μM NAA Research Articles

An effective route for the micropropagation of Medinilla formosana through ovary culture in vitro

Medinilla formosana Hayata, a beautiful endangered shrub of the family Melastomataceae endemic in Taiwan of China, is recalcitrant for micropropagation in vitro until now. And tissue browning after cutting generally occurred in the tissue culture of Medinilla species was considered to be the greatest obstacle. To avoid the serious cut-browning, immature ovaries in 0.3 cm diameter of M. formosana were taken as initial explants in this study, and its aseptic plantlets were obtained for the first time. The highest survival level (12.7 plantlets per ovary) after 3 months of culture were observed on 1/2 MS supplemented with 0.5 g l-1 active carbon and 0.4 g l-1 casein hydrolysate (pH 5.5). Furthermore, β-mercaptoethanol proved to be an effective inhibitor for its browning response of M. formosana in vitro. And 1/2 MS media supplemented with 6.67 μM BA, 0.11 μM NAA, 0.5 mM β-Met and 0.2 g l-1 AC proved appropriate for its multiplication. Finally rooted plantlets were transplanted into the greenhouse after acclimatization, and the micropropagation system of M. formosana was successfully established. This report first details an efficient method for the in vitro micropropagation of Medinilla plants.

Callus induction, high frequency shoot organogenesis and assessment of clonal fidelity in Torenia bicolor Dalzell.

An efficient protocol for the mass propagation of Torenia bicolor using internode derived callus organogenesis has been described. The optimum callusing was observed when internode explants were cultured on MS medium augmented with 4.0μM 2,4-dichlorophenoxy acetic acid (2, 4-D) and 0.5μM kinetin (Kn). The calli were subcultured on MS medium supplemented with various concentrations of N6-benzylaminopurine (BA; 1.0-5.0μM) or Kn (1.0-5.0μM) alone or in combination with naphthalene acetic acid (NAA; 0.5-1.5μM) for shoot regeneration. The highest shoot regeneration in terms of percent response (100%) and number of shoots (89.3) was observed on MS medium supplemented with 4.0μM BA and 1.0μM NAA. The best rooting was observed on half strength MS medium augmented with 0.6μM indole-3-butyric acid (IBA). On this medium, 100% shoots rooted with an average number of 6.8 roots per shoot. The rooted shoots were transplanted to soil and acclimatized successfully. Of the 80 plants transplanted to soil 72 survived. Inter simple sequence repeat (ISSR) analysis clearly indicated that callus derived plants of T. bicolor resembled the parent plant at the genetic level. The protocol described here could be used for the mass multiplication and conservation of this endemic plant.

Study of Oil Palm (Elaeis guineensis Jacq) In Vitro Embryogenesis using Young Leaves Explants

This study reported in vitro embryogenesis of oil palm using young leaves as explants. Explants were grown in solid modified MS or Eeuwens medium containing different concentrations of NAA and 2,4-D, i.e. media C1, C2, C3, C4 and C5, M1, M2, M3 and M4, to induce embryogenic calli. Compact and pearly-white, globular calli were obtained from the youngest leaf explants 28 weeks after culture.C1 media (MS medium + 107.41 µM of NAA + 100 mg.L-1 of asparagine + 100 mg.L of glutamine-1) produced the highest percentage of calli formation (30.56%), whereas C4 media (C1 supplemented with 67.86 µM of 2,-D ) was the optimal media for embryogenic callus induction. Direct embryoids were obtained from slightly older leaf explants on the C3 media containing NAA after 36 weeks of culture. However, four subcultures using the same medium with gradual reduction of auxin concentration were not successful to develop embryogenic callus and embryoid cells during the course of this study.

Management of cytokinin–auxin interactions for in vitro shoot proliferation of Althaea officinalis L.: a valuable medicinal plant

An effective, rapid and improved protocol for in vitro regeneration has been established for Althaea officinalis using nodal explants, excised from 4-week-old aseptic seedlings. Different cytokinins and auxins, alone or in combination were investigated with respect to shoot induction and development. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kn) and 2-isopentenyl adenine (2-iP) tested as supplement to Murashige and Skoog (MS) medium, BA proved to be the most effective for induction and proliferation of shoots from nodal explants. Proliferation capacity of the induced explants was differentially influenced by a combination of cytokinins [6-benzyladenine (BA), Kinetin (Kn) and 2-isopentenyl adenine (2-iP)] and auxin [indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), α-naphthalene acetic acid (NAA)]. The optimum response was observed with (7.5 μM) BA and (0.5 μM) NAA, which induced shoot regeneration in 95 % of the cultured nodal explants, with an average of 26 shoots per explant. High frequency of rooting (89 %) was obtained when shoot were transferred to ½ MS medium supplemented with (20 μM) IBA. In addition, a histological study was undertaken to better understand the origin of the regenerated shoot buds.

Genetic fidelity assessment of in vitro-regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2 ± 3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3 ± 6.3%). Maximum rooting of shoots (73.3 ± 5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.

MICROPROPAGATION AND ACCLIMATIZATION OF Aegiphila verticillata Vell.: AN ENDANGERED WOODY SPECIES

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitroacclimatization of Aegiphila verticillata, a woody species found in Brazilian rocky fields. Aseptic cultures were established by seeds and two multiplication analyses were performed. In the first, we employed 6-benzylaminopurine (BAP – 0, 2.5, 5 and 7.5 μM) + α-naphthalene acetic acid (NAA – 0, 0.2, 0.4 and 0.6 μM) and, in the second, were studied adenine sulfate, kinetin and thidiazuron (0, 5, 7.5, 10 and 12.5 μM). After 90 days, we assessed the quantitative and qualitative shoot propagation. There were more than 90% seed germination and low contamination (2%). In multiplication phase, the culture medium that promoted the best quantitative and qualitative culture development was supplemented with 7.5 μM BAP + 0.4 μM NAA. In the rooting assay, were used NAA, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3 or 0.4 μM). After 90 days, the root number and rooting quality were evaluated. In this analysis, differences were not found between the control and the other treatments. Rooted plantlets were acclimatized in styrofoam trays for 30 days, after which they were transferred to pots in the greenhouse. Only 3% of the plants subjected to initial acclimatization died and 70% of the plants transferred to the field conditions survived and showed normal development. The results founded in this work are the first involving in vitro propagation and ex vitroacclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities.

Micropropagation of Anacyclus pyrethrum and chemical profiling of the regenerated plants for pellitorine, the active principle

The paper reports the development of a protocol for large-scale micropropagation of Anacyclus pyrethrum, an invaluable medicinal plant, and the presence of pellitorine, the active principle, in leaves and roots of regenerated plants. Three explants—cotyledonary nodes, hypocotyls and cotyledons—were compared for their shoot development/differentiation ability on Murashige and Skoog’s (MS, Physiol Plant 15:473–497, 1962) medium supplemented with 1–10 µM of 6-benzylaminopurine (BAP) or kinetin (Kn). The best response (8.88 ± 0.28 shoots per explant) was elicited from the cotyledonary nodal explants on MS + 2.5 µM Kn. The in vitro regenerated shoots continued to multiply on being sub-cultured on the same medium. Elongated shoots were cultured on MS medium augmented with different concentrations (0.2–15 µM) of 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for induction of roots. The media augmented with 5 or 10 µM IAA or 5 µM NAA were the most effective for the initiation and proliferation of roots. The plantlets were successfully transferred to the following three potting mixtures: garden soil, vermiculite and garden soil (1:1), vermiculite, garden soil and perlite (1:1:1). Among these, the maximum number (75 %) of the transferred plants survived in the garden soil. The high performance liquid chromatographic analyses of roots and leaves of the regenerated plants as well as those from the native habitat revealed the presence of pellitorine in all, with the content being higher in the roots than the corresponding leaves. The developed protocol would be of use for production of A. pyrethrum plants throughout the year, and thus could become a perennial source of the herb and its active principle.

Micropropagation and genetic fidelity analysis in Valeriana jatamansi Jones

Valeriana jatamansi or Tagar is an important medicinal plant of family valerianaceae used in traditional and modern medicines. The plants of the species are harvested from the wild to meet the demand of pharmaceutical industries. Over harvesting of the species from its natural habitat put huge pressure on this species; therefore, there is the need of conservation and sustainable utilization of the species. Present study was attained to develop a highly efficient in vitro regeneration protocol for V. jatamansi using nodal explants on Murashige and Skoog (MS) basal medium supplemented with various combinations of plant growth regulators (PGRs). Medium supplemented with 1.5μM 6-benzylaminopurine (BAP), 0.5μM α-naphthalene acetic acid (NAA) and 0.1μM gibberellic acid (GA3) showed highest average shoot length (3.20cm), shoot number (2.33) and leaf number (15.33). A hundred percent rooting with significantly high mean root number (27.5±1.98) and root length (50±1.35cm) was achieved in full strength MS medium supplemented with same concentration, i.e. 1.5μM BAP, 0.5μM NAA and 0.1μM GA3 combination. A separate medium for root initiation was not required. A total of 91% plantlets survived after 1 year of acclimatization. Inter simple sequence repeat (ISSR) markers study confirmed genetic stability for in vitro raised plants by showing 100% monomorphism. High multiplication rate associated with genetic stability ensure the efficacy of the present in vitro clonal propagation protocol of this important medicinal plant. Our study revealed that entire procedure beginning from culture initiation followed by plant regeneration and field hardening required a five months period. The study has practical implications as it will be helpful to meet out industrial as well as domestic demand. In addition, it will ensure conservation of the species by providing the uniform quality planting material.

Establishment of an efficient micropropagation and callus regeneration system from the axillary buds of Bambusa ventricosa

Based on the screening of various hormone combinations, we have established an efficient micropropagation and callus regeneration system from the axillary buds of B. ventricosa. We found that 6-benzyladenine (6-BA) had a dominant role in promoting bud sprouting, multiple bud induction and proliferation in B. ventricosa. Meanwhile α-naphthaleneacetic acid (NAA) was found to be an effective factor of inducing rooting in proliferated buds. The Murashige and Skoog medium containing 22.2 µM 6-BA was optimal for bud initiation, and the MS medium containing 26.6 µM 6-BA provided a good result for multiple bud induction. However, for buds proliferation MS medium containing 22.2 µM 6-BA, 0.23 µM Thidiazuron (TDZ: N-phenyl-N-[(1, 2, 3-thidiazol-5-yl) urea]) and 0.27 µM NAA was found to be very effective. The optimal medium for rooting of proliferated bud was MS medium containing 2.7 µM NAA, 4.9 µM indole butyric acid and 4.4 µM 6-BA. Based on the establishment of an efficient micropropagation system, we investigated the frequencies of callus formation from buds of in virto raised plantlets under different culture conditions. We showed that media containing 27 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2.7 µM NAA and 0.0045 µM TDZ effectively produced callus. The callus induction rate varied between it to 60 percent. TDZ was found to be a main factor influencing the callogenesis of B. ventricosa. Medium containing 22.6 µM 2, 4-D, 2.2 µM 6-BA and 5.4 µM NAA was preferred for callus amplification among the four tested subculture media. Plants were successfully regenerated on a MS medium containing 13.3 µM 6-BA and 2.7 µM NAA, subsequently acclimatized and transplanted to an experimental pod.

In vitro plant regeneration of lotus (Nelumbo nucifera)

Abstract In this study, an efficient and reproducible plant regeneration system for lotus (Nelumbo nucifera Gaertn.) was established using shoot apical meristems from the buds and one-week-old aseptically germinated embryos as explants. Multiple shoot clumps were induced on Murashige and Skoog (MS) basal medium supplemented with various combinations of N6-Benzylaminopurine (6-BA) and α-Naphthalene acetic acid (NAA). The maximum response was obtained with 2.22 μM 6-BA, and produced 21.33 shoots per explant after four weeks. After five subcultures, multiple shoot clumps were transferred to MS basal medium supplemented with various combinations of 3-Indolebutyric acid (IBA), NAA and sucrose for root induction. After four weeks, plantlets with well-developed roots were achieved on MS basal medium supplemented with 0.54 μM NAA and 30 g/L sucrose with 100% rooting rate. The successfully acclimated plantlets were transferred to pots with the addition of 2 g/L KMnO4 into the soils, and finally fertile plants with much bigger leaves were obtained in the greenhouse. The survival rate was 97.33%.

Effects of auxins and cytokinins on tomato callus from anthers

An investigation was carried out on growth substance requirements of tomato callus derived from anthers for culture <i>in vitro</i>. Linsmaier and Skoog (1965) medium was used with various levels of auxins (IAA and NAA) and cytokinins (K and BAP). The results show that cytokinin is an absolute requirement for callus growth irrespective of the auxin level. The optimum concentration of auxin in combination with cytokinin was found to be 5 μM of NAA or 25 μM of IAA, with 5 μM of K or BAP. Callus growth on media with NAA and cytokinin was superior to that on IAA, amounting to 6.05 g per piece on medium with 5 μM of NAA and BAP. Tissues grown on this medium have the highest water content. At the onset of culture the tissue is characterized by weak growth and attains its maximal increase in fresh weight after 6 weeks.

Number of explants, culture medium and photoperiod in ornamental pineapple micropropagation

Um dos fatores limitantes da expansão do cultivo comercial do abacaxizeiro ornamental, Ananas comosus var. erectifolius (L.B. Smith) Coppens & Leal, é a disponibilidade de mudas, em quantidade e qualidade. A micropropagação permite a obtenção de maior taxa de multiplicação e garante a alta qualidade fitossanitária das mudas obtidas; porém, o custo de produção deste tipo de material propagativo ainda é relativamente elevado. Dessa forma, o presente trabalho teve como objetivo determinar uma relação mais adequada entre os fatores: número de explantes por frasco, meio de cultura e fotoperíodo, visando aumentar a taxa de multiplicação in vitro desta variedade de abacaxizeiro ornamental; e, consequentemente, otimizar os custos de produção da muda micropropagada. Utilizou-se o delineamento inteiramente casualizado, com os tratamentos envolvendo três fatores: número de explantes por frasco (4, 5 e 6); meio de cultura (MS + 0,54 μM de ANA acrescido de 2,22 μM ou 4,44 μM de BAP) e fotoperíodo (12 e 16 horas), em dez repetições. Nas taxas de multiplicação obtidas, foram registradas diferenças significativas quanto ao fotoperíodo e à interação entre os três fatores analisados. Não foram evidenciadas diferenças significativas para o número de explantes utilizados por frasco, nem para o meio de cultura isoladamente. Do ponto de vista econômico, recomenda-se a utilização de seis explantes por frasco, cultivados no meio MS + 4,44 μM de BAP + 0,54 μM de ANA, sob fotoperíodo de 12 horas de luz.

INFLUENCE OF CULTURE MEDIUM, EXPLANT LENGTH AND GENOTYPE ON MICROPROPAGATION OF Pinus taeda L.

This work aimed to establish a micropropagation protocol for Pinus taeda L. Apical shoots from 5-day seedlings, of different genotypes (F27, B05 and PC), were cultured on WV5 medium supplemented with 44 µM 6-benzylaminopurine (BAP) and 0.05 µM α-naphtaleneacetic acid (NAA), for 14 days, followed by subcultures on growth regulator-free medium. Explants were sectioned into apical shoots and nodal segments for multiplication. Different lengths of explants, BAP concentrations and cultivation periods were tested. Rooting was induced on WV5/2, WV3/2, GDm/2 and agar-water culture media supplemented with 2.68 µM NAA and 0.44 µM BAP for nine days, followed by a 4-week subculture on growth regulator-free medium. It was verified that genotype influenced shoot formation and rooting. The length of 1.0 cm is recommended for nodal segment explants to obtain a high number of axillary shoots. For apical shoots, 0.5 cm explant and WV5 medium formulation allowed the best results for elongation. The best period of subculture was eight weeks, both for nodal segments and for apical shoots. The higher percentage of nodal segments with shoots (99.2%) and the higher average number of shoots per explant (4.0) were obtained with F27 genotype in medium culture containing 2.5 µM BAP. For apical shoots, the best result for elongation was observed for the shorter explants (0.5 cm) on WV5 culture medium (218.3%). The maintenance of in vitro clonal micro garden of vigorous shoots was obtained for two years of 8-week subcultures on WV5 culture medium. The best rooting rate (55.6%) was obtained when shoots were inoculated in agar-water medium with 2.68 μM NAA and 0.44 μM BAP for nine days. Plantlets were successfully acclimatized (85% of survival), so a micropropagation protocol was established.

Induction of oil palm (Elaeis guineensis Jacq. var. Tenera) callogenesis and somatic embryogenesis from young leaf explants

Young leaf explants of oil palm (Elaeis guineensis Jacq.) var. Tenera were isolated from 9 and 13 years old trees. These explants were inoculated onto solid modified MS and Eeuwens basal media containing 107.41µM NAA and associated with 0, 22.62, 45.24 or 67.86 µM 2,4-D and onto another MS basal medium with high concentration 450µM picloram or 450 µM 2,4-D in the presence of 0.03% (w/v) activated charcoal to induce embryogenic calli. After 28 weeks of culture, compact and pearly-white, globular callus was obtained from the leaf number five (L5). The 2,4-D and NAA media concentration combination (67.86 and 107.41µMrespectively) was the optimal media for embryogenic callus induction, while the media containing only NAA (107.41µM) induced the highest percentage of calli formation (30.56%). After 36 weeks of culture, direct embryoids were obtained from leaf number six (L6) on the media containing the combination of NAA (107.41µM) and 2,4-D (45.24 µM). Highest percentage (38.33%) of direct shoot development was also obtained from the same media NAA (107.41µM).

Modified medium for micropropagation of recalcitrant potato cv. Kufri Jyoti

Experiments were conducted to devise an efficient micro-propagation medium for mass multiplication of a high yielding but recalcitrant potato cv. Kufri Jyoti using nodal explant with different concentrations and combinations of NHNOGAand NAA. Virus-indexed nodal cuttings established in full-strength Murashige and 43, 3 Skoog (1962) basal salts and vitamins, supplemented with 0.29 μM GA3 and 0.05 μM NAA was used as explants for the experiment. Among the various media tested, MS medium with increased level of ammonium nitrate (25.79 mM) supplemented with GA3 (0.58 μM) and NAA (0.1 μM) was found to be optimum as it significantly improved the shoot length, number of leaves, number of nodes and inter nodal length after 21 days of sub culturing. The next higher concentration of ammonium nitrate (30.94 mM) supplemented with GA3 (0.58 μM) and NAA (0.1 μM) also exhibited statistically at par response in most of the morphological characters but higher concentration (41.25 mM) retarded all the parameters studied.

Establishment of an efficient in vitro plantlet regeneration system from leaf explants of ornamental crabapple (Malus spp.)

SUMMARYCrabapple (Malus spp.) trees are wild relatives of apple trees and have become increasingly important as an ornamental plant germplasm resource. To establish an efficient regeneration system from leaves would be valuable for the rapid propagation of ornamental crabapple. We therefore evaluated the effects of the type and concentration of various plant growth regulators (PGRs) and carbohydrates used during the regeneration of adventitious buds, shoot multiplication, and rooting in culture. Leaf explants from three cultivars of crabapple (‘Flame’, ‘Strawberry Parfait’, and ‘Royalty’) were used. The most successful types and concentrations of PGRs for the induction of adventitious buds were 4.6 μM thidiazuron (TDZ) plus 1.0 μM α-naphthaleneacetic acid (NAA) for ‘Flame’ and ‘Strawberry Parfait’, and 4.6 μM TDZ plus 2.5 μM NAA for ‘Royalty’. Sucrose at 116 mM was the most effective carbon source to induce buds in ‘Flame’ and ‘Royalty’, while 88 mM sorbitol was the optimum sugar for ‘Strawberry Parfait’. Furthermore, using young leaves placed abaxial side-up on the medium and 10 d of dark treatment promoted leaf regeneration in all three cultivars. Moreover, 6.6 μM 6-benzyladenine (6-BA) and 176 mM sucrose promoted shoot multiplication, while the optimum concentrations for shoot growth were 2.2 μM 6-BA and 58 mM sucrose. In contrast, the best PGRs to induce adventitious rooting were 5.0 μM indole-3-butyric acid (IBA) for ‘Flame’, 4.0 μM IBA for ‘Strawberry Parfait’, and 4.0 μM IBA plus 0.5 μM NAA for ‘Royalty’. Thus, the type and concentration of PGR and carbohydrate source are critical factors in establishing an efficient in vitro regeneration system from leaf explants in crabapple. This information will be valuable when developing efficient methods to genetically transform crabapple or other fruit tree species

Optimization of efficient protocorm-like body (PLB) formation of Phalaenopsis and Dendrobium hybrids

Optimization of the protocorm-like body (PLB) formation of Phalaenopsis and Dendrobium hybrids was performed by determining the effects of plant growth regulators (PGRs) and different parts and division sizes of the PLB. For both genera, the base part was the best for the proliferation of PLBs, yielding the highest number of PLBs on a PGR-free medium for Phalaenopsis and medium containing 0.1μM NAA and 10.0μM BAP for Dendrobium. As regards the division size, four-division sections resulted in a higher PLB formation efficiency for Phalaenopsis, while two-division sections produced a higher PLB formation efficiency for Dendrobium. It is expected that these findings will be applicable to efficient PLB formation of other Phalaenopsis and Dendrobium orchids.

Effect of Physiological Age and Growth Regulators on Micropropagation of Aloe vera Followed by Genetic Stability Assessment

Effect of physiological age and growth regulators on micropropagation of Aloe vera was investigated to develop an efficient protocol for in vitro propagation using shoot bud as explant. Maximum per cent establishment of 72.22 % was observed on MS medium supplemented with 10 µM BA, 5.0 µM Kn and 1.50 µM NAA. The best medium for the multiplication of microshoots was MS medium supplemented with 10 µM BA and 7.5 µM Kn followed by subculturing on same medium for six times at an interval of 4 weeks. Increase in number of microshoots per explant was observed with the increase in subculturing passages. After sixth subculturing 22.70 number of microshoots were obtained from the single explant which was four times more as compared to the first subculturing. The percent root induction was 77.78 % in the sixth subculturing as compared to 36.11 % in the first subculturing on MS medium supplemented with 0.04 % activated charcoal. Genetic stability of the in vitro grown plants was analysed by RAPD which indicated negligible variation.

In vitro regeneration of eight cultivars of Brassica oleracea var. capitata.

Eight cultivars of Brassica oleracea var. capitata and two types of explant (hypocotyl and cotyledon) were tested for their potential to regenerate under in vitro conditions. Hypocotyl and cotyledon explants from 10-d-old seedlings were subcultured onto different callus induction media based on Murashige and Skoog (MS) basal medium supplemented with 1% sucrose and different concentrations and combinations of plant growth regulators. Hypocotyl explants were found to be more suitable for callus induction and organogenesis than cotyledon explants for all cultivars tested. In terms of regeneration, the cv. ‘Amager’ was significantly more responsive than the other cultivars tested and produced the highest number of shoots/buds per explant. Moreover, among five types of media tested, MS + 8.88 μM 6-benzyloaminopurine (BAP) + 0.53 μM α-naphthylacetic acid (NAA) was most effective for shoot regeneration. Rooting was achieved within 10–15 d on all the rooting media, but MS medium containing 5.37 μM NAA produced the maximum number of strong and healthy roots. Plantlets (95%) were subsequently established in the greenhouse, and no phenotypic variations were observed among regenerated plants. This plant regeneration protocol could be suitable for a wide range of cabbage cultivars.