Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.)

Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 μM 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyacetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 μM gibberellic acid and 1 μM indole-3-butyric acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics.     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

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 from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic 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.     

Regeneration via organogenesis in callus cultures of Argyrolobium roseum

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm⁻³ benzylaminopurine (BAP) + 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Subsequent transfer of 5 mm² callus pieces to MS medium supplemented with BAP (0.5 mg dm⁻³) alone or in combination with IAA (0.25 mg dm⁻³) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm⁻³) + IAA (0.25 mg dm⁻³) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.     

Improved plant regeneration from maize callus cultures using 6-benzylaminopurine

A new protocol for regenerating plants from cultured type I callus of the maize (Zea mays L.) inbred Pa91 includes growing the callus on medium containing 3.5 mg/l (15.5 M) of the cytokinin 6-benzylaminopurine (6BA) for 3 to 6 d and then moving the callus to medium containing no growth regulators (H medium) for an additional 15 to 21 d, where the plants actually develop. The number of plants regenerated from the 6BA treated callus was 113% to 148% greater than the number of plants produced from callus placed directly on H medium. This increased plant regeneration induced by 6BA seemed to maximize the number of plants regenerated from a gram of callus and was slightly affected by callus age or prior treatment of callus with AgNO₃. Exposure to 6BA for 9 d greatly reduced shoot and root development, and longer exposures totally prevented root formation. This inhibition of root formation could be reversed only slightly by naphthaleneacetic acid. The data indicate that high concentrations of 6BA are effective for increasing plant regeneration from maize callus cultures when short exposure times are used. This procedure has also been effective for regenerating many plants from the inbreds H99 and Mo17.Abbreviations 6BA 6-Benzylaminopurine - IAA indole-3-acetic acid - NAA Naphthaleneacetic Acid - 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - GA₃ Gibberellic acid - gfw gram fresh weight     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.)

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B₅ vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B₅ medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B₅ medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-Benzyl-aminopurine - IAA IndoIe-3-acetic acid - IBA Indole-3-butyric acid - NAA 1-Naphthalene acetic acid - Kinetin 6-furfuryl aminopurine - Zeatin 6-(4-hydroxy-3-methylbut-2-enylamino)-purine     

Improved plant regeneration from barley callus cultures by increased copper levels

Incorporation of cupric sulfate into callus induction, maintenance, and regeneration media significantly enhanced plant regeneration from callus cultures of barley (Hordeum vulgare L.) immature embryos. Embryos from the cultivars Hector and Excel were cultured on MS medium containing 0, 0.1 (MS level), 0.5, 1.0, 5.0, 10.0, 50.0, or 100.0 M cupric sulfate. Plants were regenerated beginning at 8 weeks and continuing through 36 weeks. For Hector, medium containing 50 M copper regenerated significantly more plants than any other medium, with an average of 17 plants per embryo. In comparison, medium with MS copper levels (0.1 M) regenerated only 5 plants per embryo. For Excel, medium containing 5.0 M copper was the best, regenerating 1.4 plants per embryo. No Excel regenerants were obtained on medium with MS copper levels. Increased copper levels also increased the percentage of embryos that regenerated at least one plant, in both cultivars. The results indicate that MS copper levels are not optimized for barley callus cultures, and that improved plant regeneration can be obtained at higher copper concentrations.Abbreviations MS Murashige & Skoog (1962) - 2,4-d 2,4-dichlorophenoxyacetic acid The US Government's right to retain a non-exclusive royalty-free license on and to any copyright is acknowledged     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Efficient protocol for in vitro direct plant regeneration in chickpea Cicer arietinum L

An efficient plant regeneration system was developed for two important Indian chickpea cultivars, C-235 and HC-1. Immature cotyledons (7-8 mm) directly formed shoots without an intervening callus phase on MS medium containing B5 vitamins, BAP (2.0 mg/l), IBA (0.125 mg/l), AgNO3 (1.69 mg/l) and phytagel (2.5 g/l). The regenerated shoots had normal morphology and were successfully rooted in half strength MS medium under partial dark conditions. Regenerated plants were transferred to potted soil. However, the survival rate of pot house transferred plants was 17.6 per cent.     

Efficient plant regeneration in vitro from red leaf beet via organogenesis

An efficient system for in vitro regeneration of red leaf beet, a variety of leaf beet (Beta vulgaris L. var cicla L.) generally used to decorate parterre and to prepare betacyanin, was developed for the first time in the present study. Shoot tip and petiole explants from the sterile seedlings, precultured on Murashige and Skoog (MS) medium with 15 mg/l 6-benzyladenine (BA) and 3% sucrose at 16 °C for 30 days, could form 81.02 and 17.33% translucent nodular (TN) calli, respectively. All TN calli were able to differentiate into adventitious shoots under the same culture conditions. Each explant with TN callus from the shoot tip and petiole could generate 8.65 shoots on average. It was found that both preculture of sterile seedlings and culture of explants at low temperature (16 °C) were vital for TN callus induction and adventitious bud formation of red leaf beet. The best condition for rooting was 0.5-strength MS medium with 10 g/l sucrose. After being transplanted into soil, plantlets grew well and could flower and bear fruits. Histological observation revealed that TN callus was derived from the cells of vascular tissue of the petiole and that adventitious shoots were formed through organogenesis. The factors influencing in vitro micropropagation are also discussed. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 603–608. This text was submitted by the autors in English.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Somatic embryogenesis and plant regeneration from callus cultures of Bunium persicum Boiss

Callus was obtained from mericarps of Bunium persicum Boiss. on MS medium supplemented with 2.0 mg/1 2,4-D and 4.0 mg/1 Kn. Small white clumps of compactly packed cells developed on the callus on a medium containing 1.0 mg/1 2,4-D and 0 mg/1 Kn. These cell clumps differentiated numerous globular embryos on the same medium. Embryo maturation and germination was achieved on the basal as well as on 1 mg/1 Kn supplemented medium. All regenerated plants examined were normal diploids with 2n=14.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

Plant regeneration via somatic embryogenesis in chick pea (Cicer arietinum L.)

Five genotypes of chickpea (Cicer arietinum L.) PG1, PG5, PG12, N59 and C235 were evaluated for induction of somatic embryogenesis. Somatic embryogenesis was induced from immature cotyledons of genotypes PG12 and C235 and immature embryo axes of genotypes PG5, PG12 and C235. Genotypes N59 and PG1 showed no response. The maximum frequency of globular embryo formation occurred in cotyledonary segments on MS medium with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Further embryo development was achieved only in somatic embryos derived from cotyledonary segments of genotype PG12. Globular-stage embryos derived from immature embryo axes of PG5, C235, PG12, and cotyledonary segments of C235 dedifferentiated and formed callus. The cotyledonary stage embryos of genotype PG12 germinated on half-strength MS medium supplemented with 1 mg/l zeatin. The regenerated plants were transferred to soil and grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzylamino purine - CM coconut milk - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog medium (1962) - NAA 1-napthaleneacetic acid - Picloram 4 amino-3,5,6-trichloropicolinic acid - 2,4,5-T 2,4,5-trichlorophenoxy-acetic acid - zeatin (6-[4-Hydroxy-3-methyl-2-butenylamino] purine)     

Successful plant regeneration from callus cultures of Centella asiatica (Linn.) Urban

A successful procedure was established for in vitro plant regeneration from callus derived from stem and leaf explants of Centella asiatica on semisolid modified Murashige and Skoog's [7] medium supplemented with 2.0 mg L³ kinetin and 4.0 mg L³ a-naphthaleneacetic acid. The rate of shoot-bud regeneration was the highest (42.8 and 54.3 shoots/culture in stem and leaf derived callus respectively) after 4 weeks of subculture on 4.0 mg L³ 6-benzyladenine, 2.0 mg L³ Kn, 0.25 mg L³ a-naphthaleneacetic acid and 20 mg L³ adenine sulfate. Differentiated shoots rooted within 11 days in 1/2 strength MS basal salts supplemented with 0.5 mg L³ indole-3-acetic acid and 2% (w/v) sucrose. About 85% of rooted plantlets were acclimatized and transferred to the greenhouse.     

A study on the effect of genotypes,plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus

We have developed an efficient protocol for callus induction and plant regeneration in three elite soybean cultivars (Williams 82, Loda and Newton). The technique is most novel in that the shoot buds developed from the nodal callus. Callus induction and subsequent shoot bud differentiation were achieved from the proximal end of cotyledonary explants on modified Murashige and Skoog (MS) media containing 2.26 M 2,4-dichlorophenoxy-acetic acid (2,4-D) and 8.8 M benzyladenine (BAP), respectively. Varying the carbon source optimized the regeneration system further. Among the various carbon sources tested, sorbitol was found to be the best for callus induction and maltose for plant regeneration.     

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium Communicated by S. Gleddie