Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering. Received: 25 July 1998 / Revision received: 11 November 1996 / Accepted: 30 November 1996     

Embryogenesis and plant regeneration from callus cultures derived from unpollinated ovaries of Hyoscyamus muticus L.

Unpollinated ovaries of Hyoscyamus muticus L. (commonly known as Egyptian henbane) were cultured on Murashige and Skoog and Bourgin and Nitsch media supplemented with various growth hormones to study the organogenesis, embryogenesis and regeneration of plantlets. Embryogenesis was reported for callus grown on both media containing 0.05 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Differentiation of roots and shoots from the calli also occurred in these media. Albinism or chlorophyll deficiency and variation in ploidy level were observed among the ovary-derived plantlets. Received: 7 April 1997 / Revised received: 2 August 1997 / Accepted: 2 September 1997     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l^–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l^–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed. Received: 24 February 1997 / Revision received: 10 July 1997 / Accepted: 28 July 1997     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Plant regeneration in pigeonpea [Cajanus cajan (L.) Millsp.] by organogenesis

A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media fortified with 22.2 μm N⁶-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS) basal medium supplemented with 2.22 μm N⁶-benzylaminopurine and 0.54 μm α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may be useful for improving the crop through genetic manipulations. Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998     

Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb)

Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months. Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm⁻³ indole-3-butyric acid (IBA) and 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm⁻³) and ascorbic acid (100 mg dm⁻³) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Micropropagation of Gymea Lily (<Emphasis Type="Italic">Doryanthes excelsa</Emphasis> Corrêa) from New South Wales,Australia

The physiological effects of three auxins [indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d)] and two cytokinins [thidiazuron (TDZ) and N⁶-benzylaminopurine (NAA)] on in vitro morphogenesis of Doryanthes excelsa were measured. Longitudinal bud sections derived from immature inflorescences were used as a source of explants. Callus regeneration was observed at the highest frequencies (46.2%) when grown on media containing 50 μmol L^-1 NAA and 0.5 μmol L⁻¹ TDZ. Adventitious shoot organogenesis was observed at the highest frequency (56.8%) when grown on media containing 0.5 μmol L⁻¹ NAA and 50 μmol L⁻¹ TDZ. Regenerated shoots were rooted ex vitro after 6 weeks when dipped in a solution of 50 μmol L⁻¹ NAA or no plant growth regulators were applied.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Adventitious bud induction in Eucalyptus globulus Labill

Summary Adventitious buds and shoots of Eucalyptus globulus Labill. (Tasmanian Bluegum) have been regenerated from cotyledons and hypocotyls from mature embryos and seedlings. Adventitious buds, were induced at high frequency with 0.05 μM thidiazuron in combination with 0.2 μM 2,4-dichlorophenoxyacetic acid or 5 μM α-naphthaleneacetic acid. Culture of explants in the dark inhibited bud induction, but up to 86% of cotyledons, longitudinally split just prior to culture, produced adventitious buds, in the light. Development of buds into shoots occurred only at low frequency, after transfer to media containing N⁶-benzylaminopurine.     

In vitro propagation of Agave arizonica Gentry & Weber

Agave arizonica Gentry & Weber, an extremely rare and endangered species native to Arizona, was successfully propagated in vitro using modified Murashige and Skoog media. Adventitious shoots developed from callus which formed on bulbil explants grown in a medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid. These shoots proliferated by subculture in media supplemented with 44.4 μM 6-benzylaminopurine, and either 0.5 or 5.4 μM naphthaleneacetic acid. Rooting occurred on shoots transferred to a growth regulator free medium. Rooted plants transferred to potting soil could be established under greenhouse conditions following gradual acclimatization indoors.     

A standard procedure for the micropropagation of the neem tree (Azadirachta indica A. Juss)

Micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. Regardless of their origin, shoots were successfully produced by culturing leaf explants on Murashige and Skoog medium containing benzylaminopurine (1 mg l^–1), kinetin (0.8 mg l^–1) and adenine sulphate (6 mg l^–1) in complete darkness. These shoots were further multiplied on Murashige and Skoog medium containing benzylaminopurine (0.1 mg l^–1), kinetin (0.08 g l^–l) and adenine sulphate (0.6 mg l^–1). Within 32 weeks, 80 shoots could be produced from a single leaf explant (10 mm×10 mm). Fifty-five percent of these shoots rooted on Murashige and Skoog medium containing indolebutyric acid (1 mg l^–1) and all of these grew on transfer to soil. Received: 5 May 1996 / Revision received: 23 August 1996 / Accepted: 5 October 1996     

Effect of various growth regulators on shoot regeneration of sugarcane

Summary Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.     

Effect of plant growth regulators and basal media onin vitro shoot proliferation and rooting ofMyrtus communis L.

The influence of macronutrients and growth regulators on in vitro shoot proliferation and rooting of an East Spanish population ofMyrtus communis L. were studied. Preincubation of field material on a medium without mineral salts prevented the browning from phenolic exudates. For multiplication, nodal segments of 5 mm fromin vitro produced shoots were cultured on Murashige and Skoog (MS), Schenk and Hildebrandt (SH) and Heller (H) media (full strength or diluted to 1/2 or 1/4), with 6-benzylaminopurine (BAP) at concentrations 4.4, 13.3 and 22.2 ΜM or kinetin (K) at concentrations 4.7, 14.0 and 23.2 ΜM. The optimum shoot proliferation was on quarter-strength MS medium with 4.4 ΜM BAP, whereas the maximum number of nodal segments was produced on half-strength MS medium with 4.4 ΜM BAP. Rooting of shoots was obtained by adding 2.5 – 24.6 ΜM indole-3-butyric acid (IBA) and broad range of macronutrients; Lloyd and McCown (WPM) and Gresshoff and Doy (GD) media both full strength or diluted to 1/2 were optimum. No rooting was obtained in the presence of α-naphthaleneacetic acid (NAA). Acknowledgements: This study was supported by a grant from Conselleria de Cultura, Educació i Ciència de Ia Generalitat Valenciana. The authors are grateful to Man Cannen Perea for her helpful comments.     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron     

Regeneration of Salvia sclarea via organogenesis

Summary The present work provides a system for regeneration of clary sage, (Salvia sclarea L.) via organogenesis using plant tissue culture techniques in a multistage culturing medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (9.05–181.00 μM). A higher frequency of organogenic tissue initiation was obtained from immature zygotic embryo cotyledons (IZEC) 2–3 wk after pollination on the medium supplemented with 9.05 μM 2,4-D. The organogenic tissues were then proliferated on media containing both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA). Organogenic lines were established via selection, isolation and continuous subculture of organogenic tissues on a medium containing 22.19 μM BA and 2.85 μM IAA. Shoots were regenerated from both the proliferated tissues and IZEC, and propagated in the presence of IAA or α naphthaleneacetic acid (NAA), BA and gibberellic acid (GA₃). Although roots were induced from regenerated shoots on the media containing a low concentration of IAA, IBA (0.98 μM) in combination with desiccation of regenerated shoots with a stem ∼10 mm in length promoted more and stronger root formation. After the root system was well established (20 mm in length), the regenerated plants were transferred to soil in plastic pots for further growth and production of R1 seeds in the greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.