Cut Ends Of Explants Research Articles

Exogenous implications of silver nitrate on direct and indirect somatic embryogenesis and germination of cold stored synseeds of Vanilla planifolia Jacks. ex Andrews

• Silver nitrate the medium improved somatic embryogenic efficiency in Vanilla planifolia . • Medium containing cytokinins with 2.5 mg L −1 silver nitrate induced maximum number of somatic embryos. • Developmental transition from globular to cotyledonary stages of somatic embryos were confirmed by anatomical studies. • Silver nitrate favored proliferation and rooting of shoots, and improved the survival rate of V. planifolia plantlets. Vanilla planifolia – a natural source of vanillin is the world's highest-valued spice crop with endangered status and the only hand-pollinated crop globally. The present study evaluates the effect of exogenous silver nitrate (AgNO 3 ) on effective direct and indirect somatic embryogenesis in V. planifolia for the first time. The direct and indirect somatic embryogenic capacity of leaf explants were studied using Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP), kinetin (KN), 2,4-Dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and α-naphthalene acetic acid (NAA) alone and in combinations. Additionally, the efficacy of silver nitrate (SN) in promoting somatic embryogenesis was tested with optimized growth regulators. Non-embryogenic callus was obtained from the explants when cultured on 3.0 mg L −1 2,4-D. The embryogenic potential was developed while culturing the non-embryogenic callus on 1.0 mg L −1 each of BAP and KN combined with 2.5 mg L −1 SN. On the other hand, direct somatic embryogenesis (92.0%) was obtained when culturing the explants directly on MS medium containing 1.0 mg L −1 each of BAP and KN combined with 2.5 mg L −1 SN, and a maximum of 30.0 somatic embryos (SEs) were induced per explant. Microscopic analysis revealed that SEs were originated externally as cell division initiated on the leaf surface and cut ends of explants, and have no vascular connections with the explant. Actively dividing embryonic cells possessed dense cytoplasm with prominent nuclei. The mature SEs were encapsulated and stored at - 4 °C for one year. The synseeds showed maximum germination and proliferation (17.2 shoots per SE) on MS medium containing 0.5 mg L −1 BAP, 0.25 mg L −1 KN, and 2.5 mg L −1 SN. The results indicated that AgNO 3 in the medium promoted somatic embryogenesis and plantlet regeneration in V. planifolia .

The effect of arabinogalactan proteins on regeneration potential of juvenile citrus explants used for genetic transformation by Agrobacterium tumefaciens

A possible role of arabinogalactan proteins in control of shoot regeneration from stem explants of two citrus cultivars, Carrizo citrange and ‘Duncan’ grapefruit, was investigated. Treatment of explants with (β-d-Glc)3 Yariv phenylglycoside, able to bind specifically to AGPs, led to a decrease of cumulative regeneration potential of both Carrizo citrange and ‘Duncan’ grapefruit. For Carrizo, lower cumulative regeneration potential on (β-d-Glc)3 Yariv phenylglycoside-treated explants was the result of both lower number of shoots on the explants that had shoots (explant regeneration potential) and decreased percentage of explants with shoots. In the case of ‘Duncan’, treatment with (β-d-Glc)3 Yariv phenylglycoside reduced cumulative regeneration potential only by lowering the percentage of explants with shoots, but it did not affect the number of shoots on the explants with shoots. Citrus explants treated with (α-d-Man)3 Yariv phenylglycoside, which does not bind AGPs, responded similarly to untreated explants. Transformability of cells on the cut ends of explants was also lower for both cultivars following the treatment of explants with (β-d-Glc)3 Yariv phenylglycoside. Our data suggest that arabinogalactan proteins play important role in processes controlling differentiation and genetic transformation of citrus cells by Agrobacterium.

EFFECTS OF INORGANIC NITROGEN SOURSE AND NH+4 :NO-3 RATIO ON PROLIFERATION OF DOG ROSE (ROSA CANINA)

Dog rose (Rosa canina) is one of the most important ornamental and medicinal plants which are used as rootstock for ornamental roses as Rosa hybrida and Rosa floribunda. The effects of different concentrations of NH4NO3 (10-15 and 20 mM), KNO3 (15-20 and 25 mM) and NH4:NO3ratios (10:25, 10:30, 10:35, 15:30, 15:35, 15:40, 20:35 and 20:45) was investigated on proliferation stage of dog rose cultivated on Ms media with total nitrogen levels (35; 40, 45, 50, 55, 60 and 65 mM). The relative amounts of NH4+ and NO3- ions influenced the tested parameters. The highest shoot length and most node numbers were obtained with 20:45 NH4+:NO3- ratios. Chlorosis leaf number was less with 15:35 and 20:45 NH4+:NO3- ratios. When NO3- was higher than NH4+, shoot length and node numbers were produced more and chlorosis leaf number was decreased. This study also demonstrates that with increasing nitrogen levels, the growth of shoots and node numbers were more but leaf chlorosis was decreased, therefore proliferation was better. The percentage of axillary shoot was affected by NH4NO3 concentrations and it was highest in 20 mM concentration, but with increasing KNO3, the leaf necrotic and feeble callusing percentage at the cut ends of explants were more and lower in row. Key words: Dog rose, in vitro culture, MS nitrogen source, NH4+: NO3- ratio, proliferation.

Somatic embryogenesis, organogenesis, and regeneration from leaf callus culture of Decalepis hamiltonii Wight & Arn., an endangered shrub

A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N6-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA3) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.

Establishment of Plantlets and Evaluation of Differentiated Roots for Alkaloids in Rauwolfia serpentha

Different explants of Rauwolfia serpentina were tested for their capacity and root differentiation. MS sedium containing 2.0 mg I-1 BAP or 1.5 mg I-1 BAP with 0.5 mg I-* NAA gave the best shoot proliferation from shoot apices (84.12%). Multiple shoots subcultured on the same media gave higher number of shoots (6–9). Callus formed at the cut ends of explants produced shoots when subcultured on the above media. Rooting was achieved after transfer of shoots either to hormone free or half strength or full strength MS medium with different concentrations of IAA (0.5,1.0 mg I-1 and IBA (0.5,1.0 mg I-1).The complete regenerated plantlets have been successfully transferred to earthen pots in green house. Maximum root differentiation from leaves, stem and nodal cuttings derived calli was obtained on MS medium supplemented with NAA (2.0 mg I-1) and BAP (1.5 mg I-1). Identification of reserpine and other alkaloids from differentiated roots holds an interesting alternative for controlled production of alkaloids from this endangered, overexploited medicinal plant species.

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 α-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 μM) and BA (10.0 μM) 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.

IN VITRO REGENERATION OF SHOOTS ON STEM EXPLANTS OF HAPLOID AND DIPLOID FLAX (LINUM USITATISSIMUM)

Shoots were regenerated from stem explants of haploid (2n = x = 15) and diploid (2n = 2x = 30) flax (Linum usitatissimum L. cv. Rocket 4) on a modified Murashige and Skoog (1962) medium supplemented with benzyladenine and napthaleneacetic acid. Segments of 15 mm lengths, cultured from donor plants at early flowering, regenerated 6.1 (mean) shoots per mm which were epidermal in origin and retained the chromosome number of the donor. The rate and frequency of shoot regeneration was greater at 30 °C as compared to 25 °C. Callus originated from the cut ends of explants and was more vigorous at the lower temperature. Shoots of callus origin were infrequent and had the doubled chromosome complement (2n = 2x = 30 and 2n = 4x = 60).

The Effects of Varying Oxygen Concentrations on Osteogenesis and Embryonic Cartilage in Vitro

1. An improved method of organ culture, employing embryonic chick tibiae, demonstrated consistent patterns of osteogenesis. With all other factors constant, the amount of bone and collagen formed or destroyed could be controlled solely by altering the amount of oxygen available to the explants from the gaseous phase of the culture. 2. Maximum osteogenesis was observed with an oxygen concentration of 35 per cent. Collagen-fiber formation and osteogenesis were suppressed by lowering the oxygen concentration to 5 per cent and, to a lesser degree, by raising it to 95 per cent. High oxygen concentrations also appeared to cause osteoclasia. 3. Cartilage matrix and cells near the cut ends of explants also demonstrated significant alterations under various oxygen concentrations. High oxygen concentrations (95 per cent) caused extensive chondroclasia. Intermediate oxygen concentrations (20 to 65 per cent) produced numerous large collagen fibers in and about the lacunae of degenerating chondrocytes, while other chondrocytes, undergoing alterations of morphology, came to resemble osteoblasts. Metachromasia of cartilage matrix was lost. Low (5 per cent) and high (95 per cent) oxygen concentrations did not produce similar collagen fiber production, alterations of chondrocyte morphology, or loss of matrix metachromasia.