Explants For Callus Induction Research Articles

An Efficient Callus Induction and Regeneration Protocol for a Drought Tolerant Rice Indica Genotype AC39020

Most of the cultivated indica rice genotypes are less amenable to genetic modifications due to their poor callus induction and regeneration potential. The prerequisite for genetic enhancement of indica rice genotypes by biotechnological approach is to develop an efficient protocol for callus induction and plant regeneration. In the present study, we established an optimized regeneration protocol for rice genotype AC39020, which is moderately drought tolerant with high root growth and biomass. To use this genotype in the crop improvement program the prerequisite is callus induction and regeneration protocol in this indica rice genotype. The mature seeds of AC39020 used as explants for callus induction on LS, MS and N6 media with different hormones and amino acid concentrations. LS basal media with 2.5 mgL-1, 2, 4-D and 500 mgL-1 glutamine showed 91.3% callus induction frequency. Subsequently the embryogenic callus was cultured on MS media supplemented with BAP, Kinetin, NAA, and TDZ. The MS medium supplementedwith 4mg L-1 BAP and 0.5 mgL-1 NAA showed 75% regeneration efficiency. Since regeneration in indica rice varieties is tedious, far-reaching, highly genotype-specific, we exposed the embryogenic calli for mild desiccation stress for 24 h and 48 h. The desiccation treatment for 48h increased shoot regeneration frequency from 16.7 % to 40.2 % compared to non-desiccated calli. The protocol developed was highly reproducible and this protocol can also be used for further improvement of this rice genotype through genetic modification.

OPTIMIZATION OF CALLUS INDUCTION AND SUBCULTURE CONDITIONS OF TAXUS CHINENSIS

Taxus chinensis (Pilger) Rehd.var. mairei (Lemee et Levl.) Cheng et L. K. Fu is an evergreen plant, which belongs to the Taxaceae family. These slow growing gymnosperms were harvested in large amounts to provide taxol for clinical trials. Due to the relative scarcity of the natural resources and the low yield of Paclitaxel (taxol), it is very necessary to produce alternate sources of taxol by callus cultures. In this work, we established conditions for callus induction and subculture of T. chinensis . The experimental results showed that B5 medium supplemented with 1 mg/L 2,4 - dichlorophenoxy acetic acid (2,4 D), 30 g/L sucrose and 7.5 g/L agar (pH 5.5) was effective for inducing callus and the better explants for callus induction was petiole. When the callus was sub cultured on seven different media and tamed several times, it could grow fast and anti - browning on the B5 medium containin g 1 mg/L α - naphthaleneacetic acid (NAA), 0.5 mg/L 2, 4 - D, 0.05 mg/L kinetin (KT), 0.05 g/L ascorbic acid (Vc), 0.1 g/L activated carbon (Ac), 30 g/L sucrose and 7.5 g/L agar (pH 5. 75).

<i>In Vitro</i> Callus Induction and Shoot Regeneration from Leaf Explants of <i>Glinus lotoides</i> (L.)—An Important Medicinal Plant

G. lotoides L. is a threatened plant that is frequently harvested for medicinal purpose. However, its distribution in the world is limited because of short period of seed viability and poor seed germination. The objective of this study was to develop in vitro propagation protocol for G. lotoides through callus induction. For callus induction, different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid) and BAP (6-benzyl amino purine) were used. Seeds were sown on growth regulator-free MS medium and shoots from the in vitro germinated seedlings were excised and cultured on MS medium containing 0.5 mg/l BAP. Young leaves from these shoots were used as explant for callus induction and shoot regeneration. Maximum callus induction (100%) was observed on medium containing 2,4-D (0.5, 2.0, 3.5 mg/l) or NAA (2.0, 2.5 mg/l) in combination with 0.5 mg/l BAP. However, 2,4-D was the best in overall callus induction. The highest regeneration (20%) frequency was achieved on the medium containing 0.5 mg/l BAP. Highest number of shoot (2.83 ± 1.22) and shoot length (2.16 ± 0.87 cm) per explant were obtained in the presence of 0.25 mg/l BAP + 0.5 mg/l KIN (Kinetin). In shoot multiplication media, maximum mean (6.43 ± 0.87) of shoot was observed on MS medium containing 0.5 mg/l BAP. The best shoot length (1.70 ± 0.14 cm) was recorded on control medium. The highest (95%), maximum root number (14.10 ± 1.47) and root length (1.01 ± 0.10 cm) were obtained on a medium supplemented with 1.5 mg/l Indole-3-butyric acid (IBA). All the plants (100%) were survived after acclimatization in greenhouse. The present study can be useful for callus induction and indirect shoot regeneration form G. lotoides.

Leaf-derived organogenesis in vitro for mass propagation of lisianthus (Eustoma grandiflorum (Raf.) Shinn

Lisianthus (Eustoma grandiflorum (Raf.) Shinn) is an important ornamental commodity in South-East Asia. However, mass propagation of the plant at a commercial scale to satisfy market demands is faced by limited availability of high quality and uniform seedlings as planting material. Using different regeneration media and leaf explants for callus induction, regeneration, proliferation, root formation and acclimatization were studied. High callus induction and adventitious shoot formation were possible from leaf explants of E. grandiflorum ‘White Lavender’ cultured on Murashige and Skoog (MS) medium supplemented with 3.0 mg/l thidiazuron (TDZ) and 0.3 mg/l α-naphthalene acetic acid (NAA), but high quality shoots (8.0) was established on MS medium containing 0.5 mg/l N6-benzyladenine (BA) and 0.002 mg/l NAA. In the same medium, adventitious shoots could be multiplied up to the fourth subculture at a rate of 1.74 which decreased to a 1.57 multiplication rate in subsequent subcultures. Shoots rooted easily on MS medium containing 0.1 mg/l BA and 0.02 mg/l NAA with 3.9 roots per shoot. The plantlets, which were successfully acclimatized in a mixture of burned-rice husk and organic manure (1:1, v/v) with 90% survival, grew well after repotting.

Somaclonal variation of cardenolide content in Heywood's foxglove, a source for the antiviral cardenolide glucoevatromonoside, regenerated from permanent shoot culture and callus

Cardiac glycosides are plant natural compounds used to treat cardiac insufficiency in humans. Recent findings suggest a further medical use in oncology and virology. Since species of the genus Digitalis are the major source of these compounds, in vitro culture techniques can be useful tools for the propagation and selection of elite genotypes. Digitalis mariana ssp. heywoodii, an endemic and endangered species in Portugal, is known to have a very high content of cardiac glycosides, especially glucoevatromonoside. Here, D. mariana ssp. heywoodii plants were cloned from a seed-derived shoot explant through direct or indirect in vitro propagation. Multiple shoot formation was induced from a seed grown on agar-solidified Murashige and Skoog (MS)-based medium supplemented with 9.8 μM indole-3-butyric acid (IBA). Shoots were used to regenerate plants directly, and some were then used as a source of root explants for callus induction and growth by transferring to MS medium supplemented with 9.3 μM kinetin (KIN), 5.7 μM indoleacetic acid (IAA), and 1.1 μM 2,4-dichlorophenoxyacetic acid (2,4–D). Callus that was 3, 6, or 24 mo old efficiently developed adventitious shoots when placed on MS medium containing 13.3 μM 6-benzylaminopurine (BAP) and 1.7 μM IAA. Plants regenerated from either callus (R plants) or shoot cultures (S plants) were transferred to the greenhouse and propagated for 3, 4, or 10 mo. Regenerated plants were analyzed for cardenolide content and accumulation pattern. The highest cardenolide content, about 2.0 mg/100 g DW in terms of digitoxin equivalents (Dteq), was found in S10 plants (10 mo in the greenhouse), whereas plants regenerated from 24-mo-old callus (R24/10; 10 mo in the greenhouse) had the lowest cardenolide content of about 1.2 mg Dteq/100 g DW.

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L−1, and for the ovary, it is MS + picloram 1.5 mg L−1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L−1 + TDZ 0.1 mg L−1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.

Micropropagation ofMucuna pruriens(L.) DC and determination of tyrosinase

The protocol for in vitro propagation of Mucuna pruriens (white seed) and the extraction of laccase-like enzyme –tyrosinase; has been standardized. Root, internode, axillary buds, seed and leaf were used as explants for callus induction. Maximum compact green calli were produced by using different concentrations of NAA and combinations of IBA+BAP. Maximum white friable calli were produced by using different concentrations of 2, 4-D and combination with Kinetin. Direct organogenesis and indirect organogenesis were derived from various explants of M. pruriens by using different combinations of auxins and cytokinins. The high phenolic content of M. pruriens caused initial browning which was avoided by applying 0.01% of acid washed charcoal into 1 liter MS medium. Instead of charcoal, ascorbic acid (50 mg/L), citric acid (25 mg/L) and L-arginine (25 mg/L) were added into 1 L MS medium and kept in dark condition to overcome browning effect. The laccase-like enzyme tyrosinase was determined by qualitative analysis and quantified.

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96% callus induction from cotyledonary node explants on MS medium supplemented with 1.5mgl(-1) 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5mgl(-1) 1-naphthalene acetic acid (NAA), (2) 97% shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0mgl(-1) N6-benzylaminopurine (BA) and 1mgl(-1) NAA,37 (3) 98% rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5-4mgl(-1)) after 45days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5%).

In Vitro Host-Free Seed Culture, Callus Development and Organogenesis of an Obligatory Root-Parasite Striga hermonthica (Del.) Benth: The Witch-Weed and Medicinal Plant

Striga hermonthica (Del.) Benth a well-known hemi-parasitic weed, it also has been used widely in African folk medicine to remedy broad spectra of diseases. The current contribution is an attempt to establish reproducible in vitro callusing system. In vitro seedling’s stem segments were used as an explant for callus induction, in 1.5% or 3.0% sucrose added into Murashig and Skoog medium (MS) and supplemented with different auxins, α-Naphthalene-3-acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), Indole-3-acetic acid (IAA), or Indole-3-butryic acid (IBA) at different concentrations each alone or in combination with cytokinin 0.5 mgL−1 6-benzyl aminopurine. The most effective auxin was NAA with maximum 79% callus induction rate. All auxin treatments induced callus in all concentration when used alone or combined with BAP, except 2,4-D, which induced the callus only when combined with BAP. A high sucrose concentration was required for high callus induction rate by all auxin treatments. IAA and IBA auxins induced direct shoot regeneration and had low callus induction rates. NAA, IAA and IBA induced organogenic calli, whereas 2,4-D in combination with BAP induced non-organogenic callus. We further screened preliminarily the phytochemical contents of the callus and intact plant, which was revealed the presence of flavonoids, terpenes, saponins, cardiac glycosides, alkaloids, tannins and coumarins. Experimental data of both seed culture and callus induction could provide a route to further enhance the efficiency of callus initiation of S. hermonthica for medicinal purposes and understanding the infection mechanism of the witch-weed plant.

Effect of different plant growth regulators on callus induction in Catalpa bungei

The present study describes a reliable method for callus induction of Catalpabungei plant. Calli were in vitro initiated from leaves, stem and petiole segments using DKW basal media. The effect of combinations of BA (6-benzyl adenine) and NAA (α-naphthalene acetic acid) on callus induction was investigated. It was found that growth regulators and their concentrations have obvious effects on the callus induction, the increment of callus index and callus physical appearance. Moreover, callus was formed at different times among three kinds of explants that is, leaves, stem segment and petiole. The best callus induction capabilities were obtained with low levels of NAA in combinations with BA. However, supplemented medium with high concentrations of NAA led to relatively low callus frequency and the callus obtained turned to brown color. Generally, the highest frequencies of callus formation from leaves, stem and petiole explants were observed in the medium containing 0.05 mg l-1 of NAA and 5.0 mg l-1 of BA. However, petiole is the most potential explant for callus induction. Key words: Catalpa bungei, tissue culture, plant growth regulators, callus.

In vitro propagation of an endangered medicinal herb Chlorophytum borivilianum Sant. et Fernand. through somatic embryogenesis

Tuberous roots of Chlorophytum borivilianum Sant. et Fernand. which are a source of steroidal saponins, possess immunomodulatory, adaptogenic, aphrodisiac, antipyretic, diuretic, hemostatic and anti-tumour properties. Poor seed setting and germination and slow growth in conventional vegetative propagation are major constraints in the large-scale cultivation of this commercially important medicinal plant. In the present study, a procedure for in vitro propagation of this endangered herb through somatic embryogenesis has been established. Seeds of Chlorophytum borivilianum were germinated on MS medium supplemented with 57.74μM gibberellic acid and hypocotyl portion from germinated seedling was used as explant for callus induction. Moderate to good callus induction was observed on MS medium containing 1.16μM kinetin and 1.13-2.26μM 2,4-dichlorophenoxyacetic acid. Regular subculturing of callus on kinetin (1.16μM) and 2,4-dichlorophenoxyacetic acid (1.13μM) supplemented medium induced somatic embryogenesis. In modified MS medium, 1.79mM NH4NO3 and 10.72mM KNO3 was optimal for somatic embryogenesis. 7.38μM 2-isopentenyladenine supplemented to modified MS medium, showed best response for somatic embryogenesis while proline (0.76mM) as an amino acid supplement gave better response than glutamine. 30% germination of mature somatic embryos was achieved on MS medium supplemented with 15.54μM 6-benzylaminopurine. Multiplication of C. borivilianum through somatic embryogenesis may offer a better approach compared to organogenesis for developing scale-up technology employing bioreactors for its mass propagation.

CALLUS INDUCTION AND PHYTOCHEMICAL CHARACTERIZATION OF <i>Cannabis sativa</i> CELL SUSPENSION CULTURES

Callus of Cannabis sativa has been successfully induced from C. sativa explants and seedings. It seems that flowers are the best explant for callus induction and induction under light also give better results than induction in dark. Four cell culture lines were established from flower induced callus. Phytochemical profiles of C. sativa suspension cell cultures were investigated using HPLC and 1H-NMR. Cannabinoids and phenolic compounds related to cannabinoids such as flavonoids could not be found in the cell suspension cultures and there is no major chemical difference between the cell lines though they can visually be distinguished by their colors. Only in one cell line some aromatic compounds in the water/methanol extract could be observed in the 1H-NMR. Further investigations showed that none of these compounds are flavonoids. It seems that lack of cannabinoids in the cell cultures is related to lack of polyketide synthase activity. Keywords: Callus, Cannabis, phytochemical

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou ( Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou ( Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l −1 6-benzylaminopurine (BA) and 1.0 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l −1 BA and 1.0 mg l −1 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l −1 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l −1 BA and 1.0 mg l −1 a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L −1 indole-3-acetic acid (IAA), 0.3 mg L −1 kinetin, 30 g L −1 sucrose and 8 g L −1 agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L −1 IAA, 0.5 mg L −1 zeatin, 30 g L −1 sucrose and 8 g L −1 agar; (3) 96.67% rooting on MS medium containing 30 g L −1 sucrose and 8 g L −1 agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.

Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deepwater rice (Oryza sativa L.) cultivars of Bangladesh

Experiments were carried out to induce embryogenic callus and plant regeneration from five different deepwater rice cultivars. Using mature embryos as explant, all the cultivars demonstrated high callus induction and plant regeneration frequencies. When coleoptile and root segments used as explants, they had low frequencies of embryogenesis. Depending on different genotypes, the best plant regeneration was obtained on LS (Linsmaier and Skoog, 1965) based medium supplemented with 2 mg l -1 BAP + 1.5 mg l -1 2,4-D. Large variabilities in callus growth and plant regeneration potential were revealed among the cultivars tested. Cultivar HA-8 formed a high frequency (78%) of callus than that of other cultivars. In contrast cv. HA-1 produced the highest percentage (72%) of plant regeneration. The callus growth potential was not correlated with the plant regeneration potential. Coleoptiles and root segments produced calli, which did not develop any shoot bud in regeneration media. Moreover, the calli turned blackish, watery and translucent after 25-28 days of culture. It is clear that mature seed scutellum (MSS) is the best explant for callus induction and plant regeneration.

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g−1 DW, valtrate (VAL) 10.2mgg−1 DW, and didrovaltrate (DID) 2.9mg g−1 DW were observed in root cultures after 7–8wk of culture.

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem)

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5-1 mg l(-1) 2,4-dichlorophenoxyacetic acid and 0.5-2 mg l(-1) 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.

Induction of Callus from Seedling Explants of Citrus macrophylla W. Rootstock

Citrus macrophylla is an important citrus rootstock for Mexican lemon (Citrus aurantifolia S.). This study was conducted to select explant type and to optimize cultural requirements for induction callus of C. macrophylla in vitro. The explants tested were leaf, epicotyl, cotyledon, and root segments excised under sterile conditions from 4-week-old nucellar seedlings. The various medium comprising either basal of Murashige and Skoog (MS) or Murashige and Tucker (MT) salts suplemented with various concentrations of plant growth regulators, including naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylaminopurine (kinetin) were used for the establishment of the explants. All cultured explants initiated callus from the cut ends after 2 weeks, when cultured on a modified MT medium suplemented with 6 mg NAA and 0.2 mg kinetin; cotyledon segments were the best explant for callus induction and development (43 mm2). Root segments were the lowest explants for callus induction.

Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 µM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM.

Somatic embryogenesis and plantlet regeneration in mandarin ( Citrus reticulata Blanco)

Callus cultures were initiated from leaf, epicotyl, cotyledon and root segments of in vitro grown nucellar seedlings of Citrus reticulata Blanco ‘Local Sangtra’. Callus initiation was seen on cut ends of all the explants within 10 days of culture. The epicotyl segments were the best explant for callus induction. Best callus induction occurred on MS medium containing NAA (10 mg l −1) and kinetin (0.5 mg l −1). MS medium supplemented with NAA (10 mg l −1), kinetin (1 mg l −1) and vitamins (10 × normal MS levels) were best for induction of somatic embryogenesis. The epicotyl-derived callus exhibited significantly higher (83.0%) mean somatic embryogenesis than calli derived from other explants. Most of the somatic embryos germinated like normal embryos, but some only produced shoots. The time taken for root initiation from separated shoots was 7–10 days. The number of roots per shoot and root length also varied with culture media. Plantlet survival in vitro was directly proportional to the number of roots produced per shoot. In vitro derived plantlets were successfully transplanted to soil.