Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

Effect of medium osmotic potential on callus induction and shoot regeneration in flax anther culture

Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is the prerequisite for the application of doubled haploid technology in a practical breeding program. Pre-culture of anthers on a medium containing 15% sucrose for 2–7 days before transfer to the same medium containing 6% sucrose for a total of 28 days culture period significantly increased shoot regeneration for all four genotypes evaluated. Moreover, pre-culture of anthers on medium containing 15% sucrose for 2–7 days was sufficient to dramatically reduce the frequency of shoot regeneration from somatic tissues and thereby to increase the frequency of microspore-derived plants in flax anther culture. Furthermore, replacing 15% sucrose with 6% sucrose and 9% polyethylene glycol (PEG), or 3% sucrose and 12% PEG, in pre-culture medium did not significantly affect callus induction and shoot regeneration. The results indicate that sucrose may act as carbon/energy source as well as an osmotic regulator in flax anther culture. Sucrose as an osmotic regulator may be replaced by a non-metabolizable osmoticum: PEG. The implication of this study in flax anther culture and breeding is discussed.     

影响油菜下胚轴外植体芽高频率再生的因素

以芥菜型油菜DB3 的下胚轴为外植体 ,分析了苗龄、激素、AgNO3 对油菜外植体的高频率再生的影响。结果发现 ,3～ 5d的苗用于诱导愈伤组织较佳 ,6 BA 1 .5mg/L +NAA 0 .1mg/L的组合对下胚轴芽的分化较好 ,AgNO3 可提高下胚轴芽的分化率     

Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.     

Plant regeneration from shoot tips and callus of papaya

Summary Two methods of in vitro culture were employed to regenerate papaya plants. One involved regeneration of plants from callus and the other, production of multiple plants from single shoot-tip explants. Callus was induced from stem sections of papaya seedlings in a medium containing 1 mg per 1 NAA and 0.1 mg per 1 kinetin. The callus regenerated shoots and/or embryoids when transferred to a medium of lower auxin, 0 to 0.05 mg per 1 IAA, and higher cytokinin, 1 to 2 mg per 1 kinetin Multiple shoots were produced when the excised shoot-tip explants were cultured in a medium supplemented with 0.05 mg per 1 IAA and either 5 mg per 1 kinetin or 0.5 to 1.0 mg per 1 benzyladenine. Root formation of the shoots or embryoids that derived from callus or shoot tips occurred in a medium containing 5 mg per 1 IAA and in a light intensity of 3000 to 4000 Ix. The rooted plants could be established in soil and under standard greenhouse conditions after they had been acclimated by initially growing them in moist vermiculite contained in polyethylene-covered pots. This research was supported by the National Science Council, Republic of China.     

Transcriptome profiling reveals cytokinin promoted callus regeneration in Brassica juncea

Plant Cell, Tissue and Organ Culture (PCTOC) - Plant hormones not only play important roles in regulating plant growth and development, but they also promote cell dedifferentiation and...     

Improved shoot regeneration system through leaf derived callus and nodule culture of Sansevieria cylindrica

Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 μM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5μM 6-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 μM BA plus 1 μM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 μM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

Optimization of callus culture and shoot multiplication ofAsparagus densiflorus

The effects of different growth regulators on induction and growth of callus ofAsparagus densiflorus cv. Sprengeri were studied. Calluses grew more rapidly on Murashige and Skoog basal medium supplemented with 5.4 μM p-chlorophenoxyacetic acid (pCPA) and 4.4 μM 6-benzylaminopurine (BA) (medium 1) as compared to the same medium with 11.3 μM 2,4-dichlorophenoxyacetic acid (2,4-d) and 4.6 μM kinetin (medium 2). Calluses on medium 1 were soft and friable, whereas, compact, hard calluses originated on medium 2. Different concentrations and combinations of BA and/or kinetin were also used to study their effects on shoot regeneration. Kinetin was found to be less effective than BA in the initiation of shoots (1.8 shoots/callus). High numbers of shoots were produced in the presence of 0.4 μM BA alone (3.3 shoots/callus). The addition of ancymidol (5 μM) in MS with 0.4 μM BA enhanced multiplication of shoots (9.8 shoots/explant) and also produced well-developed crowns.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell

 Adventitious shoot buds were induced from leaf and stem explants of Bacopa monnieri on Murashige and Skoog medium supplemented with benzyladenine or kinetin. The source of the explants as well as different gelling agents in the medium were found to influence shoot induction and eventual shoot growth. The best response was obtained in leaf explants taken from shoot cultures grown in medium supplemented with 2 μM benzyladenine and gelled with 0.2% gelrite. A transverse section of the leaf explant incubated in this medium showed several shoot primordia emerging from the leaf surface. This system exhibited a potential for repeated harvesting of the shoots from the original leaf explant as the latter continued to expand and regenerate new shoots, upon repeated periodical subculturing onto fresh medium. However, the callusing response of the plant was very low. Qualitative TLC studies of the regenerated shoots revealed a phytochemical profile similar to that of the field grown-plants. Received: 20 March 1998 / Revision received: 1 December 1998 / Accepted: 12 December 1998     

Plant regeneration from cocoyam callus derived from shoot tips and petioles

The effects of various growth regulators on morphogenesis from cocoyam tissues (Xanthosoma sagittifolium) were investigated. Calluses were initiated from shoot tip and petiole explants and proliferated on medium containing 1.36 μM dicamba. Callus production was significantly greater from petioles than from shoot tips. Thidiazuron (0.045 μM) enhanced callus production when dicamba (13.5 μM) was used, and was more favorable to petioles than shoot tips. Friable shoot tip callus was subcultured into liquid media containing either 1.36 μM dicamba alone, 1.35 μM 2,4-D + 0.46 μM kinetin or 1.36 μM dicamba + 0.46 μM kinetin to induce adventive regeneration. Tissues producing single or aggregated shoot buds were subcultured into media containing 0, 0.049 and 0.49 μM 2-isopentenyladenine where bud multiplication and shoot regeneration were observed. Bud aggregates were formed from callus in liquid cultures containing 1.36 μM dicamba, 1.36 μM dicamba + 0.46 μM kinetin or 1.35 μM 2,4-D + 0.46 μM kinetin. Shoot bud clumps which remained green produced shoots, daughter buds, and plantlets in stationary and agitated liquid media containing 0, 0.049 and 0.49 μM 2iP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tropane alkaloid production by shoot culture of Duboisia myoporoides R. Br

This work demonstrates the presence of hyoscyamine and scopolamine at different stages of shoot regeneration from non-organogenic and organogenic calli. The 11-week-old non-organogenic calli contained 0.41+/-0.03 and 0.23+/-0.02 microg g(-1) dry wt hyoscyamine and scopolamine respectively. However, no root meristem was found in the calli. The alkaloids were absent in 2-week-old organogenic calli. The shoot-buds induced on the non-organogenic and organogenic calli did not contain these alkaloids. Hyoscyamine and scopolamine contents of the 6-week-old non-rooted shoots regenerated from non-organogenic calli were 7.8+/-0.1 and 6.5+/-0.4 microg g(-1) dry wt respectively and those in the 9-week-old non-rooted shoot regenerated from organogenic calli were 38.5+/-0.4 and 3.6+/-0.1 microg g(-1) dry wt respectively. Hyoscyamine and scopolamine contents of the 4-week-old roots regenerated from non-organogenic and organogenic calli were higher than those in the non-rooted shoots. Since the presence of hyoscyamine and scopolamine in the non-rooted shoot depends on the stage of differentiation, manipulation of culture environment may improve hyoscyamine and scopolamine contents of the non-rooted shoots.     

Efficient plant regeneration from in vitro leaves and petioles via shoot organogenesis in Sapium sebiferum Roxb.

Sapium sebiferum Roxb. is a widespread and economically important multipurpose tree due to its high value in ornamental, and biodiesel production as well as medicine. A highly efficient in vitro plant regeneration system through direct shoot organogenesis was established for the first time from leaves and petioles of S. sebiferum. The results showed that plant growth regulators (PGRs), mechanical damage, explant orientation, explant source, and developmental stage had a strong influence on the in vitro morphogenesis of S. sebiferum. For shoot organogenesis from leaves, the highest adventitious shoot induction rate (96.67%) with 25.67 shoots per explant was obtained when mechanically damaged leaves (the first three leaf explants at the top, leaf #1–3) were cultured with the abaxial surface placed down on Murashige and Skoog (MS) medium containing 0.5 mg L^?1 thidiazuron (TDZ). For in vitro morphogenesis of petioles, the combination of 1-naphthylacetic acid (NAA) and 6-benzylainopurine (6-BA) played a key role in cell fate determination. All of the in vitro petioles produced adventitious shoots on MS medium containing 1.0 mg L^?1 6-BA and 0.1 mg L^?1 NAA, while they produced green calli on medium fortified with 0.5 mg L^?1 6-BA and 1.0 mg L^?1 NAA. The shoots were subcultured in medium fortified with 0.5 mg L^?1 6-BA and 0.1 mg L^?1 NAA for multiplication and elongation. The elongated shoots successfully rooted on half-strength MS (1/2 MS) medium fortified with 0.5 mg L^?1 indole-butyric acid (IBA) and 0.25 mg L^?1 indole-3-acetic acid (IAA), and the regenerated plantlets successfully acclimatized with a survival rate of 92.56% in the greenhouse. The genetic fidelity of in vitro regenerated plants was evaluated using inter simple sequence repeat molecular markers. The in vitro regenerated plants were found to be the true to their mother plant. This study will be beneficial for the large-scale propagation as well as the genetic improvement of S. sebiferum.

     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Growth and alkaloid production of callus culture induced from Securinega suffruticosa

The growth of, and production of alkaloids by, callus derived from budding stem explants of the germinated seeds of Securinega suffruticosa has been studied. The major alkaloids produced were securinine and allosecurinine with the latter being present in the greatest amount. The effects of pH, growth hormones, sucrose concentration and light and dark on callus growth and alkaloid production have been examined in detail. The pattern of alkaloid production in the callus culture appeared to be similar to that in the root of the securinega plant.