Embryogenic suspension cultures of Pinus nigra Arn.: growth parameters and maturation ability

Suspension cultures have been established from embryogenic tissues of Pinus nigra initiated from immature zygotic embryos. The growth of tissues in liquid medium has been influenced by initial tissue weight used for the establishment of the cultures as well as by genotype. In most of the cases initial tissue weight 0.5 g was insufficient and the cultures showed poor growth and later degeneration. Higher amount of initial tissues (1 or 2.5 g) was more efficient for the establishment and proliferation of somatic embryos in liquid medium. The growth of suspension cultures was also cell line dependent. Somatic embryo maturation in liquid medium was very limited and no plantlet regeneration occurred. Cotyledonary somatic embryos developed and produced emblings when the suspension was plated on filter paper discs and cultured on solid maturation medium. Based on our experiments we can state that the embryogenic tissues are able to grow and proliferate in liquid medium but somatic embryo maturation and plantlet regeneration occur only on solid medium.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

Optimized somatic embryogenesis in Pinus strobus L.

Summary Somatic embryogenesis (SE) initiation in Pinus strobus was optimized by the manipulation of plant growth regulator (PGR) concentrations in the culture medium. Modified Litvay medium (MLV) of Litvay et al. (1985) supplemented with lower than routinely used PGR concentration increased initiation of established embryogenic cultures from approximately 20 to 53%. The original developmental stage of zygotic embryos had a pronounced effect on the SE response. The optimum stage was the pre- to shortly post-cleavage stage. A substantial genetic influence on initiation of SE was indicated by a significant variance component due to families. Genotype X collection date and genotype X media interactions had large effects on initiation of SE. The PGR levels in the culture medium prior to maturation had a significant effect on subsequent production of mature somatic embryos. Embryogenic tissue initiated and proliferated on medium with a low level of PGR consistently produced a high number of somatic embryos, indicating that optimized initiation protocol also enhanced somatic embryo production. Somatic embryos of 93 embryogenic lines (representing five families) that were initiated on media with different PGR concentrations were converted to plants at an overall frequency of 76%, and grown in the greenhouse. With these improved protocols, application of P. strobus SE in commercial clonal forestry is feasible as an alternative to traditional breeding and reforestation.     

Progress towards initiation of somatic embryogenesis from differentiated tissues of radiata pine (Pinus radiata D. Don) using cotyledonary embryos

Green cones of radiata pine were collected from two open-pollinated, elite families in two successive years at weekly intervals, and initiation of embryogenic cultures was investigated as a function of sampling date, initiation medium, explant type, and developmental stage. A combination of dissected embryos and a modified Litvay medium, Glitz, was best. This combination gave the highest rate of initiation, and it was possible to initiate somatic embryogenesis (SE) from differentiated cells in the epicotyledonary region of post-cotyledonary zygotic embryos from the two tested families with an average initiation rate of approximately 24% and 7% from stage five and six embryos, respectively. This is different from established initiation protocols of embryogenic cultures in radiata pine, which has traditionally been based on embryo rescue and continued proliferation of immature zygotic embryos. A further implication of initiation of SE from excised post-cotyledonary embryos was that the period of initiation of embryogenic cultures was extended from 4 to 12 wk.     

Development of a cyclic somatic embryogenesis regeneration system for leek (Allium ampeloprasum L.) using zygotic embryos

Summary In leek (Allium ampeloprasum L.) a cyclic system of somatic embryogenesis was developed. Somatic embryos used for cyclic embryogenesis were able to develop the same type of embryogenic callus as zygotic embryos in the primary cycle. For the first time a comparison of the efficiencies of both expiants was made. Ten families were investigated for somatic embryogenesis. There was a genetic relationship with respect to somatic embryo production between the reciprocal crosses. From each family one genotype was selected for investigating cyclic somatic embryogenesis. Different levels of somatic embryo production were found between the expiants of zygotic and somatic embryos. The two best genotypes, 92.001-03 and 92.002-33 produced twice as many somatic embryos as the overall average. On average, 56% of the somatic embryos finally developed into greenhouse plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MS medium Murashige and skoog (1962) basal medium     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

Callusogenesis and somatic embryogenesis induction in hybrid embryos from the seeds of Pinus sibirica

The results of long-term work on the induction of somatic embryogenesis in Siberian pine (Pinus sibirica Du Tour) growing in a natural stand of trees and in clone grafting plantation located in the Western Sayan are shown. Controlled pollination of the clones of Siberian pine had a positive influence on the state of callus cultures. The cytological analysis of embryonal-suspensor mass made it possible to identify embryological structures morphologically close to zygotic embryos at early developmental stages; as a result, the callus tissue was recognized embryogenic. We revealed donor plants (clones), whose zygotic embryos in vitro can serve as a source of embryogenic callus tissue.     

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening of large numbers of seed families of Pinus strobus L. for somatic embryogenesis from immature and mature zygotic embryos

The somatic embryogenic process was investigated using immature and mature zygotic embryos originating from 13 open-pollinated seed families selected for their embryogenic capacity from over 128 seed families of Pinus strobus. In a first step, intact megagametophytes with precotyledonary embryos from these families were placed on modified Litvay medium. Embryogenic tissues (ETs) were obtained for 12 of them, with initiation rates varying from 2.6% to 23%. On average, 14% of the ETs (36/258) formed stable embryogenic cell lines (ECLs) after 4–6 months of subculture. Mature somatic embryos were produced for 30 out of 52 cell lines, and plants were regenerated. Later, initiation of ETs from mature zygotic embryos was also tested for the selected families. ECLs were obtained for five of them, with a maximum initiation rate of 2.7%, and plants were produced for four ECLs. Received: 19 March 1998 / Revision received: 22 May 1998 / Accepted: 5 June 1998     

Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra- and inter-species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four-cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l^?1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.     

Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

火炬松胚性细胞悬浮培养物的生长参数变化

以火炬松（PinustaedaL.）成熟合子胚来源的胚性愈伤组织为材料建立了胚性细胞悬浮系,测定了其培养物的鲜重、干重、细胞体积和胚数及培养液中的pH值、电导率和蔗糖浓度等生长参数在培养过程中的变化动态。结果表明,在培养周期内,培养液中的pH值、电导率和蔗糖浓度的逐步降低与培养物的鲜重、干重、细胞体积和胚胎数的逐步增加保持一致性。在培养至18—21d,pH值、电导率和蔗糖浓度均接近或降到最低点,而胚数及细胞体积的增长都达到最高点。     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Somatic embryogenesis from different tissues of Spanish populations of maritime pine

The somatic embryogenesis (SE) capacity of megagametophytes belonging to Continental and Mediterranean Spanish provenances of maritime pine (Pinus pinaster Aiton) was studied, noting factors (megagametophyte developmental stage and culture medium) that enhanced the induction and establishment of SE lines. In both provenances, initiation and establishment of embryogenic calli was higher on megagametophytes in which the dominant zygotic embryo had begun to develop. In the Mediterranean provenance, however, SE lines were also established from megagametophytes enclosing zygotic embryos with well-developed cotyledons. A modified Litvay medium (mLV) containing 9.9???M 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4???M 6-benzyladenine (BA) was superior to DCR medium containing 13.6???M 2,4-D and 4.4???M BA for SE induction, but there were no differences between media in terms of the number of SE lines established after 4?months in culture (153 vs. 155 established SE lines, for mLV and DCR media respectively). Of the 26 embryogenic lines tested for maturation, 15 (58?%) produced cotyledonary somatic embryos and 75?% of these gave rise to plants on germination medium. SE-like cultures from adult maritime pine trees were also initiated, but embryogenic lines could not be established. This is the first report on the production of SE in maritime pine of Continental and Mediterranean origin. The micropropagation protocols presented here provide an important tool for the vegetative multiplication of selected families and breeding programs for maritime pines from Spain.