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.     

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.     

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.     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus

High frequency somatic embryogenesis in papaya (Carica papaya L.) tissue cultures was achieved by culturing hypocotyl sections from ten-day-old seedlings on half-strength Murashige and Skoog salts (MS) medium containing modified MS vitamins, 2.3 to 112.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), 400 mg l^-1 glutamine, and 6% sucrose. Four hermaphroditic Hawaiian cultivars produced embryogenic calluses after ten to 14 weeks of culture at 27°C in the dark. Efficiency in embryogenic response of genotypes differed, Kapoho > Sunset > Sunrise > Waimanalo. The frequency of embryogenesis in induction medium containing 4.5 M 2,4-d was lowest with 3% sucrose and highest with 7% sucrose. Somatic embryos developed directly from embryogenic calluses on induction medium, or, more often, they differentiated from calluses subcultured on a medium devoid of growth regulators. Between 50 and 500 embryos were produced from each 2-mm hypocotyl section after at least two months on induction medium and two months on maturation medium. Embryos subsequently developed into normal-looking plants on MS medium. Shoot cuttings from germinated embryos and micropropagated plants were rooted with 5.0 M indole-3-butyric acid (IBA), grown in the greenhouse, and transferred to the field.Journal Series no. 3732 of the Hawaii Institute of Tropical Agriculture and Human Resourees     

Somatic embryogenesis in Simarouba glauca

Frequency of somatic embryogenesis from callus cultures derived from immature cotyledon explants of Simarouba glauca Linn. was highest on solid MS medium supplemented with 11.1 M benzyladenine and 13.42 M -naphthaleneacetic acid. On transfer of the somatic embryos into maturation medium containing half-strength MS medium supplemented with 1.89 M abscisic acid (ABA) and 2% (w/v)sucrose, 20–25 % of embryos germinated within 20 days of culture with distinct cotyledon, hypocotyl and radicle.     

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     

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and -naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA. Treatment with zeatin (22.8 M) and BA (10.0 M) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.     

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 M 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 M BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 M BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l^–1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Callus induction and plant regeneration from mature zygotic embryos of a tetraploid Alstroemeria (A. pelegrina × A. psittacina)

Summary A simple procedure was developed to induce callus growth and whole plant regeneration for a tetraploid cultivar of Alstroemeria. The callus, induced from mature zygotic embryos cultured on a medium supplemented with 20 M kinetin with 10 or 20 M NAA, could be maintained for one year without any loss of regeneration potential. Maximum frequency of regeneration (40%) was obtained with calli maintained on the medium containing 20 M kinetin and 20 M NAA. Whole plant regeneration occurred via somatic embryogenesis in the absence of growth regulators and the plantlets grew to maturity and flowered in the greenhouse conditions.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - MSO Basal medium devoid of any plant growth regulator - NAA -Naphthaleneacetic acid - TDZ N-phenyl-N 1,2,3,-thiadiazol-5-ylurea (thidiazuron) - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid     

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH₄ ⁺ and NO₃ ^– ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 M) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH₄ ⁺ and NO₃ ^– concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 M) enhanced the conversion of somatic embryos to plantlets.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Somatic embryogenesis in perennial statice Limonium bellidifolium,Plumbaginaceae

Cotyledon explants from perennial statice Limonium bellidifolium (Statice caspia Willd.) were cultured on Murashige and Skoog's basal medium (MS) supplemented with various levels of 2,4-D, kinetin and sucrose. Embryogenic calluses developed over a period of 10 days with the highest response at 4.5 M (1 mg l^–1) 2,4-D, 0.5 M (0.1 mg l^–1) kinetin and 117 mM sucrose. Following induction, the calluses were transferred to MS media supplemented with 88 or 117 mM sucrose and 0 or 0.5 M kinetin. Somatic embryos at the globular, heart-shaped, torpedo, and cotyledonary stages developed. Fully germinated plantlets developed with the best response in medium supplemented with 117 mM sucrose and 0.5 M kinetin. Direct somatic embryogenesis without a callus phase was observed with some of the cotyledon explants. Induction, maturation and germination of somatic embryos on the optimized media were equally effective using cotyledon, hypocotyl and root explants. Serial sections of L. bellidifolium cotyledon explants cultured for two weeks indicated that pro-embryogenic masses originated from parenchyma cells below the epidermis. Further histological observations of embryogenic calluses confirmed the initiation and development of globular and heart-shaped embryos and repetitive somatic embryogenesis. Ultrastructural observations indicated that the embryogenic cells were less vacuolate with abundant organelles compared to the cells of the explant. This is the first report of somatic embryogenesis in the Plumbaginaceae.     

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.     

Stepwise decrease of 2,4-D and addition of BA in subculture medium stimulated shoot regeneration and somatic embryogenesis in buffalograss

Plant regeneration of buffalograss `Texoka' was achieved through both somatic embryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 M 2,4-D combined with either 0.44 M or 1.32 M BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D (4.5 or 9 M) resulted in higher percentages of embryogenic callus while 2,4-D at 2.25 M generated shoot-producing callus but with a lower percentage of embryogenic callus. Transfer of calluses from medium containing 4.5 M 2,4-D and 0.44 M BA to the somatic embryo initiation medium containing 0.9 M 2,4-D gelled with either 7 g 1^–1 agar or 3 g 1^–1 Gelrite led to the formation of somatic embryos. Somatic embryo initiation medium gelled with 3 g 1^–1 Gelrite led to significantly higher frequency of somatic embryo formation than in medium gelled with 7 g 1^–1 agar. Callus of a female genotype `315' generated under similar treatments did not produce shoots or somatic embryos.     

Changes in nutrient composition and pH of the culture medium during in vitro shoot proliferation of crabapple and pear

Immature soybean (Glycine max (L.) Merr) embryos, or cotyledons isolated from them, were cultured on modified MS medium containing B5 vitamins and NAA (50 M) to induce somatic embryogenesis. The effects of media variables, dissection treatments and light conditions were investigated in this system. The efficiency of embryogenesis increased as sugar concentration decreased from 12 to 1.5%; sucrose and glucose were similarly effective as carbon sources. In an examination of the effects of medium pH, values between pH 5.0 and 7.0 gave similar embryogenesis efficiencies, but the frequency of normal embryos was greater in media with low pH values. In buffered medium (10 mM MES), a pH of 5.0 was inhibitory to embryogenesis, and most normal embryos were produced at pH 5.5. Under various dissection treatments, embryogenesis efficiency and root and callus production were increased by tissue damage. Somatic embryogenesis was observed both in darkness and in light, although embryo development was impaired under high light (80 E m^-2 s^-1) conditions. The ability of somatic embryos to germinate was closely correlated with embryo normality, and was influenced little by the hormone content of germination media. Of various media tested for their ability to support the growth of germinated embryos, a medium based on hydroponic nutrient salts, supplemented with yeast extract, and gelled with Difco-Bacto agar gave the best plantlet growth.Abbreviations E m^-2 s^-1 microEinsteins per square meter per second - NAA -napthalene acetic acid - N50 MS salts with B5 vitamins and 50 M NAA (Napthalene acetic acid) - MES 2(n-morpholino) ethanesulphuric acid - BAP benzylamino purine - IBA indole butyric acid This paper (No. 86-3-97) is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of palmarosa grass (Cymbopogon martinii)

A cell suspension culture was established from nodal callus ofCymbopogon martinii (Roxb.) Wats in a liquid medium containing Murashige and Skoog (1962) basal salts, vitamins, 100 mg 1^–1 myo-inositol and 20 g l^–1 of sucrose (MS) that was supplemented with 13.6 M 2,4-dichlorophenoxyacetic acid and 1.15 M kinetin. An initial inoculum density of 2 x 10⁴ cells ml^–1exhibited optimum cell growth. Calli were obtained 12–15 days after the suspension was plated onto semisolid medium of a similar composition. When calli were transferred to semisolid regeneration medium containing MS + 6.7 M N ⁶-benzyl-adenine + 1.15 M kinetin, somatic embryogenesis and plantlet regeneration occurred after 10–25 days. There was no significant decrease in the regeneration potential of the calli even when the cultures were initiated from 47-week-old cell suspensions. Chromosome counts of cells in suspensions, calli and somatic embryos derived from cultures of different ages revealed the presence of diploids, tetraploids and octaploids. However, the 33 regenerated plants tested were all diploid, indicating that only diploid cells were capable of regeneration in vitro.Abbreviations MS Murashige and Skoog (1962) basal salts with vitamins (100 mg1^–1 myo-inositol, 20 g1^–1 sucrose) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA N ⁶-benzyl-adenine - Kn kinetin - MSC MS + 13.6 M 2,4-D + 1.15 M Kn - MSR MS +6.7 M BA + 1.15 M Kn     

Asparagus somatic embryos: Production in suspension culture and conversion to plantlets on solidified medium as influenced by carbohydrate regime

Methods were developed for the production of somatic embryos of asparagus (Asparagus officinalis L.) in suspension culture and subsequent conversion to plantlets on solidified medium. Stem-derived callus that was subcultured twice on Murashige and Skoog (MS) medium + 0.54 M naphthaleneacetic acid (NAA) and 1.4 M 2-isopentenyladenine (2iP) was used to initiate suspension cultures. Six out of 15 such cell suspensions (MS medium with 54 to 107 M NAA) had a high embryogenic capacity. These cell suspensions consisted primarily of single elongated cells (about 90% of all single cells), embryogenic cell clusters (2571/ml), and globular translucent embryos (32/ml). The latter converted to plantlets within four weeks on embryo development medium (EDM), which was solidified MS medium containing 0.54 M NAA and 0.98 M 2iP. Suspension-derived embryos formed secondary globular embryos at high frequencies (251 to 258/g callus) when placed on EDM with a low carbohydrate (sucrose, glucose or fructose) level (2%). In contrast, EDM with a high carbohydrate level (10%) caused a reduction in the frequency of secondary embryos (30 to 85/g callus), while resulting in the promotion of embryo growth and conversion, 3.6 to 8.5 times higher than 2% carbohydrates. Transfer of globular somatic embryos from cell suspension to EDM with high carbohydrate levels (4 to 10%) for two weeks followed by transfer to EDM with a low carbohydrate level (2%) resulted in a 2 to 4 times higher conversion rate to plantlets than those that remained at the 4 to 10% levels.Abbreviations ANOVA analysis of variance - EDM embryo development medium - NAA naphthaleneacetic acid - MS Murashige and Skoog - RCBD randomized complete block design - 2iP 2-isopentenyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid