Soybean Regeneration Research Articles

Analysis of Soybean Somatic Embryogenesis Using Chromosome Segment Substitution Lines and Transcriptome Sequencing.

Soybean is an important cash crop that is widely used as a source of vegetable protein and edible oil. The regeneration ability of soybean directly affects the application of biotechnology. In this study, we used the exogenous hormone 2,4-D to treat immature embryos. Different levels of somatic incidence were selected from the chromosome segment substitution lines (CSSLs) constructed by SN14 and ZYD00006. Transcriptome sequencing of extreme materials was performed, and 2666 differentially expressed genes were obtained. At the same time, a difference table was generated by combining the data on CSSL rearrangement. In the extreme materials, a total of 93 differentially expressed genes were predicted and were then analyzed by cluster analysis and Gene Ontology (GO) annotation. After screening and annotating the target genes, three differentially expressed genes with hormone pathways were identified. The expression patterns of the target genes were verified by real-time quantitative PCR (qRT-PCR). Haplotype polymorphism detection and linkage disequilibrium analysis were performed on the candidate gene Glyma.09g248200. This study provided more information on the regulation network of soybean somatic embryogenesis and regeneration processes, and further identified important genes in the soybean regeneration process and provided a theoretical basis for accelerating the application of biotechnology to soybean for improving its breeding efficiency.

The Influence of Seed Viability on the Germination and In Vitro Multiple Shoot Regeneration of Soybean (Glycine max L.)

The moisture status of seeds is usually high during the period of harvest and deterioration (loss of viability) starts to occur when seeds are stored for longer periods. In the present study, soybean seeds were evaluated using a standard germination test, in vitro germination, and for efficient multiple shoot induction, following storage under ambient conditions for 0, 3, 6 and 9 months. Results showed that seeds stored for more than 3 months had reduced moisture content and decreased germination percentages in LS677, LS678, Dundee, Peking, TGx1740-2F and TGx1835-10E of the tested genotypes. In particular, seeds stored for 9 months showed significantly poor seed viability and less than 50% overall seed germination (Dundee—42%, LS678—49%, TGx 1740-2F—44%, TGx 1835-10E—48%), except for LS677 and Peking, with 52 and 55%, respectively. The efficiency of multiple shoot induction also decreased with prolonged seed storage, with all genotypes recording an overall decline from about 96% to 40% regeneration efficiency within 9 months. The results obtained clearly indicated that high germination rates and efficient in vitro shoot induction depended largely on seed viability and storage duration, and significantly differed according to genotypes.

Phenotypic traits of Mexican soybean seeds and their correlation with in vitro shoot induction and susceptibility to Agrobacterium infection

Background and Aims: Soybean regeneration and transformation are considered highly genotype-specific; however, little is known about desirable seed traits that could be useful indicators of their regeneration and transformation capacities. In the present study, eight commercially available Mexican soybean varieties, as well as the Jack genotype as a control, were phenotypically characterized to determine the seed traits that are important factors for their in vitro performance and susceptibility to Agrobacterium tumefaciens infection.Methods: Grain dimensions, seed weight, moisture, oil, protein, ash, carbohydrate content and macro and micro elements (Mg, K, Ca, P, Na, Mn, Fe, Cu and Zn) were measured and correlated with the corresponding direct shoot organogenesis capability and Agrobacterium infection susceptibility using the cotyledonary node as explant.Key results: Notably, the ash content was the only important factor that inversely correlated with the capacity for shoot organogenesis, whereas carbohydrate and phosphorus content were positively correlated with susceptibility to Agrobacterium infection. The best response in terms of multiple shoot formation and Agrobacterium transient transformation was observed with the soybean varieties Huasteca-100, Nainari and Suaqui-86, which have lower ash content and a higher carbohydrate and phosphorus content.Conclusions: In most reported studies, seed phenotypic traits have been overlooked as factors that influence their regeneration and transformation potential. In the present study, we present evidence of associations between some specific seed traits and regeneration and transient transformation of soybean.

Functional analysis of the GmESR1 gene associated with soybean regeneration.

Plant regeneration can occur via in vitro tissue culture through somatic embryogenesis or de novo shoot organogenesis. Transformation of soybean (Glycine max) is difficult, hence optimization of the transformation system for soybean regeneration is required. This study investigated ENHANCER OF SHOOT REGENERATION 1 (GmESR1), a soybean transcription factor that targets regeneration-associated genes. Sequence analysis showed that GmESR1 contained a conserved 57 amino acid APETALA 2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) DNA-binding domain. The relative expression level of GmESR1 was highest in young embryos, flowers and stems in the soybean cultivar ‘Dongnong 50’. To examine the function of GmESR1, transgenic Arabidopsis (Arabidopsis thaliana) and soybean plants overexpressing GmESR1 were generated. In Arabidopsis, overexpression of GmESR1 resulted in accelerated seed germination, and seedling shoot and root elongation. In soybean overexpression of GmESR1 also led to faster seed germination, and shoot and root elongation. GmESR1 specifically bound to the GCC-box. The results provide a foundation for the establishment of an efficient and stable transformation system for soybean.

Improvement of callus production and shoot regeneration using various organs of soybean (Glycine max L. Merr) by response surface methodology

Soybean (Glycine max L. Merr) is a commercially important crop that is sensitive to most abiotic stresses. Successful genetic transformation that has been widely used for yield improvements of many commercial crops needs efficient and reproducible plant regeneration protocols, in which the optimum concentrations of plant growth regulators play crucial roles. Here, response surface methodology was successfully employed to find proper combinations of plant growth regulators that facilitate more callus induction and shoot regeneration of soybean. Five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) were selected as the two independent variables in a central composite design, and quadratic models were generated and evaluated by proper statistical measures. The best combination of plant hormones, which facilitate more callus induction and shoot regeneration, using shoot, root, and leaf explants, was predicted and evaluated. These findings suggest that statistical modeling can be employed to estimate the approximate range of plant growth regulators needed for successful in vitro plant regeneration of various soybean cultivars, with potential applications in plant genetic transformation practices.

In Vitro Regeneration Potential of Seven Commercial Soybean Cultivars (Glycine max L.) for Use in Biotechnology

Abstract This work is aimed to evaluate in vitro regeneration potential of seven commercial soybean varieties Bohemians, Cardiff, Gallec, Merlin, Moravians, Naya and Silensia (Glycine max L.) cultivated in Central Europe. Our results showed the half-seeds could be effectively used as an explant source for all tested cultivars. The regeneration was initiated on the media containing growth regulators 1.67 mg.l-1 BAP and 0.25 mg.l-1 GA3. Within the first five days culture, green chlorophyll-containing explants were observed with frequency from 18.3% to 55.9%. Two weeks later, the explants responded by production of calli with the efficiency up to 83.0%. First shoots appeared after 2–3 weeks of subculture on the media. The soybean regeneration showed to be genotype-dependent with variable efficiencies from 5.7% (cv. Naya) to 37.7% (cv. Gallec). The cultivars Cardiff, Merlin and Gallec appear to be the most promising candidates for further biotechnological use. Application of antioxidants such as L-cysteine, dithiothreitol and sodium thiosulfate does not have effect on the explant regeneration for the first five days.

DNA METHYLATION ANALYSIS DURING THE OPTIMIZATION OF Agrobacterium-MEDIATED TRANSFORMATION OF SOYBEAN.

Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (Decrease in DNA methylation 1 and DNA methyltransferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.

High frequency regeneration in soybean [Glycine max (L.) Merrill.] through direct somatic embryogenesis from immature cotyledons

A simple and efficient system of soybean regeneration protocol was standardized through direct somatic embryogenesis by using immature cotyledons as explant. Three genotypes, namely, DS 2706, Pusa 5 and PS 1477 were selected for the evaluation of their capacity for in vitro somatic embryogenesis and plantlet regeneration. The medium MSB5 (MS macro + micro + B5 organics) supplemented with different concentrations of plant growth regulators (2, 4-D, NAA, BAP and ABA) were evaluated for somatic embryo initiation and subsequent development. Somatic embryos were initiated in the shortest duration (25.1 days) with the highest response (86.5 %) on MSB5 medium supplemented with 40 mg l −1 2, 4-D compared to lower concentrations or combination treatments with NAA and BAP. Somatic embryos proliferation was highest (76.25 %) in the MSB5 medium supplemented with 20 mg l−1 2, 4-D and 13 mg l−1 ABA. Somatic embryos and their morphological development were noted on medium supplemented with 6 % maltose and 0.5 % activated charcoal (79.47 %) compared to sucrose. Maltose at 6 % gave good maturation of somatic embryos (92.67 %). The highest germination (53.03 %) of somatic embryos into plantlets was achieved in the medium containing 1.5 % sucrose. Amongst the genotypes screened, DS 2706 was the most responsive. The protocol developed can be used for direct regeneration via somatic embryogenesis in different genotypes, which could also be used for attempting genetic transformation.

Construction and analysis of a suppression subtractive hybridization library of regeneration-related genes in soybean.

The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

Protective role of pulsed magnetic field against salt stress effects in soybean organ culture

Pulsed magnetic field (PMF) effects on soybean plant regeneration under salt stress conditions were investigated. Seedlings were raised from seeds pre-treated with 0.1, 1.0, 10.0 and 100.0 Hz PMF. Cotyledonary nodal (CN) explants from PMF exposed and unexposed seedlings were cultured in media containing different concentrations of NaCl (0, 10, 20, 30 and 40 mM). In CN explants from unexposed seedlings, increasing salt concentration progressively suppressed the regeneration and development of shoots and roots. Plantlets were regenerated only on medium containing 0, 10, 20 and 30 mM NaCl. The highest dose of NaCl (40 mM) failed to induce shoot formation and strongly reduced the number of roots which also exhibited reduced length. Cotyledonary nodal explants from PMF exposed seedlings, cultured at 10, 20 and 30 mM NaCl, exhibited a higher frequency of shoot and root regeneration, as well as a higher number and length of shoots and roots compared to unexposed ones, with 1.0 Hz frequency resulting the most efficient in promoting regeneration. At 40 mM NaCl, the promotive effect of different PMFs frequencies was related to the induction of a greater number of roots and the enhancement of root length. Our results suggest that PMF pre-treatment could help the regeneration of soybean under salt stressed condition.

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 µM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 µM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.

Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (Glycine max. L)

Thidiazuron (TDZ), primarily a cotton defoliant, has been later accepted as a plant growth regulator. In spite of extensive studies, the physiological function of TDZ is still uncertain. The aim of the present experiment was to study the activity of TDZ in in vitro regeneration of soybean. The seeds of soybean were cultured separately on MS and B5 medium supplemented with TDZ. The hypocotyls, cotyledons, cotyledonary nodes without axillary buds and cotyledonary nodes with axillary buds were used as explants and their capacity to direct regeneration was tested on both MS and B5 media containing TDZ (0.9–5.4 μM). Shoot formation was observed only on cotyledonary nodes with axillary buds cultured on MS and B5 basal media with TDZ (0.9–5.4 μM). All tested explants cultured on B5 medium with TDZ produced roots. Root formation was not observed on MS basal media supplemented with TDZ. Results show that TDZ functions as cytokinin (to produce the shoots) and auxin (to produce the roots) on various explants depending on the basal medium used.

Somatic embryogenesis and plant regeneration in Chinese soybean (Glycine max (L.) Merr.)—impacts of mannitol, abscisic acid, and explant age

From a preliminary experiment on 98 Chinese soybean varieties, 12 varieties with somatic embryogenesis frequency ranging from 0.0% to 85.7% were selected for further study in order to enhance the efficiency of somatic embryogenesis and plant regeneration. The effects of different mannitol concentrations, abscisic acid (ABA) concentrations, and embryo explant ages (sizes) were investigated. Significant differences in somatic embryogenesis were found among the 12 soybean varieties, with initiation frequencies varying from 22.1% to 89.0% under suitable mannitol concentration, and with N25281, N25263, and N06499 having the highest somatic embryogenic capacity. The results showed that all three factors were relevant for raising rates of callus initiation and somatic embryogenesis, but with differential responses among the genotypes. The treatment of 3.0% (w/v) mannitol, 5 mg l−1 ABA, and a 4- to 5-mm-sized explant was found to be optimal for somatic embryogenesis, generating the highest explant-based regeneration rate at 83.0%. The greatest average number of plantlets regenerated per explant (1.35) was observed in N25281. The above results provide a basis for efficient regeneration of soybean and are informative for the development of genetic transformation systems in Chinese soybean germplasm.

Factors influencing regeneration of soybean from mature and immature cotyledons

Soybean (Glycine max L.) plantlets were e?ciently regenerated from mature and immature cotyledons of?ve different genotypes by studying various parameters affecting regeneration. Green organogenic noduleswere induced at the proximal end, which subsequently differentiated into shoot buds on modi?ed MS(Murashige T. and Skoog F. 1962. Physiol. Plant. 15: 473-497) medium. The presence of 6-benzylaminopurine(BAP) and thidiazuron (TDZ) in the medium exerted a synergistic effect, in that regenerationeffciency was higher than for either cytokin alone. The regenerated shoot buds elongated and rooted onMS medium containing 0.29 lM gibberellic acid (GA3) and 2.69 lM a-naphthaleneaceticacid (NAA),respectively. Rooted plants were established in the greenhouse with 87% success and produced viable seeds.Preliminary studies with Agrobacterium show great promise for soybean transformation based on theregeneration protocol reported here.

대두 품종에 따른 자엽절에서의 다신초 형성

대두의 재분화 체계를 확립하고자 품종별 shoot 형성률, 적정배지 및 적정조직을 구명하고자 하였다. 유묘의 조직부위별 비교에서는 1개의 자엽을 포함한 절에서 multiple shoot 형성률이 높았다. 기본배지에서는 1/2 B5배지가 효율적이었으며, TDZ 처리시에 multiple shoot 형성률이 높았으며, 처리농도는 2mg/L가 효율적이었다. Shoot 형성시 자엽 네 전분은 배양 1주까지 증가 후 감소하였으며, 수용성 당은 배양 2주까지 증가 후 shoot가 형성되는 배양 4주에는 관찰되지 않았다. 품종 간 비교에서는 품종에 따라 shoot 형성 양상이 구분되어, '흑청콩' 등 11품종에서는 multiple shoot 형성률이 높았고, '만리콩' 등 16품종에서는 pair shoot 형성률이 높았다. 자엽절에서 shoot가 형성되는 양상에 따른 조직화학적 관찰 결과 pair shoot의 경우 내생적으로 잠재한 액아에 의한 것이며, multiple shoot의 경우는 새로이 형성된 shoot로 확인되었다. For the plant regeneration of soybean (Glycine me L. Merr.), the shoot formation rate, optimal medium and tissue conditions were examined using Korean soybean cultivars. Among the parts of seedling, a node that includes one cotyledon showed the highest shoot formation rate among other tissues. Half-strength B5 medium was more efficient than full strength medium. Formation rates of pair shoots (1 to 2 shooting) were higher in the when benzyl adenine was supplemented. The formation rates of multiple shoots, that is, 4 to 5 in shooting, were high when thidiazuron was supplemented. Multiple shoot was de novo formed in cutting side of cotyledonary node. The effective concentration of thidiazuron for shoot induction treatment was 2 mg/L. Among the 27 cultivars, multiple shoot formation rates were high in the 11 cultivars including 'Heugcheongkong, and pair shoot formation rates were high in the 16 cultivars including 'Malikong'.

Adventitious Shoot Formation from Hypocotyl Sections of Mature Soybean Seeds.

A new system for simple and efficient shoot regeneration of soybean (Glycine max (L.) merr.) is reported using the hypocotyl of mature seeds. Two transverses of 1-mm sections of the hypocotyl were cut from mature seeds: sections containing a cotyledonary node and sections 1 to 2 mm from the cotyledonary node. The effects of thidiazuron (TDZ) concentrations, plating methods, and genotypic differences were examined. Shoots were formed from cotyledonary node ends in all conditions examined. Meanwhile, shoots were formed from the ends of hypocotyl sections on B5 media containing TDZ. The optimal TDZ concentration for organogenesis was 2~10 μM. The efficiency of organogenesis varied with the method of plating of explants. The ends of the hypocotyl sections in contact with the media only produced adventitious shoots. Adventitious shoots were formed effectively by placing explants on the media oriented so that the hypocotyl axes were perpendicular to the surface of the media. The efficiency of organogenesis differed with the position of the hypocotyl ends. It was important to use the ends that were 1 mm from the cotyledonary nodes. Genotypic differences were observed in the organogenesis of the hypocotyl ends.

Regeneration of soybean via embryogenic suspension culture

In an attempt to establish an alternative plant regeneration system for soybean [Glycine max (L.) Merrill] cultivars used in Brazilian breeding programs, ten genotypes were tested for their embryogenic potential. Cotyledons were removed as explants from immature seeds harvested from field-grown plants. After 45 days on induction medium, the number of responding cotyledons and the number of somatic embryos per immature cotyledon were evaluated. The percentage of explants that produced somatic embryos varied from 1 to 70% among cultivars. The average number of somatic embryos produced per cotyledon pair ranged from 0.01 to 10.3 with a mean of 3.4. Suspension cultures were initiated with three Agrobacterium tumefaciens susceptible cultivars. Suspensions were successfully developed from Bragg and IAS5 cultivars. The packed cell volume, in one-month growth, increased 8.1 fold for Bragg and 3.5 fold for IAS5 and the fresh weight increased 6.6 and 2.8 fold, respectively. The cultivars differed for the analysed parameters. All tissue from each cultivar was transferred to the maturation medium and subsequently to the germination medium. The germination frequency was 45.7 and 54.9% for Bragg and IAS5, respectively. Plants were gradually exposed to ambient humidity over one week and then planted in soil. All plants yielded seeds in the greenhouse.

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

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.

Stable transformation via particle bombardment in two different soybean regeneration systems

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.