In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.     

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.     

Benzyladenine induction of buds and somatic embryogenesis in Picea omorika (Pančić) Purk.

Somatic embryogenesis and adventitious bud formation, initiated from shoot explants of Picea omorika is described. Benzyladenine (BA) as the only growth regulator, added to modified Von Arnold and Eriksson medium, induced formation of both adventitious buds and embryogenic tissue. Optimal BA concentration for bud induction was 4.5 M and further bud development and plantlet formation was achieved on growth regulator-free medium. The embryogenic tissue formation was induced when the explants were first grown on the medium with high BA content (22.5 M) and then transferred to medium without growth regulators. Subsequent proliferation of embryogenic tissue was accomplished by subculturing on medium containing 9 M 2,4-dichlorophenoxyacetic acid and 4.5 M BA, and further embryo development was achieved on medium with 12 M abscisic acid. Embryos cultured on growth regulator-free medium formed roots and rooted plantlets were successfully established in soil in the greenhouse.     

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Somatic embryogenesis and plant regeneration inMedicago suffruticosa

A successful procedure has been designed for the regeneration of plantlets from leaf sections of the self-pollinating species,Medicago suffruticosa. Callus growth was promoted by a 4-week culture period on liquid Kao's medium containing 4.9 M benzyladenine and 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), followed by a 4-day treatment in which the benzyladenine was elevated to 44.4 M. Shoots/plantlets were observed after 3–4 weeks culture on growth regulator-free agar-solidified medium. Under these conditions, the regeneration frequency from callus was 18% and a histological study showed that this regeneration was through somatic embryogenesis. The growth regulator treatment, with a relatively high concentration of growth regulators (44.4 M benzyladenine) for a short time period (4 days), is important for inhibiting polyphenol compounds and for stimulating callus growth and plant regeneration.Abbreviations 2,4-d 2,4-dichiorophenoxyacetic acid - BA benzyladenine - NAA -napthaleneacetic acid     

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.     

Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu-a multipurpose leguminous tree

Plant regeneration via somatic embryogenesis was achieved from callus derived from immature cotyledons of Acacia catechu Willd. on Woody Plant Medium (WPM) supplemented with 13.9 M kinetin and 2.7 M 1-naphthaleneacetic acid. The addition of 0.9–3.5 mM L-proline to the medium influenced development of somatic embryos and also promoted secondary somatic embryogenesis. The light-green somatic embryos germinated on half-strength MS medium supplemented with 2% (w/v) sucrose. Somatic embryos germinated into plantlets that were acclimatized in the greenhouse and subsequently transferred to the field.     

Somatic embryogenesis and plant regeneration in Gymnema sylvestre

Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from hypocotyl, cotyledon and leaf explants excised from seedlings of Gymnema sylvestre. Embryogenic callus was induced on Murashige and Skoog (MS) medium containing 2,4-D (0.5–5.0 M) +BA (0.5–2.0 M) and 2% (w/v) sucrose in 6–8 weeks of culture. Globular/heart stage embryos developed on induction medium. These embryos produced torpedo and cotyledon stage embryos upon sub-culturing on embryo maturation medium EM8 (medium containing MS salts, B5 vitamins, 0.5 M BA and 2% sucrose). Embryo germination and plantlet formation was achieved by sub-culturing mature embryos on fresh EM8 medium. The plantlets were acclimatized in the greenhouse.     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.     

Somatic embryogenesis and plant regeneration from zygotic embryo culture in blue spruce (Picea pungens Engelman.)

Summary Somatic embryogenesis and plantlet formation have been achieved from cultured mature zygotic embryos of blue spruce (Picea pungens Engelman.). The effect of three basal media LP, LM, and BLG, all used at half-strength, was tested at the induction phase. LM medium induced somatic embryogenesis to a higher extent than LP whereas BLG did not produce any embryonal-suspensor mass representing stage 1 somatic embryos. The embryonal-suspensor mass was induced on a wide range of auxin/cytokinin ratios. However, media containing either 2 M NAA and 10 M BA, or 10 M NAA and 5 M BA produced somatic embryos that gave the highest frequency of plantlets. The level of ABA required in the maturation medium for somatic embryos to mature properly varied with the auxin/cytokinin levels in the induction medium on which the somatic embryos were derived. Inclusion of AgNO₃ (10 – 100 M) in the induction medium reduced somatic embryogenesis and embryo conversion.Abbreviations NAA naphthalene-acetic acid - BA N⁶-benzylaminopurine - ABA abscisic acid     

Micropropagation of Frazinus angustifolia from mature and juvenile plant material

Micropropagation of Fraxinus angustifolia Vahl has been successfully achieved both from mature and juvenile plant material using shoot tip and nodal explants. Several basal media supplemented with benzyladenine (BA) and indolebutyric acid (IBA) were tested for shoot proliferation. The most new explants per mature explant (5.3) was obtained on DKW medium plus 4.4 M BA+0.98 M IBA. The most new explants per juvenile explant (5.6) was produced on QL medium plus 8.9 M BA+0.49 M IBA. Rooting was achieved on WPM supplemented with 0.98–4.9 M IBA. Rooted plantlets were transferred to soil and acclimatized with 85% survival.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA 1-naphthaleneacetic acid     

Direct plant regeneration from leaf explants of Plumbago species

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 M 6-benzylaminopurine (BA), 1.4 M indole-3-acetic acid (IAA), 370 M adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 M indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.     

Somatic embryogenesis from ovaries,developing ovules and immature zygotic embryos,and improved embryo development of <Emphasis Type="Italic">Castanea sativa</Emphasis>

Somatic embryogenesis of European chestnut (Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 M 2,4-dichlorophenoxyacetic acid plus 0.5 M 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 M BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 M BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Adventitious shoot organogenesis and plant regeneration from cotyledons of Dalbergia sissoo Roxb., a timber yielding tree legume

A procedure is outlined to induce adventitious shoot organogenesis from semi-mature as well as mature cotyledons lacking the embryonic axis of Dalbergia sissoo Roxb., an economically important leguminous tree. Shoot buds were induced in the proximal region of the semi-mature cotyledons on Murashige and Skoog's (MS) medium supplemented with 4.44 M 6-benzyladenine (BA) and 0.26 M -naphthaleneacetic acid (NAA). These buds elongated into shoots following transfer to a similar medium containing half-strength macro-nutrients. Adventitious shoot bud formation was also induced in the mature cotyledons. However, unlike the semi-mature explants, the mature cotyledons exhibited shoot bud differentiation on MS medium containing 22.20 M BA without NAA. Pre-culture of mature cotyledons in liquid MS medium containing 8.88 M BA for a duration of 48 h improved shoot bud regeneration up to six-fold. Regenerated shoots, derived from semi-mature and mature cotyledons, rooted on half-strength MS medium containing 1.23 M and 4.92 M indole-3-butyric acid (IBA), respectively. Plantlets were acclimatized and established in soil.     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.