Morphogenesis in Long-term Maintained Immature Embryo-derived Callus of Wheat (Triticum aestivum L) - Histological Evidence for Both Somatic Embryogenesis and Organogenesis

Callus induced from immature embryos of wheat cv Kharchia 65 on Murashige and Skoog’s medium containing 2.5 mg l¹ 2,4-D was maintained In the regenerable state by subculturing every 5-6 weeks on medium supplemented with 2,4-D (2.5 mg l^-1) and proline (10 mg l^-1). Addition of proline helped maintain morphogenic competence for over four years. The regenerating callus was analysed histologically about one year after first induction. Both somatic embryogenesis and shoot organogenesis were seen in the same callus tissue that contained typical stages of somatic embryoid development and evidence for the de novo shoot bud formation.     

Plant Regeneration from Cultured Immature Embryos and Inflorescences of 25 Cultivars of Wheat (Triticum aestivum)

Immature embryos and inflorescences of wheat (Triticum aestivum)were cultured on agar media. The presence of the auxin 2, 4-Dinduced the formation of multiple shoots in a proportion ofcultures. In some cases the shoots developed from organized,embryoid-like structures. The morphogenetic capacities of 25different cultivars of spring and winter wheat were compared,and clear differences were found between genotypes. Shoots werecultured separately, and the resulting plants grown to maturityin the glasshouse. Over a thousand plants were regenerated inthis way, and phenotypic variation was observed amongst theregenerants. Key words: Wheat, Somatic embryogenesis, Regeneration     

幼胚长度、2，4-D浓度、光强度等对花生体细胞胚发生的影响及高效再生系统的建立

花生幼胚在含2,4-D的诱导培养基中,形成近球状的致密的胚性愈伤组织、杆状两极结构及子叶期体胚。继代培养也有体胚发生。光照明显抑制体胚发生类似于自然栽培的情况。成熟培养基中诱导体胚根、芽两极发育完全。光下,具有根、芽的体胚于再生培养基中长成小植株后移栽于盛沙土的盆中正常生长、结实。在较好的影响因素(光照、幼胚长度、激素、切分方式、接种密度)组合下,体胚发生频率达75％以上,每子叶形成体胚3个以上。该体细胞胚高效再生系统与合子胚的发育相似,是遗传转化和胚发育研究的良好系统。     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Seed Maturation Regulators Are Related to the Control of Seed Dormancy in Wheat (Triticum aestivum L.)

In Arabidopsis, the regulation network of the seed maturation program controls the induction of seed dormancy. Wheat EST sequences showing homology with the master regulators of seed maturation, LEAFY COTYLEDON1 (LEC1), LEC2 and FUSCA3 (FUS3), were searched from databases and designated respectively as TaL1L (LEC1-LIKE), TaL2L (LEC2-LIKE), and TaFUS3. TaL1LA, TaL2LA and TaFUS3 mainly expressed in seeds or embryos, with the expression limited to the early stages of seed development. Results show that tissue-specific and developmental-stage-dependent expressions are similar to those of seed maturation regulators in Arabidopsis. In wheat cultivars, the expression level of TaL1LA is correlated significantly with the germination index (GI) of whole seeds at 40 days after pollination (DAP) (r = –0.83**). Expression levels of TaFUS3 and TaL2LA are significantly correlated respectively with GIs at 40 DAP and 50 DAP, except for dormant cultivars. No correlation was found between the expression level of TaVP1, orthologue of ABA INSENSITIVE3 (ABI3), and seed dormancy. DELAY OF GERMINATION1 (DOG1) was identified as a quantitative trait locus (QTL) for the regulation of seed dormancy in Arabidopsis. Its promoter has RY motif, which is a target sequence of LEC2. Significant correlation was found between the expression of TaDOG1 and seed dormancy except for dormant cultivars. These results indicate that TaL1LA, TaL2LA, and TaFUS3 are wheat orthologues of seed maturation regulators. The expressions of these genes affect the level of seed dormancy. Furthermore, the pathways, which involve seed maturation regulators and TaDOG1, are important for regulating seed dormancy in wheat.     

Somatic Embryogenesis in Cassava: The Anatomy and Morphology of the Regeneration Process

Anatomical and morphological studies demonstrated that somaticembryos developed similarly on mature seed and clonal leaf explantsof cassava (Manihot esculenta Crantz) cultured for 20–24d on Murashige and Skoog (MS2) basal medium supplemented with4.0 mg l^–1 2,4-D (Stage 1) before transfer to MS2 basalmedium supplemented with 0–01 mg l^–1 2,4-D and 0–1mg l^–1 6-benzylaminopurine (Stage II medium). Within 7d of inoculation onto Stage I medium, cell divisions occurredin the adaxial tissues of cotyledon-piece and leaf-lobe explants,and associated with this was the development of embryogeneticprotusions and ridges on the adaxial surface. Foliose structuresand somatic embryo initials developed from these tissues oncotyledon, embryonic axis and leaf-lobe explants and, when cultureswere transferred to Stage II medium, further somatic embryodevelopment occurred. Somatic embryos apparently originatedfrom groups of cells and were identified by the presence ofa closed root axis, a shoot axis and cotyledons of similar shapeand venation to those of zygotic embryos. Somatic embryos hadno vascular connection with parental cultures. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, anatomy, morphology, morphogenesis     

The Effect of High-Concentration Auxin and Plant Regeneration in the Immature Embryo Culture of Soybean

Using immature embryos of soybean as explants, green structures and somatic embryoids were able to be induced on higher auxin-containing media. Genotypes, developmental degree of the embryos, origin of the explants and medium compositions all affected the occurrence of the structures and calli. After the green structures were transferred to high 2,4-D containing medium (30 mg/l) calli were reinduced. These calli were maintained on the same medium without being subcultured for 2 months and then transferred to lower hormone-containing media. After 2 weeks, a great number of new green structures in the same shapes were induced. It was shown that high level of 2,4-D played a unique role in lasting the morphogenesis ability of the cultures. When the green structure were cultured on low hormone-containing media they developed new leaves and formed leaf clusters while the apical did not develop. In order to stimulate the apical development the medium containing 2 mg/l GA3 and 0.1 mg/l IBA was used and some plantlets were obtained. The different effects of NAA and 2,4-D on the explants and calli were studied. Calli induced from the cotyledon of immature seeds (416 mm) had a regeneration ability stronger than that from the seedlings. The calli induced by use of the medium containing high concentration of 2,4-D (5–30 mg/l) have higher potentialities in producing green structures. In contrast, the calli induced by high concentration of NAA (10 mg/l) were highly root-morphogenetic. The explants and the calli cultured on the medium containing 5 mg/l 2,4-D could be maintained for a long term without being subcultured frequently.     

亚麻品种'双亚5号'的胚性愈伤组织诱导和体细胞胚胎发生

亚麻品种'双亚5号'的种子在MS 1.0 mg·L-1 2,4-D 30 g·L-1蔗糖培养基上可诱导出愈伤组织,将其转入MS 0.5mg·L-1KT 0.5mg·L-1NAA培养基上培养10周后诱导出大量胚性愈伤组织,结构较致密,浅黄色,表面有成团的紧密粘附在一起的小颗粒形态.但其继代周期不宜太长,继代次数不宜多,否则易回到非胚性化状态.胚性愈伤组织转A.MS 1.5 mg·L-1(T 1.0 mg·L-1 2,4-D分化培养基上培养40 d后可获得大量的球形胚状体.少量球形体胚可萌发形成正常的子叶胚初期形态,较多的球形体胚形成次生体胚或仅有单极性的畸形胚状体.组织解剖学观察表明,诱导出的是亚麻胚性愈伤组织和胚状体.     

Somatic Embryogenesis and Morphogenetic Potential of Spring Barley (Hordeum vulgare L.) in the System of Technological Genetic Transformation

We studied somatic embryogenesis and morphogenetic potential in young embryos of 17 spring barley cultivars. A considerable effect of the genotype as well as of certain biologically active compounds in the growth medium on the frequency of embryogenic callus formation and regenerative capacity was observed. The Murashige and Skoog medium complemented with myoinositol and casein hydrolysate is suitable for embryogenic callus induction. The regeneration medium should be complemented by hormones, 2.5 mg/l auxin 2,4-D and 0.1–0.5 mg/l cytokinin 6-BAP, to increase the frequency of somatic embryo maturation. The presence of cefotaxime and high level of copper are desirable to increase the efficiency of callogenesis, regeneration, and callus quality. Three cultivars with economically valuable characters and high morphogenetic potential were recommended for development of efficient technology for barley transformation.     

皇冠草体细胞胚胎发生及其体胚发生过程中内源激素的变化

皇冠草是一种具有重要经济价值的大型观赏水草。以其幼叶为外植体,在MS培养基上,短期内(25d),直接诱导获得了体细胞胚。对不同的细胞分裂素及其不同组合的体胚诱导效应进行测试,发现：6-苄氨基嘌呤(6-BA,1mg/L)和玉米素(Zt,1mg/L)结合使用效果最好,诱导频率达100%,平均每个外植体可形成4.87个体细胞胚。当在含有Zt(1mg/L)的MS培养基中加入萘乙酸(NAA 0.5mg/L)时,外植体不能形成体细胞胚,而是形成大量的根。在含有吲哚乙酸(IAA,1mg/L)的MS培养基上,成熟的体胚5d后即可发育成完整的小植株。72%的小植株被成功地移栽到了水族箱中。采用HPLC法,对自外植体培养之日起到体胚发生和成熟的各个时期的内源激素含量进行了测定。发现各种内源激素的含量均不断增加,10d后达到对照的2倍左右。IAA含量在培养第10天时达到第一个峰值,第二个峰值出现在25d后;玉米素和玉米素核苷(Zt+ZR)含量在15d后达到最大值(为对照的8倍多),然后逐渐下降;赤霉素(GA-3)的变化趋势与细胞分裂素基本相同,但到体胚萌发时,其含量已恢复到对照水平;培养10d后的脱落酸(ABA)含量一直保持到了体胚成熟期。     

The Role of Endogenous Ethylene in Carbohydrate Metabolism of Medicago sativa L. Somatic Embryos in Relation to Their Regenerative Ability

Effects of the ethylene biosynthesis inhibitors salicylic acid (SA) and aminoethoxyvinylglycine (AVG) on germination of Medicago sativa L. somatic embryos and their conversion to seedlings in relation to carbohydrate content and α-amylase activity were studied. Both SA, an inhibitor of ACC oxidase, and AVG, an inhibitor of ACC synthase, when present in the regeneration medium (0.1 and 1 μM) were found to drastically reduce the embryo germination rate. In addition, SA and AVG were found to almost completely or completely, respectively, arrest the process of embryo conversion to seedlings. The inhibitory effects of SA and AVG on germination and conversion may indicate that the processes required endogenous ethylene. AVG and SA clearly slowed down starch disappearance during the 48-h imbibition in the regeneration medium prior to radicle elongation, which was correlated with inhibition of the activity of α-amylase, an enzyme responsible for starch hydrolysis. It is probable that ethylene may activate α-amylase in the germinating alfalfa somatic embryos. In contrast, the disappearance of soluble sugars in the embryos in the presence of the inhibitors tested was accelerated. The disappearance of soluble sugars (to null or almost null) in embryos was faster in the presence of SA in the regeneration medium after 24 and 48 h compared to the disappearance rate with AVG present in the medium. Only glucose was present after a 48-h incubation in the regeneration medium in the presence of the two ethylene biosynthesis inhibitors, in contrast to the control embryos in which glucose was not detected.     

Changes of Soluble Protein Components in Winter Wheat (Triticum aestivum L.) Shoots During Vernalization and its Relation to Morphogenesis

Winter wheat “Nong Da 139” and spring wheat “Zhong 8022” were used for this experiment. The effect of low temperature treatment at different periods on protein content and composition of the seedling shoots as well as subsequent development was studied. The main results are as follows: 1. More than 40 days of vernalizing treatment were required for a rapid and uniform earing by winter wheat “Nong Da 139”. After 14–21 days vernalization, the winter wheat is possible to ear in summer, but in an irregular manner. The results indicate that during the whole vernalization process, the effect of low temperature on earing development varies with the duration of treatment. At the earlier stage, it is likely that the Iow temperature induces a change in physiological process, and in the later stage, it only accelerates the development. It is there- fore, suggested that there are two distinct processes existing during vernalization. The transformation from the former to the latter state appears to occur at the middle period of vernalization process. 2. The protein metabolic inhibitors, such as ethionine and p-fluorophenylalanine, interfere with the vernalizing process of winter wheat also at its middle period. 3. With low temperature treatment for different periods, the soluble protein content and composition are found to be changed in winter wheat shoots. At the middle-stage of vernalization (after 14 days low temperature treatment), not only is the content of protein increased twice as compared with the control, but new proteins (on electrophorestic gel) are also produced. On the contrary, there was no difference in protein bands for spring wheat. Spring wheat not to be treated by low temperature, has already possessed the proteins that appeared in vernalized winter wheat shoots. These results demonstrated that the days of 14–21 are the critical time for the vernalization of the winter wheat. The new proteins synthesized at this stage might be the factor of the determination whether or not the plants will transform from vegetative stage into reproductive phase.     

Somatic karyotype,heterochromatin distribution,and nature of chromosome differentiation in common wheat,Triticum aestivum L. em Thell

Heterochromatin distribution and structural differentiation of somatic chromosomes of five common wheat cultivars — Chinese Spring, Wichita, Cheyenne, Timstein, and Hope — were studied by an acetocarmine/N-banding technique. Detailed morphological observations on acetocarmine stained somatic chromosomes of Chinese Spring were made on all A genome chromosomes (except 1A), all B genome chromosomes, and chromosomes 1D, 2D, and 7D. N-banding patterns of chromosomes 2A, 3A, 5A, 6A, 1D, 2D, and 7D were described for the first time. Substitution lines of 21 individual chromosomes each of Cheyenne, Timstein, and Hope in Chinese Spring were analyzed by N-banding. A high frequency of N-band polymorphism was observed, especially for most of the B genome chromosomes. Chromosomes 3A, 5A, 2D, and 7D showed a constant banding pattern. Three cases of doubtful substitutions, Hope 2A, 2B, and Timstein 7A, and several cases of incomplete and chromosomally modified substitutions were observed. The reduced level of chromosome pairing that is often observed in intercultivar hybrids of wheat may be due to heterochromatic differentiation, genic and structural heterozygosity, or hybrid dysgenesis.     

Pollen Dimorphism and Its Relation to the Formation of Pollen Embryos in Anther Culture of Wheat (Triticum aestivum)

The anomalous pollen grains in vivo at the frequencies of 2.61% and 6.81% respectively were found in anthers of two hybrid materials of wheat from which we had obtained the higher frequencies of induction of pollen embryos in anther culture in the past few years. This indicated that the pollen dimorphism also presented itself in the anthers on wheat plants. A comparative cytological study of development of anomalous pollen grains in vivo and of the early stages of the formation of pollen embryos in vitro was carried out and the results suggest that in cytological details there is an obvious similarity between them. This reveals the possibility that the pollen embryos night derive itself from the anomalous pollen grains in wheat as in the other plants. Therefore, it was considered that the appearance and frequency of the anomalous pollen grains could show the senibility of the development of microspores to the changes of various internal and external factors. The significance of the sensibility for the formation of pollen embryos in anther culture of wheat is discussed.     

Transverse Relaxation Time of Leaf Water Protons and Membrane Injury in Wheat (Triticum aestivum L.) in Response to High Temperature

The relationship between high temperature stress injury andtemperature dependence of the transverse relaxation time (T₂)of leaf water was examined using NMR in four cultivars of wheatdiffering in their sensitivity to high temperature stress. TheT₂declined with increasing temperature between 25 and 35 °C.A comparison of relative injury based on electrolyte leakageand T₂, between 40 and 50 °C, indicated that while membranepermeability increased with increasing temperature there wasan increase in T₂until 44 and 48 °C in susceptible and tolerantcultivars respectively, followed by a sharp decline. This patternof change in T₂with increasing temperature was consistent whetherthe same or different samples were used for each treatment temperature.Loss of temperature dependence of T₂after heat killing indicatedirreversible changes in T₂, probably due to the loss of membraneintegrity. Heat tolerant varieties, which suffered less membraneinjury, had a higher T₂compared to susceptible varieties. Tolerantvarieties also maintained the T₂of leaf water protons to highertemperatures than did sensitive varieties. This NMR-based, non-invasive,rapid technique could be used to efficiently detect heat injuryin leaf tissues. Copyright 1999 Annals of Botany Company Membrane integrity, transverse relaxation time, high temperature stress, Triticum aestivum L.