Influence of antibiotic cefotaxime on somatic embryogenesis and plant regeneration in indica rice

An antibiotic, cefotaxime (Omnatax) has been found to promote somatic embryogenesis and subsequent plant regeneration in vitro in indica-type basmati rice cultures. Response was highly genotype specific. The number, mass and morphology of the calli formed on the scutellar tissues were dependent on the growth medium (with or without cefotaxime). The embryogenic nature of nodular calli was confirmed through histological analysis and their plant regeneration ability. The calli of variety Pusa basmati 1 grown on medium supplemented with cefotaxime (100 mg/L) exhibited up to 70.5% plant regeneration as compared to control (51.51%). Plants regenerated from emryogenic calli were phenotypically normal and identical to seed-derived plants and exhibited normal fertility. A limited humidity and an optimal aeration of the culture tubes further enhanced the frequency of somatic embryogenesis and plant regeneration.     

The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeum vulgare L.)

Recently it has been reported that the cephalosporin antibiotic cefotaxime increases growth, regeneration and embryogenesis in wheat calli. We investigated the effect of cefotaxime on callus initiated from immature embryos of four barley (Hordeum vulgare L.) varieties. In calli cultured in the presence of antibiotic callus growth was up to 45% greater than in controls and the frequency of regenerating calli was increased by up to 80%. There was an apparent interaction of the antibiotic with genotype and the 2,4-D in the medium.     

A novel method to induce direct somatic embryogenesis,secondary embryogenesis and regeneration of fertile green cereal plants

A direct somatic embryogenesis and secondary embryogenesis protocol was developed for seven cereal species, thus providing a new vista for in vitro plant genetic transformation or propagation. This paper describes a novel process that has been successfully developed for efficient regeneration of a wide range of cereal species and genotypes. This tissue culture and regeneration system does not require formation of callus tissues and takes approximately 2 months to complete, shorter than any of the currently available systems requiring 3-4 months. Rapid induction of direct somatic embryogenesis in barley (Hordeum vulgare), common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu), rye (Secale cereale) and oats (Avena sativa) was induced from excised immature scutellum on DSEM medium. Newly developed globular embryos were cultured on SEM medium for a second cycle of embryogenesis followed by germination (GEM medium) and regeneration of embryos into normally growing green and fertile plants. In vitro techniques to induce direct somatic embryogenesis, secondary embryogenesis and plant regeneration from these cereals require a specific sequence of defined media and controlled environments. The sequence and the timing of the media used, as well as their hormonal composition and balance are critical aspects of this process. The organic and mineral compositions of these media are not new but are important for supporting and sustaining rapid growth of the tissues.     

Impact of cefotaxime on somatic embryogenesis and shoot regeneration in sugarcane

A cephalosporin antibiotic, cefotaxime (Omnatax™) promoted somatic embryogenesis and subsequent shoot regeneration in vitro from spindle in sugarcane irrespective of the genotypes as (CoJ 83, CoJ 88 and CoJ 64) culturered on MS medium with 2,4-D (2.5 mgl⁻¹) and kinetin (0.5 mgl⁻¹). Seven different concentrations of cefotaxime (100, 200, 300, 400, 500, 600 and 700 mgl⁻¹) were tested to find the optimal concentration of cefotaxime for somatic embryogenesis from callus cultures. Among the three varieties, calli of variety CoJ 83 incubated on MS medium with 2,4-D (2.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) + cefotaxime (500 mgl⁻¹) exhibited maximum somatic embryogenesis. To improve shoot regeneration, the callus was transferred to MS medium with BAP (0.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) in combination with different levels of cefotaxime. Highest frequency of shoot regeneration was observed in callus of CoJ 83 in the presence of 500 mgl⁻¹ cefotaxime. The plantlets could be successfully hardened in polybags and transferred to soil, where they exhibited normal growth. Our results convincingly demonstrated that cefotaxime improves somatic embryogenesis from spindle and regeneration from embryogenic calli of sugarcane and hence can be strongly recommended for rapid and large scale multiplication of sugarcane.Key words: Saccharum officinarum L., leaf segments, callus, plant regeneration, antibiotic     

小麦幼胚培养中的体细胞胚胎发生

小麦品种崇阳红麦和鄂思一号杂种一代幼胚培养具有再生植株的潜力。从一个幼胚经200天左右的连续培养获得530多株再生植株,并从中获得了典型的具有两极性的与愈伤组织块仅局部相连的胚状体。体细胞胚胎发生是小麦幼胚培养的主要途径,但受培养条件的影响,以MS培养基作基本培养基,低浓度2,4-D(0.4mg/1)和水解酪蛋白(1000mg/l)有利于体细胞胚胎发生。     

Microspore culture of small grain cereals

Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis.In wheat, the effect of basic media (W₁₄, A2, CHB₃, P4-m) was compared and among them the W₁₄, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB₃ and MS_m media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB₃ media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.     

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.     

Comparative Effects of Rye Chromosomes 1R and 5R on Androgenesis in Cultured Anthers of Wheat–Rye Substitution Lines as Dependent on the Line Origin

The effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat–rye substitution lines was studied as dependent on the cultivar origin of the rye chromosomes and on the wheat genome (A or D) subjected to substitution. Chromosome 1R stimulated embryogenesis in anther cultures, while chromosome 5R suppressed it regardless of whether the corresponding wheat chromosomes were substituted in the A or D genome. The effect of chromosome 1R on embryogenesis proved to depend on its cultivar origin. Along with rye chromosome 1R, wheat chromosome 1A was shown to substantially affect total seedling regeneration. Regeneration of green seedlings was dramatically affected both by rye chromosome 1R and by wheat chromosome 1D. The results supported the published data that individual androgenesis parameters (embryogenesis, total plant regeneration, green plant regeneration) are controlled by different genetic mechanisms.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Comparative effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat-rye substitution lines dependending on the line origin

The effects of rye chromosomes 1R and 5R on androgenesis in cultured anthers of wheat--rye substitution lines was studied as dependent on the cultivar origin of the rye chromosomes and on the wheat genome (A or D) subjected to substitution. Chromosome 1R stimulated embryogenesis in anther cultures, while chromosome 5R suppressed it regardless of whether the corresponding wheat chromosomes were substituted in the A or D genome. The effect of chromosome 1R on embryogenesis proved to depend on its cultivar origin. Along with rye chromosome 1R, wheat chromosome 1A was shown to substantially affect total seedling regeneration. Regeneration of green seedlings was dramatically affected both by rye chromosome 1R and by wheat chromosome 1D. The results supported the published data that individual androgenesis parameters (embryogenesis, total plant regeneration, green plant regeneration) are controlled by different genetic mechanisms.     

Improved microspore embryogenesis induction and plantlet regeneration using putrescine,cefotaxime and vancomycin in Brassica napus L.

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l^?1) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l^?1 putrescine. Putrescine treatment at 0.5 mg l^?1 for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish^?1) was possible when induction medium was supplemented with 50 mg l^?1 cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l^?1 at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l^?1 cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l^?1 during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish^?1) in contrast to untreated cultures (93.6 embryos Petri dish^?1) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l^?1 for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.     

Effect of ticarcillin/potassium clavulanate on callus growth and shoot regeneration in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.)

Ticarcillin/potassium clavulanate is a very effective combination of antibiotics to eliminate Agrobacterium tumefaciens during tomato transformation. It shows no toxicity to tomato tissues at a concentration of 150 mg/l and significantly promotes callus formation and shoot regeneration. The transformation frequency was raised more than 40% in comparison to cefotaxime. Cefotaxime itself did not inhibit callus growth in culture medium, but it clearly decreased shoot differentiation. Together with kanamycin, cefotaxime shows a strong negative effect on callus growth, shoot regeneration and transformation efficiency. Unlike the widely used carbenicillin and cefotaxime, ticarcillin/potassium clavulanate is light stable and resistant to inactivation by β-lactamase. Furthermore, ticarcillin/potassium clavulanate is more economical than carbenicillin and cefotaxime. In conclusion, ticarcillin/potassium clavulanate is a very good alternative to eliminate Agrobacterium tumefaciens in plant transformation and has the potential to be widely used for plants which are sensitive to carbenicillin and cefotaxime. Received: 22 September 1997 / Revision received: 7 November 1997 / Accepted: 15 December 1997     

Influences of cefotaxime and carbenicillin on plant regeneration from wheat mature embryos

The influences of cefotaxime and carbenicillin on regeneration potential of wheat (Triticum aestivum L.) mature embryos were investigated. Filter-sterilized cefotaxime enhanced regeneration capacity although it did not affect the average number of shoots per explant. The highest regeneration capacity of 55.4 % was obtained on regeneration medium supplemented with 100 mg dm^?3 cefotaxime. Filter-sterilized carbenicillin did not stimulate plant regeneration. However, higher concentration (100 mg dm^?3) accelerated callus browning and inhibited the following regeneration. Autoclaved antibiotics at all tested concentrations showed detrimental effects on callus morphogenesis and plant regeneration.     

Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat

Cellular totipotency is one of the basic principles of plant biotechnology. Currently, the success of the procedure used to produce transgenic plants is directly proportional to the successful insertion of foreign DNA into the genome of suitable target tissue/cells that are able to regenerate plants. The mature embryo (ME) is increasingly recognized as a valuable explant for developing regenerable cell lines in wheat biotechnology. We have previously developed a regeneration procedure based on fragmented ME in vitro culture. Before we can use this regeneration system as a model for molecular studies of the morphogenic pathway induced in vitro and investigate the functional links between regenerative capacity and transformation receptiveness, some questions need to be answered. Plant regeneration from cultured tissues is genetically controlled. Factors such as age/degree of differentiation and physiological conditions affect the response of explants to culture conditions. Plant regeneration in culture can be achieved through embryogenesis or organogenesis. In this paper, the suitability of ME tissues for tissue culture and the chronological series of morphological data observed at the macroscopic level are documented. Genetic variability at each step of the regeneration process was evaluated through a varietal comparison of several elite wheat cultivars. A detailed histological analysis of the chronological sequence of morphological events during ontogeny was conducted. Compared with cultures of immature zygotic embryos, we found that the embryogenic pathway occurs slightly earlier and is of a different origin in our model. Cytological, physiological, and some biochemical aspects of somatic embryo formation in wheat ME culture are discussed.     

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.     

A novel method for increasing the frequency of somatic embryogenesis in wheat tissue culture by NaCl and KCl supplementation

The effect of NaCl, KCl and LiCl on the growth and morphogeneis of tissue cultures originating from immature embryos of four wheat (Triticum aestivum L.) and one triticale (Triticosecale)varieties was investigated. The morphogenetic pathway to plant regeneration in Chinese Spring wheat was determined as incomplete somatic embryogenesis because the differentiation and subsequent germination of the shoot apices happened in the early phase of embryo development. Culture medium supplemented by NaCl suppressed the differentiation of shoot apices resulting in the development of more typical somatic embryoids. Forty mM concentrations of both NaCl or KCl increased the formation of somatic embryos in Chinese Spring. Arthur and GK Kincso wheat varieties while Lasko triticale regenerated well without the addition. The salts inhibited plantlet formation from somatic embryoids so the salts supplement should be omitted. Forty mM LiCl inhibited growth while 10mM LiCl had no effect on growth or embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5mg/L benzylamino purine (BAP), 100mg/L kanamycin and 250mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.     

The effects of 2ip and 2,4-D on rice calli differentiation

The role of isopentenyl adenine (2ip) in cereal tissue and plant regeneration was analyzed by addition of this compound into various cultural media. Preculture of rice calli in the presence of certain levels of 2ip before transferring onto a normal tissue culture regeneration medium increased significantly the rate of embryogenesis as well as plant regeneration. Different combinations of 2ip and 2,4-D in preculture media had profound effects on cell differentiation. The procedures of embryogenesis and plant regeneration can be separated by manipulating the ratio of 2,4-D to 2ip in the culture media. Various possible functions of 2ip, 2,4-D and KT in cereal regeneration are also suggested.Abbreviations 2ip isopentenyl adenine - 2,4D 2,4-dichlorophenoxyacetic acid - KT kinetin - IAA indole acetic acid     

Zygote implantation to cultured ovules leads to direct embryogenesis and plant regeneration of wheat

Direct embryogenesis and plant regeneration were obtained by implantation of individual wheat ( Triticum aestivum L.) zygotes into cultured ovules of wheat or barley. The zygotes were isolated mechanically from emasculated spikes, 3–9 h after hand-pollination. In 13 independent experiments, a total of 186 zygotes were implanted into excised ovules obtained from emasculated spikes which had been treated previously with 2,4-dichlorophenoxyacetic acid to induce parthenocarpic, embryoless ovary development. On average, 17.2% of the implanted zygotes gave rise to dorsiventrally differentiated embryos. The embryos resembled those growing in planta with no obvious deviation from the zygotic embryogenesis pathway. In contrast to previously described regeneration systems from individual zygotes of higher plants, this is the first study in which direct embryo formation is reproducibly obtained without intermediate tissue dedifferentiation. Most embryos germinated when transferred to regeneration medium, and later formed phenotypically normal, fully fertile plants. Regenerants were confirmed to be derived from the implanted zygotes by means of AFLP and/or morphological analyses. Although zygote implantation has long been established as a useful method in sexual animal reproduction, an equivalent technique for plants is described here for the first time. Since the zygotes enter the embryogenic pathway directly, the genome is presumably as stable as during embryogenesis in planta . With this new approach, isolated wheat zygotes are accessible to micromanipulation without affecting their subsequent embryonic development.