Cloning mature holm oak trees by somatic embryogenesis

Key message

Integuments from holm oak developing ovules were suitable initial explants to obtain embryogenic lines from which plants could be regenerated.

Abstract

The implementation of multivarietal forestry as part of breeding strategies is expected to provide more productive forest plantations. To achieve this, a reliable and effective method for mass production of clonal plants is needed. Somatic embryogenesis is considered the enabling technology for implementing multivarietal forestry. The holm oak (Quercus ilex L.) is a Mediterranean evergreen tree of economic interests because of the acorn production for animal feed and edible fungi mycorrhization. The aim of this work was to obtain clonal plants by inducing somatic embryogenesis in tissues of female flowers from mature trees. The influence of the developmental stage of the explant, the genotype and medium composition, and the effect of arabinogalactan proteins on the induction frequency, were assessed. Somatic embryogenesis induction (frequency ranging from 1.2 to 3.2 %) was restricted to ovules excised at an advanced stage of development, when they were at least 3–4 mm wide and the rest of the ovules within the ovary had aborted. Somatic embryos arose from the integuments of those fertilized ovules. Embryogenic response was obtained on media with and without plant growth regulators. All the genotypes that were cultured on medium containing “as reported by Schenk and Hildebrandt (Can J Bot 50:199–204, 1972)” SH macronutrients could be captured. Treatments including Larix arabinogalactan proteins did not improve induction, while those from Acacia inhibited the embryogenic response. Several embryogenic lines were multiplied by repetitive embryogenesis on medium lacking plant growth regulators. Mature somatic embryos of three genotypes were germinated at frequencies ranging from 41 to 58 %, and converted into plants at frequencies from 11 to 30 %, depending on the genotype.     

Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis

Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2–4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes.     

Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii sarg, trees

Summary The present investigation reports optimized parameters for somatic embryogenesis and cryopreservation of embryogenic cultures using shoot apical domes from mature trees of Pinus roxburghii Sarg. Embryogenic tissue of P. roxburghii Sarg. was cryopreserved for 24 h, 10 d, and 8 wk using sorbitol and dimethylsulfoxide (DMSO) as cryoprotectants. Results indicate that 0.2M sorbitol and 5% DMSO had the best cryoprotecting effect. The recovered tissue showed luxuriant growth on maintenance medium (II). Partial desiccation of thawed embryogenic tissue for 24 h prior to transfer to maturation medium enhanced the maturation of somatic embryos. Maturation frequency increased from 1.3 to 18.3% after 12 h desiccation treatment, and from 18.3 to 61.8% after 24 h of desiccation. However, non-desiccated embryogenic tissue produced the least number of somatic embryos (1.3%) on the maturation medium with the same abscisic acid and Gellan gum concentration. All the three embryogenic lines produced plantlets and had the same appearance and normal growth as compared to unfrozen controls.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

Vegetative propagation of Quercus suber L. by somatic embryogenesis. I. Factors affecting the induction in leaves from mature cork oak trees

Somatic embryogenesis was induced in expanding leaves from epicormic shoots forced to sprout from segments of branches collected from several hundred-year-old cork oak trees. Following a basic protocol previously defined for leaves taken from seedlings of this species, several factors were studied to improve the response. The induction frequency was significantly higher when the length of exposure to growth regulators was increased from 7 to 30 days. The combined application of NAA and BAP was essential for induction. Although both regulators had a very significant influence, their interaction was not significant, suggesting independent roles. Leaf size had a crucial effect, because beyond a certain threshold, embryogenesis could not be obtained. Embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium for more than 2 years.     

Cryopreservation of embryogenic tissues from mature holm oak trees

The development of a vitrification method for cryopreservation of embryogenic lines from mature holm oak (Quercus ilex L.) trees is reported. Globular embryogenic clusters of three embryogenic lines grown on gelled medium, and embryogenic clumps of one line collected from liquid cultures, were used as samples. The effect of both high-sucrose preculture and dehydration by incubation in the PVS2 solution for 30–90 min, on both survival and maintenance of the differentiation ability was evaluated in somatic embryo explants with and without immersion into liquid nitrogen. Growth recovery of the treated samples and ability to differentiate cotyledonary embryos largely depended on genotype. Overall, high growth recovery frequencies on gelled medium and increase of fresh weight in liquid medium were obtained in all the tested lines, also after freezing. However, the differentiation ability of the embryogenic lines was severely hampered following immersion into LN. Two of the embryogenic lines from gelled medium were able to recover the differentiation ability, one not. In the lines with reduced or no differentiation ability, variation in the microsatellite markers was observed when comparing samples taken prior to and after cryopreservation. The best results were achieved in the genotype Q8 in which 80% of explants grown on gelled medium differentiated into cotyledonary embryos following cryopreservation when they were precultured on medium with 0.3 M sucrose and then incubated for 30 min in the PVS2 solution. Explants of the same genotype from liquid medium were unable to recover the differentiation ability. A 4-weeks storage period both in liquid nitrogen and in an ultra-low temperature freezer at −80 °C was also evaluated with four embryogenic lines from gelled medium using the best vitrification treatment. Growth recovery frequencies of all lines from the two storage systems were very high, but their differentiation ability was completely lost.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Cloning adult trees of Arbutus unedo L. through somatic embryogenesis

Arbutus unedo L. is a perennial tree, native in the Mediterranean area, and tolerant to stress conditions. Due to its economic potential, there is an increasing demanding for plants by producers and farmers. In order to offer cloned material with assured quality, several micropropagation protocols have been developed, including somatic embryogenesis induction. However, little is known about this process on strawberry tree and a great deal of work is still necessary to successfully clone in A. unedo through somatic embryogenesis. Thus, the main goals of this work were: (i) to test the effect of the genotype on somatic embryogenesis induction, (ii) to analyse the role of adult and young materials on induction, and (iii) to perform a comparative histological analysis between somatic embryos and their zygotic counterparts. Somatic embryogenesis was induced on apical expanded leaves from in vitro shoots of several genotypes in Andersson medium with 3% sucrose and different concentrations of BAP (2.0 mg L^?1) and NAA (0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 mg L^?1). Embryogenesis induction rates ranged from 0 to 94.5%. Higher induction rates were achieved on the medium with 2 mg L^?1 BAP and 2 mg L^?1 NAA, and are genotype dependent. After a 3-month induction period, the highest somatic embryogenesis induction rate was 94.5% on genotype AU4. Embryos at different developmental stages were found, as well as abnormal somatic embryos. SEM images showed different anomalies being the most common embryos displaying more than two cotyledons or fused embryos. Embryo germination was not genotype dependent and the maximum embryo conversion rate achieved was 73.5%. However, only 39.21% of the embryos were able to grow into plantlets which displayed a normal chromosome number (2n?=?26). Histological analysis showed differences in the cell organization between somatic and zygotic embryos, as well as several morphological anomalies. Overall, the developed somatic induction protocol proved to be very efficient, with high induction rates achieved, both from seedling and adult material, but its genotype dependent. However, embryo conversion still needs to be improved, in order to fully seize the potential of this micropropagation technique.

     

Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Plant regeneration through somatic embryogenesis from mature leaf explants of Eryngium foetidum,a condiment

Eryngium foetidum L. is an important plant cultivated as a leafy vegetable and for its essential oil, which are of high economic value in international trade market. Plants were regenerated through somatic embryogenesis from mature leaf explants of field grown plants. Leaf explants produced dark brown, compact callus on Linsmaier and Skoog (LS) medium with the combination of 1.0 mg l^-1 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.0 mg l^-1 benzylaminopurine (BAP). Somatic embryos were induced from embryo-forming callus cultures on Murashige and Skoog (MS) medium supplemented with 0.1 mg l^-1 2,4-D, 2.0 mg l^-1 BAP and 1.0 mg l^-1 gibberellic acid (GA₃). Subsequently, conversion of these somatic embryos into plantlets occurred on MS medium supplemented with 1.0 mg l^-1 GA₃ and/or 0.1 mg l^-1 BAP. The regenerated shoots were rooted and elongated on MS medium supplemented with 0.1 mg l^-1 IAA and 1.0 mg l^-1 GA₃. These plantlets were hardened and transferred to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 μM naphthalene acetic acid and 2.22 μM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.     

Induction of somatic embryogenesis from different explants of shoot cultures derived from young <Emphasis Type="Italic">Quercus alba</Emphasis> trees

The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old white oak (Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage. Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7–8 weeks after culture initiation, although most emerged after 9–12 weeks in culture. The sequence of application of media for somatic embryo induction was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and 2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%) were achieved after cold storage for 2 months.     

Characteristics of Bupleurum falcatum plants propagated through somatic embryogenesis of callus cultures

Various characteristics including the saponin content in the root of Bupleurum falcatum plants propagated in vitro through somatic embryogenesis of callus cultures were compared with those of the plants propagated by seeds. The asexually propagated plants had an aerial part of more uniform characteristics than those of sexually propagated ones. However, both the mean and variance of root weight of the former were significantly larger than those of the latter. As for the saponin content of the root on a dry weight basis, there was little difference between the two groups. The amounts of saikosaponins c and d in a root were significantly larger in the asexually propagated plants than in the sexually propagated ones.     

Vegetative propagation of Quercus suber L. by somatic embryogenesis. II. Plant regeneration from selected cork oak trees

The regeneration of somatic seedlings from selected 100-year-old cork oak trees is reported. The induction of somatic embryogenesis from leaves of epicormic shoots was significantly affected by genotype, harvesting time and their interaction. Leaves from all five selected trees produced somatic embryos when the segments of branches used as sources of epicormic shoots were collected in May. Genotype, but not the level of photosynthetically active radiation, affected the proliferation of the embryogenic lines and the number of detachable embryos that could be obtained from them. Genotype also affected several steps leading to conversion of somatic embryos, from germination to complete acclimatisation of somatic seedlings. Almost 40% of the somatic embryos from all lines germinated, showing coordinated root and shoot growth. Although the mean percentage of recovery for the whole process was low, plants could be regenerated from four of the five trees tested.     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5???M). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15?d followed by cycles of 8?h dark and 16?h light, under white fluorescent lamps with a light intensity of 3,000?lm/m² and at temperature of 28?±?2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9?mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.     

Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants

Somatic embryos of cassava (Manihot esculenta Crantz) cultivar ‘Nanzhi 188’ were isolated and cut into fragments to be cultured on media with various cytokinins and auxins. Cytokinin induced of cassava shoot organogenesis, while auxin stimulated somatic embryogenesis. The effectiveness on organogenesis was different based on different cytokinins and in combination with auxins. Benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and N-isopentenyladenine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 M 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid