Showing papers on "Micropropagation published in 1998"

Somatic embryogenesis and plant regeneration from floral explants of cacao (theobroma cacao l.) using thidiazuron

Abstract: A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.

Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high-frequency axillary shoot proliferation.

Abstract: A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal segments from mature plants. Of the three different cytokinins – N6-benzyladenine (BA), kinetin, and thidiazuron – evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot formation was a function of cytokinin activity alone, faster bud break coupled with an enhanced frequency of shoot development (92%) and internode elongation were dependent on the synergistic influence of gibberellic acid (GA3) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly influenced by the explanting season. By repeated subculturing of nodal segments harvested from the in vitro-formed axenic shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA3, prolific shoot cultures free from proximal callusing and showing a high-frequency multiplication rate were established. The percentage shoot multiplication (98–100%) as well as the number of shoots per node (six to eight) were highest during the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%) in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic acid and indole-3-butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transfer. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics as well as vegetative and floral morphology.

Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis.

Abstract: A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases.

Culture on sugar medium enhances photosynthetic capacity and high light resistance of plantlets grown in vitro

Abstract: The significance of photosynthetic photon flux (PPF) and sugar feeding for the production of plants in vitro is only poorly understood. Nicotiana tabacum L. plantlets were grown photoautotrophically and photomixotrophically (3% sucrose) at two different PPFs (60 μmol m -2 s -1 and 200 μmol m -2 s -1 ) to investigate the effect of these culture parameters on photosynthetic performance and growth. Photomixotrophically-grown plantlets showed an increase in carbohydrate content, mainly in glucose and fructose. Plant growth, dry matter accumulation and total leaf area were higher under photomixotrophic than photoautotrophic conditions. Not only biomass formation but also photosynthesis was positively affected by exogenous sucrose; the chlorophyll (Chl) content and the light-saturated rate of photosynthetic oxygen evolution were higher in photomixotrophic plantlets. Photoinhibition occurred in plantlets that were grown photoautotrophically at the higher PPF. It became apparent as a loss in Chl content and photochemical efficiency. Photoinhibited plantlets showed a decrease in the D2/LHCII and CP47/LHCII ratios, suggesting a preferential loss of proteins from the photosystem II (PSII) core. The increased content of xanthophyll cycle pigments in photoinhibited plantlets indicated that also protective mechanisms were activated. Photomixotrophic growth of the plantlets prevented the occurrence of photoinhibitory symptoms. Therefore, we conclude that culture on sugar medium increases not only the photosynthetic potential but also the high light resistance of plantlets grown in vitro.

Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture

Abstract: A protocol for micropropagation of Lilium longiflorum using pseudo-bulblets from in vitro shoot-tip-derived stem nodes was developed. Shoot tips from stems of dormant bulbs were cultured on one-half Murashige and Skoog (MS) medium. Stems from plantlets derived from the shoot tips were cut into nodal segments, which were then cultured on one-half MS medium supplemented with 1 μM 6-benzyladenine (BA). Pseudo-bulblets formed on each node after 1 month. An average of 32 pseudo-bulblets were formed on all nodes of the plantlet. Pseudo-bulblets gave rise to multiple shoots when cultured on one-half MS medium supplemented with 2.3 μM BA. Shoots were rooted on one-half MS medium containing 1.1 μM α-naphthaleneacetic acid. A continuous system of propagation by multiplication of pseudo-bulblets with no dormancy period was developed. The 80 flowering plants produced from shoot tip culture were acclimatized in the greenhouse for 3 months and then grown in the experimental garden for 8 months.

Proliferation of meristematic clusters in disposable presterilized plastic bioreactors for the large-scale micropropagation of plants

Abstract: Proliferation of meristematic clusters of several plants in an inexpensive airlift bioreactor system, consisting of a disposable presterilized light transmittable plastic film vessel is described. The optimal shape, size, and structural function of the disposable plastic bioreactor are based on the bubble column and airlift glass bioreactors. The disposable bioreactors are designed in a conical configuration with a single inoculation and harvest port and multiple use dispensing and mixing accessories. Shearing damage and foaming problems known to exist in bioreactors due to the plant's rigid cell wall and size were greatly reduced in the disposable plastic bioreactors. The disposable bioreactors were used for propagule proliferation and growth, using meristem and bud clusters of potato, fern, banana, and gladiolus. The clusters' biomass increased five-to eightfold over a period of 26–30 d, depending on the species. The clusters were separated mechanically by a chopper made of a grid of knives. The chopped propagules were inoculated to agar medium for further growth and developed into transplantable plants. In the case of gladiolus and potato, corms and tubers developed in a sucrose-elevated storage organ induction medium, respectively, after the initial formation of small shoots. The plantlets and storage organs were transplanted to an acclimation greenhouse and continued to grow with a 95–100% survival, depending on the species. Plant development was followed for a period of 16 wk in fern and 12–14 wk in potato, banana, and gladiolus and normal shoot and leaf growth was observed. The feasibility of large-scale liquid cultures for plant micropropagation is discussed.

In vitro propagation of Holostemma annulare (Roxb.) K. Schum., a rare medicinal plant

Abstract: A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.

Successful plant regeneration from callus cultures of Centella asiatica (Linn.) Urban

Abstract: A successful procedure was established for in vitro plant regeneration from callus derived from stem and leaf explants of Centella asiatica on semisolid modified Murashige and Skoog's [7] medium supplemented with 2.0 mg L3 kinetin and 4.0 mg L3 a-naphthaleneacetic acid. The rate of shoot-bud regeneration was the highest (42.8 and 54.3 shoots/culture in stem and leaf derived callus respectively) after 4 weeks of subculture on 4.0 mg L3 6-benzyladenine, 2.0 mg L3 Kn, 0.25 mg L3 a-naphthaleneacetic acid and 20 mg L3 adenine sulfate. Differentiated shoots rooted within 11 days in 1/2 strength MS basal salts supplemented with 0.5 mg L3 indole-3-acetic acid and 2% (w/v) sucrose. About 85% of rooted plantlets were acclimatized and transferred to the greenhouse.

Micropropagation of Bambusa edulis through nodal explants of field-grown culms and flowering of regenerated plantlets

Abstract: Nodal explants obtained from 10-year-old field-grown culms of Bambusa edulis produced multiple shoots on a Murashige-and-Skoog-based medium supplemented with 0.1 mg/l of thidiazuron (TDZ). Hundreds of regenerated shoots rooted well on a medium supplemented with 0.01 mg/l TDZ and 0.5 mg/l 2,4-dichlorophenoxyacetic acid and were successfully transferred to soil for field trials. Albinism occurred at the rate of about 30% among the regenerated shoots, and isolated albino shoots also proliferated on the medium containing TDZ. Some of the green and albino shoots also flowered on the medium containing TDZ. A potted plant also flowered and survived after flowering.

Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.).

Abstract: A restricted part of the undeveloped stem of the garlic clove, called the “stem disc”, which is just under the basement of the immature foliage leaves, proved to be a very potent explant for the micropropagation of garlic. Twenty to thirty tissue-cultured shoots consistently were differentiated from a single clove during 1 month of culture on phytohormone-free Linsmaier and Skoog medium. In addition, more than 90% of the shoots formed bulblets in vitro during an additional 1 month of culture. Pretreatment of the garlic bulbs at 4 °C for approximately 8 weeks before preparing the stem discs enhanced both shoot development and bulblet formation. This novel method for culturing garlic was designated the stem-disc culture method. Shoot development in this type of in vitro culture apparently is divided into four stages: expansion of tissue zones surrounded by the basal parts of the immature foliage leaves, formation of dome-shaped structures, bud differentiation directly from each dome, and development into shoots and bulblets. The dome-shaped structures appeared within 5 days of the onset of culture and had developed independently into shoots approximately 1 cm high 3 weeks later. Histological observations showed that both the internal cell organization and formation process of the dome-shaped structures were similar to those in the meristem. In addition, events leading to the formation of these dome-shaped structures appeared to be initiated by vigorous cell division in the epidermis of concentric tissue zones surrounded by the basements of immature foliage leaves. The results of several field trials showed that the stem-disc culture method is useful for the production of garlic seed plants, including virus-free plantlets. Furthermore, it is a novel field cultivation system for garlic in that the seedlings produced by in vitro-induced bulblets are used as seed instead of the usual cloves.

Natural light as an alternative light source for the in vitro culture of banana (Musa acuminata cv. ‘Grande Naine’)

Abstract: The concept of using sunlight for micropropagation systems is proposed as a way of reducing tissue culture costs. Shoot tips of Musa acuminata cultivar ‘Grande Naine’ were cultured in a non-controlled natural light environment at the IAEA Laboratories, Austria during summertime. Significantly more shoots were produced by plantlets cultivated in a sunlit room with photosynthetic photon flux densities (PPFD) fluctuating up to 570 μmol m-2 s-1, temperatures between 23 and 30 °C and photoperiods of 12 to 16-h, than by plantlets under artificial light in a growth chamber providing controlled conditions of a constant PPFD of 65 μmol m-2 s-1, temperatures ranging from 23 to 29 °C and a 16-h photoperiod. Highest multiplication rates were achieved in a greenhouse with PPFD reaching 860 μmol m-2 s-1 and temperatures of 18 – 43 °C, but browning of leaves and loss of turgor occurred. Nevertheless, rooted plantlets showed 100% survival during acclimatisation and normal development. Photoperiods of 12 – 16 h did not affect the multiplication rates.

Genotypic Response of Cowpea Vigna unguiculata (L.) to In Vitro Regeneration from Cotyledon Explants

Abstract: A plant regeneration system applicable to 17 cowpea genotypes was developed. Cotyledons were initiated on 1/3 MS medium containing 15 to 35 mg N6-benzyladenine (BA) per 1 (66.6 to 155.3 µM) for 5 to 15 d. For shoot regeneration, the explants were transferred to a medium containing 1 mg BA per 1 (4.4 µM). Within 1 wk, shoot formation was visible at the proximal end of the cotyledons. Regeneration percentages (1% to 11%) and the numbers of shoots (4 to 12 per explant) were significantly influenced by genotype. Culture duration and BA concentration in the initiation stage significantly affected regeneration capacity. Explants initiated on media containing 15 mg BA per 1 for 5 d resulted in the highest percentage of explants capable of regeneration. Conversely, the highest number of shoots was obtained from explants initiated on media supplemented with 35 mg BA per 1. Whole plants were obtained on a plant growth regulator-free medium. To our knowledge, this is the first report of plant regeneration from U.S. commercial cowpea cultivars and breeding lines. This system is adaptable to diverse cowpea genotypes and will facilitate cowpea genetic transformation.

Factors influencing induction, propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

Abstract: A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots.

A protocol for micropropagation of Alpinia galanga

Abstract: Emerging buds of rhizome of Alpinia galanga Willd produced shoots and roots simultaneously when cultured in MS medium supplemented with kinetin 3.0 mg l-1. Each explanted shoot bud produced 8 shoots in average and roots simultaneously within 8 weeks. Shoot proliferation could be continued even after a year by transferring each divided shoot explant to the same medium. Regenerated plantlets could be sucessfully transferred to soil where they grew well within 10–12 weeks with 80% survivality.

Somatic Embryogenesis and Plant Regeneration in Different Organs of Euterpe edulis Mart. (Palmae) : Control and Structural Features

Abstract: Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L−1). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L−1) and NAA (0.1 mg L−1). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L−1). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.

Micropropagation of 21 species of Mexican cacti by axillary proliferation

Abstract: We have developed micropropagation systems for 21 species of Mexican cacti using explants from seedlings germinatedin vitro or shoot segments of juvenile 2–3-yr-old greenhouse plants. The species propagated belong to the generaAstrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, andStenocactus. Multiple shoot formation from areoles was achieved in Murashige and Skoog (MS) medium supplemented with either 1 or 2 mg N6-benzyladenine (BA) per 1 (4.44 or 8.87 μM) or BA at 1 or 2 mg/l plus naphthaleneacetic acid at 0.1 or 1 mg/l (0.54 or 5.37 μM). The requirements of growth regulators for optimal shoot proliferation, the velocity of the response, and the number of buds produced by explant were different among the genera and species studied. Rooting of the shoots generatedin vitro was achieved in MS medium supplemented with indoleacetic acid at 0.5–1 mg/l (2.85–5.71 μM) or indolebutyric acid at 0.5–1 mg/l (2.46–4.90 μM). Finally, 70–95% of the rooted plants transferred to potting medium survived.

Variability among Vitis vinifera cultivars in micropropagation, organogenesis and antibiotic sensitivity

Abstract: Susceptibility to numerous diseases, pests and abiotic stresses make it necessary to use a high level of chemical In vitro culture of 32 Vitis vinifera cultivars and intras- control and to adopt specific cultural practices. Genetic pecific hybrids was initiated from axillary buds. The improvement through conventional methods, i.e. hybriddevelopment of roots and shoots was followed during ization, does not appear to be practical in order to obtain 14 subcultures on two hormone-free micropropagation less susceptible scion cultivars with similar wine qualities media. One medium (M64) was used until the 8th sub- to those of classic cultivars. Non-conventional approaches culture, after which it was replaced by G90 medium have therefore been proposed (Bouquet, 1989; Meredith, which was found more suitable for plantlet growth and 1996). A prerequisite to develop such approaches for permitted an increase in the time between subcul- grapevine is the ability to perform eYcient in vitro techtures. Large differences in plantlet growth between niques (Monette, 1988), but genetic variability of the cultivars were demonstrated on both media. The material has to be taken into account (Bouquet et al., number of roots had greatest variability between culti- 1982; Stamp et al., 1990; Torregrosa and Bouquet, 1996). vars (CV=39%) as compared with stem length (CV= Micropropagation in grapevine was first performed 21‐22%) and number of nodes (CV=12‐14%). The using entire plantlets obtained from microcuttings (Galzy, number of nodes was positively correlated with shoot 1961). More recently, the induction of bud proliferation length whereas root number appeared to be poorly has been shown to provide an alternate pathway to positively correlated with shoot development. grapevine micropropagation (Jona and Webb, 1978). Adventitious bud regeneration from leaves was studied Micropropagation produces suitable tissues for regenerafor 20 cultivars and averaged 36.7% of regenerative tion through organogenesis and embryogenesis (Stamp explants with large differences between cultivars and Meredith, 1988). In addition, in vitro culture of (CV=47%). However, organogenic competence was plantlets permits the rapid propagation of the genotypes not correlated with micropropagation ability. High obtained by non-conventional methods. sensitivity of Vitis vinifera plantlets to kanamycin and Regeneration of whole plants by somatic embryogenesis hygromycin was demonstrated with a strong inter- and organogenesis has been intensively studied (for action between cultivar and antibiotic. At 0.8 mg dm’ review, Torregrosa, 1995). The genetic engineering of 3, hygromycin was lethal to plantlets. This effect was somatic embryos was first reported by Mullins et al. only observed at 4 mg dm’3 for kanamycin, whereas (1990) and has been used to transfer genes of potential 1m g dm’3 stimulated the development of plantlets. interest through Agrobacterium-mediated transformation (Le Gall et al., 1994; Krastonova et al., 1995). Colby

Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron

Abstract: The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10μM) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate.

In vitro induction of tetraploids in colchicine-treated cocoyam plantlets

Abstract: Colchicine was applied in liquid B5 medium supplemented with 0.49 μm N6(δ2-isopentenyl)adenine to tissue culture derived cocoyam plantlets. Tetraploids (4n=52) and octoploid (8n=104) were produced by treating plantlets with 1.25 mM and 2.50 mM colchicine. Of all the plantlets treated with 1.25 mM or 2.5 mM, 16.7% and 20.0% respectively were mixoploids. These mixoploids were of the 2x-4x, 2x-4x-8x and 4x-8x types with 4x cells being dominant. Non-treated plantlets and plantlets treated with dimethylsulphoxide (DMSO) had typical diploid chromosome counts, 2n=26. Flow cytometric analysis showed a significant increase in relative amounts of DNA in nuclei of colchicine-treated tissues corresponding to 4C levels and peaks appearred at excitation channels of 100.05 ± 4.4. Nuclei of non-treated plantlets had 2C levels of relative DNA amounts, peaks occurring at fluorescence channels of 52.8 ± 2.7. Most of the cells of non-treated plantlets were arrested at the G1 phase (90.5%) with negligible amounts at S (6.9%) and G2/M (2.6%). Colchicine-treated plantlets had relatively lower number of nuclei at G1 (33.6 or 33.3% for 1.25 mM or 2.50 mM) and significantly higher number of nuclei at G2 (47.1 or 46.0% for 1.25 mm or 2.5 mm). Non-treated tissues had significantly lower nuclei at S-phase compared to colchicine-treated plantlets.

Protocol for in vitro propagation of Excoecaria agallocha L., a medicinally important mangrove species.

Abstract: An in vitro propagation protocol has been developed for Excoecaria agallocha L. (Euphorbiaceae), a mangrove species. Nodal segments were used for axillary shoot proliferation. One shoot from each node of binodal explants was observed 3 weeks after inoculation. The best axillary sprouting was seen on a newly formulated medium containing BA, Zeatin and IBA in concentrations of 13.3 μM, 4.65 μM and 1.23 μM, respectively. The new medium, first used in this study, has a specific composition of major nutrients, MS micronutrients and iron compounds. Nodal segments from rooted cuttings and seedlings responded better than those of mature tree explants. Multiple shoot induction was complemented with efficient shoot elongation, and repeated subculture of binodal segments from axillary shoots resulted in 10–12 shoots per explant in 3 months. Rooting was achieved by growing shoots in the new medium with 0.23 μM IBA. Regenerated plants were successfully acclimatized to the natural environment, and about 85% of plantlets survived under ex vitro conditions. This is the first report of micropropagation in the genus Excoecaria and also in mangrove tree species.

Rapid vegetative multiplication in Colocasia esculenta (L) Schott (taro)

Abstract: Initial multiplication of taro was achieved by dividing corms into pieces, each with several buds, and planting them in sterile potting mix. Surface planting and decapitation of the new primary meristem on each sucker forced additional shoot meristems to grow. Meristems of six cultivars were cultured on a modified Murashige and Skoog medium plus TDZ. In experiments with the cultivar Niue, explants cultured on modified MS medium plus 2.6 μM (0.6 mg L-1 TDZ grew more vigorously than on media including BA. Transfer to 4.3 μM (1.0 mg L-1 TDZ subsequently gave a 15–25 fold increase in production of plantlets per four week culture period compared to four-fold with BAP. The two stage propagation technique described in this paper is an efficient way to produce taro plants for commercial planting or the production of pathogen-tested material.

Effect of different growth regulators and genotype on in vitro propagation of jojoba

Abstract: Explants of jojoba (Simmondsia chinensis (Link) Schn.) from randomly selected plants were propagated in vitro. On modified Murashige‐Skoog (MS) medium supplemented with 1 mg/litre BA (N6‐benzyl‐adenine) there was a 4.6‐fold increase in shoot numbers at 30 days. After 15 days on modified MS medium supplemented with 3 mg/litre IBA (indolebutyric acid) c. 25% of the shoots had developed roots. The best exflasking survival rate of the rooted plantlets (90%) was observed when the plantlets were transferred to an organic mixture, Agrosoil™. Subsequent growth rates in Agrosoil™ were higher than in other potting media tested. On the other hand, the response of each clone was highly variable, some exhibiting 75% root formation at 60 days while others displayed little response.

Effect of genotype on micropropagation of walnut trees (Juglans regia)

Abstract: Embryos of Juglans regia originating from elite trees, selected for their wood quality were cultured on DKW medium supplemented with 4.44 μM 6-benzylaminopurine (BA) and 0.005 μM indole-3-butyric acid (IBA). Results on the effects of cytokinin (BA) and auxin (IBA) on shoot development indicated that elongation is enhanced with 2.22 μM BA while the formation of new axillary shoots is favored by 4.44 μM BA and 0.005 μM IBA. It was, also, observed that different genotypes had different requirements for growth regulators. Significant differences were observed among the multiplication rates of twelve different clones of J. regia. The effect of genotype is obvious in the rooting phase, as well. Some clones exhibit high rooting ability (95 %) and some low (5 %).

Teak propagation by in vitro culture

Abstract: Possibilities of propagating teak by in vitro culture were assessed from a realistic standpoint of production within the framework of a Research and Development proiect in Sabah (East Malaysia). The tissue culture conditions developed noticeably improved the germination rate of poor germination capacity seed lots. Those obtained in vitro could be further micropropagated. The micropropagation protocol was conceived as simply as possible to multiply vegetatively, through axillary budding-derived shoots, teak genotypes issuing from in vitro germination or of different ages growing outdoors. For this latter type of plant material, the cultures were initiated using l to 2 cm long mononodal portions collected from preferably actively growing shoots. Cultures could also be initiated from shoot apical meristems with a success rate of 60 %. Regardless of their origin, the genotypes were micropropagated according to an exponential multiplication rate of 3n, with n as the number of the 2-month-duration subcultures. Acclimatization was carried out under mist-system in nursery conditions with more than 90 % success. The tissue-culture produced plants developed vigorously during 3 to 4 months of culture in nursery until they reached a suitable size for planting out. To date, 50,000 in vitro plants have been produced on an experimental scale and at low cost applying this protocol that is suitable for intensive production of superior quality teak planting stock. Considering these achievements, the prospects of in vitro culture for propagating selected teak genotypes compared to what can be expected from propagation by rooted cuttings are discussed, mainly from a development point of view. (Resume d'auteur)

Micropropagation studies in Ceropegia SPP

Abstract: The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l−1) N6-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l−1) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l−1) BA and 23.2 μM (5 mg·l−1) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l−1) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l−1) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l−1) BA. Rooting of the shoots was possible both byin vitro andex vitro means.

Efficient in vitro shoot regeneration responses of Phaseolus vulgaris and P. coccineus

Abstract: Phaseolus vulgaris L. is the most important economic species within the genus Phaseolus, and it is grown in all parts of the world. Genetic improvement by conventional breeding has met considerable success, although production of hybrids between species within the genus has been limited due to sexual incompatibility or other evolutionary lethalities. Recent advances in tissue culture have offered the opportunity to produce cultivars which could not be obtained by conventional breeding methods, but regeneration protocols are influenced by the genotype. A standard regeneration procedure was assessed for its applicability to elite breeding lines of P. vulgaris L. and landraces of P. coccineus L. from seedling explants containing a cotyledon and a small portion of the split embryonic axis. In vitro culture response and regeneration ability varied significantly between species and amongst genotypes. P. coccineus produced more shoots per explant with a higher rooting efficiency than P. vulgaris. These significant genotype effects suggest that genetic factors are important in the response to in vitro tissue culture. Different genotypes were identified, which were more competent to in vitro culture and could produce highly responsible hybrids. This in vitro culture system carry a high potential for propagation of P. vulgaris and P. coccineus, and subsequent exploitation of hybrid forms, which could also be incorporated into somatic cell approaches to improve these species.

High frequency of plant regeneration from anther culture in flax, Linum usitatissimum L.

Abstract: The effects of induction medium compositions on flax anther culture were investigated in order to improve the efficiency of callus induction and plant regeneration. Anthers were inoculated onto the modified MS medium supplemented with five different combinations of plant growth regulators. The medium containing the combination of 2 mg/l 2,4-dichlorophenoxy-acetic acid (2,4-D) and 1 mg/l 6-benzylaminopurine (BAP) produced a significantly higher number of calli forming shoots/100 responded anthers and a significant increase in overall efficiency of regeneration than the same basal medium containing 1 mg/l α-naphthalene-acetic acid (NAA) and 2 mg/l BAP (CK). Among the five levels of thiamin hydrochloride tested, the modified MS medium containing 10 mg/l thiamin hydrochloride significantly increased the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration compared with the same basal medium containing 0.1 mg/l thiamin hydrochloride. Maltose concentration had a significant effect on the percentage of anthers producing calli, the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration. The medium containing 6% or 9% maltose produced the highest overall efficiency of regeneration among the five levels of maltose evaluated. Sucrose concentration significantly affected the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration, and dramatically affected the frequency of microspore-derived plants and the frequency of spontaneous chromosome doubling in microspore-derived plants. The efficiency of doubled haploid line production obtained in this study appears adequate for applied breeding programmes.

Optimization of the mineral composition of in vitro culture media

Abstract: Plant tissue culture requires the optimization of growth media. The effect of eight different macronutrient formulations on the in vitro culture of Gatharanthus roseus nodes was investigated: the caulogenesis rate decreased with the overall ion concentration and the ammonium content of the growth medium. The methodology proposed for the optimization of the mineral composition of the growth medium is based upon the adjustment of the macronutrients contents to the plant requirements. Such an approach was used for the comparison between the effects of the optimized growth medium and those of the usual Murashige and Skoog solution in the growth of the multiple shoots issued from Solatium paludosum root calluses. The new formulation MH1 [with a diminution of the ionic concentration, nitrogen (N) supplied in the form of nitrate and calcium (Ca) in larger amounts than potassium (K)] doubled the biomass production and tripled the yield of solamargine, a secondary metabolite.

Hormonal activities of 5-aminolevulinic acid in callus induction and micropropagation

Abstract: The role of 5-aminolevulinic acid (5-ALA) as a precursor of chlorophyll and a herbicide is well documented. 5-ALA as a Plant Growth substance is also proven in recent times. In the present report, to elucidate the physiological effects of 5-ALA, the compound was used in in vitro studies using MS medium supplemented with 5-ALA at 2, 5 and 10 mg l-1. Leaf and cotyledonary node were used as the explants. In vitro studies confirmed the hormonal role of 5-ALA by striking proliferation of callus paripassu induction of rooting and shooting with a profound effect of the former than the latter. Thus, 5-ALA has the dual properties of auxin and cytokinin in the induction of callusing and rhizogenesis, and shooting respectively.