Showing papers on "Kinetin published in 2006"

Seed enhancement with cytokinins: changes in growth and grain yield in salt stressed wheat plants

Abstract: Cytokinins are often considered abscisic acid (ABA) antagonists and auxins antagonists/synergists in various processes in plants. Seed enhancement (seed priming) with cytokinins is reported to increase plant salt tolerance. It was hypothesized that cytokinins could increase salt tolerance in wheat plants by interacting with other plant hormones, especially auxins and ABA. The present studies were therefore conducted to assess the effects of pre-sowing seed treatment with varying concentrations (100, 150 and 200 mg l−1) of cytokinins (kinetin and benzylaminopurine (BAP)) on germination, growth, and concentrations of free endogenous auxins and ABA in two hexaploid spring wheat (Triticum aestivum L.) cultivars. The primed and non-primed seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) were sown in both Petri dishes in a growth room and in the field after treatment with 15 dS m−1 NaCl salinity. Both experiments were repeated during 2002 and 2003. Among priming agents, kinetin was effective in increasing germination rate in the salt-intolerant and early seedling growth in the salt-tolerant cultivar when compared with hydropriming under salt stress. Thus, during germination and early seedling growth, the cytokinin-priming induced effects were cultivar specific. In contrast, kinetin-priming showed a consistent promoting effect in the field and improved growth and grain yield in both cultivars under salt stress. The BAP-priming did not alleviate the inhibitory effects of salinity stress on the germination and early seedling growth in both cultivars. The increase in growth and grain yield in both cultivars was positively correlated with leaf indoleacetic acid concentration and negatively with ABA concentration under both saline and non-saline conditions. The decrease in ABA concentration in the plants raised from kinetin-primed seeds might reflect diminishing influence of salt stress. However, the possibility of involvement of other hormonal interactions is discussed.

Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker--an endangered medicinal herb.

Abstract: An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.

Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus corniculatus L.

Abstract: We determined the effects of zeatin (ZEA), isopentenyl adenine (2iP), kinetin (KIN), benzyladenine (BA), and thidiazuron (TDZ) on seed germination, elongation of seedling shoots and roots, frequency of regeneration, and the number of regenerants per seedling in Lotus corniculatus L. Sterilized seeds were cultured in vitro on Murashige and Skoog (1962) medium containing 3% sucrose, 0.7% agar, and various cytokinins (0, 0.08, 0.22, 0.35, 0.80, 2.20, and 3.50 μM). After 30 days, seedlings were transferred to cytokinin-free medium for another 60 days. All cytokinins stimulated the rate and percentage of seed germination at least twofold in optimum concentrations; TDZ and ZEA were the most active, followed closely by BA, whereas KIN and 2iP stimulated germination in higher concentrations only. Elongation of shoots and roots was strongly inhibited at the lowest TDZ and BA concentrations, whereas ZEA, KIN, and 2iP exerted moderate, dose-dependent inhibition. The frequency of regenerant-producing seeds was highest on ZEA and BA, whereas the greatest number of regenerants per seedling was found on TDZ. It is concluded that the culture of seeds on cytokinin-containing media, followed by transfer to cytokinin-free medium, is a suitable procedure for rapid production of a large number of uniform regenerants. The presumed role of particular cytokinins is discussed.

Optimizing tissue culture media for efficient transformation of different indica rice genotypes

Abstract: An efficient system was established for high frequency embryogenesis and regeneration of indica rice, Oryza sativa L. cv. MDU 5. Basic media, carbohydrate sources and concentrations, agar concentrations, amino acids, cytokinins and auxins were evaluated in callus induction and regeneration media to establish the most efficient regeneration protocol for MDU 5 indica rice. The optimized callus induction and regeneration media consisted of the basic MS salt mixtures and vitamin solutions supplemented with 4% maltose, 1g/L casein hydrolysate and 50 mg/L tryptophan, solidified with 1% agar. The callus induction medium was supplemented with 2,4−D 2 mg/L, kinetin 0.5 mg/L, indole acetic acid 1 mg/L, and 6−benzyl aminopurine 0.5 mg/L whereas the media for regeneration phase comprised kinetin 2 mg/L, indole acetic acid 1 mg/L and 6−benzyl aminopurine 2 mg/L. The media optimized for MDU 5 was analyzed for response of 73 other indica genotypes. Of these indica varieties 64 genotypes yielded 98.5% callus induction within eight days, 59% embryogenic calli formation, initiation of multiple green buds within eight days and a regeneration rate of 90%. The embryogenic calli derived were used for transformation with Agrobacterium tumifaciens (LBA 4404 or EHA 101 strains). Two binary vectors (pKHG4 and pIG121Hm) containing hph and GUS genes were used in these transformation studies. Fifty-one genotypes responded to the optimized media by producing hygromycin−resistant calli. The -histochemical test for s−glucuronidase activity was positive from 32 genotypes with the transformation efficiencies ranging between 7.0% and 8.3%.

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens : a valuable medicinal plant

Abstract: A rapid and efficient protocol for the large-scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15-day-old aseptic seedlings is described. Of the three different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half-strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α-naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole-3-butyric acid (IBA). The in vitro-raised plantlets with well-developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.

In vitro studies on effects of plant growth regulators on callus and suspension culture biomass yield from Gymnema sylvestre R.Br

Abstract: Callus cultures were initiated from nodal segments and leaf explants of Gymnema sylvestre on Murashige and Skoog (1962) medium containing basic salts and 30 g/l sucrose supplemented with different concentrations (0.10, 0.25, 0.5, 1.0, 2.0 and 5.0 mg/l) of 2,4-dichlorophenoxy acetic acid (2,4-D), �-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), kinetin (KN) and 6-benzyladenine (BA). Callus induction was observed in 0.5 mg/l of 2, 4-D supplemented medium for both explants. At the initial stage, some parts of explants enlarged and gave raise to pale yellowish calli after 2-3 weeks of incubation. The harvested cell biomass was subjected to extraction of active principles. In this study, cell biomass extracts were compared with extracts from leaves of naturally growing gymnema plants. HPLC analysis of these extracts showed that the main components of the active principles namely gymnemic acids and gymnemagenin were present in sufficiently large amounts in the cultured undifferentiated cells.

Somatic embryogenesis and plant regeneration from different wild diploid cotton (Gossypium) species

Abstract: Calli were successfully induced from hypocotyls of eight wild diploid cotton species (Gossypium) on MSB (MS salts and B5 vitamins) medium supplemented with 0.09 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.32 μM KT (kinetin). Plant growth regulator (PGR) combinations, adding GA3 (Gibberellic acid), high inorganic salt stress, and PGR-free media were used to induce embryogenic calli from nonembryogenic calli. Embryogenic cultures were induced from G. aridum S. (D4 genome), G. davidsonii K. (D3-d genome), G. klotzschianum A. (D3-k genome), G. raimondii U. (D5 genome), and G. stocksii M. (E1 genome). We then observed somatic embryogenesis in the five species while calli of G. africanum V. (A1-2 genome), G. anomalum W. (B1 genome), and G. bickii P. (G genome) remained nonembryogenic. Somatic embryogenesis was adjusted by changing sugar sources, regulating combinations of PGRs, and using cell suspension culture. Embryos at various developmental stages produced mature and germinating embryos when cultured on filter paper placed on the media containing different sugar sources. The utility of different sugar sources promoted globular embryos developing into cotyledonary stage and increased the frequency of cotyledonary embryos developing into normal plants. Normal plantlets were regenerated from G. davidsonii, G. klotzschianum, G. raimondii, and G. stocksii. Only abnormal plantlets were obtained in G. aridum. This work will contribute to broadening the number of regenerable cotton species and provide foundations for somatic hybridization in cotton to create new germplasm.

Direct plant regeneration from cultured young leaf segments of sugarcane

Abstract: Young leaf segments (1.0–1.5 cm) excised from spindle explants of three commercial sugarcane varieties viz. Co J 64, Co J 83 and Co J 86 were cultured on different media compositions based on Murashige and Skoog (1962) salts. Cultured explants exhibited swelling followed by direct shoot regeneration on media containing naphthaleneacetic acid, in all the three varieties. Highest frequency 83.12% shoot regeneration occurred on Murashige and Skoog medium supplemented with naphthaleneacetic acid (5.0 mg l−1) and kinetin (0.5 mg l−1) in variety Co J 83. Medium devoid of naphthaleneacetic acid and supplemented with only kinetin did not induce direct shoot regeneration in any of the varieties thus tried. Subsequently profuse rooting of shoots was observed on the same medium and complete plantlets were recovered within 6 weeks. The plantlets were hardened and transferred to soil. Tissue culture derived field-grown plants were normal and exhibited faster growth and better tillering. This developed single step method of direct plant regeneration can be used for rapid mass cloning and genetic transformation of sugarcane.

In vitro clonal propagation through mature nodes of Tinospora cordifolia (Willd.) Hook. F. & Thoms.: An important ayurvedic medicinal plant

Abstract: A protocol was developed for rapid clonal propagation of the important medicinal climber, Tinospora cordifolia, through in vitro culture of mature nodal explants. Shoots were initiated on both Murashige and Skoog (MS) medium and woody plant medium (WPM) supplemented with 2.32 μM kinetin (KIN). Of the two basal media tested, WPM was found to be superior to MS medium for the induction of multiple shoots. Among the cytokinins tested, N6-benzyladenine (BA) was more effective than KIN for axillary shoot proliferation. KIN was superior to BA in terms of shoot elongation. An average multiplication rate of 6.3 shoots per explant was obtained with WPM supplemented with 8.87 μM BA. Shoot clumps harvested from this medium were transferred to WPM supplemented with 2.22 μM BA and 4.65 μM KIN for shoot elongation. Elongated shoots were rooted in half-strength MS medium supplemented with 2.85 μM indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to sand and established with 80% survival.

Rapid clonal propagation of Vitex trifolia

Abstract: This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 - 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.

An efficient plant regeneration system for mucuna pruriens l. (dc.) using cotyledonary node explants

Abstract: The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.

In vitro multiplication of Ocimum gratissimum L. through direct regeneration

Abstract: The objective of this study was to develop a rapid system for regeneration of the important medicinal plant, Ocimum gratissimum L, from nodal explant. Single node explants were inoculated on basal MS (Murashige and Skoog, 1962) medium containing 3% (w/v) sucrose, supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for direct plant regeneration. Maximum numbers of shoot (14.3± ± ± ±1.5) were observed on the medium containing 0.5 mg/l BAP and 0.25 mg/l IAA after four weeks of culture. Regenerated shoots were separated and rooted on same half strength MS medium supplemented with 0.5 mg/l of IAA alone for three weeks. Well-developed complete plantlets were transferred on to specially made plastic cup containing soilrite. Acclimatized plantlets were successfully grown in garden soil.

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Abstract: Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm−3 kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm−3 NAA. Rooted shoots were successfully acclimatized and established in soil.

In vitro organogenesis and plant formation in cucumber

Abstract: In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 µM sucrose, 0.8 % agar, 3.62 µM 2,4-dichlorophenoxy acetic acid and 2.22 µM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant−1) was achieved on MS medium supplemented with 8.88 µM BA, 2.5 µM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 µM) favoured shoot elongation and indole 3-butyric acid (7.36 µM) induced rooting. Rooted plants were hardened and successfully established in soil.

Efficient regeneration system from wheat leaf base segments

Abstract: Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm−3 2,4-dichlorophenoxyacetic acid + 1 mg dm−3 naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm−3 kinetin (6.3 plantlets per segment).

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

Abstract: High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 µM N6-benzyladenine (BA) + 1 µM 2,4-dichlorophenoxyacetic acid (2,4-D) Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d Of the two cytokinins, 5 µM BA was optimum for eliciting morphogenic response in 8333 and 7083 % cultures with an average of 416 ± 047 and 370 ± 056 shoots in cotyledon and leaflet derived calli, respectively The addition of 05 µM α-naphthaleneacetic acid (NAA) to MS + 5 µM BA further elevated the maximum average number of shoots to 1208 ± 104 and 537 ± 052 for cotyledon and leaflet calli, respectively The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA Nearly 95 % shoots developed an average of 54 ± 041 roots on half strength MS medium supplemented with 10 µM IBA

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

Abstract: A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L-1 IAA and 0.5 mg L-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g-1 dry weight) and the mother plants (12.01 mg g-1 dw).

Análise de crescimento de plantas de manjericão tratadas com reguladores vegetais

Abstract: GROWTH ANALYSIS OF BASIL PLANTS SUBMITTED TO PLANT GROWTH REGULATORS The objective of this study was to evaluate the effect of plant growth regulators on the development of basil plants (Ocimum basilicum L.). The experiment was seeded in 12-liter pots and carried out in a greenhouse. The experimental design was a randomized block with four treatments, four replications, and five harvest times. Treatments consisted of the following plant growth regulators applied as foliar sprays: gibberellic acid (GA3), 2-chloroethyl phosphonic acid (ethephon), and kinetin at 100 mg L -1 , which were prepared in aqueous solution. Plant growth regulator applications were performed at 40, 60, and 80 days after planting (DAP) and plant development was evaluated during harvest time, at 14-day intervals, 50, 64, 78, and 106 DAP. Plants treated with kinetin showed the highest physiological index values, and exhibited greater development due to increases in leaf area and dry matter provided by this plant growth regulator.

Shoot initiation and multiplication from a mature tree of Terminalia arjuna roxb

Abstract: This study describes a protocol for the regeneration of complete plantlets of Terminalia arjuna from nodal explants of mature trees. Shoot multiplication from nodal explants was achieved by culturing on Murashige and Skoog (MS) medium containing different concentrations of 6-benzyladenine (BA), thidiazuron or kinetin, or BA in combination with α-naphthaleneacetic acid (NAA). The best shoot multiplication response was obtained from nodal explants grown on modified MS (half-strength major salts and Fe-EDTA) medium containing 4.44 μM BA and 0.53 μM NAA. Seasonal variations significantly affected the proliferation potential of nodal explants and best proliferation was observed from explants collected during April to May. Microshoots were rooted on half-strength MS medium with 4.92 μM IBA. The rooted shoots were acclimatized successfully.

Use of plant growth regulators in micropropagation of Kappaphycus alvarezii (Doty) in airlift bioreactors

Abstract: The effects of plant growth regulators on cal- lus induction rate and regeneration of K. alvarezii ex- plants was evaluated. K. alvarezii calluses were induced in vitro with kinetin (K), 6-benzylaminopurine (B), 1- naphtalene acetic acid (N) and spermine (S). After 30 days, K. alvarezii explants produced filamentous cal- luses and isolated crystalline filaments growing from the medullar region and from cortical cells at the cut edge. The plant growth regulators 1-naphtalene acetic acid (1 mg L −1 ) and 6-benzylaminopurine (1 mg L −1 ) and the 1-naphtalene acetic acid + kinetin + spermine (1, 1, 0.018 mg L −1 respectively) combination pro- duced 85 to 129% more calluses, with significant differ- ences versus the control ( p < 0.05). Spermine at 0.018 mg L −1 produced calluses in the apical, intercalary and basal regions of explants. Spermine also reduced callus induction time to 7 days, which is faster than previ- ously reported induction times with other plant growth regulators. An airlift bioreactor was designed and char- acterized to micropropagate K. alvarezii calluses. The bioreactor had mixing times ranging from 4.6-10.3 sa tT90 and T95, which is shorter than those for the

In vitro propagation of Notocactus magnificus

Abstract: Most commercially grown cacti can be easily propagated by seed and/or cuttings. A group of rare and endangered species does not fit into this category and is therefore a good candidate for in vitro propagation productions as a tool to overcome habitat and plant-destruction. The number of rare and endangered species of Cacti goes into about 100. Many show a low production and germination of seeds and plantlets are prone to damping-off, making the in vitro propagation a feasible alternative for the multiplication and conservation of their germplasm. The aim of the present investigation is to establish a protocol for the in vitro culture and plant regeneration of Notocactus magnificus, the blue cactus, a highly ornamental species, native to Brazil. The surface sterilization of the explants was achieved with immersion for 10 min in sodium hypochlorite solution for either seeds (0.25% v/v) or ribs segments (1% v/v). Callus formation was observed when explants were cultured on MS medium supplemented with sucrose at 2% (w/v), 2,4-dichlorophenoxyacetic acid 0.5 μM, benzylaminopurine 4.4 μM, thiamine HCl 0.4 mg l−1 and i-inositol 100 mg l−1. The regeneration of shoots was carried out on MS medium supplemented with either different concentrations of benzylaminopurine and 1-naphthaleneacetic acid, or kinetin and indole-3-acetic acid. The highest number of shoots occurred when MS medium was supplemented with benzylaminopurine 22.2 μM, sucrose 3% (w/v) and agar 0,6% (w/v). In vitro spontaneous rooting of shoots was observed after eight months under culture on MS medium. Only in vitro rooted shoots developed into normal plants under glasshouse culture conditions. This in vitro protocol should be useful for the conservation as well as mass propagation of Notocactus magnificus.

Kinetin-induced differentiation of normal human keratinocytes undergoing aging in vitro.

Abstract: Kinetin (N(6)-furfuryladenine) is a cytokinin growth factor having several anti-aging effects reported for human cells and fruit flies. We have observed that short-term culturing of human keratinocytes in the presence of 40 to 200 microM kinetin results in a significant inhibition of cell growth. Studies were undertaken to analyze the process of differentiation as a reason for growth inhibition. Keratinocytes at different passage levels were treated with fetal calf serum (FCS) and calcium as differentiation-inducing positive controls, with different concentrations of kinetin, and with a combination of kinetin and calcium. The induction and progression of differentiation was monitored by morphological observations and by using several differentiation markers, including keratins (K10 and K14), involucrin, epidermal transglutaminase, and some new keratinocyte-specific antibodies isolated by the phage display method. In young keratinocytes, two days of calcium treatment reduced the K14 level by 78%, and increased the levels of K10 and involucrin by 40% and 29%, respectively. In comparison, 40 microM kinetin had no effect on the K14 level, but increased the K10 level by 28% and that of involucrin by four-fold. The combination of calcium and 40 microM kinetin led to a decrease by 23% in the K14 level, to an increase in the level of K10 by 55%, and to a two-fold rise in the involucrin level. These results suggest that the rate, extent, and quality of differentiation depend on the inducing agent, and that kinetin may be useful in promoting the differentiation of human keratinocytes, especially in the presence of calcium.

In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture.

Abstract: Rapid in vitro propagation of the terrestrial orchid, M. khasiana through immature seed culture was achieved. Immature seeds of 8-9 week after pollination (WAP) cultured on MS medium (2% sucrose) supplemented with 500 mgl(-1) casein-hydrolysate and 1 microM N6-benzyladenine (BA) exhibited germination of 75% seeds after 107 days of culture and subsequently supported the development of PLBs. Subsequent culture on MS medium enriched with 6 microM of indole-3-acetic acid (IAA), 18 microM each of BA and kinetin induced multiple shoots and plantlets. Transfer of PLBs to MS medium with 0.1% activated charcoal (AC) facilitated rapid proliferation of PLBs, while AC at 0.2% favored shoot bud induction and rhizome enlargement. The plantlets, developed on medium with IAA, BA and kinetin, after hardening in vitro for 8-10 weeks were planted in community pots and transferred to poly-house. The plantlets showed 65% survival under field conditions.

In vitro micropropagation and ex vitro acclimation of bupleurum kaoi – an endangered medicinal plant native to taiwan

Abstract: Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi ,a n endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l 21 BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shoot multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l 21 indole-3-butyric acid and 0.1– 0.2 mg l 21 a-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2 þ DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4 wk) and ex vitro-acclimated plantlets.