Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Comparative analysis of embryogenic potential of soybean cultivars zoned in different eco-geographic world regions

Embryogenic potential of 19 soybean cultivars originated from different eco-geographic regions (Europe, China, America and Far East) has been established and analyzed. Cultivars TSZ-14, Amurskaja-111, Gribovskaja mestnaja were emphasized as highly embryogenic genotypes. Cultivar Mantherova biela vel. revealed the highest embryogenic potential (80%). The comparison of embryogenic potential of the Ukrainian cultivars with given data indicated various parameters which could affect the induction of soybean somatic embryogenesis. It had also been supposed that cultivars from the Far East and Europe could have the potential for further screening of new highly embryogenic genotypes. The data obtained enlarge the data base pool of the soybean somatic embryogenesis research and enable using the most appropriate cultivars for further genetic improvement.     

Improved somatic embryogenesis of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.     

不同荔枝品种采后果实衰老的比较

比较了5个荔枝(Litchi chinensis Sonn.)品种("怀枝"、"糯米糍"、"桂味"、"红蜜荔"和"水晶球")果实采后在常温下的衰老表现.其中,"糯米糍"果实衰老褐变快,而"桂味"衰老最为缓慢.果实褐变过程伴随失水,但品种间脱水速度与果实衰老的速度无显著相关性.果皮的褐变潜力、果皮细胞壁糖醛酸含量、果胶甲酯化程度、果皮和果肉总钙含量、果皮水溶性钙含量与果实衰老均无显著的相关性,但膜透性与坏果率呈显著的正相关,而果皮结构钙与之有显著的负相关性.     

Embryogenic response of multiple soybean [Glycine max (L.) merr.] cultivars across three locations

Summary Nine soybean [Glycine max (L.) Merr.] cultivars representing midwestern, mid-south, and southern US growing regions were evaluated at each of three locations (Athens, GA; Lexington, KY; and Wooster, OH) using uniform embryogenic induction and proliferation protocols in order to evaluate the portability of soybean somatic embryogenic protocols to different locations. The experimental design minimized variation between locations by having all cultivars present at all locations on all days. A quantitative weighted score for primary embryo induction was developed on average embryo number per explant and was used to describe non-embryogenic, poorly embryogenic, moderately embryogenic, and highly embryogenic responses. Ranking of cultivars remained similar across all locations, indicating a uniform transportability of the protocol, at least as far as embryo induction is concerned. Continued proliferation of embryogenic cultures was also measured using a repetitive growth measure but few meaningful conclusions could be made due to the high level of variability including inconsistent growth of cultures between each subculture. Overall, several cultivars were identified as being uniformly embryogenic or non-embryogenic at the primary induction phase at all locations, and we predict that those embryogenic cultivars could be used by any laboratory for high-efficiency induction of embryogenesis. The best of these cultivars, ‘Jack’, was uniformly responsive across all locations and should be selected as the genotype most likely to yield positive results when attempting to culture and genetically engineer soybeans via embryogenic protocols.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted. The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6). Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction. Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 3.0 mg l^–1 of 2,4-D and 30 g l^–1 of sucrose. Agar concentration in the regeneration medium was also critical for the shoot induction. Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (2.0 mg l^–1) of NAA. The optimal concentration of kinetin for the highest shoot regeneration frequency (6777%) was different among the cultivars tested. The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds.     

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.)

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

An assessment of somatic embryogenesis and cryo-preservation methods with a wide range of Sitka spruce breeding material from the UK

High variability in regeneration capacity has previously been observed within a population of seedlings in several spinach cultivars. The cultivar “Matador” exhibits particularly low regeneration potential, and the majority of lines obtained in our previous study displayed a stable embryogenic capacity only for a limited period of time. In order to shorten the time required for embryogenic capacity assessment for individual lines, a model system for the rapid evaluation of embryogenic capacity was developed. This model system was based on the expression of a gene encoding spinach ribosome-inactivating protein (SoRIP2), which showed low expression levels in roots grown under non-inductive conditions. Induction of globular somatic embryos (SEs) resulted in a 285-fold increase in SoRIP2 expression that dropped to the control level beyond cotyledonary-stage SEs. The model system was tested by comparing the expression of SoRIP2 and the index of embryo-forming capacity (EFC), which integrates the frequency of regeneration and the mean SE number per root explant. Comparisons were always made within the same line, and the expression of SoRIP2 and the EFC index were determined 4 and 12 weeks after starting induction treatment, respectively. High positive correlations between SoRIP2 expression and EFC were obtained for the two factors that influenced embryogenic capacity the most: genotype (r²?=?0.81) and photoperiod (r²?=?0.92). The results indicate that the expression of SoRIP2 can be successfully used for early evaluation of regeneration capacity of individual lines, before SEs can be seen with the aid of a stereomicroscope, even 8 weeks earlier than by the conventional method.     

Tissue position,explant orientation and naphthaleneacetic acid (NAA) affect initiation of somatic embryos and callus proliferation in Norway spruce (Picea abies)

Sections from mature zygotic embryos of Norway spruce exhibited different capacities for somatic embryo initiation. The upper hypocotyl part (Zone 2) was the most embryogenic, followed by the lower hypocotyl (Zone 3) and the apical zone (Zone 1); the root part (Zone 4) never initiated embryonal-suspensor masses (ESM). The embryogenic capacity of mature zygotic embryo is narrowly located in the vicinity of Zone 2. The frequency of embryos differentiating simultaneously ESM on Zones 1 and 3 is very low (0.6%) compared to those initiating ESM on Zones 1 and 2 (7%) or Zones 2 and 3 (16%). Elevated concentrations of naphthalene acetic acid (40 and 80 M) reduced ESM initiation and callus proliferation on all sections but Zone 1. Highest initiation rate was obtained when explants were cultured with an apical-end-up orientation. Placing the explant basal-end-up partially inhibited the expression of embryogenic capacity, as well as decreasing the callus proliferation on Zone 3. A weak positive correlation (r=0.19, p < 0.001) was found between embryogenic capacity of the explant and proliferation rate of the derived callus.     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.     

Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums

A procedure was established for the induction of regenerable calli from immature inflorescence segments of high-tannin cultivars of sorghum (Sorghum bicolor (L.) Moench). Murashige & Skoog's medium with several components altered was utilized for inducing, maintaining, and regenerating the cultures. Embryogenic calli formed at a frequency of 8–70% depending on the genotype. During a ten-month period, 3600 plants were regenerated from eight genotypes tested. Among the developmental stages of immature inflorescence tested (from differentiation of secondary branch primordia to floret formation) no critical differences were found in potential for callusing, embryogenesis or regeneration. Genotypic differences were observed in pigment production, embryogenic callus formation, shoot differentiation, and in maintenance of regeneration capacity.Abbreviations 2,4-D dichlorophenoxyacetic acid This is Journal Paper Number 11972 from the Purdue University Agricultural Experiment Station     

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Enhancement and repression of somatic embryogenesis in cell cultures of carrot by cold pretreatment of stock plants

Roots of nine cultivars of carrot (Daucus carota L.) were exposed to one, two or three months cold treatment and cells isolated from cold-treated and control roots were assayed for the production of somatic embryos. Cells obtained from the one- or two-months cold treatments formed embryos earlier, produced embryos over a longer period of time and produced more embryos per callus than the controls. In contrast, cells obtained from roots exposed to three months cold displayed a reduction in all parameters of embryogenic competence and for some cultivars this treatment resulted in production of cells with no embryogenic competence. The relationship of cold treatment of stock plants to the induction of metastable and stable patterns of gene expression and the induction of somatic embryos are discussed.     

Taxonomic Study of Dimocarpus longan by Random Amplified Polymorphic DNA Technique

The classification and relationship among 35 samples of longan (Dimocarpus longan Lour. ) have been studied by RAPD (random amplified polymorphic DNA) and statistical cluster analysis. These results showed that it was unlikely possible that "Lizhiben" and "Lizhi kongyan" were hybrid progeny of longan and litchi ( Litchi chinensis Sonn. ) as what some people believe. The "Lizhiben", and "Nanhu Jiaohe" were highly mutant varieties. There were some variance among the "Jiaohe" wilted-nut longan cultivars. Different cuhivars given the same name, such as "Dongbf', "Honghezi" and some other cultivar, were also possible. It was concluded that longan cultivars which have been tested in this research could be classified into 6 groups. The first group, including 12 cultivars such as "Shuizhang", "Youtanben", "Wulongling" etc., which were the well known best cultivars appropriate for producing dry longan, was named as Shuizhang-Wulongling group. The second group, including 11 cultivars like "Chike", "Jiaoyan", "Fuyan", "Chushuben" etc., was named as Chike-Chushuben group. The Dongbi series cuhivars were named after their cultivars as the Dongbi group. The special cuhivars, "Lidongben", "Nanhu Jiaohe" and "Lizhiben" were named respectively as Lidongben group, Nanhu Jiaohe group, and Lizhiben group, because of their more characteristic DNA polymorphism tested in all respective cuhivars. The results indicate that the application of RAPD technique in the taxonomic study has gained some resolution which could not be obtained by morphological analysis and has provided available data for developing longan production and for breeding new longan cultivars.     

Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.)

Improved suspension cell culture systems are needed to facilitate the application of recombinant DNA technology for wheat germplasm enhancement. This study evaluated three wheat (Triticum aestivum L.) cultivars, and the effects of medium basal salts, 2,4-D, sucrose, and L-proline concentrations on the establishment of rapidly growing and highly embryogenic callus and suspension cultures. Percent embryogenic calli was visually estimated and verified with light and scanning electron microscopy. The most highly embryogenic callus was produced by cultivar Bobwhite on medium with MS basal salts, 5.6 M 2,4-D, 58 mM sucrose, and zero proline. The suspension cultures that produced the greatest number of regenerated plants utilized callus tissue produced on solid medium with MS basal salts, 87 mM sucrose, 9 M 2,4-D, and no proline.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid; RG, relative growth - %EC percent embryogenic calli - RV Redway and Vasil medium (1990a) - DPA days postanthesis