In this study we establish an efficient method for the regeneration for
via somatic embryogenesis. Immature cotyledons from siliques of 4–6 month-old plants were cultured on MS medium supplemented with plant growth regulators (BA — 6-benzylaminopurine; NAA — α-naphthaleneacetic acid; TDZ — 1-phelyl-3-(1,2,3-thiadiazol-5-yl) urea: 2,4 D-2,4-dichlorophenoxy-acetic acid). A high frequency of embryogenic callus was produced after two weeks in culture. The somatic embryos were obtained with a frequency of 10% of explants on MS medium supplemented with 1.34 μM NAA + 8.87 μM BA and 2.68 μM NAA + 17.74 μM BA within 3 weeks in culture. The alternative regime of MS medium supplemented with 1.34 μM NAA + 4.44 μM BA produced somatic embryos at a frequency of 38%.
Authors:Anna Resetár, Zita Demeter, Emese Ficsor, Andrea Balázs, Ágnes Mosolygó, Éva Szőke, S. Gonda, L. Papp, G. Surányi, and C. Máthé
In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L−1 or 2–10 mg L−1 NAA with 1 mg L−1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L−1 NAA and 1 mg L−1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.
Authors:M. Bobák, J. Šamaj, A. Blehová, M. Ovečka, A. Hlavačka, P. Illéš, and Z. Kutarňová
The possibility of plant regeneration via direct
somatic embryogenesis from leaf tissue of Drosera spathulata Labill. has been
studied, using MS medium supplemented with various concentration of NAA and
BAP. Scanning electron microscopy investigations in combination with
histological analysis verified direct somatic embryogenesis by formation of
globular, torpedo-shaped, heart-shaped and cotyledonary embryo like structures.
Optimal somatic embryo formation occurred when leaves were pre-treated with low
concentration (0.005 mg l-1) of 2,4-D within 6 h. Prolonged
influence of 2,4-D to 24-48 h led to the expression of morphological and
Authors:A. Bakrudeen, G. Subha Shanthi, T. Gouthaman, M. Kavitha, and M. Rao
An efficient protocol was standardized using axillary bud and shoot tip explants of Catharanthus roseus — an anticancer medicinal plant. The highest number of shoots (19.6 shoots / auxiliary node) was observed after 45 days of culture in the MS medium supplemented with NAA (4.0 mg l−1) + BA (4.0 mg l−1). Shoots were proliferated and elongated in the same medium. High frequency of rooting (82.5%) was obtained in half strength MS + IBA (4.0) from axillary bud derived shoots. The rooted plantlets were successfully established in soil.
Among the different concentrations of Thidiazuron (TDZ) and between the two media Gamborg (B5) and Murashige and Skoog (MS), the highest frequency of shoot formation could be seen in the MS medium with TDZ concentration of 4.54 μM. Among the different concentrations of Naphtalene acetic acid (NAA) and Benzyl adenine (BA) in the two aforementioned media, the maximum proliferation and rooting of saffron shoots were obtained in a B5 medium containing 2.22 μM NAA and 2.68 μM BA. Peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), esterase (EST) and polyphenoloxidase (PPO) measurements proved that all the enzymes had a similar pattern of changes, according to which their concentrations increased in the first stages of development and then decreased. The same pattern was observed for polyphenoloxidase in a B5 medium while in the MS medium a reverse pattern was observed. The enzyme concentration decreased and then increased during shoot formation. The results show the principal role of antioxidant enzymes in the complicated process of organogenesis.
The effect of exogenous application of growth regulators (IAA, NAA, GA3 and BAP) on xylogenesis has been investigated in actively growing shoots of Leucaena leucocephala by anatomical methods. Auxins are (both IAA and NAA) found to be the key regulators of differentiation of narrow lumen vessels and tracheids. These elements were derived not only from fusiform cambial cells, but also from axial parenchyma through transdifferentiation. The exogenous feeding of GA3 induced differentiation of gelatinous fibres, deformed vessels, elongated thin walled fibres and a high fibre/vessel ratio. BAP application decreased the cambial activity and cell differentiation process resulting in development of thick walled fibres and vessel elements, and also delayed lignification of cell walls. The application of combination of hormones showed both synergistic and antagonistic nature of interaction among different hormones. Combination of GA3 and auxins induced cambial cell division and relatively fast differentiation of xylem, while increasing concentrations of GA3 over auxins induced differentiation of gelatinous fibres. The combination of high concentration of BAP either with auxins or with GA3 slowed down cambial activity and xylem differentiation. Moderate concentrations of BAP and GA3 induced gelatinous fibres with thick gelatinous layer indicating the synergistic effect of both hormones.
Poddar K, Vishoni RK, Kothari SL (1997) Plant regeneration from embryogenic callus of finger millet
(L.) Gaertn. on high concentration of NH
as a replacement of NAA in the medium. Plant Sci 129
Authors:Tingzhang Hu, Hua Zeng, Zaigang Chen, Xiaoyun Huang, Yongwei Yang, and Guixue Wang
Using uniform random design optimization and the mathematical model equation we optimized the regeneration tissue culture system of the chilli pepper. An efficient and detailed plant reproducible protocol in vitro has been established using different explants and induction media for three chilli pepper cultivars. The result displayed that the seedlings at the curved hypocotyl stage were the best choice to prepare for explants, the genotype of explants affected shoot buds induction frequency and number of shoot buds per explant, and the cotyledon explant was more responsive than hypocotyl explant. The optimal media for maximum shoot initiation and regeneration and the optimal elongation medium were obtained. For Capsicum annuum var. annuum (cv. Xinsu), Capsicum annuum var. annuum (cv. Neimengchifeng) and Capsicum frutescens (cv. Xingfu), the induction rates were 99.17%, 97.50 and 96.11%, respectively; the elongation rates of shoot buds were 86.67%, 85.19% and 82.96%, respectively. The MS medium with 0.57 μM IAA and 0.69 μM NAA is the best choice for root induction. The frequency of their root emergence was 95.00–98.33%. Regenerated chilli peppers were successfully acclimatized and cultivated with 100% survival. This work will help to improve multiplication process and the genotype of chilli pepper, and may have commercial impact.
Rapid differentiation of multiple shoots was observed in 94% of nodal explants of one year old Nyctanthes arbor-tristis L. plants. Shoot bud induction and multiplication took place on Murashige and Skoog (MS) medium supplemented with two cytokinins, i.e. Benzyladenine (BA) or Kinetin (Kn) either alone or in combination with different auxins, indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Between different media, pH levels and growth regulators tried, the optimum condition for maximum regenerative response was obtained on MS + Kn (2.5 μM) + N AA (0.5 μM) media at 5.8 pH, forming cultures with 23.26 ± 0.89 number of shoots and 6.36 ± 0.80 cm shoot length after 8 weeks of culture. Histological sections confirmed the formation of multiple buds from nodal explants. Rooting was achieved ex vitro by dipping the basal ends of microshoots in 200 μM IBA for 30 min followed by their transplantation in sterile soilrite. The plantlets with well-developed shoot and root system were successfully established in garden soil and grown outside in a greenhouse with a 80% survival rate.