Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation

2009 ◽  
Vol 39 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
Cathy L. Hargreaves ◽  
Cathie B. Reeves ◽  
Jens I. Find ◽  
Keiko Gough ◽  
Puthiyaparambil Josekutty ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Pinus Radiata ◽  
Zygotic Embryo ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Embryogenic Tissues ◽  
Preparation Techniques ◽  
Embryo Maturity ◽  
Collection Time ◽  
Excised Embryos

The principal aim of this investigation was to improve somatic embryogenesis initiation and to enhance representation of families and genotypes within those families of Pinus radiata D. Don. A total of 19 open-pollinated seed families, many with unrelated and weakly related parents, were tested. Optimum stage of cone maturity for initiation success was tested by five collections made at 1 week intervals, spanning the developmental period from pro-embryo to cotyledonary embryos. Two media were compared; embryo-development media (EDM6) and a modified Litvay medium (Glitz). Two zygotic embryo explant-preparation techniques were tested; embryos with retained megagametophytes and excised embryos. Proliferating embryogenic tissues were obtained from all four treatments (2850 explants per treatment, 570 per collection time) for the 19 families. The best initiation rates were achieved with a combination of Glitz medium with excised zygotic embryos, with 55% of explants from all collections and all families combined giving rise to proliferating embryogenic tissue. At the optimal collection time for each of the families, this treatment gave a range of 47%–97% initiation success with an average of 70% per family.

scholarly journals Physiological and Structural Aspects of In Vitro Somatic Embryogenesis in Abies alba Mill

Forests ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1210
Author(s):  
Terezia Salaj ◽  
Katarina Klubicová ◽  
Bart Panis ◽  
Rony Swennen ◽  
Jan Salaj
Keyword(s):  
Somatic Embryogenesis ◽  
Cell Lines ◽  
Somatic Embryos ◽  
Abies Alba ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Peg 4000 ◽  
Initiation Frequency ◽  
Embryogenic Tissues

Initiation of somatic embryogenesis from immature zygotic embryos, long-term maintenance of embryogenic tissue in vitro or by cryopreservation, as well as maturation, of somatic embryos of Abies alba Mill. are reported in this study. For the initiation of embryogenic tissues, a DCR medium containing different types of cytokinins (1 mg.L−1) were tested. During three consecutive years, 61 cell lines were initiated out of 1308 explants, with initiation frequencies ranging between 0.83 and 13.33%. The type of cytokinin had no profound effect on the initiation frequency within one given year. Microscopic observations revealed presence of bipolar somatic embryos in all initiated embryogenic tissues. Besides the typical bipolar somatic embryos, huge polyembryonal complexes, as well as “twin” embryos, were observed. Maturation of somatic embryos occurred on a DCR medium supplemented by abscisic acid (10 mg.L−1), polyethylene glycol (PEG-4000, 7.5%) and 3% maltose. The maturation capacity was cell-line dependent. All of the four tested cell lines produced cotyledonary somatic embryos, though at different quantities, of 16 to 252 per g of fresh weight. After germination, seedlings developed, but their further growth soon stopped after the formation of a resting bud. Altogether, seven cell lines were cryopreserved, using the slow-freezing technique. After rewarming, all tested cell lines showed regrowth rates between 81.8 and 100%.

scholarly journals Induction of somatic embryogenesis in Pinus heldreichii culture

2007 ◽  
Vol 59 (3) ◽  
pp. 199-202 ◽  
Author(s):  
Dragana Stojicic ◽  
Branka Uzelac ◽  
Dusica Janosevic ◽  
Ljubinka Culafic ◽  
Snezana Budimir
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Dichlorophenoxyacetic Acid ◽  
Immature Zygotic Embryos ◽  
Induction Frequency ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Further Development

The potential for somatic embryogenesis in zygotic embryo and megagametophyte cultures of Pinus heldreichii was examined. Somatic embryogenesis was initiated from megagametophytes containing immature zygotic embryos at early stages of development. An induction frequency of up to 6.7% was obtained on Gresshoff and Doy medium in the presence of 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l benzyladenine (BA). Formation and further proliferation of embryogenic tissue were achieved upon transfer of explants to a medium with reduced levels of growth regulators. Somatic embryos are being cultured for further development. .

scholarly journals Pinus canariensis plant regeneration through somatic embryogenesis

2020 ◽  
Vol 29 (1) ◽  
pp. eSC05
Author(s):  
Ander Castander-Olarrieta ◽  
Paloma Moncaleán ◽  
Itziar A. Montalbán
Keyword(s):  
Somatic Embryogenesis ◽  
Cell Lines ◽  
Somatic Embryos ◽  
Embryogenic Tissue ◽  
Established Cell Line ◽  
Zygotic Embryos ◽  
Pinus Canariensis ◽  
Embryogenic Cell ◽  
Embryogenic Cell Lines ◽  
Established Cell

Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis.Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain).Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions.Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth.Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful biotechnological tool for both research purposes and massive vegetative propagation of this species.Keywords: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo.Abbreviations used: embryogenic tissue (ET); established cell line (ECL);  somatic embryogenesis (SE); somatic embryos (Se’s).

scholarly journals Enhanced Somatic Embryo Induction of a Tree Peony, Paeonia ostii ‘Fengdan’, by a Combination of 6-benzylaminopurine (BA) and 1-naphthylacetic Acid (NAA)

Plants ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 3 ◽  
Author(s):  
Xiuxia Ren ◽  
Ya Liu ◽  
Byoung Ryong Jeong
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Zygotic Embryos ◽  
Ms Medium ◽  
Hypocotyl Explants ◽  
Cotyledon Explants ◽  
Embryo Induction ◽  
Naphthylacetic Acid ◽  
Better Than

Somatic embryogenesis is a preferred method for vegetative propagation due to its high propagation efficiency. In this study, zygotic embryos, cotyledons, and hypocotyls of Paeonia ostii ‘Fengdan’ were used as the explant to induce somatic embryogenesis. The results showed that a combination of 0.5 mg·L−1 thidiazuron (TDZ) and 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) was effective in inducing somatic embryos from the zygotic embryo and cotyledon explants. Hypocotyls only formed somatic embryos on Murashige and Skoog (MS) medium supplemented with both 0.5 mg·L−1 TDZ and 0.5 mg·L−1 1-naphthylacetic acid (NAA). Moreover, the compact callus was effectively produced from zygotic embryo, cotyledon, and hypocotyl explants in medium supplemented with a combination of 3.0 mg·L−1 6-benzylaminopurine (BA) and 1.0 mg·L−1 NAA, and then converted into somatic embryos in the same medium, and the ratio of the explants with embryo induction and number of embryos induced per explant were much higher than those induced by 0.5 mg·L−1 TDZ and either 0.5 mg·L−1 2,4-D or 0.5 mg·L−1 NAA. The MS medium was better than the woody plant medium (WPM) for inducing somatic embryos from zygotic embryo and hypocotyl explants, whereas the WPM was better than the MS medium for somatic embryogenesis induction from cotyledon explants. All of the somatic embryos developed well into mature embryos on their respective media supplemented with both 3.0 mg·L−1 BA and 1.0 mg·L−1 NAA. Overall, the protocols for indirect somatic embryogenesis from zygotic embryo, cotyledon, and hypocotyl of P. ostii ‘Fengdan’ were successfully established, which can greatly facilitate their propagation and breeding processes.

scholarly journals Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

2014 ◽  
Vol 65 (1-2) ◽  
pp. 37-41 ◽  
Author(s):  
Maria G. Ostrolucká ◽  
Diana Krajmerová
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Casein Hydrolysate ◽  
Zygotic Embryos ◽  
Embryo Germination ◽  
Repetitive Somatic Embryogenesis ◽  
Secondary Embryos ◽  
Somatic Embryo Conversion

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l<sup>-1</sup> glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l<sup>-1</sup>) and BAP (0,5-1,0 mg•l<sup>-1</sup>). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l<sup>-1</sup>) or on medium with ABA and GA<sub>3</sub> (each 0,2 mg•l<sup>-1</sup>) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l<sup>-1</sup> BAP when cultures were mantained at 2<sup>o</sup>C for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

scholarly journals COMPARISON OF ZYGOTIC AND SOMATIC EMBRYOGENES1S IN CERCIS CANADENSIS

HortScience ◽  
1992 ◽  
Vol 27 (11) ◽  
pp. 1167d-1167 ◽  
Author(s):  
L.G. Buckley ◽  
E.T. Graham ◽  
R.N. Trigiano
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Ammonium Ion ◽  
Zygotic Embryos ◽  
University Of Tennessee ◽  
Cercis Canadensis ◽  
Developmental Sequences ◽  
The University ◽  
Shoot Meristems

Zygotic and somatic embryos are purported to follow similar developmental sequences, but few investigations have thoroughly compared the two processes. Developing pods of Cercis canadensis L. (redbud) were collected from trees on the Knoxville campus of the University of Tennessee once or twice per week from 28 March to 8 August 1991. At least 10 ovules/sample date were fixed in FAA to evaluate zygotic embryo ontogeny. A minimum of 40 ovules/sample date were aseptically excised and placed on SH medium supplemented with 9.0 μM 2,4-D and 5 mM ammonium ion to initate somatic embryogenesis. Zygotic and somatic embryos were prepared for histological examination using standard paraffin techniques. Somatic embryos developed primarily from cotyledons and epicotyls of zygotic embryos mat were cultured between 6 June and 19 July. Somatic and zygotic embryos were subtended by multiseriate suspensors and progressed through recognizable globular, cordate and cotyledonary stages of development. Cotyledon morphology was similar for both embryo types. However, many somatic embryos failed to differentiate dome-shaped shoot meristems exhibited by their zygotic counterparts.

scholarly journals Somatic embryogenesis of selected spruce species (Picea abies, P. omorika, P. pungens 'Glauca' and P. breweriana)

2011 ◽  
Vol 77 (3) ◽  
pp. 189-199 ◽  
Author(s):  
Teresa Hazubska-Przybył ◽  
Krystyna Bojarczyk
Keyword(s):  
Somatic Embryogenesis ◽  
Picea Abies ◽  
Sucrose Concentration ◽  
Medium Composition ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Embryogenic Cell ◽  
Nursery Production ◽  
Embryogenic Cell Lines

Somatic embryogenesis was studied in four spruce species (<em>Picea abies</em>, <em>P. omorika</em>, <em>P. pungens</em> 'Glauca' and <em>P. brewenana</em>) to determine if this method can be used for in vitro propagation of coniferous trees. The highest frequency of initiation of embryogenic tissue was obtained when mature zygotic embryos were used as explants. It ranged then from 10.8% (<em>P. brewenana</em>) to 23.75% (<em>P. omorika</em> and <em>P. pungens</em> 'Glauca'). The frequency of embryogenic tissue initiation was strongly affected by medium composition, i.e. addition of appropriate auxins (2,4-D, NAA, Picloram) and sucrose concentration (10-20 g<sup>-1</sup>"1). A lower frequency was obtained in <em>Picea omorika</em> (10%) when megagametophytes (endosperms with immature zygotic embryos) were used as explants. No emryogenic tissue was produced from hypocotyls, cotyledons and needles. A satisfactory frequency was achieved with the use of somatic embryos of <em>Picea abies</em> (30%). The proliferation of embryogenic cell lines of spruces was affected by medium type. The experiments resulted in production of somatic plantlets of <em>P. abies</em> and <em>P. omorika</em>. This enables the application of this method of spruce micropropagation for genetic and breeding research or for nursery production.

Diploid and haploid embryogenesis in Larixleptolepis, L. decidua, and their reciprocal hybrids

1990 ◽  
Vol 20 (1) ◽  
pp. 9-14 ◽  
Author(s):  
P. von Aderkas ◽  
K. Klimaszewska ◽  
J. M. Bonga
Keyword(s):  
Growth Regulators ◽  
Zygotic Embryo ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Immature Zygotic Embryos ◽  
Binucleate Cells ◽  
Meristematic Cells ◽  
Reciprocal Hybrids ◽  
Morphological Differences ◽  
Haploid Embryogenesis

Diploid and haploid embryogenesis was induced in two Larix species (L. decidua and L. leptolepis) and their reciprocal hybrids. Diploid embryogenic tissue was initiated in immature zygotic embryos isolated with the micropylar half of the megagametophyte left attached. These were placed either on modified LM or MSG medium supplemented with the growth regulators 2,4-D and 6-benzyladenine. MSG medium was solidified with either gellan gum or agar. There was no appreciable difference in response between the two. Haploid embryogenesis was induced in isolated megagametophytes placed on modified LM medium supplemented with 2,4-D and 6-benzyladenine. Diploid embryogenic tissue was subcultured on medium with growth regulators, but haploid embryogenic tissue grew well on medium without growth regulators. There were few morphological differences between the diploid and haploid embryogenic tissue. In all species and hybrids, haploid cultures contained more coenocytic long cells. Binucleate cells were most common, but tetranucleate and octanucleate cells were also present. Haploid cultures showed poorer organization than the diploid ones, with only a few cultures having well-developed embryoids. Haploid tissue originated from expanded cells of the megagametophyte. Diploid tissue originated from the suspensor region of the zygotic embryo; it proliferated from isolated clusters of meristematic cells in early embryoids. Diploid and haploid cultures differed not only from the outset, but also in the mature embryoids they produced.

Plant regeneration by somatic embryogenesis in Pinus thunbergii resistant to the pine wood nematode

2019 ◽  
Vol 49 (12) ◽  
pp. 1604-1612
Author(s):  
Tingyu Sun ◽  
Yanli Wang ◽  
Lihua Zhu ◽  
Xiaoqin Wu ◽  
Jianren Ye
Keyword(s):  
Somatic Embryogenesis ◽  
Plant Regeneration ◽  
Somatic Embryo ◽  
Cell Line ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Pinus Thunbergii ◽  
Embryogenic Tissue ◽  
Embryo Production ◽  
Somatic Embryo Production

Pine wilt disease (PWD) is a severe threat to pine forests in East Asia. Screening and breeding of resistant varieties is a very effective way to prevent and control PWD; however, no reliable somatic embryogenesis system has yet been developed for the elite nematode-resistant Pinus thunbergii Parl. line. In this study, we studied the plant regeneration via somatic embryogenesis of nematode-resistant P. thunbergii. Initiation of embryogenic tissue was significantly affected by seed family (p = 0.017), immature zygotic embryo stage (p = 0.032), and initiation medium (p = 0.004). Seed family 37 was the most favorable female parent for initiation of P. thunbergii. Furthermore, the initiation rate increased from the pre-embryonic stage to the cleavage polyembryonic stage. The optimal medium was I2, containing 2,4-dichlorophenoxyacetic acid (9 μmol·L−1) and 6-benzyladenine (4.4 μmol·L−1). A statistically significant interaction between cell line and subculture time (24 months) was observed in the influence on proliferation rate, somatic embryo production, and percentage germination (p < 0.001). In this study, the highest somatic embryo production was achieved using cell line 37-1 (1983 somatic embryos per gram fresh mass), with approximately 83.5% of somatic embryos germinating after transferring to germination medium, of which 77.6% converted into plantlets.

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