degree of variation in chromosomal number of somatic embryos regenerated melon plants increased with the time of culture,and those cultured in one to two months had the least variation.It was also found that degree of chromosomal number variations varied with melon varieties.     

Variation in chromosome number in the seedling progeny of a somaclone of Paspalum dilatatum

The somaclone,C39,derived by tissue culture from the obligate apomict Paspalum dilatatum cv Raki(2n=50),had 50 chromosomes and a karyotype apparently identical to Raki.SC2 seedlings of C39 showed a high degree of phenotypic variation which was often associated with increased chromosome numbers,but some of the variant seedlings were karyotypically indistinguishable from Raki or C39.Plants with increased chromosome numbers exhibited a high degree of intraplant chromosome variation(aneusomaty).In one of the SC2 seedlings,the chromosome number of root tip cells varied from 58 to 82 and in several other seedlings the range was more than 10.The results suggested that the ability to form seed apomictically was much reduced in C39 and that this plant showed some capacity for sexual reproduction and the resulting seedlings,with a chromosome number of about 70,were genetically unstable.Of 11 SC2 seedlings examined cytologically,6 did not produce any viable seed.Seedlings grown from seed of the remaining 5 plants showed that aneusomaty persisted in the SC3 generation.SC3 seedlings which were phenotypically similar to their maternal parent showed a similar range of chromosome numbers to that parent.Some of the SC3 seedlings exhibited an even wider range of chromosome numbers(e.g.56-136),and these plants were all dwarfs.     

节节麦×大麦杂种胚再生植株的细胞遗传学研究

以节节麦(2n=14)为母本和大麦(2n=14)进行杂交,两组合平均结实率为31 .11%。对31个幼胚进行愈伤组织诱导培养,其中2个形成全能性愈伤组织进而分化出再生杂种植株。细胞学观察表明,杂种胚再生植株均是染色体加倍的节节麦-大麦双二倍体(2n=28)或染色体再加倍的多倍体。节节麦-大麦双二倍体在减数分裂中期Ⅰ染色体配对平均构型为14.79Ⅰ+1.95Ⅱ+ 4.53Ⅱ′+0.01Ⅲ,表现为自交不育。杂种胚再生植株染色体数目再加倍的多倍体有少数产生了自交种子。 Abstract:Hybridizations between Aegilops tauschii(2n=14)and Hordeum vulgare(2n=14)were made using Ae.tauschii as female.The mean frequency of seed set was 31.11% in two cross combinations,13 hybrid embryos were cultured for inducing callus,of which 2 produced totipotent callus,and plants were regenerated from them.These plants were Ae.tauschii-H.vulgare amphidiploids with doubling chromosome number(2n=28)and octoploids with repeatedly doubling chromosome number(2n=50~56).The Ae.tauschii-H.vulgare amphidiploids were sterile and their average chromosome pairing at MI were 14.79I+1.95II+4.53II′+0.01III.Some octoploid(2n=56)plants produced seeds after selfing.     

Effects of Temperature Acclimation Pretreatment on the Ultrastructure of Mesophyll Cells in Young Grape Plants （Vitis vinifera L. cv. Jingxiu） Under Cross-Temperature Stresses

Leaves from annual young grape plants （Vitis vinifera L. cv. Jingxiu） were used as experimental materials. The ultrastructural characteristics of mesophyll cells in chilling-treated plants after heat acclimation （HA） and in heat-treated plants after cold acclimation （CA） were observed and compared using transmission electron microscopy. The results showed that slight injury appeared in the ultrastructure of mesophyll cells after either HA （38℃ for 10 h） or CA （8℃ for 2.5 d）, but the tolerance to subsequent extreme temperature stress was remarkably improved by HA or CA pretreatment. The increases in membrane permeability and malondialdehyde concentration under chilling （0℃） or heat （45℃） stress were markedly inhibited by HA or CA pretreatment. The mesophyll cells of plants not pretreated with HA were markedly damaged following chilling stress. The chloroplasts appeared irregular in shape, the arrangement of the stroma lamellae was disordered, and no starch granules were present. The cristae of the mitochondria were disrupted and became empty. The nucleus became irregular in shape and the nuclear membrane was digested. In contrast, the mesophyll cells of HA-pretreated plants maintained an intact ultrastructure under chilling stress. The mesophyll cells of control plants were also severely damaged under heat stress. The chloroplast became round in shape, the stroma lamellae became swollen, and the contents of vacuoles formed clumps. In the case of mitochondria of control plants subjected to heat stress, the outer envelope was digested and the cristae were disrupted and became many small vesicles. Compared with cellular organelles in control plants, those in CA plant cells always maintained an integrated state during whole heat stress, except for the chloroplasts, which became round in shape after 10 h heat stress. From these data, we suggest that the stability of mesophyll cells under chilling stress can be increased by HA pretreatment. Similarly, CA pretreatment can protect chloroplasts, mitochondria, and the nucleus against subsequent heat stress; thus, the thermoresistance of grape seedlings was improved. The results obtained in the present study are the first, to our knowledge, to offered cytological evidence of cross-adaptation to temperature stresses in grape plants.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.     

Rapid micropropagation of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium

An efficient protocol has been developed for rapid micropropagation of Ocimum basilicum. Multiple shoots were induced by culturing shoot tip explants excised from mature plants on a liquid Murashige and Skoog (MS) medium supplemented with 5–100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplementation to the culture medium was 50 μM for 8 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (78 %), mean number of shoots (11.6 ± 1.16) and shoot length (4.8 ± 0.43 cm) per explant. A culture period longer than 8 d with TDZ resulted in the formation of fasciated or distorted shoots. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA). The micropropagated shoots with well developed roots were successfully established in pots containing garden soil and grown in greenhouse with 95 % survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Plant regeneration from protoplasts of hydroxyproline resistant cell line in Onobrychis viciaefolia

An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onobrychis viciaefolia was established.In SH medium supplemented with 1mg/L2,4-dichlorophenoxy-acetic acid(2,4-D),0.5mg/L kinetin(KT)and 0.2mg/L naphthalene acetic acid(NAA),the division frequency of protoplastderived cells reached up to over 60%,and microcalli were obtained in 5-6wk.Upon transferring them on agar solidified MS medium plus 2mg/L indole-3-acetic acid (IAA),shoots were induced.After cultivating them on MS medium with or without IAA,roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal(2n=28).The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP.     

Genetic transformation of gentian using wild-type Agrobacterium rhizogenes

Leaf sections of greenhouse-grown Miscanthus x ogiformis Honda 'Giganteus' plants and leaf sections or shoot apices of in vitro shoot cultures were grown on Murashige and Skoog medium containing various concentrations of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid. On leaf sections, the callus induction decreased with increasing BA concentration. The percentage of embryogenic callus was increased, the percentage of root-forming callus decreased, and a new shoot-forming callus type was formed by inclusion of BA during callus induction. A higher percentage of shoot-forming callus was formed on shoot apices compared with leaf sections of in vitro-grown shoots when cultured on 0.4 μM BA. The largest number of plants per callus piece was regenerated from shoot-forming callus, but maintenance of the high regeneration capacity proved difficult. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chromosomal instability in Lathyrus sativus L.

Summary In Lathyrus sativus (2n=14), variety LSD-1 shows an instability of somatic chromosome number which can be observed in root tip and shoot tip mitoses. In this variety, approximately 54% of the seedlings showed intra-individual variation in chromosome number ranging from 2n=14–3. This variability in chromosome number was recorded in approximately 60% of the dividing cells. Two seedlings were triploid with 21 chromosomes. Variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result in spindle abnormalities, chromosome degradation and minute chromosomes. The variation in chromosome number is probably responsible for the pollen polymorphism noted in this particular strain. The possible mechanism of intra-individual variability and the occurrence of the phenomenon vis-a-vis its applications are discussed.     

Some notes on the cytotaxonomy of two Ruppia species in South Australia

Chromosome counts were made from root tip cells of Ruppia megacarpa Mason (2n = 20 chromosomes) and of Ruppia tuberosa Davis and Tomlinson (2n = 20 and 3n = 30 chromosomes). The plants were collected in South Australia from coastal lakes, lagoons and the sea. The salinity in these waters varied from 6.9 to 67.9% total dissolved solids (TDS). We report the occurrence of Ruppia tuberosa in the tidal zone of the sea, and the occurrence of a triploid population of this species in a lagoon.     

Somaclonal Variation in Chromosome Number and Nuclei Number of Regenerated Plants from Protoplasts of Actinidia eriantha

By counting the chromosome number of root tip cells in 18 regenerated plants derived from protoplasts of Actinidia eriantha Benth., the authors found 12 euploid plants and 6 mixoploid plants. Of the 12 euploid plants, 6 were diploid (2n = 2x = 58) and the other 6 were tetraploid (2n =4x = 116). The chromosome numbers of the mixoploid plants varied from 59 to 203. Mttltinueleate phenomenon was also observed in the interphase cells of 10 protoplast-derived plants. Cells with binuclei or trinuelei were common and cells having heptanuclei were also seen occassionally. Muhinucleate phenomenon did not occur in the control, i. e., the donor plant, where the chromosome number was 2n = 2x = 58.     

毛花猕猴桃原生质体再生植株根尖染色体数目变异与细胞多核现象

对18株毛花猕猴桃(Actinidia eriantha Benth.)原生质体再生植株的体细胞染色体数做了观察,其中12株为整倍体:二倍体(2n=58)和四倍体(2n=116)各6株;另外6株为混倍体,其染色体数目变化在59～203之间。还发现原生质体再生植株有丝分裂间期细胞存在多核现象,有多核细胞的共10株,细胞核数目以双核和三核较常见,最多的有7个核。对照植株为2n=2x=58,未发现多核细胞。     

Chromosome number variations in micropropagated true-to-type and off-type banana plants (Musa AAA Grande Naine cv.)

Summary The objective of the present study on banana plants (Musa AAA Grande Naine cv.), obtained byin vitro shoot tip culture, was to determine whether modifications in chromosome number could account for the appearance of the off-types with mosaiclike leaf defects or dwarf stature, the most frequent off-types observed after micropropagation. Chromosome counts were conducted on shoot tip samples treated with 8-hydroxyquinoline, digested in pectinase and stained with Schiff's reagent. On average, 160 counts were made for each treatment. Four types of plant material were studied: phenotypically true-to-type plants, dwarf off types, mosaiclike off-types obtained by micropropagation, as well as true-to-type plants obtained by standard propagation techniques of suckers with no micropropagation history. Some cells from all four types of plant material were found to have an abnormal chromosome number (i.e., 2n = 3x = 33), characteristic of triploid banarias. The percentages of aneuploid cells were 14%, 22%, 35%, and 5%, respectively. Descending aneuploidy was noted in micropropagated plants derived from true-to-type and dwarf off-type suckers. The statistical analysis revealed that the two latter types of plant material had the same percentage of aneuploid cells. Thus, the dwarfism could not be correlated with a change in the chromosome number. Conversely, ascending aneuploidy was observed in the mosaiclike material, with 34 or 35 chromosomes in almost 28% of the cells. This percentage was significantly higher than in true-to-type plants and highlight the genetic origin of the mosaiclike variation.     

Rapid detection of aneuploidy in <Emphasis Type="Italic">Musa</Emphasis> using flow cytometry

We report a procedure for the rapid and convenient detection of aneuploidy in triploid Musa using DNA flow cytometry. From a population of plants derived from gamma-irradiated shoot tips, plants were selected based on aberrant morphology and their chromosome numbers were counted. Aneuploids plants with chromosome numbers 2n=31 or 32 were found as well as the expected triploid plants (2n=3x=33). At the same time, the nuclear DNA content of all plants was measured using flow cytometry. The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as an internal reference standard. The relative DNA content of individual plants was expressed as a ratio of DNA content of CRBC and Musa (DNA index). In order to estimate the chromosome number using flow cytometry, the relative DNA content of plants with unknown ploidy was expressed as a percentage of the DNA content of triploid plants. The classification based on flow cytometry fully agreed with the results obtained by chromosome counting. The results indicated that flow cytometry is a convenient and rapid method for the detection of aneuploidy in Musa.     

Establishment of Trisomic Series of Millet (Setaria italica L. Beauv.)

The young-ears of “Yugu No. 1”, a millet (Setaria italica (L.) Beauv. ) variety of good quality, high yielding and stress-resistance, were chosen to induce callus on N6 medium. After 3—5 times of subcultures, the callus was treated with colchicine (0.02%) for 48 hours and then transferred hack to subculture medium to restore growth, after which the callus was transferred to differentiation medium and subsequently, tetraploid plants were obtained. Through crossing, using tetraploid plants as female parent and diploid plant as male parent, 5 triploid plants were found among 2100 offspring plants. The triploid plants were fertilized with pollens from diploid plants. Among the plants developed from the seeds harvested on triploid plants, 9 kinds of trisomics (totally 283 plants) were identified according to chromosomal number and morphologic feature. Each kind of the trisomics has specific marker feature: (Figures in parentheses represent the number of plants obtained) Triplo-1 (52) has rolling leaves, Triplo-2 (18) is dark green, Triplo-3 (43) is bushy and has degenerated spikelets at the tip of panicles: Triplo-4 (58) has long bristle: Triplo-5 (16) has slender stalks: Triplo-6 (36) has twisted panicle neck; Triplo-7 (44) is semi-erect; Triplo-8 (8) has thick panicles; Triplo-9 (8) is pseudonormal (Similar with diploid of “Yugu No. 1”).     

The genotoxic effects of Logran on Hordeum vulgare L. and Triticum aestivum L

In the present study, the cytogenetic effects of the herbicide Logran on root tip cells of Triticurn aestivum L. and Hordeum vulgare L. and changes of total protein content in root tip meristems were studied. The seeds of plants were treated with various concentrations of Logran (125, 250, 500 microg/ml) for 3 and 6 h. The percentages of abnormal cells were seen to increase with increasing treatment period and concentrations. The most dominant types of observed abnormalities were C-mitosis, distributed metaphase and anaphase, stickiness. All the used concentrations of Logran significantly induced a number of chromosomal aberrations in root tip cells of Hordemrn vulgare L. and Triticum aestivum L. Logran also decreased mitotic index. The decrease of protein content in root tips of Triticum aestivum L. is significant at all the treated concentrations and treatment periods when compared with control.     

美味猕猴桃原生质体再生植株细胞遗传学研究 Ⅰ．体细胞染色体数目的变化

研究了美味猕猴桃叶愈伤组织原生质体再生植株和母株（ＡｃｔｉｎｉｄｉａｄｅｌｉｃｉｏｓａｌｉｎｅＮｏ．２６）茎尖体细胞染色体数目。结果表明：母株２ｎ＝６ｘ＝１７４。所测２９株再生植株的茎尖体细胞染色体数目差异显著,多为非整倍体类型,占所测植株的７２．４％左右;体细胞染色体数目介于１４２－３１０条之间,其中２ｎ＝６ｘ＝１７４约占２０．７％,少于１７４条染色体的植株约占３１．０％,超过１７４条染色体的植株则占４８．３％左右。个别单株部分茎尖体细胞在有丝分裂后期出现染色体桥、断片和落后染色体等异常现象。并对以上现象进行了扼要的讨论。     

Nuclear dynamics during the simultaneous and sustained tip growth of multiple root hairs arising from a single root epidermal cell

Nuclear dynamics in root hairs, which depends upon the actin cytoskeleton, appears to be an important factor in root-hair tip growth. Previous evidence suggests that there is an absolute requirement for the nucleus to be a fixed distance from the growing root-hair tip for tip growth to proceed. To test this hypothesis, nuclear dynamics were examined in root-hair cells bearing multiple root hairs. The majority of root-hair cells of transgenic plants overexpressing the ROP2 GTPase (ROP2 OX) bear multiple root hairs. Simultaneous and sustained fast tip growth occurred in multiple root hairs of ROP2 OX, with the continual presence of tip-localized cytoplasm in these growing hairs. Nuclear dynamics were imaged in ROP2 OX by co-expressing a transgene encoding a nuclear localization signal (NLS)-green fluorescent protein (GFP) fusion protein. The nucleus was in continual proximity to one of the growing root-hair tips, whilst the other tip elongated at a similar rate but in the absence of the nucleus from the shank of that root hair. To test whether this phenomenon was an artefact of ROP2 overexpression, nuclear dynamics were examined in wild-type and NLS-GFP transgenic plants. Multiple root hairs on the same cell underwent simultaneous and sustained fast tip growth, with the nucleus lying deep within the shank of only one of these hairs. The nucleus was also moved into the root-hair tip during the severe root-hair tip branching which is characteristic of ROP2 OX transgenic plants. These results suggest that fast tip growth can proceed in some multiple root hairs at extreme distances from the nucleus.