Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)     

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

Induction of somatic embryogenesis in cultured cells of <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants resulting from breeding processes. An in vitro protocol has been developed for somatic embryogensis from calluses and cell cultures of Chenopodium quinoa. Callus was induced from hypocotyl explants within 2 weeks of culture on a modified Murashige and Skoog (MS) medium supplemented with 0.45 M 2,4-D. Calluses were cultured on solid or liquid MS medium and later the development of somatic embryos was observed on both employing the same MS medium without 2,4-D. To our knowledge this is the first report of somatic embryogenesis in Chenopodium quinoa.     

Effect of exogenous plant growth regulators on in vitro seed growth,embryo development,and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.)

Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA₃ and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar Klages × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA₃ alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA₃ produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

Comparative study of somatic embryogenesis from immature and mature embryos and organogenesis from leaf-base of Triticale

Immature and mature zygotic embryos of hexaploid, Triticale var. DT-46 formed an embryogenic callus, with subsequent somatic embryo formation upon subculture to MS (Murashige and Skoog, 1962) or N₆ (Chu et al., 1975) nutrient medium supplemented with various concentrations (9.0–22.5 M) of 2,4-dichlorophenoxyacetic acid (2,4-D). Of the two types of explants, embryogenic tissue from immature embryos responded at a higher frequency, to form somatic embryos over the callus surface. Leaf-base segment cultured on to 2,4-D-containing medium formed a tissue which did not form somatic embryos and instead differentiated into shoot-buds. N₆ medium proved to be more effective than MS in support of somatic embryogenesis or shoot-bud formation. Regeneration of plantlets occurred on 2,4-D-free basal medium. These in vitro-formed plantlets were successfully transferred to soil and set seed.     

Anther culture of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis>: callus formation and the development of multicellular and embryo-like structures

Androgenesis is an important technique to generate double haploid plants. Anther and microspore cultures are the methods to induce haploid embryogenesis. For culture initiation, it is necessary to select anthers with the appropriate developmental stage of microspores. For lupins, limited reports about the establishment of initial cultures for androgenesis are available. In this study, different parameters of anther culture of three genotypes of Lupinus angustifolius were investigated. For all genotypes, a considerable correlation was observed between the buds and the anthers, depending on their location in the inflorescences. Buds from the central segment of inflorescences had yellowish green anthers that contained the maximum number of microspores at uninucleate stage. Cytological investigation shows that the anthers containing these microspores were the most responsive to induction. Two types of developmental pathways were observed for microspores. In case of cold pre-treated and untreated inflorescences, microspores developed into multicellular and embryo-like structures, respectively. Effects of different factors showed significant differences among: genotypes, pre-treatment, growth regulators (GRs) and genotypes × GRs interaction. Among three genotypes, Emir showed the highest number of multicellular and embryo-like structures on MS medium + 2.0 mg/l 2,4 D + 0.5 mg/l Kinetin (Kin). For all genotypes, anthers produced calli on MS medium containing 2.0 mg/l 2,4 D + 0.5 mg/l Kin. These calli continued their growth on regeneration medium (MS + 2.0 mg/l BA + 0.5 mg/l NAA) and produced roots. Taken together, these results provide a good basis for further research towards the development of haploid plants for L. angustifolius.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

Endogenous phytohormone profile during oat (Avena sativa L.) haploid embryo development

The development of embryos requires interaction of many endogenous hormones. The aim of the study was to determine which endogenous phytohormones are involved in the process of oat (Avena sativa L.) haploidization. Oat haploids were obtained by wide crossing with Zea mays L. The hormonal profiles of the ovaries with (OE) and without developed embryo (OWE) were compared. Phytohormone contents were measured by UHPLC coupled with mass spectrometer. The total content of indole-3-acetic acid (IAA), trans-zeatin (tZ), and kinetin (KN) in OE was significantly higher than in OWE. 4-Chloroindole-3-acetic acid was detected only in OWE. There were no differences between OE and OWE in the content of gibberellins (GA₁, GA₃, GA₄, GA₆, GA₇) and stress hormones (abscisic, salicylic, jasmonic acids). Content of endogenous KN was highly negatively correlated with the percentage of haploid embryos, germinated haploid embryos, haploid plants on MS (in vitro), haploid plants in soil (ex vitro), and doubled haploid lines. The tZ content negatively correlated with the frequency of haploid embryo formation, germination, and haploid plants obtained in vitro, as opposed to GA₁, which correlated positively. A positive correlation was found between IAA and tZ in OE, whereas in OWE it was a negative correlation. The profiles of phytohormones in OE and OWE were determined; however, their mode of action needs to be clarified.

     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

The inheritance of genetic markers in microspore-derived plants of barley Hordeum vulgare L.

Summary Biochemical, molecular and morphological markers have been used to monitor the segregation of alleles at major gene loci in microspore-derived lines of four spring barley crosses and their parents. Significant deviations from the expected Mendelian ratios were observed for four of the ten markers studied in the cross. Distorted ratios were associated with loci located on chromosomes 4H and 6H. The differential transmission of alleles was in favour of the responsive parent (Blenheim) used in the anther culture studies. For the -Amy-1 locus on chromosome 6H, the preferential transmission of Blenheim alleles was most pronounced in the haploid regenerants that were colchicine treated. These results are discussed in relation to the genetic control of androgenetic response in barley and with respect to the exploitation of another culture in barley improvement.     

Reflexions sur nos cultures d'antheres de plantes cultivées

Abstract

Considerations about our anther cultures of cultivated plants. – One of the main activities performed at the Casaccia Nuclear Centre, in the framework of a contract between CNEN and the European Communities, centers on the induction of haploid plants by anther culture and the subsequent chromosome doubling in order to obtain completely homozygous diploid plants. In tobacco, it is now possible to obtain haploid plants from any cultivar; we perform in vitro culture of internodes from which homozygous diploid plants are regenerated, taking advantage of natural phenomenon of endopolyploidy. In order to try to generalize this method of producing haploid plants in other plant species, we are studying the mechanism involved in haploid embryogenesis which occurs in vitro in the microspores. Datura, Nicotiana and Atropa are among the genera in which a direct embryogenesis from the microspore is observed; it is interesting to note that all three genera belong to the family Solanaceae and are very rich in alkaloids. In almost all the other cases of in vitro induction of haploids, microspores produce calli from which plantlets can be differentiated, but this way of plant regeneration is less interesting because only few plantlets are obtained and it is not sure that each haploid comes from a single microspore. We examined the factors which could influence the transformation of microspores into embryoids in tobacco, namely: the developmental stage of microspore, the degeneration of tapaetal cells, the genotype of microspore, the composition of cultural media, the physiological conditions of the plant from which the anthers were taken. From a practical point of view, it would be desirable to have informations on methods giving a maximum number of haploid plants from one embryogenic anther and the greatest number of embryogenic anthers from the cultured anthers. Our recent experiments on anther culture in liquid shaken medium have yielded good results (about 7,000 embryoids from 25 embryogenic anthers). Further, we are conducting several experiments in order to synchronize the development of the microspores in the anthers; to this end, we analyse the effect of cold treatment, ionizing radiation and gravity force. Experiments are being performed with other cultivated species, beside tobacco, in order to solve some problems of plant breeding more easily and quickly through haploidy. With the aim of introducing, in cultivated tomato, some desirable characters from the wild species, Lycopersicum peruvianum, (self-incompatibility, disease resistance, simultaneous flowering), we have obtained the interspecific hybrid through in vitro culture of young embryos. Haploid production from this hybrid could allow to quickly obtain various genetic recombinations from these two species. For this purpose we are carrying out anther cultures as well as single microspore cultures. In rice, strawberry and L. peruvianum, several diploid and tetraploid plantlets were obtained from our anther cultures. Work is in progress to ascertain the mode of their origin.     

Direct somatic embryogenesis and shoot regeneration from leaves and internodes of Pluchea lanceolata (DC.) C.B. Clarke

Pluchea lanceolata (DC.) C.B. Clarke is a threatened native medicinal plant. Increasing the propagation of this plant will preserve the wild population and provide material for medicinal use. In vitro and field-collected shoots and leaves were tested for response to 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), for initiation of direct shoot regeneration (DSR), or direct somatic embryogenesis (DSE). Leaves and internodes collected from field-grown plants produced only callus, while in vitro–raised shoots exhibited DSR and DSE on Murashige and Skoog (MS) medium with 2,4-D and TDZ. Direct shoot regeneration occurred on medium with TDZ from internode and leaf segments obtained from in vitro–developed shoots. In vitro–grown shoots were rooted on half-strength MS medium with 2 mg L⁻¹ indole-3-butyric acid and acclimatized. Survival in natural conditions was 62.5% for DSE and 79% for DSR plantlets.

     

<Emphasis Type="Italic">In vitro</Emphasis> studies to produce double haploid in <Emphasis Type="Italic">Indica</Emphasis> hybrid rice

The aim of this investigation was to improve in vitro the technique of production of double haploid in Indica hybrid rice by combining anther culture, hormone shock and doubling chromosome. It was discussed how to avoid somaclonal variation during culturing and to reduce the time of this process. The anthers of KDML 105 × SPR 1 (Indica × Indica) were cultured in Linsmaier and Skoog (LS) medium, which contained nutrients, growth regulators [(2,4,-dichlorophenoxy acetic acid (2,4-D) and naphthalene acetic acid (NAA)] and organic compounds, and then subcultured by inducing embryo-like structure (ELS) LS media. During 4 weeks used LS media supplemented with 10 μM KNO³ + 2 mg/L 2,4-D + 2 mg/L NAA + 20% coconut water + 1 mg/L of activated charcoal had induced high embryogenic frequent callus with length of 4–5 mm. The supplementation of 0.2 g/L colchicine and 100 μM 2,4-D was the most efficient in LS media. Over 70% of viable double haploid ELS were produced in 8 weeks and subcultured only twice compared with conventional anther which takes more than 12 weeks. This new technique can therefore be applied to rice in order in shorten time to produce higher number of double haploid plantlets.     

Somatic embryogenesis in <Emphasis Type="Italic">Buchanania lanzan</Emphasis> spreng

Summary We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species.     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.