μM Kinetin Research Articles

An efficient somatic embryogenesis system for velvet bean [Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug

The Mucuna pruriens Linn. is an important medicinal legume cover crop. Almost all the parts of the plant are reported to contain l-3,4-dihydroxy phenylalanine (l-DOPA), a non-protein amino acid that acts as precursor for the neurotransmitter dopamine. Here we report a rapid and reliable protocol for high fidelity regeneration of M. pruriens plants via somatic embryogenesis. Embryogenic callus was induced from cotyledon segments of in vitro grown seedlings on Murashige and Skoog medium supplemented with 6.7 μM 2,4-dichlorophenoxy-acetic acid (2,4-D). High-frequency somatic embryogenesis was achieved after transfer of embryogenic callus clumps to MS medium supplemented with 2.3 μM Kinetin (Kn) and 5.4 mM α-naphthaleneacetic acid (NAA) supplemented with 13.6 μM adenine sulphate. The maximum number of cotyledonary-stage embryos (60.5 ± 12.7) was obtained after 10 weeks. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. The protocol should provide an efficient means for large-scale cultivation and in vitro manipulation of M. pruriens, an important green manure cover-crop with medicinal properties.

Enhanced micropropagation of Dendrobium huoshanense C.Z. Tang et S.J. Cheng through protocorm-like bodies: The effects of cytokinins, carbohydrate sources and cold pretreatment

The effects of cytokinins, carbohydrate sources and cold pretreatment on the conversion of protocorm-like bodies (PLBs) to shoots were investigated for the enhancement of micropropagation of Dendrobium huoshanense C.Z. Tang et S.J. Cheng, an endangered medicinal plant in China. The effects of cytokinins and carbohydrate sources on the conversion of PLBs to shoots depended on their types and concentrations. The best results in terms of shoot development from PLBs occurred on 1/2 MS medium supplemented with 20 μM kinetin and 10 g l −1 maltose. Cold pretreatment at 10 °C for 1–2 weeks significantly enhanced the conversion of PLBs to shoots, and over 1300 shoots were obtained from one gram of PLBs after 3 months of culture. The developed shoots were rooted on growth regulator-free MS medium supplemented with 8 g/l banana paste to give complete plantlets, which were successfully acclimatized with a survival rate of approximately 65%. The results indicate that a suitable cold pretreatment (10 °C for 1 week) followed by the use of 20 μM kinetin and 10 g/l maltose in 1/2 MS medium would produce a large number of shoots from PLBs for plantlet regeneration of D. huoshanense.

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.

Somatic Embryogenesis in Hedychium bousigonianum

An efficient primary somatic embryo (SE) and secondary somatic embryo (SSE) production system was developed for the ornamental ginger Hedychium bousigonianum Pierre ex Gagnepain. Addition of two ethylene inhibitors, salicylic acid (SA) and silver nitrate (AgNO3), to the culture media improved the system. Callus was initiated and proliferated on a medium containing Murashige and Skoog (MS) basal salts supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid and 4.6 μM kinetin. Friable callus was transferred to a liquid medium containing MS basal salts, B5 vitamins, 0.6 μM thidiazuron, and 8.9 μM 6-benzylaminopurine to induce somatic embryogenesis. The effects of various concentrations of SA (0, 25, 50, 75, 100, 125, 150 μM) and AgNO3 (0, 10, 20, 30, 40, 50, 60 μM) on callus growth, SE, and SSE development was further evaluated. The rate of callus growth decreased as the concentrations of SA or AgNO3 increased. AgNO3 and SA at all concentrations stimulated SE and SSE development better than the control although a decrease in embryo production was observed at higher concentrations of both SA and AgNO3. The best concentrations for SA were 75 and 100 μM, whereas for AgNO3, they were 30 to 50 μM for both SE and SSE production.

Callus formation, plant regeneration, and transient expression in the halophyte sea aster (Aster tripolium L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l−1 casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 105 protoplasts ml−1, 25 μg batch−1 of plasmid vector, and 30% polyethylene glycol 4,000.

An Efficient Procedure for Regeneration from Leaf-derived Calluses of Lonicera macranthoides ‘Jincuilei’, an Important Medicinal Plant

‘Jincuilei’ is a mutant selected from Lonicera macranthoides Hand.-Mazz. It produces abundant flowers that never open with a chlorogenic acid (CGA) content up to 6.0%. Propagation through rooting or grafting has only a 30% survival rate. This study was undertaken to establish an efficient protocol for rapidly regenerating this mutant. Leaf explants were inoculated on Gamborg's B5 medium supplemented with different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenozyacetic acid (2,4-D). The optimal combination for callus induction was 4.4 μm BA with 2.26 μm 2,4-D, which resulted in 86.7% of leaf explants producing calluses in 4 weeks. Calluses produced from this optimal medium were cultured on B5 medium containing different concentrations of kinetin (KT) and α-naphthalene acetic acid (NAA). The best formulation for shoot induction was B5 medium containing 0.9 μm KT and 5.4 μm NAA in which 73.4% of cultured calluses produced shoots in 8 weeks, and shoot numbers ranged from three to six per callus piece (1 cm3). Adventitious shoots were cut and rooted in half-strength Murashige and Skoog medium supplemented with 14.8 μm 3-indolebutyric acid. Roots initiated 10 d after culture, and rooting percentages ranged from 98% to 100%. Plantlets grown in a container substrate in a shaded greenhouse had over a 95% survival rate. During the last 6 years, over four million plantlets were regenerated using this established procedure, and there was no somaclonal variation. Fresh and dry weights of 1000 flowers, CGA contents, and dry flower yields of the regenerated plants were not significantly different from those of the stock ‘Jincuilei’ propagated by cutting, indicating that plants regenerated from this established procedure were stable. This established in vitro culture method has led to rapid commercial production of this medicinal plant on more than 1500 ha of production field.

Pengaruh Pemberian Sitokinin Terhadap Pertumbuhan Palea dan Lemma Padi Melaui Kultur In Vitro

A research has been carried out with the aim s to: 1. study the influence of both the kind and concentration of cytokinins on palea and lemma growth in in vitro culture; 2. study the influence of palea and lemma age towards external application of cytokinin in in vitro culture ; and 3. determine the best palea-lemma age, kind and concentration of cytokinin which resulted the best growth of palea and lemma in in vitro culture . The used experimental method was Completely Randomised Design (CRD) with factorial treatment pattern. The applied treatment consisted of three factors i.e. palea-lemma age (U): (55, 60, and 65 days after planting); kind of cytokinin (S): (BAP and Kinetin); and Cytokinin Concentration (K): (0, 5, 10, and 15  M) with 3 replications. The observed variables were the palea and lemma growth with the parameters were palea-lemma length and width. The results showed that the kind of cytokinin had influenced the growth of palea-lemma in in vitro culture, and kinetin has better influence on the palea-lemma growth. The age of the palea-lemma determined the responsiveness of the palea-lemma towards external application of cytokinin. Older palea-lemma showed less responsive than younger ones towards external application of plant growth regulators. Moreover, the treatment combination (U1S2K2) (10 µM Kinetin applied to 55-day-old rice palea-lemma) had the best effect on increasing the size of palea-lemma of IR 64 rice.

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA3) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.

In vitro organogenesis and antioxidant enzymes activity in Acanthophyllum sordidum

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 µM 2,4-dichlorophenoxyacetic acid + 4.65 µM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 µM 1-naphthalene acetic acid (NAA), 2.69 µM NAA + 4.54 µM thidiazuron and 2.46 µM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.

In vitro regeneration of clonally uniform plants of Crataeva magna: a high value medicinal tree by axillary branching method

Crataeva magna (Lour.) DC (synonym C. nurvala) is a high-value Indian medicinal tree. The multiple uses of C. magna have resulted in its over-exploitation. Erratic seed germination combined with destructive harvesting and habitat destruction in the form of deforestation has added to the enormity of the problem. Therefore, the need for conservation of this plant is vital. Development of an efficient micropropagation protocol will play a significant role in meeting the requirement of quality planting material for commercial cultivation thereby conserving the species in its natural habitat. In the present study, shoot multiplication was achieved by culturing single node segments derived from a field grown tree on Murashige and Skoog’s (MS) medium supplemented with 2.66 μM N6-benzyladenine, 1.39 μM Kinetin (Kn), 0.57 μM indole-3-acetic acid (IAA), 3% sucrose and 0.2% gelrite. 96% rooting was achieved within 22 days by culturing the in vitro formed shoots on half strength MS medium with 11.42 μM IAA, 9.8 μM indole-3-butyric acid, 0.46 μM Kn and 198.25 μM phloroglucinol. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%. To this date 500 plants have been produced. Inter simple sequence repeat analysis has confirmed genetic uniformity of the tissue-cultured plants.

In vitro germination and micropropagation of Givotia rottleriformis Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA3). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.

Estabelecimento de cultura de células em suspensão e identificação de flavonóides em Cordia verbenacea DC.

O trabalho teve como objetivos o estabelecimento de culturas de células em suspensão, extração, separação e identificação de flavonóides em extratos de folhas e de células em suspensão de Cordia verbenacea. Células dessa espécie, após terem sido subcultivadas três vezes no meio MS suplementado com 2,32 µM de cinetina + 10,74 µM de ANA a intervalos de 28 dias, apresentaram cinco estágios de crescimento: as fases lag, exponencial, linear, desaceleração e estacionária. O maior percentual de crescimento (37%) ocorreu no período exponencial entre o quarto e o décimo segundo dia e o menor (3%) na fase lag até o quarto dia. Para identificação de flavonóides, foram usados extratos submetidos à separação e purificação por CCD e CCL e os componentes obtidos submetidos à Espectroscopia de Ultravioleta, Espectrometria de Infravermelho e Massa e Ressonância Magnética Nuclear de Hidrogênio. Após as frações das amostras de folhas e células terem sido separadas pelo eluente ácido acético, foram identificados os componentes 7,4'-diidróxi-5'-carboximetóxi isoflavona e 7,4'-diidróxi-5'-metil isoflavona. Foi detectado maior concentração dessas substâncias nas células cultivadas in vitro.

High frequency plant regeneration from protoplasts in cotton via somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM8P medium with 0.45 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 µM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 µM indole-3-butyric acid (IBA) and 0.93 µM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.

In vitro regeneration of Leucaena leucocephala by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 µM N6-benzylamino-purine (BAP) and 5.37 µM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 µM indole-3-butyric acid (IBA) and 0.23 µM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 µM NAA and 12.24 µM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 µM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 µM BAP, 2.5 to 5.0 µM IBA and 0.5 mM spermidine.

Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis

Picea koraiensis, called Korean spruce, is an evergreen tree and found mostly in northeast Asia. In this study, plant regeneration via somatic embryogenesis from open-pollinated immature zygotic embryos of nine genotypes of elite trees was established. Immature zygotic embryos were cultured onto RJW medium modified from 505 medium with 21.48 μM NAA, 2.22 μM BA, and 2.32 μM KT. The average frequency for all nine genotypes was 74.2%. Embryogenic calluses of the nine genotypes of elite trees were subcultured on RJW basal medium containing 8.06 μM NAA, 1.11 μM BA, and 1.16 μM kinetin. The calluses of three lines, 3#, 9#, and 2#, were actively proliferated but others were not. Somatic embryogenesis was induced from the embryogenic callus in genotypes of 3#, 9#, and 2# on RJW medium with ABA and 60 g l−1 sucrose. Cotyledonary somatic embryos were subjected to a drying process. The drying of embryos by uncapping the culture bottle for 5 days on a clean bench resulted in a high frequency of germination of somatic embryos (87% in RJW medium). However, plantlet conversion from germinated embryos was greatly reduced and the optimal medium for plant conversion was 1/2 WPM or 1/2 BMI medium. In conclusion, we have, for the first time, established a plant regeneration system via somatic embryogenesis in the Korean spruce, which can be applied for rapid micropropagation of elite trees.

High-frequency direct somatic embryogenesis from leaf tissues of Drimiopsis kirkii Baker (giant squill)

Whole plants were regenerated from excised leaves of Drimiopsis kirkii Baker (Lily of the Valley) through direct somatic embryogenesis. An initial exposure to a low level of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 μM) in the medium was essential in inducing the direct formation of somatic embryos. A high concentration of 2,4-D (4.52 μM) in the proliferation medium reduced embryogenesis and enhanced callus formation. The presence of kinetin in the medium enhanced the somatic-embryogenesis-inducing effect of 2,4-D (0.45 μM). The maximum embryogenesis rate (4,026 somatic embryos per gram of leaf) was obtained in explants cultured for 30 d in medium supplemented with 2.33 μM kinetin and 0.45 μM 2,4-D (embryo induction medium). Kinetin (4.65 μM) also enhanced embryo germination (97.6%), but the presence of α-naphthalene acetic acid in the medium drastically reduced embryo germination. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.

Efficient somatic embryogenesis and plant regeneration from shoot apex explants of different Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn.)

An efficient somatic embryogenesis and plant regeneration system was developed from shoot apex explants of finger millet, Eleusine coracana. Eight genotypes, CO 7, CO 9, CO 13, CO 14, GPU 26, GPU 28, GPU 45, and GPU 48, were assessed in this study. The maximum somatic embryogenic induction, at 98.6%, was obtained from explants cultured on Murashige and Skoog medium supplemented with 18.0 μM dichlorophenoxyacetic acid and 2.3 μM kinetin. The highest number of shoot induction (26) was observed after transfer of embryonic callus to regeneration medium supplemented with 4.5 μM thidiazuran and 4.6 μM kinetin. Significant differences were observed between genotypes for somatic embryogenesis and plant regeneration. GPU 45 gave the best response, while CO 7 was the least responsive under the culture conditions tested in this study. Regenerated plants were successfully rooted and grown to maturity after hardening in soil.

Optimizing Culture Conditions for in vitro Propagation of Trichosanthes cucumerina L.: An Important Medicinal Plant

ABSTRACT A simple and efficient, single medium–based protocol for rapid in vitro propagation was developed for Trichosanthes cucumerina L., an important medicinal plant of the family Cucurbitaceae. The effects of culture vessel, medium quantity per vessel, and inoculation density were investigated, and optimum culture conditions for vigorous growth for large-scale propagation were standardized. Axillary bud proliferation coupled with rooting from nodal explants was obtained in MS medium supplemented with 0.46 μM kinetin and 2.46 μM indole 3-butyric acid. The highest culture response (97 %) and multiplication rate of 1:7 (seven shoots per explant for next subculture in 4 weeks) coupled with rooting was achieved in this medium. Inoculation density of five nodes per culture bottle containing 30 ml of the medium was the optimum. Rooted plantlets could be established in sand with an average of 90% survival. The micropropagated plants exhibited morphological similarity with the mother plants. The results indicate the key culture conditions can be regulated to improve the multiplication rate and quality of planting material.

Influence of Auxins and Sucrose in Monoterpenoid Oxindole Alkaloid Production by Uncaria tomentosa Cell Suspension Cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize ( Zea mays L.)

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines (Liao 7980, Dan 9818, Dan 340, and Dan 5026). The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 μM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L-proline (12 mM) in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6-benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.