Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.
Karim H. Al-Juboory, J. Al-Naimi, L.K. Al-Amiry, R. Shibli, and R.M. Skirvin
Monica E. Figueroa-Cabanas and Dennis P. Stimart
Direct shoot organogenesis (DSO) on Antirrhinum majus L. (snapdragon) was evaluated in vitro to determine the inheritance of genes conditioning this response. One-centimeter-long hypocotyls excised from 2-week-old seedlings started in vitro in the dark on Murashige and Skoog medium served as explants. Optimal conditions for DSO on explants included hypocotyl excision from 10-day-old seedlings, 2.22 μmol BA in the culture medium, and a 21-day culture duration. An adventitious shoot was counted once it developed a stem terminated by at least one leaf appearing to have originated from an apical meristem. Seven populations were evaluated for DSO: parent 1 (P1) with lowest DSO (0.3 shoots); parent 2 (P2) with highest DSO (13.9 shoots); F1 (P1 × P2); F1 (P2 × P1); F2 (self-pollination of F1); P1 × [P1 × P2]; and P2 × [P1 × P2]. P1 and P2 were chosen as parents based on DSO counts being lowest and highest, respectively, of inbreds evaluated. DSO appears to be a trait under nuclear genetic control. High DSO appears to be dominant over low DSO. The trait appears to be simply inherited through one or two genes.
Eugenio Pérez-Molphe-Balch and Neftalí Ochoa-Alejo
An efficient system for in vitro regeneration by organogenesis starting from internodal stem segments from seedlings of Mexican lime (Citrus aurantifolia Christm. Swing.) and mandarin (C. reticulata Blanco cv. Monica) was developed. The best results were obtained when the wounded edges of internodal stem segments cut longitudinally were placed downward on the surface of the culture medium. The optimal culture medium from both species was Murashige and Skoog with vitamins from B5 medium, 5% sucrose, 33.3 μm BA and 5.4 μm NAA. The best response was obtained when the segments were incubated at 25 ± 2 °C for 21 d in darkness, followed by 29 d on a 16/8-h light/dark cycle (fluorescent light, 54 μmol·m-2·s-1). The best regeneration system tested allowed the attainment of adventitious shoots from 96% and 88% of the explants in Mexican lime and mandarin, respectively. In Mexican lime an average of 7.8 well-differentiated shoots per explant was obtained, and in mandarin the yield was 5.1. Rooting of 70% of the shoots was achieved in culture medium with NAA (2.7–5.4 μm) or IBA (2.5–4.9 μm). Of the rooted plants, 85% adapted well to soil conditions. Chemical names used: 6-benzylaminopurine (BA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA).
Martín Mata-Rosas, Ángel Jiménez-Rodríguez, and Victor M. Chávez-Avila
Plants of Magnolia dealbata were regenerated from zygotic embryos through somatic embryogenesis and direct organogenesis. Medium and incubation conditions were determinating factors for the development of morphogenetic responses. Photoperiodic exposure was a limiting factor in the general development of the explants, and incubation in darkness allowed their development. The highest formation of shoots per responding explant were obtained on woody plant (WP) medium supplemented with 13.3 μM or 22.2 μM 6-benzylaminopurine (BA) in combination with 2.26 μM or in absence of 2,4-dichlorophenoxyacetic acid (2,4-D) from which 2.5 shoots per explant were induced. Subcultures on WP medium, supplemented with polyvinylpyrrolidone (PUP) 40,000 1 g·L–1) avoided necrosis of explants. Somatic embryos were formed in 85% of explants cultivated on WP medium with 2,4-D (2.3 μM or 4.5 μM); 20% induced indirect embryogenesis and 65% formed direct somatic embryogenesis. The plants were transferred to soil to acclimatize under greenhouse conditions, achieving 90% survival. Somatic embryo conversion to plantlets was obtained with subculture on WP basal medium without growth regulators. In vitro culture can play a key role in the propagation and conservation of this endangered species.
Hak-Tae Lim, Eun-Ae Lee, and Won-Bae Kim
This study was conducted to investigate the possibility of obtaining plantlets via somatic embryogenesis and organogenesis as means of in vitro mass propagation in Allium victorialis var. platyphyllum Makino, one of the most popular wild vegetable plants in Korea. Shoots formed directly when bulb explants of A. victorialis were cultured on MS medium containing 0.2 mg·L–1 NAA and 2.0 mg·L–1 zeatin under 16 hours (light)/8 hours (dark) illumination. The use of leaf and shoot tip explants was not successful, largely due to explant senescence in the present of plant growth regulators. Embryogenic calli were obtained from the bulb explants of A. victorialis on MS medium supplemented with 0.2 mg·L–1 NAA, 0.2 mg·L–1 BAP, and 1.0 mg·L–1 picloram after 4–5 weeks of culture in the dark at 27°C. Upon transfer to shoot-induced MS medium containing 0.2 mg·L–1 NAA and 2.0 mg·L–1 zeatin, embryogenic calli gave rise to numerous somatic embryos, which subsequently developed into multiple shoots after 3 months of culture under 16 hours(light)/8 hours (dark) illumination. For root induction, regenerated shoots were transferred to MS medium added with 1.0 mg·L–1 NAA. Regenerants with well-developed roots were potted in an artificial soil mixture of vermiculite (1) and perlite (1).
Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson
Plant regeneration ability of ginseng (Panax ginseng) via organogenesis was studied morphologically and anatomically. Compact callus was introduced from four different types of explants—leaf, petiole, flower stalk, and root—of in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (1.0 mg·L–1) and kinetin (0.1 mg·L–1) were conditioned for 2 weeks on the same medium. These calli differentiated into adventitious shoots when cultured on half-strength MS basal medium plus kinetin at 1.0 mg·L–1 and STS at 2.5 mg·L–1. An addition of GA3 (1.0 mg·L–1) and BA (1.0 mg·L–1) to MS basal medium, however, induced high-frequency in vitro flowering (86.1%) and multiple shoot budding, which affected the normal, complete development of plantlets. Plantlets with well-developed root systems were obtained 6 weeks after regenerated shoots had been transplanted to half-strength MS20 medium containing IBA at 0.25 mg·L–1. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all of the regenerants were typically diploids as were the mothers plants, indicating that nuclear DNA content remained stable during cell differentiation.
Hak Tae Lim, Y.S. You, and E.J. Park
In order to regenerate explants of Brassica campestris ssp. pekinensis, known to be one of the most difficult crops to regenerate via organogenesis, three different explants, cotyledon, hypocotyl, and leaf, were cultured on MS basal medium supplemented with several plant growth regulators. In the medium containing NAA at 0.5 mg/L and BAP at 3.0 mg/L, the shoot regeneration, when hypocotyl was used as explant, was found to be quite effective. In the case of cotyledon, the most suitable combination of plant growth regulators was NAA at 1.0 mg/L and BAP at 3.0 mg/L. Treatment of AgNO3 (1.0 mg/L) for shoot regeneration gave positive results in general. Zeatin at 2.0 mg/L was very effective in shoot induction of leaf explant, especially when combined with BAP at 2.0 mg/L, NAA at 1.0 mg/L, and AgNO3 at 0.5 mg/L. A system to produce transgenic plants in Brassica spp. has also been developed using hypocotyl and cotyledonary-petiole segments and shoot-tips. An explants from 4-day-old seedlings were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimaeric bacterial gene encoding hygromycin and kanamycin resistance, along with other genes of interests. The explants were co-cultured for 2 to 6 days before transfer to hygromycin and kanamycin selection media. Shoots regenerated directly from the explants in 1 to 4 weeks and were excised, transferred to shoot elongation medium, rooted in root induction medium, and planted in soil. Genetic transformation was confirmed by kanamycin or hygromycin resistance, GUS activities, and Southern blotting.
L. Xu, G.F. Liu, and M.Z. Bao
m 1-naphthalene acetic acid (NAA). However, results were not satisfactory. Therefore, our objective was to develop a regeneration system from leaf explants of L. formosana via organogenesis induced by TDZ, which could be applicable for
Mohammed Elsayed El-Mahrouk, Mossad K. Maamoun, Antar Nasr EL-Banna, Soliman A. Omran, Yaser Hassan Dewir, and Salah El-Hendawy
·L −1 2,4-D after 8 weeks, ( E ) elongation of embryos on Murashige and Skoog (MS) medium without plant growth regulator (PGR), and ( F ) elongation of embryo-like structures on MS medium without PGR (bar = 1 cm). Organogenesis of responded ovules
Yuanyuan Miao, Qiaosheng Guo, Zaibiao Zhu, Xiaohua Yang, Changlin Wang, Yuan Sun, and Li Liu
) Tulipa edulis with elongated stolon; ( B ) T. edulis stolon developed into a new bulb; and ( C , D , and E ) T. edulis stolon formation at the initial, middle, and later stages. Stolon formation is a process of organogenesis, which is defined as