Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures. Received: 14 July 1998 / Revision received: 2 November 1998 / Accepted: 6 November 1998     

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.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 M of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.Abbreviations BA 6-benzyladenine - GD Gresshoff and Doy Medium - IBA indole-3-butyric acid     

Shoot development in Quercus suber L. somatic embryos

Summary N⁶-Benzyladenine (BA; 0.04–4μM) application to germinated Quercus suber somatic embryos considerably increased caulinar apex elongation frequency and maintained active growth in the plantlets, although it did not have a significant effect on the percentage of shoots with normal morphology. The addition of 0.5 μM indoleacetic acid together with the cytokinin did not have any effect. The use of a low concentration (0.04 μM) of BA allowed the appropriate radicle elongation in all germinating somatic embryos, but higher concentrations arrested this elongation.     

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.     

Comparison between somatic and zygotic embryo development in Quercus robur L.

ABSTRACT

Somatic embryogenesis from juvenile explants as an efficient way for oak clonal propagation is drastically limited by the low rate of embryo germination. A comparison of the development of immature somatic and zygotic embryos, and a study of the changes in sugar content and lignin accumulation during somatic versus zygotic embryo development were conducted in view of understanding the effect of reserve substance deficiency upon somatic embryo maturation. A morphological comparison of somatic and zygotic embryos led to the identification of 4 to 7 similar developmental stages in both types of embryos, thus indicating that the accumulation phase in both zygotic and somatic embryos occurs at the same stage, when the cotyledons became thicker and opaque. Carbohydrate analysis showed the presence of glycerol, inositol, mannitol, galactose, trehalose, xylose, arabinose, glucose, fructose and sucrose in all stages of zygotic and somatic embryo development, but in different amounts. The amount of glycerol, inositol, glucose and sucrose during the early stages is larger in zygotic embryos than in somatic ones, but the time course of their accumulation is similar in both types of embryos. Lignin content, which increased continuously during development, showed a similar behaviour in zygotic and somatic embryos. In somatic embryos which were able to germinate, lignin content was higher than in nongerminating embryos at the same stage.     

In vitro root induction in axillary microshoots of Quercus robur L.

Whilst considerable efforts have been made to optimise shoot multiplication and rooting in oak, little attention has been paid to the impact of conditions used for multiplication on subsequent root formation. An optimised technique for rooting of oak microshoots has been developed to assess the effect of cytokinin treatments applied to shoot multiplication cultures on the subsequent rooting of microshoots. We found IBA to be more effective at inducing root formation in microshoots than NAA. Efficient rooting of oak microshoots (80%) was achieved after 35 days on medium supplemented with 1.0 mg litre^-1 IBA. Lower concentrations of IBA reduced the frequency of root formation and significantly increased the time taken for microshoots to form roots. High concentrations of IBA (3.0 mg litre^-1) produced similar rooting frequencies but with significantly increased numbers of roots formed by each microshoot. However, high concentrations of IBA stimulated the production of basal callus. Rooting of microshoots was unaffected by the concentration of BA used during shoot multiplication, although basal callusing was greater in microshoots taken from multiplication medium supplemented with the highest concentration of BA (1.0 mg litre^-1) and rooted on medium supplemented with 3.0 mg litre IBA. Reducing the period of exposure to auxin to 7 days by transferring microshoots to auxin-free medium increased the frequency of root formation (84%), led to more rapid root formation and a reduction in basal callus formation.     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

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.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

A comparison between Theobroma cacao L. zygotic embryogenesis and somatic embryogenesis from floral explants

Summary In order to improve the late phases of Theobroma cacao L. embryogenesis from tissues of maternal origin, zygotic embryogenesis and somatic embryogenesis were compared, with respect to morphological, histological, and physiological parameters. Zygotic embryogenesis could be divided into three steps: (a) embryogenesis sensu stricto, (b) a growth period in which cotyledonary embryos reached their final dimensions, and (c) a maturation period in which embryos accumulated protein and starch reserves, dehydrated to a water content equal to 30%, and underwent a modification in soluble sugar composition. Monosaccharides and sucrose contents decreased to the benefit of the oligosaccharides raffinose and stachyose. The formation of somatic embryos by use of basic protocols was studied to define the limiting factors that could lie behind their poor development. Morphological abnormalities of somatic embryos, which represented 80% of the total population, were described. A histological study showed that somatic embryos lacked starch and protein reserves; moreover, their water content was much higher than that of their zygotic counterparts. Introducing a growth period into the culture protocol made for better embryo development. Adding sucrose and abscisic acid to the maturation medium was effective in increasing reserve synthesis and resulted in higher germination, conversion, and acclimatization rates.     

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.

     

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

Summary Somatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl⁻¹ sucrose, 3 gl⁻¹ gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.     

Propagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. (holm oak) trees by somatic embryogenesis

Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L^?1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1–3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L^?1) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4 °C for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21 to 66.7% of the SE generated for both genotypes.     

Morphohistological analysis of the origin and development of somatic embryos from leaves of mature Quercus robur

Summary In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.     

Regeneration of transgenic plants by Agrobacterium-mediated transformation of somatic embryos of juvenile and mature Quercus robur

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

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.