Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Somatic embryogenesis in callus culture of Mussaenda erythrophylla cvs. Queen Sirikit and Rosea

Somatic embryogenesis was achieved from mid-rib and internodal calluses of Mussaenda erythrophylla L. cvs. Queen Sirikit and Rosea cultured on Murashige and Skoog basal medium containing 8.9 M BA+0.57 M IAA+10 mg l^-1 ascorbic acid. Clumps of somatic embryos were separated and grown into complete plantlets when transferred to 1/2 MS medium+37 M adenine sulphate with 2% (w/v) sucrose.     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey)

Summary The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01–120 M) alone or in combination with BAP (8 M). Somatic embryogenesis was induced by both PAA and IAA at 0.01–20 M with 8 M BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1–2.5 M, whereas PAA gave best results at 10 and 20 M under identical culture conditions. Higher concentrations (30–120 M) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - PAA phenylacetic acid     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture

Embryogenesis from isolated microspore cultures of wheat was improved by ovary co-culture when compared to a completely defined medium. This indicates that essential factor(s) in addition to PAA or its analogs may be supplied by the ovaries. Isolated microspores cocultured with 20 ovaries of wheat on the top of semi-solid MMS3 induction medium for 21–30 days gave the best response. Both the number and quality of the embryos was significantly increased. The maximum frequencies of dividing microspores and of embryogenesis were 94% and 2.4%, respectively. Up to 2583 embryos were formed per 100 anthers of cv Chris and between 18% and 43% of the larger embryos regenerated into green plants upon transfer. Genotype differences for both induction and embryogenesis phases were reduced using ovary co-culture. However, there was still a strong genotype influence on plant regeneration with cv Chris, with the F1 of Chris × Sinton displaying the highest frequencies. These results are important with respect to enhancing haploidy applications in wheat biotechnology and plant breeding.Abbreviations PAA Phenylacetic acid - MMS modified MS medium - MS Murashige and Skoog's medium 1962 - FHG Hunter's FHG medium 1988     

High-frequency somatic embryogenesis from small suspension-cultured clusters of cells of an interspecific hybrid of Oryza

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F₁ plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200m in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with -naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 M) and sucrose (30 gl^-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthalenacetic acid - 4-PU 4-pyridylurea - MS Murashige and Skoog (1962)     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Triazole treatment of explant source provides stress tolerance in progeny of Geranium (Pelargonium hortorum Bailey) plants regenerated by somatic embryogenesis

Thehypothesis that chemically induced stress tolerance in plants can betransferredto a larger clonal population regenerated by somatic embryogenesis wasevaluatedusing the triazole compound paclobutrazol as a chemical inducer of stresstolerance in Geranium (Pelargonium horturum Bailey). Seedswere imbibed in 3.4, 10.2 or 17.0 M (1, 3, 5 mgL^–1) paclobutrazol for 24 h and germinatedfor 7 days. Hypocotyl explants were cultured in vitro toinduce somatic embryogenesis. Plants regenerated from somatic embryos wereexposed to heat stress at 56°C. Explants treated with3.4 M paclobutrazol yielded a substantially higher number ofsomatic embryos compared with untreated explants. In contrast, 17.0M paclobutrazol treatment inhibited embryogenesis producing asignificantly lower number of somatic embryos. There was no difference in theembryo number between control and 10.2 M treatment. Somaticembryos derived from 3.4 and 10.2 M paclobutrazol treatedexplants developed into plants at a faster rate than the control and 17.0M treatments. Plants derived from paclobutrazol-treatedexplants displayed a greater tolerance to heat stress compared with thecontrols. Observations in this study provide a technique for regeneratingplantsin tissue/cell culture with additional desirable traits such as stresstolerancewith minimal chemical contamination of the environment.     

A novel type of somatic embryogenesis in Coffea arabica

A novel type of somatic embryogenesis characterized by an efficient and highly synchronized embryo formation was observed in embryogenic callus of Coffea arabica initiated on Murashige and Skoog medium containing kinetin (4 mg/l) and 2,4-D (1 mg/l). It occurs in suspension and goes along with the suppression of High Frequency Somatic Embryo Induction (HFSE). This is achieved by favoring during cultivation senescence-or necrosis-like processes which apparently do not impair the competence for embryogenesis. Since the resulting embryos germinate at a rate of 94.5 % without the need of a maturation step, we propose the term Self-Controlled Somatic Embryogenesis (SCSE).In addition, HFSE was optimized using half-strength liquid medium with 0.1 mg/l kinetin and 0.25 mg/l 2,4-D for proliferation of embryonic tissue, and 2.6 mg/l ABA for maturation of embryos. Yields as well as germination rates of HFSE embryos were markedly lower as compared to SCSE.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - NAA 1-naphthaleneacetic acid - HFSE high frequency somatic embryo induction - LFSE low frequency somatic embryo induction - SCSE self-controlled somatic embryogenesis     

Effect of plant growth regulators on the cellular growth and levels of intracellular protein,starch and polyamines in embryogenic suspension cultures of Pinus taeda

Somatic embryogenesis is the most important in vitro culture system for conifer propagation. However, Pinus taeda has been considered recalcitrant to somatic embryogenesis in commercial scale-up. The study of biochemical and physiological aspects of cell growth could lead to a better understanding of somatic embryogenesis in this species. In the present work, we investigated the cell growth dynamics, intracellular levels of proteins, starch and polyamines in suspension cultures of Pinus taeda established in plant growth regulator-free medium (BM0) and in medium supplemented with 2 M 2,4-dichlorophenoxyacetic acid, 0.5 M 6-benzylaminopurine and 0.5 M Kinetin (BM2). Cell cultures growing in BM0 medium showed an increase in the sedimented cell volume from 3.77 to 17.73 ml after 24 days of culture. Those cultured in BM2 medium showed an increase in the sedimented cell volume from 4.23 to 25.17 ml after 20 days of culture. Intracellular proteins levels increased during the exponential growth phase and starch levels decreased until the exponential phase, followed by a synthesis up to the stationary phase, in both BM0 and BM2 media. Highest putrescine levels occurred in cultures growing in BM0 medium and this was associated with the low cellular growth.     

Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare X Triticum aestivum hybrids

Summary Embryogenic callus cultures were obtained by culturing young inflorescence tissues of Hordeum vulgare cv. PF51811 (2x)XTriticum aestivum cv. Chinese Spring (6x) hybrids on 2,4-D-containing N₆ medium. After subculture for about 10 months the calli retained a high potentiality for somatic embryogenesis and plant regeneration. Of about 300 regenerated plants, approximately 100 were transplanted to potting soil. Eight embryoids and three regenerated plants examined had 28 chromosomes identical to the original hybrid plants, while one regenerated plant was found to be a mixploid composed of cells with 28 and 56 chromosomes. The possibility for obtaining amphiploid hybrids through tissue culture is discussed.     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.