Viable Plantlets Research Articles

Using float hydroculture for acclimatization of in vitro plantlets in some pyrus genotypes

In this study, the potential of float hydroculture for the acclimatization of in vitro plantlets of two wild pear types (Pyrus elaeagrifolia Pall.) and Old Home x Farmingdale 333 (OHxF 333) pear rootstock (P. communis L.) was investigated. The wild pear types are obtained from our dwarf clone rootstock breeding program and are resistant to drought and chlorosis stresses. In vitro plantlets were generated by rooting micro cuttings with 250 mg L– 1 IBA by quick-dip method for 10 s under aseptic conditions. After this treatment, the micro cuttings were cultured on MS basal medium with ½ strength of macro and micro elements without growth regulators for 4 weeks. The cultures were incubated in the dark for the first 5 days. For acclimatization by float hydroculture, in vitro plantlets were placed in the cells of a styrofoam tray on plastic tubs (25 cm x 25 cm x 12 cm) filled with tap water. The water was not replaced during acclimatization. The float hydrocultures were incubated for 4 weeks in a climate room at 25±2 °C with 16 h light (35 μmol∙m– 2∙s– 1) and 8 h dark photoperiod for 4 weeks. At the end of this period, the percentage of viable plantlets removed from float hydroculture was 84.0%, 62.7% and 64.7% in OHxF 333, AH-32 and AH-75, respectively. These percentages are quite high in wild pear types that could not acclimatize to external conditions with known methods. There was no significant difference between leaf mesophyll thickness of Pyrus plantlets removed from float hydroculture and the mature plants in the orchard. These values varied between 153.0 ± 18.2 µm and 202.4 ± 4.8 µm. In conclusion, float hydroculture is identified as an easy and successful acclimatization technique for genotypes that are particularly difficult to acclimatize in vitro plantlets such as wild pear types.

Optimization of an In Vitro Embryo Rescue Protocol for Breeding Seedless Table Grapes (Vitis vinifera L.) in Italy

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness.

Why so Complex? The Intricacy of Genome Structure and Gene Expression, Associated with Angiosperm Mitochondria, May Relate to the Regulation of Embryo Quiescence or Dormancy-Intrinsic Blocks to Early Plant Life.

Mitochondria play key roles in cellular-energy metabolism and are vital for plant-life, such as for successful germination and early-seedling establishment. Most mitochondria contain their own genetic system (mtDNA, mitogenome), with an intrinsic protein-synthesis machinery. Although the challenges of maintaining prokaryotic-type structures and functions are common to Eukarya, land plants possess some of the most complex organelle composition of all known organisms. Angiosperms mtDNAs are characteristically the largest and least gene-dense among the eukaryotes. They often contain highly-variable intergenic regions of endogenous or foreign origins and undergo frequent recombination events, which result in different mtDNA configurations, even between closely-related species. The expression of the mitogenome in angiosperms involves extensive mtRNA processing steps, including numerous editing and splicing events. Why do land-plant’s mitochondria have to be so complex? The answer to this remains a matter of speculation. We propose that this complexity may have arisen throughout the terrestrialization of plants, as a means to control embryonic mitochondrial functions —a critical adaptive trait to optimize seed germination. The unique characteristics of plant mtDNA may play pivotal roles in the nuclear-regulation of organellar biogenesis and metabolism, possibly to control embryos quiescence or dormancy, essential determinants for the establishment of viable plantlets that can survive post-germination.

Insights into the conversion potential of Theobroma cacao L. somatic embryos using quantitative proteomic analysis

Abstract Somatic embryogenesis (SE) has been routinely used as mass micropropagation technique, and as a model system for investigating the structural, physiological, and molecular events occurring during somatic embryo development. Successful in vitro SE is related to the quality and yield of somatic embryos obtained. In cacao (Theobroma cacao L.) SE, the efficiency of somatic embryo production has improved with secondary SE. However, the low number of somatic embryos able to conversion into viable plantlets is still low. Then, two morphological types of normal mature somatic embryos can be identified during cacao secondary SE. The first type shows white appearance and high conversion potential (75%), while the second type shows translucent appearance and exhibit low conversion potential (15%). In order to investigate the proteins that can be associated to conversion potential in cacao somatic embryos, the mass spectrometry HDMSE proteomic approach was used. At least 60 proteins showed differences in abundance levels in cacao white somatic embryos, when compared to translucent. An increased abundance of Beta-glucosidase, NAD(P)-linked oxidoreductase and Electron transfer flavoprotein proteins were observed in white somatic embryos. Moreover, in translucent somatic embryos were observed an increased abundance of Cytochrome P450 and Pathogenesis-related proteins. Using white somatic embryos as a model, we suggest that carbohydrate metabolism process and the redox regulation are involved in the control/regulation of somatic embryo quality. These new findings may improve cacao SE protocol, as well as the understanding of the role of pivotal metabolic pathways associated to this in vitro morphogenetic route.

Viability and susceptibility of propagation material from coffee plants to Colletotrichum sp.

This study aimed to verify the viability of propagation material from coffee plants descended from germplasm susceptible to blister spot disease as well as its susceptibility to Colletotrichum sp. relative to commercial coffee cultivars. In the first experiment, fruits were harvested from plants with and without symptoms of blister spot and sowed in trays containing a commercial sterilized substrate. The percentages of germinated seeds, viable plantlets and seedlings were evaluated. In diseased tissues, pathogens were isolated and identified though a pathogenicity test. In the second experiment, ten commercial cultivars and one cultivar originating from plants with blister spot were inoculated with the pathogens to assess the severity of anthracnose. Significant differences were not observed with respect to seed germination. However, the viability of plantlets and seedlings was reduced in the cultivar originating from plants with blister spot (Genotype Originated from Diseased Plants-GODP). These plants showed characteristic symptoms of blister spot, including necrosis in the leaves and hypocotyls, wilting and death. In the necrotic lesions, we observed characteristic sporulation of Colletotrichum sp. The cultivar most susceptible to anthracnose in cotyledonary leaves was Catuaí Vermelho (GODP), which presented the highest area under the disease progress curve (AUDPC). In conclusion, the viability of propagation material from coffee plants that had descended from plants with symptoms of blister spot (GODP) was reduced compared with plants from other genotypes, although seed germination was not affected. Moreover, GODP species are more susceptible to Anthracnose on the cotyledonary leaves relative to the other analyzed commercial cultivars. This work is the first to report on different symptoms exhibited by seedlings originating from the seeds of plants with symptoms of blister spot.

Truncation of LEAFY COTYLEDON1 Protein Is Required for Asexual Reproduction in Kalanchoë daigremontiana

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

Effectiveness of tissue culture media components on the growth and development of cauliflower (Brassica oleracea var. Botrytis) seedling explants in vitro

A key factor in the application of in vitro techniques to cauliflower improvement is the development of efficient protocols for regeneration of plants from tissue for use in breeding programs for the selection of the desirable genotypes under biotic and abiotic stress. Experiments were conducted to study the effect of different media components (agar or agar + sucrose or agar + Murashige and Skoog (MS) salts or agar + sucrose + MS) on callus induction and regeneration from five explants types (cotyledon, hypocotyls, shoot apex, primary root and root tip) and also the effects of auxin and cytokinin were carried out using one F 1 hybrid cauliflower cv. Medallion. The results show that cotyledons, mid roots and root apices grown on agar + MS + sugar were the most developed compared with explants on the other media. This medium was the most productive in terms of lateral root number and root length. The presence of 2,4-dichlorophenoxyacetic acid increased callus production compared to 6-benzylamino purine. Within the selected explants, a significant difference was indicated between different explants under different treatments. Liquid culture was more successful at producing viable plantlets than solid culture. Key words: Cauliflower, explants, in vitro , growth regulator, growth characters, solid and liquid culture.

Carrot (Daucus carota L.) In Vitro Regeneration

Carrot (Daucus carota L.) In Vitro Regeneration Experiments were carried out for optimization of conditions for morphogenic callus production and regeneration of carrot (Daucus carota L.) using four cultivars cultivated in Iran. Hypocotyl segments were put on media supplemented with 0.2 mg·L-1 2,4-D for callus initiation and proliferation. Small portions of callus (25 mg) were cut and transferred to new media with different concentrations of 2,4-D including 0.2, 0.5 and 1 mg·L-1. MS (Murashige & Skoog 1962) and MSm (Masuda et al. 1981) media were used in the experiments to identify the differences between these two media according to callus, embryoid and plantlet production. Obtained results showed that low level of 2,4-D (0.2 mg·L-1) was more effective in morphogenic callus production although higher amounts of it (0.5 mg·L-1 and 1 mg·L-1) caused more callus growth. Kinetin in concentration of 0.1 mg·L-1 was more effective for regeneration than BAP used in concentration 1 mg·L-1 especially according to embryoid production. MSm medium was more useful than MS for callus production and regeneration of carrot plantlets. Among tested cultivars Nantes Improved had more capability to produce viable plantlets.

In Vitro Asymbiotic Germination, Protocorm Development, and Plantlet Acclimatization of Aplectrum Hyemale (Muhl. ex Willd.) Torr. (Orchidaceae)

A method to significantly improve the germination and development of plantlets of Aplectrum hyemale was developed. Seeds of this terrestrial orchid were collected in a natural stand, surface disinfected and grown in vitro on a gellified growth medium in absence of symbiotic fungi. Seeds were sowed whole or after excision of the seed coat. Embryos that were removed from the seed coat showed a significant increase in germination and survival percentages compared to whole seeds. Embryos grew to form a ramified protocorm, irrespective of seed treatment, followed by plantlet formation. Plantlets were successfully acclimatized and transferred to soil conditions. Asymbiotic in vitro culture is therefore shown as a potential tool to produce viable plantlets for use in natural site restoration.

Endogenous ascorbic acid modulates meristem reactivation in white spruce somatic embryos and affects thymidine and uridine metabolism

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.

THE BENEFICIAL ROLE OF DIFFERENT AUXINS AND POLYAMINES AT SUCCESSIVE STAGES OF SOMATIC EMBRYO FORMATION AND DEVELOPMENT OF PANAX GINSENG IN VITRO

The production of viable plantlets via somatic embryogenesis in Panax ginseng requires different culture media corresponding to successive developmental stages. The effects of several auxins and polyamines have been tested at various steps. Multiplication of the embryogenic root-derived callus has been optimized on half-strength MS medium supplemented with 3-(benzo[b]selenyl)acetic acid (BSAA, a synthetic auxin) and kinetin; exogenously applied polyamines were deleterious at this stage, causing browning of the callus, diminished capacity of embryo initiation, and an increased tendency to hyperhydricity. BSAA again appeared to be the most favourable auxin at the initiation stage, but here its action was reinforced by the presence of polyamines, spermidine being the most efficient. Among the auxin needed at the next step, i.e., for the regeneration of embryos, the two seleniated auxins BSAA and seleniated 2,4-dichlorophenoxyacetic acid (2,4-D-Se) were the more efficient. For the harmonious development of plantlets, i.e., the simultaneous outgrowth of shoots and roots, the polyamines were favourable, with a greater efficiency for spermine.

Histological investigation of the multiplication step in secondary somatic embryogenesis of Quercus robur L.

Standardized explants composed of hypocotyl and root-tip were prepared from embryonic structures obtained from one em- bryogenic line of Quercus roburL. maintained by regular transfer onto a solidified reference medium composed of the MS mineral solu- tions, glucose (0.12 M), casamino acid (0.1%), and NAA (10.74 µM). The regeneration capacity from these explants were tested on the reference medium and on 2 alternative media in which the NAA (10.74 µM) was omitted or substituted for a combination of IBA (9.80 µM)/BAP (8.90 µM). Within 30 days, 4 types of responses were observed including direct and indirect secondary embryogenesis. In the direct pathway, somatic embryos arose from 3-4 epidermal cells following two different modes, depending on whether or not the formation of a meristematic mass preceded the initiation of the embryogenic process. In the indirect pathway the embryos were formed from clumps of mitotically active cells included in callus developed within the cortical tissues. Depending on their histological origin, the embryos exhibited differences in their structural organization which could influence their potential for further maturation and conversion into viable plantlets. Explants prepared from small translucent embryonic structures were more embryogenic and expressed the direct pathway of secondary embryogenesis at higher frequency than explants prepared from more advanced embryonic structures. On the culture medium without growth regulator, direct secondary embryogenesis was the exclusive response whereas on the culture medium with growth regulators added both direct and indirect secondary embryogenesis occurred. NAA favoured the direct secondary embryogenesis, while conversely, the IBA/BAP combination stimulated the indirect secondary embryogenesis. The results are discus- sed in reference to the PEDC concept (pre-embryogenic determined cells). secondary embryogenesis / growth regulators / histology / oak / PEDC

Auxin-regulated gene expression and embryogenic competence in callus cultures of sweetpotato, Ipomoea batatas (L.) Lam.

High levels of 2,4-dichlorophenoxyacetic acid (2,4-D, 10 µM) and sucrose (3%) are required for both the induction and maintenance of callus for somatic embryogenesis in sweetpotato. Newly inducted embryogenic callus lines in sweetpotato cv. White Star produce competent embryos that convert readily into plantlets. With age, these embryogenic callus lines produce a greater proportion of incompetent embryos with poor conversion potential. One hypothesis for this change is that auxin- and/or sugar-responsiveness may be altered with aging. A comparison of two older embryogenic lines (K592 and M892) to two new lines (K1194 and K195) addressed the relationship between extent of embryo conversion and relative abundance of mRNAs hybridizing with heterologous auxin- or sugar-responsive gene probes. The respective cDNAs utilized were the auxin-responsive pJCW1 and pJCW2 and the sugar-responsive Ivr2 and Sh1. Embryos from new callus lines formed more shoots, roots, and viable plantlets than embryos from older callus lines. In addition, new callus lines had greater relative levels of mRNAs hybridizing to auxin-responsive cDNAs. These sweetpotato mRNAs were themselves found to be 2,4-D-responsive in dosage analyses. In contrast, differences between young and old cultures were not evident for mRNAs hybridized to sugar-regulated genes. Our results support the suggestion that desensitization of auxin-responsiveness is a central feature of reduced embryogenic competence in callus lines following prolonged exposure to 2,4-D and elevated sucrose levels.

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos').

St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N'-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l-1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders.

Precociously germinating somatic embryos of Vitis vinifera have lower ABA and IAA levels than their germinating zygotic counterparts

Somatic embryos of Vitis vinifera (cv. Grenache noir) develop normally up to the torpedo stage, but they germinate precociously and form viable plantlets with very low frequency. Because a peak in abscisic acid (ABA) in mid‐embryogenesis could be one factor preventing precocious germination during normal seed development, we followed the development of ABA content concurrent with that of the somatic embryos. Additionally, we measured changes in indoleacetic acid (IAA) levels. We also compared the levels of both hormones during precocious germination of somatic embryos and during normal germination of their zygotic counterparts. Somatic embryos were able to accumulate ABA and IAA throughout their development but no peak in ABA concentration was detected during embryogenesis. This suggests that the switch from mid‐ to late‐embryogenesis is not triggered. Furthermore, during precocious germination, i.e. from the torpedo stage onwards, the concentrations of ABA and IAA in somatic embryos were much lower than during normal germination of zygotic embryos. Thus, it is likely that when precocious germination occurs, grape somatic embryos do not accumulate ABA and/or IAA in sufficient concentrations to support normal plantlet development. Therefore, for grape somatic embryos we propose that prevention of precocious germination, i.e. triggering late‐embryogenesis, is attainable by an ABA treatment followed by slow desiccation, as already shown for conifer somatic embryos. Our results also suggest that the role of ABA and IAA for improving normal germination after imposed quiescence should be investigated.

Somatic embryogenesis on Thin Cell Layers of a C4 species, Digitaria sanguinalis (L.) Scop

Somatic embryogenesis was obtained from transverse thin cell layers (tTCLs) of Digitaria sanguinalis. tTCLs (0.2 - 0.4mm thick, 1mm in diameter) were excised from 4-week-old seedlings and placed onto Murashige and Skoog media supplemented with a varying concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (from 1 µM to 100 µM) and sucrose (from 3% to 24%). Somatic embryos were obtained in the dark 7-10 days after inoculation from tTCLs excised at specific levels on the seedling and cultured in the presence of 2,4-D (5 µM to 10 µM) and sucrose (3 to 6%). The exposure of the tTCLs to light decreased the percentage of tTCLs forming somatic embryos. Viable plantlets were obtained 2 weeks after transfer onto a cytokinin-containing medium.

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine ( Vitis viniferaL.): Embryonic Organogenesis

Somatic embryos of Vitis viniferaL. ‘Grenache noir’ develop abnormally and form viable plantlets at very low frequencies. They grow continuously and, after the torpedo stage, they form giant structures which do not undergo further organogenesis. Morphological, histological and cytochemical data were used to study development from the globular to the giant-embryo stage. Histological organization of somatic embryos until the torpedo stage was similar to that of zygotic embryos. Somatic embryos formed bipolar axes, which differentiated precociously and simultaneously a root and a shoot meristem. However, they differed from their zygotic homologues by forming a cotyledonary crown or multiple cotyledons and by their rapid cellular differentiation. At the end of the torpedo stage and up to the giant-embryo stage, somatic embryos underwent some characteristic events of germination: the radical grew, tannins accumulated, and protodermal cells suberized. However the shoot apex was rapidly disorganized and disappeared. This peculiar behaviour is discussed in comparison with the phenomenon of precocious germination often observed for immature zygotic embryos in in vitroculture.

Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridization

Interspecific crosses were made by hand-pollination of Allium cepa with pollen of 19 species belonging to nine sections of two subgenera of the genus Allium. In all cases viable plantlets were obtained from ovary culture. The efficiency depended on the relationship of the pollen donor to A. cepa. The hybrid character of the regenerants was checked by morphological comparisons with the parents and/or by one or more cytological methods such as flow cytometric DNA measurement, karyotype analysis, and genomic in situ hybridization (GISH). Hybrids were confirmed for 18 new species combinations. The viable hybrid of the most distant cross resulted from crossing A. cepa with A. sphaerocephalon. The relevance of the verification methods and the potential use of the hybrids for breeding purposes are demonstrated.

Plantlet Regeneration from Encapsulated and Non-encapsulated Desiccated Somatic Embryos of a Forest Tree: Sandalwood (Santalum album L.)

Somatic embryos obtained from embryogenic tissues of sandalwood (Santaium album) were grown on White’s medium containing abscisic acid, (ABA, 1.89, 3.78 or 18.92 μM) and various concentrations of sucrose (87.6 to 350.4 mM) to induce maturation. The embryos were isolated and desiccated for 10, 20 or 30 days: One lot of the desiccated somatic embryos was encapsulated in sodium alginate gel and the other lot was not encapsulated. Both encapsulated and nonencapsulated desiccated embryos showed revival of growth upon rehydration on White’s medium and developed into plants. The desiccation tolerance and regeneration of viable plantlets depended upon the pretreatment given to somatic embryos. Embryogenic tissue subjected to dry state for 30 days showed revival of somatic embryogenesis upon transfer to a fresh nutrient medium. Implications of maturation and desiccation of somatic embryos on its germinability are discussed.