Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

In vitro response and pedigree analysis for somatic embryogenesis of long-day photoperiod adapted soybean

Northern-adapted soybean cultivars were screened for their ability to form somatic embryos in vitro in response to exposure to 180 M 2,4-dichlorophenoxyacetic acid in a modified Murashige & Skoog (1962) medium containing B5 (Gamborg et al. 1968) organics and 43.5 mM sucrose. The 20 cultivars formed between 1 and 7 embryos per cotyledon with between 18% and 98% of the cotyledons responding. The response was genotype dependant. Three ancestral lines, 840-7-3, A.K. Harrow and Mandarin were prominent in the genetic background of long-photoperiod adapted lines and showed a high degree of somatic embryogenesis.Abbreviations 2,4-d 2,4 dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Towards normalization of soybean somatic embryo maturation

Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media.     

Auxin-orientation effects on somatic embryogenesis from immature soybean cotyledons

Summary Development of somatic embryos of soybeanGlycine max (L.) Merr. has been studied using cultivars J103 and McCall and five auxin-sucrose treatment: naphthalene acetic acid at 10 mg/liter with 1.5% sucrose (N10); 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.25, 0.5 or 1.0 mg/liter with 1.5% sucrose (D.25, D.5 D1); and 2,4-D at 25 mg/liter with 3% sucrose (D25). Cotyledons were excised aseptically from immature embryos 3 to 5 mm in length, and placed on the media with either the adaxial or abaxial side down (adaxial or abaxial orientiation, respectively). After 30 d, numbers of normal or total somatic embryos, or both, were counted. For J103 explants on D25 or N10 media, the greatest number of embryos was produced from the central region of abaxially oriented explants on D25, but only 5% of these embryos were normal. The greatest number of normal embryos was produced from the marginal region of adaxially oriented explants on D25. For McCall explants on D.25, D.5, D1, or N10 media, the greatest numbers of normal embryos were produced from the marginal regions of abaxially oriented explants on D1 or N10. Embryo induction was examined histologically for the N10, D25, and D.5 treatment, using serial section of paraffin-embedded cotyledons stained with hematoxylin and safranin. On N10 medium, embryos were produced unicellularly, or more frauently, multicellularly, from homogeneous embryogenic tissue arising from epidermal and subepidermal cells at the distal periphery of the cotyledon. On D25 medium, embryos were produced multicellularly from a geterogeneous embryogenic tissue formed in the central region of the cytoledon, and consisting of embryogenic cells interspersed with large, vacuolate cells. The D.5 treatment showed an intermediate response. Embryo initiation in this soybean system is predominantly multicellular. This work was supported in part by a grant from Lubrizol Genetics, Inc., and by the Agricultural Experiment Station, University of Kentucky. Paper number 88-3-13 from the Kentucky Agricultural Experiment Station.     

A quantitative nature of somatic embryogenesis in soybean

Summary The objectives of this research were to investigate the genetic parameters associated with the in vitro formation of somatic embryos in soybean and to determine the effect of light intensity on the embryogenic capability of F₁, F₂, and backcross (RC₁P₁ and RC₁P₂) progenies derived from crosses between embryogenic (IAS-5 and Embrapa-1) and non-embryogenic (Paraná) cultivars. Immature cotyledons (4–6 mm in length) derived from the parental lines, F₁, F₂, RC₁P₁, and RC₁P₂ were grown for 90 d on the inductive N10 medium, after which the number of somatic embryos was recorded. Chi-square tests for goodness of fit showed that the genetic component of the somatic embryogenesis trait is controlled in a quantitative manner by approximately 10 genes. A normal distribution for somatic embryo formation in the F₂ generations was observed reinforcing the quantitative nature of the trait. Variation in light intensity (8–12 and 27–33 μmol m⁻²s⁻¹) had no effect on somatic embryo formation in the parental material tested.     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Continuous long-term somatic embryogenesis in alfalfa

Summary Somatic embryogenic callus was induced on two induction media, B5h and SH4K. Embryos formed on the callus induced on B5h medium when the callus was still on the induction medium. On the other hand, embryos could not form on the callus induced on SH4K medium unless the callus was transferred to a growth regulator-free medium. Callus induced and maintained on B5h medium lost embryogenic capability quickly during the subculture. Callus induced and maintained on SH4K medium, however, consistently remained highly embryogenic. The callus mass showed steady increase during its maintenance on SH4K medium. The embryos induced on SH4K medium showed vigorous germination. Normal and fully fertile plants were recovered from the embryos developed from the callus maintained on SH4K medium.     

Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups

In the US, soybean genotypes are classified into maturity groups (MG; total of 13) that represent areas of adaptation generally correlated with latitude bands. To determine if one regeneration procedure could regenerate representatives from diverse areas of adaptation, 18 soybean genotypes representing nine MG were compared for organogenic adventitious regeneration and plant formation from hypocotyl explants following the procedure previously tested on representatives from only three MG. Responding explants were those capable of producing shoots on the acropetal end of the explant from either the outer edge plus central region or the central region only. This enabled determination of the contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by discounting those explants, it also enabled estimates of true adventitious regeneration. All 18 genotypes were capable of producing meristemoids/shoots solely from the central region with responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also capable of producing elongated shoots that could be successfully rooted. No morphological differences were noted among regenerants, or between them and seed-initiated plants. All regenerants produced viable seed which germinated and produced morphologically normal plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-based organogenic regeneration procedure and provided conservative estimates for responses that were truly/solely adventitious in nature.     

Induction and development of somatic embryos from cell suspension cultures of soybean

Glycine max (L.) Merr. (soybean) andGlycine soja Sieb. and Zucc. cell suspension cultures were grown and used as inoculum sources for growing callus on agar-solidified nutrient media. Concentrations and chemical forms of the growth regulators in liquid and solidified media were altered in an attempt to achieve in vitro plant regeneration. Numerous embryoids, particularly ofG. soja, were produced on basal nutrient media supplemented with 100 ppm casein hydrolysate, 0.1 μM abscisic acid, 2.25 μM 2,4-dichlorophenoxyacetic acid, and 15 μM adenine or 0.46 μM kinetin. Often the roots of the embryoids elongated. This was enhanced in the presence of an inhibitor of gibberellin synthesis (1 to 20 μM Amo 1618). Callus recovered from aG. soja suspension culture produced one shoot structure when grown on a solid medium containing 0.2 μM Amo 1618 and 80 μM glutathione. The shoot structure consisted of two distinct buds, one producing two leaves. The shoot did not develop into a plant. Although regeneration of soybean plants was not achieved, these observations suggest that it may be achievable. The investigations reported in this paper (no. 81-3-100) were performed in connection with a project of the Kentucky Agriculture Experimental Station and the paper is published with the approval of the Director.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Effect of polyethylene glycol and sugar alcohols on soybean somatic embryo germination and conversion

The effect of different vitamins and inorganic micronutrients on callus growth and the induction and proliferation of somatic embryos from young mature, fully expanded leaves of chilli pepper (Capsicum annuum L.) was investigated. Explants were cultured on a solid Murashige and Skoog (MS) medium supplemented with 8% (w/v) sucrose, 12.9 M 6-benzyladenine, 9 M 2,4-dichlorophenoxyacetic acid and 0.5 mg l^–1 thiamin.HCl in various combinations of eleven different vitamins. Alternatively, explants were cultured onto a solid medium containing MS macro- and micronutrients except for the salts of Mn, Zn, I, Cu and Co which were added at either the standard MS concentration or at a tenfold increased (Cu, Co) or decreased (Mn, Zn, I) concentration. The results indicated that somatic embryogenesis from pepper leaves is favoured by the addition of nicotinic acid to the culture medium and the increase of copper concentration (an average induction of 70.2 globular embryos/mm² of explant surface, 9.2% higher than control), without reducing embryo maturation and germination.     

Cotransformation frequencies of foreign genes in soybean cell cultures

Summary Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%–27%. Similar rates of cotransformation were observed among various marker pairs.     

Development of an embryogenic suspension culture of soybean (Glycine max Merrill.)

A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1^–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration.     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Fatty acid composition of soybean (Glycine max (L.) merr.) somatic embryos

Summary Somatic embryos from four soybean cultivars were matured for 30 and 45 d. Success of embryo germination was evaluated for each length of maturation. The percentage of somatic embryos undergoing successful germination, as defined by rooting and shoot emergence, was greater for embryos matured 45 d than for embryos matured 30 d. Therefore, embryos matured for 45 d are probably physiologically more mature than embryos matured for 30 d. Relative percentages of fatty acids comprising oils and lipids of somatic embryos were determined for each length of maturation and for each cultivar. Variation in relative percentages of palmitic acid, oleic acid, and linoleic acid was affected by length of maturation. However, these changes were genotype dependent. A significant interaction between the cultivars Clark and Maple Arrow and stage of maturation was observed for levels of oleic acid. No other interactions were observed. These data suggest that if changes in relative percentages of certain fatty acids are associated with soybean somatic embryo maturation the changes are genotype dependent. This is journal paper No. J-12870 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2763. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that also may be suitable. This research was supported in part by grants from the American Soybean Association Development Foundation and the Iowa Soybean Promotion Board.     

Somatic embryo initiation and germination in diploid cotton (<Emphasis Type="Italic">Gossypium arboreum</Emphasis> L.)

Summary The diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A₂-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M₂ [0.04M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)] On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M₁ (0.2M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M₂ medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.     

Secondary somatic embryogenesis of cassava on picloram supplemented media

The object of this study was to evaluate different strategies for the production of secondary somatic embryos of cassava on picloram-supplemented media. Embryogenically competent calli maintained on double-strength Murashige and Skoog (1962) (MS) medium supplemented with 1 mg l⁻¹ picloram were used as starting material. Secondary embryogenesis from this callus was tested using various basal salt media in either the solid or the liquid state and containing two different concentrations of picloram. Some of the factors effecting the conversion of the embryos into plantlets were also studied. A liquid Schenck and Hildebrand (1972) medium containing 60 g l⁻¹ sucrose and 12 mg l⁻¹ picloram favoured the continual production of a highly embryogenic nodular callus. The normal development of somatic embryos from this tissue was dependant on the use of a picloram-free MS basal salt medium. The embryos were desiccated over a saturated salt solution of K₂SO₄ (RH 97.5% at 25 °C) and allowed to develop into plantlets on a MS medium containing 0.1 mg l⁻¹ BA. This procedure allowed for the normal elongation of the embryonic hypocotyl and formation of vigorous and viable shoots and roots. This revised version was published online in June 2006 with corrections to the Cover Date.