激素对转基因雪莲毛状根植株再生及类黄酮产生的影响

为了研究外源激素GA3和IAA对3个转基因新疆雪莲类黄酮高产毛状根系C17、C27、C46的植株再生及其总黄酮含量的影响,在培养基中添加不同浓度的GA3和IAA,结果发现,GA3浓度高于1.0 mg/L时,可诱导毛状根系产生不定芽,其中以GA3浓度为2.0 mg/L时,转基因毛状根系C17的不定芽再生率最高,可达82％。高压液相色谱以及紫外分光光度法测定结果表明,与未用激素处理的毛状根和它的再生植株相比,外源激素GA3和IAA能显著提高毛状根培养物中芹菜素和总黄酮的含量。毛状根系的组织干重与类黄酮的含量没有相关性,但毛状根系的再生率与类黄酮的含量几乎呈反相关性。     

Kinetics of littorine content in various developing stages of regenerates of Atropa belladonna

 Aseptically propagated regenerates were cultivated in a hydroponic apparatus, a phytotron or in the field, and their growth and littorine contents were investigated. No littorine was detected in aseptic regenerates cultured on solidified Murashige and Skoog medium, nor was it found in leaves under the three conditions tested. In roots, it was common features to all three conditions tested that littorine increased dramatically after transplantation from culture tubes and was a major alkaloid up to week 4; subsequently the littorine contents varied depending on the cultivation conditions. Roots cultivated in the field showed a marked thickening and rapid disappearance of littorine; those cultivated in the hydroponic apparatus were thin and maintained a high level of littorine for a long time. In a plant cultivated for 16 weeks in a pot, littorine content in the roots decreased with increasing root diameter. Received: 1 October 1999 / Revision received: 16 March 2000 / Accepted: 24 March 2000     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Insect-resistant plants with improved horticultural traits from interspecific potato hybrids grown in vitro

Summary Plants were regenerated from petiole calli of interspecific hybrids of Solanum tuberosum x S. berthaultii, an insect-resistant wild species. Callus culture was used to generate genetic changes to overcome the restricted recombination between the two genomes. Two plants out of 58 (3.5%) from calli of hybrid J114-1 showed stable and heritable differences from the hybrid over two cycles of evaluations in the field. Replicated trials were conducted in 1987 and 1988, using two populations of plants propagated by nodal cuttings from the original regenerates maintained in vitro. One regenerate showed insect resistance and increased marketable yield (approximately two fold) in the field. The other had higher levels of phenolic exudate in one of the two types of foliar trichomes associated with the insect resistance mechanism. Some desirable changes were discernible only in sexual progeny of regenerates, not in the regenerates themselves. In a backcross to S. tuberosum, 7 of 14 (50%) regenerates from hybrid F743-4 showed more progeny (up to 15-fold) with improved trichome traits and horticultural characteristics than the original hybrid. The variations were not associated with changes in ploidy. Fifteen plants obtained from these crosses are currently being incorporated into breeding lines. These results suggest that a period of callus culture followed by plant regeneration may aid in the introgression of desirable traits from wild species into crop plants.     

Effects of hypophysectomies and thyroxine replacement upon the initiation of tail regeneration in the lizard, Anolis carolinensis

A histological evaluation of the effects of hypophysectomy and throxine therapy in young tail regenerates was carried out in the small iguanid lizard, Anolis c. carolinensis. Hypophysectomy caused a delay but did not inhibit blastema formation. The growth of the ependyma into the wound region was delayed in hypophysioprivic regenerates by about a week. Growth and differentiation of hypophysioprivic regenerates after blastema formation was variable, ranging from virtually no growth to the formation of a differentiated but very small protuberance. However, actual tail elongation was inhibited by hypophysectomy. In those hypophysioprivic regenerates that did show signs of differentiation, muscle groups were poorly defined, scanty in appearance and not as well differentiated as the cartilage tube. Thyroxine treatment in the young hypophysioprivic regenerates stimulated normal growth and normal appearance and differentiation of promuscle and procartilage aggregates as well as the growth of the ependymal tube into the blastema.     

Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regeneration of<Emphasis Type="Italic"> Acer caudatifolium</Emphasis> Hayata plantlets from juvenile explants

Juvenile and fully mature Acer caudatifolium Hayata explants were assayed for their organogenic capacity. A protocol for multiple shoot culture formation and in vitro plant regeneration was developed for juvenile axillary bud cultures. Mature explants failed in shoot regeneration. Shoot multiplication was achieved by releasing apical dominance of the single elongated shoot on woody plant medium (WPM) supplemented with 0.7 mg l(-1) 6-benzylaminopurine and 0.05 mg l(-1) alpha-naphthaleneacetic acid. The highest rooting percentage was recorded on half-strength WPM containing 1.0 mg l(-1) indole-3-butyric acid. Regenerated plantlets were successfully hardened to ex vitro conditions and continued to grow after transfer to soil. No morphological aberrations were observed in the regenerates.     

Regeneration of symmetrical forelimbs in the axolotl,Ambystoma mexicanum

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

Tissue Differentiation of the Regeneration Rind in Jerusalem artichoke Stem

In trees, after removal of the bark, the vascular tissues of the newly-formed bark usually developed as a continuous layer. However, the stem of the herbaceous Jerusalem artichoke, after girdled, gives rise to regeneration of many irregularly arranged vascular bundles. Early July is the best time for girdling as the vascular bundles are well-developed, One week after girdling, some small groups of vascular tissues appeared in callus. Later on the vascular bundles eventually grew close together sooner or later, yet there were some wide pith rays which separated the various sized vascular bundles and exhibited irregularly contours. From these experiments, it is further evidenced that tile stem of herbaceous plants can also be girdled and regenerates a new rind. Furthermore, the girdled portion of this plant regenerates the vascular tissues which in a rather different way from all the plants that previously studied.     

Pattern-deficient forelimb regeneration in adult bullfrogs

This study was designed to test the ability of adult bullfrogs (Rana catesbeiana) to regenerate forelimbs, both with and without various experimental treatments. Distal humerus-level forelimb amputations provided with additional deviated (sciatic) nerve and/or repeated soft-tissue injury exhibited considerable outgrowth. However, control sham-operated forelimbs also produced regenerates with comparable frequency, size, and morphological complexity. The lengths of the regenerates ranged from 0.4 to 2.6 cm, representing an outgrowth of 10-65% of the portion removed by the distal humerus amputation plane; some regenerates exhibited an external morphology indicative of digitlike structures. Some outgrowths were flexible but only one was capable of independent movement. Victoria Blue staining of whole regenerates revealed a variety of internal cartilage elements. Staining showed a single solid mass of cartilage in some regenerates while others had several individual and variably shaped cartilages projecting distally. Histological analysis also revealed the presence of connective tissue, striated muscle, and abundant nerve fibers in addition to the individual cartilage elements. We have tentatively termed these responses pattern-deficient regeneration.     

Proportion regulation in the planarian,Dugesia tigrina following regeneration of structures

Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reorganization of regenerates of the dermal type in rats

Skin regenerates being formed on the stomach of rats were exposed to dosed injuries by needles. These injuries have significantly hastened formation of definitive regenerates.     

Morphogenetic changes during newt tail regeneration under changed gravity conditions

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals.     

Tissue culture in Haworthia

Summary Plants regenerated on two different media (NK and I) from the calluses of simple or cloned subcultures, which were originated from a single stock callus of Haworthia setata derived from its flower bud, were observed for eight characters, i.e., somatic chromosome number in root tips, growth vigor, leaf shape, leaf color, number of stomata per unit leaf area, esterase zymogram, chromosome association at meiotic metaphase I in pollen mother cells, and pollen fertility. From these regenerates plants with different characters from those of the parental plant were obtained. With regards to chromosomal aberrations, tetraploids, aneuploids, plants with a part of the chromosome segment deleted, with reciprocal and non-reciprocal translocations, or with paracentric inversions and those showing sub-chromatid aberrations at meiosis were obtained. The NK medium tended to regenerate more tetraploids and less plants carrying translocation than the I medium.Chromosome variabilities in somatic cells of the regenerates correlated with those of the calluses, from which they regenerated, while they did not correlate with either the meiotic irregularities (chromosome association at MI) or pollen fertility of the regenerates. From these facts, it was concluded that a rather large number of callus cells participate in the regeneration of an individual plant, although, however, only a few limited types of the cells form its germ line.Polyploidy affected growth vigor, leaf shape, stomata number and chromosome association at MI, but its effects were not detected on other characters. Chromosomal aberrations at the diploid level produced no clear changes in the regenerate's phenotype except in meiotic chromosome configuration and pollen fertility.Most chromosomal variants obtained in the present study are already reported in plants collected from wild populations, but plants with the deletion of a whole chromosome (karyotype 7L+6S) or chromosome segment (7L+1M+6S and 14L+2M+12S) have never been reported: this fact suggests that tissue culture is a powerful tool for producing plants with novel karyotypes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 436     

Asymmetric protoplast fusions between wild species and breeding lines of potato – effect of recipients and genome stability

 The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation. Received: 30 April 1998 / Accepted: 24 July 1998     

Morphogenetic interactions between rotated skin cuffs and underlying stump tissues in regenerating axolotl forelimbs

Rotation of skin cuffs 180° around the longitudinal axis of the underlying tissues in the axolotl forelimb results in a high percentage of multiple regenerates after amputation through the rotated skin. Similar results occur after rotation of only the anteroposterior (A-P) axis of the skin. Rotation of only the proximodistal (Pr-Ds) axis of the skin results in normal regenerates whereas dorsoventral (D-V) axial skin rotation results in single regenerates with some disturbances in symmetry. Rotation of anterior or posterior half cuffs of skin produces results similar to those obtained after A-P rotation of full skin cuffs, and rotation of dorsal or ventral skin halves duplicates the results obtained by rotating full skin cuffs about the D-V axis. Skin cuffs rotated for periods from 6 months to over 2 years before amputation are also capable of causing multiple regenerates to form. No significant difference in the percentage of multiple regenerates was seen after skin rotation and limb amputation through shoulder, upper arm, and forearm levels. X-Radiation (4000 r) of either the skin or underlying tissues before skin rotation resulted in single regenerates after amputation. If a strip of normal skin was turned perpendicularly to the long axis of the irradiated underlying stump tissues, the regenerative response was blocked. In some of the above experiments, regenerates with longitudinally duplicated upper arm and forearm segments appeared. It is postulated that normally both the skin and the underlying limb tissues can influence morphogenesis during regeneration and that they work in harmony. In contrast, rotated skin and the underlying tissues each exert a morphogenetic influence upon the regenerating limb, and the regenerate is not able to integrate these disharmonious influences. This is reflected in the highly abnormal morphology of the regenerates. The nature of the morphogenetic influence disrupted by skin rotation is not yet known.     

In vitro propagation and chromosome doubling of a Triticum crassum x Hordeum vulgare intergeneric hybrid

Summary In vitro culture of inflorescence tissue of a Triticum crassum (6x) x Hordeum vulgare cv. Bomi (2x) intergeneric hybrid resulted in the proliferation of totipotent callus from which plants were regenerated. Regeneration was also achieved from immature inflorescence callus of T. crassum but not from H. vulgare. T. crassum x H. vulgare regenerates had a somatic chromosome number of 28, identical to that of the original hybrid. Four chimeric plants with a partially doubled chromosome number were obtained by in vitro colchicine treatment of hybrid callus prior to induction of plant regeneration. All T. crassum regenerates had 35 chromosomes rather than the expected number of 42. Meiotic analysis of a 35-chromosome plant revealed an extremely abnormal meiosis which might be attributable to a complete disturbance in meiotic control system(s) including that of meiotic pairing.Dedicated to Professor Georg Melchers on the occasion of his 75th birthday.Contribution No. 624 Ottawa Research Station, Research Branch, Agriculture Canada, Ottawa, Ont. K1A OC6 (Canada)     

The possible effect of the mechanical factor on the completeness of the regeneration of the spinal skin in mice

In mouse tissues, regenerates forming in the place of full-thickness skin wounds on the back, were exposed to dosed mechanical injuries. As a result the structure of final regenerates in some animals differed from the scar, that is usually formed after healing of this type of wound.     

Analysis of the progress of hormone-receptor amplification during differentiation in regenerating planarians.

Planarians developing from isolated regenerates, when pretreated with epinephrine at different periods of their second regeneration, show a higher rate of sugar uptake than the controls, which were not treated with epinephrine. The hormone receptor development can be influenced most effectively by pretreatments performed during the 1--3 and 6--9 day periods, though some effect can be evoked at any time in the course of the second regeneration. The head regenerates developing from the anterior end of the original midpiece (and regenerating tails the second time, B1) were found to be most sensitive, while the similar regenerates developing from the hindpiece of the original planarian (C) showed the weakest reactivity. The experiments provide further evidence that the hormone receptors' activity can be amplified in the course of regenerative development, leading thus--at a later stage--to a more intense sugar uptake response of the cells.