Addition Of Kinetin Research Articles

IN VITRO MICROPROPAGATION OF CG41 APPLE ROOTSTOCK

The apple production area in Uruguay is of approximately 4000 ha, with an average fruit production of 62,000 t per year. Apple is the most important crop within deciduous fruits, and has behaved as the most demanding, in crop technology, during the last years. Local climate and soil conditions in the south of the country, where the major apple producing area is located, can generate sanitary problems to apple trees. New rootstocks of CG series were introduced from Cornell University in 1996 by the Fruit Program of the National Agricultural Research Institute (INIA - Uruguay) with the purpose of evaluating new tools to solve some of these soil problems as Eriosoma lanigerum and Phytophthora sp. The rootstocks introduced were CG 16, CG 3041 and CG 5935. CG41 was selected through the results of the first trials since this rootstock showed to be well adapted, very precocious and productive, with similar vigor to M9, tolerant to Eriosoma lanigerum, and resistant to Phytophthora sp. This work was carried out to develop a protocol of in vitro propagation of CG41 rootstock, in order to generate faster plant availability to nurseries. Cytokinin (benzyladenine and thidiazuron) effect was evaluated during the multiplication phase. The multiplication rate obtained was higher than three and there was significant difference between the treatments compared. Thidiazuron was the most effective hormone in the phase of multiplication. However, thidiazuron used in successive sub-cultures generated deformations in explants and leaves of plantlets. Therefore its incorporation had to be alternated with other growth regulators. Also different elongation media were compared and the most effective included kinetin's addition. Finally, different rooting substrates were evaluated. At this level the cut in V at the base of the plant was critical, independently of its size.

English

Optimization of tissue culture conditions for Sorghum bicolor L. through somatic embryogenesis from immature embryos is important for the genetic manipulation and improvement of this agronomically valuable crop. In an attempt to develop a successfully reproducible in vitro regeneration protocol for a group of diverse sorghum genotypes, 10 sorghum lines including locally adapted and commercially important elite genotypes were assessed for their regeneration potential on different culture media–containing adequate growth regulators combinations. The maximum response of embryogenic callus induction was obtained from explants cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.7 mgL-1 L-proline. The addition of kinetin to the MS-based culture media had a negative effect on the formation of embryogenic calli. The results reveal that embryogenic callus formation and regeneration were highly genotype dependent. The line LG3 revealed the highest mean number of embryogenic callus (47.5 ± 6.0%) across the media tested. On the other hand, SPP462 was the least responsive (13.4 ± 3.3%) to embryogenic callus induction. The regeneration percentage of the different genotypes ranged from zero to 22.1%. The lines LG3, LG4, Dorado, SPGM94021 and SPMD94001 succeeded to form shoots on the three tested regeneration media. Nevertheless, the genotypes LG8 and TX2794 produced shoots on only two out of the three media. Three lines failed to regenerate on any of the tested media. Adding 0.5 mgL-1 naphthalene acetic acid and 0.5 mgL-1 of indole-3-butyric acid (IBA) did not enhance the root induction. Regenerated shoots developed into normal mature plants. Regeneration of sorghum genotypes could be improved through the use of different auxins and cytokinins in callus induction and shoot formation media. The auxin, 2,4-D was critical for the induction of embryogenic calli. However, the addition of the cytokinin (kinetin) adversely affected the formation of embryogenic callus. On the other hand, the shoot induction was more influenced by the addition of indole-3-acetic acid (IAA), 6-benzylaminopurine (BA) and thidiazuron (TDZ).   Key words: Sorghum, immature embryos, callus induction, regeneration, auxin, cytokinin.

The effect of indole-butyric acid and kinetin on rooting of rose cuttings in winter and summer

The effect of indole-butyric acid, both alone and with a low concentration of kinetin, on the rooting of rose cuttings in winter and summer, is presented in this paper. The experiments were conducted using 500 and 1000 mg/l IBA with or without the addition of 5 mg/l kinetin. The growth regulators were applied by dipping the base of a cutting for 5 s in an aqueous solution of these substances. Cuttings 5-6 cm in length were made from the mid-part of a stem of a rose grown in a greenhouse. The experiments were carried out using 'Queen of Bermuda' and 'Baccara' cuttings. The investigations showed that treating rose cuttings rooted in winter with an IBA solution had a significant promotive effect on the quantity of rooted cuttings, number of formed roots on the cutting, as well as on the length of the longest root. A distinctive increase in the number of breaking buds was also seen on the cuttings treated with IBA. The IBA solution applied to cuttings rooted in the summer significantly decreased the number of rooted cuttings and breaking buds. However, no significant influence on the number and length of formed roots was found. Addition of kinetin to the IBA solutions did not have any effect on the rooting of rose cuttings either in winter or summer.

European orchid cultivation – from seed to mature plant

We describe a method for growing orchids of the genera Dactylorhiza and Ophrys, two European members of the subfamily Orchidoideae, from seeds to mature plants using asymbiotic in vitro cultures and glasshouse pot cultures. Four media were used: two new media 1/4-2 and Mo2 and two modifications of Michl medium (Michl 1988). We also describe a highly efficient technique for seed disinfection using a syringe. We tested the effects of ethanol treatment on Anacmaptis morio (L) R.M. Bateman, Pridgeon & M.W. seeds, sugar media composition on Dactylorhiza majalis (Rchb.) P.F. Hunt & Summerh., Oeceoclades decaryana (H. Perrier ex Guillaumin & Manguin) Garay & Taylor and Ophrys lojaconoi P. Delforge and the effect of kinetin on Dactylorhiza majalis protocorm growth. Sucrose was the best carbon source, while hexose resulted in the inhibition of protocorm development at early stages. The addition of kinetin at 10 mg/l resulted in the formation of the largest protocorms. Ethanol can have positive effect on seed germination when applied for a short time (2 min), while long-time ethanol exposure (60 min) can kill the seeds.

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (Mesembryanthemum crystallinum L.)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO3, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO3 applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.

High frequency embryogenic callus induction and plant regeneration from mature caryposis of big bluestem and little bluestem

Big bluestem ( Andropogon gerardii Vitman) and little bluestem [ Schizachyrium scoparium (Michaux) Nash.] are native to the North America and are important forage grasses and ornamental grasses. Both grasses are proposed as ideal biomass producers for cellulosic ethanol production. To apply genetic transformation, which is an important tool for incorporating desirable agronomic traits into plants to both species, however requires an efficient and reproducible regeneration protocol. We used mature caryopses from big and little bluestem as explants and tested the effect of various combinations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 3, 4 or 5 mg l −1) and kinetin (KT) (0, 0.1 or 0.2 mg l −1) on embryogenic callus induction with LS as the basal medium. The highest percentage of embryogenic calli induction occurred on medium containing 2, 4-D alone at 2 mg l −1 for ‘Bison’ and on medium containing 4 mg l −1 2, 4-D alone for ‘Bonilla’ big bluestem. For little bluestem, the highest percentage of embryogenic callus induction occurred on medium containing 3 mg l −1 2, 4-D plus 0.1 mg l −1 kinetin, suggesting that addition of KT is beneficial. Shoot regeneration took place on LS basal medium without any plant growth regulator for both species, although the addition of KT increased both regeneration frequency and the number of shoots produced per callus. Rooting of shoots reaching about 2 cm long occurred readily with or without α-naphthaleneacetic acid (NAA). Rooted plantlets were all successfully established in the soil.

Agrobacterium-medlated high-efficiency transformation of creeping bentgrass with herbicide resistance

We performedAgrobacterium-mediated genetic transformation of creeping bentgrass(Agrostis stolonifera L.) and produced herbicide-resistant transformants from commercial cultivars Crenshaw and Penncross. Seed-derived embryogenie calli were infected withA. tumefaciens EHA105 harboring pCAMBIA 3301, which includes an intron-containinggus reporter and abar selection marker. To establish a stable system, we examined various factors that could potentially influence transformation efficiency during the pre-culture, infection, and co-cultivation steps. The addition of kinetin to the callus pre-culture media increased efficiency about three-fold. Once the optimum infection and co-cultivation conditions were identified, this protocol was used successfully to bulk-produce herbicide-resistant transgenic plants whose herbicide resistance was confirmed using the BASTA® resistance test. Southern blot analysis demonstrated integration and low copy numbers of the integrated transgenes, and northern blot analysis verified their expression. Thus, we have established an efficient genetic transformation system for creeping bentgrass and confirmed a high frequency of single-copy transgene integration and functional gene expression.

Axillary proliferation and tuberisation of Dioscorea cayenensis–D. rotundata complex

Yams (Dioscorea spp) are tuber crops used as staple food in Africa because of their nutritional value. However agronomic constraints, phytosanitary problems and the lack of good healthy planting material restrict their production. In contrast to the inefficiency of traditional method of planting, tissue culture techniques allow to increase the multiplication and the rapid production of pathogen- free plant material. This work was undertaken to provide farmers in African countries with healthy microplants and microtubers as seeds. In vitro nodal segments of two varieties of local yams D. cayenensis–D. rotundata complex (cv. ‘Singo’, cv. ‘Singou’ and cv. ‘Gnidou’) were micropropagated on the modified medium of Murashige and Skoog. The morphogenesis, the growth of microplants and microtuber formation have been found to be controlled by external factors that act individually and synergistically. Addition of kinetin (2 mg l−1) to the culture media could reduce multiplication rate (node number) of some clones. An increase of the sucrose concentration from 3% to 5% induced no change in the multiplication and tuberisation parameters. An important reduction of the multiplication (shoot number, height and node number) and the tuberisation (tuber number and length) was observed with 8% sucrose. Multiplication (shoot and node number) was increased in the presence of jasmonic acid (10 μM). JA also induced an increase of tuber number in the absence of Kin. Multiplication of yam by in vitro growth of nodal segments is a way for rapid clonal multiplication and could allow solving the problem of lack of seed material faced by farmers. This method could also be used for multiplication of elite cultivars, independently of the growing season.

Modification of cadmium toxicity in pea seedlings by kinetin

The effect of foliar application of kinetin on the growth and some physiological processes of pea plants growing in soil supplemented with 25 or 50µM Cd were studied. Cadmium treatment inhibited the growth rate, chlorophyll (Chl) content, net photosynthesis (P<sub>N</sub>), content of soluble sugars and free amino acids of either shoots or roots. The application of kinetin (kin) enhanced the growth rate, Chl content, P<sub>N</sub>, soluble sugars and free amino acids content of shoots and roots; dark respiration rate (R<sub>D</sub>), contents of soluble protein and proline were increased by cadmium treatment. The addition of kinetin to Cd-stressed plant reduced R<sub>D</sub>, soluble protein and proline content. Considerable variations in the content of Na<sup>+</sup>, K<sup>+</sup>, Ca<sup>2+</sup> and Mg<sup>2+</sup> were induced by Cd-treatments. Foliar application of kinetin exhibited a favorable effect on the accumulation of some ions and antagonized or ameliorated the inhibitory effect of Cd stress on some others.

The effects of growth regulators in proliferation of Sorbus redliana 'Burokvölgy'

The Hungarian cultivar Sorbus redliana 'Burokvölgy' was proliferated on Murashige and Skoog (MS, 1962) medium with half-strength macroelements and 100 mg/1 meso-inositol, 20 g/1 sucrose, 11 g/1 agar-agar. Different combinations of kinetin (KIN), metatopolin (mT), benzyladenine (BA), benzyladenine-ribosid (BAR) and indolebutiric acid (IBA) were tested, and pH was adjusted to 5.6 every case using KOH. The cultures were incubated at 20-24 °C in 8/16 hours dark/light photoperiod for 50-52 days. The main aim of our research was to find the optimal growth regulator and its optimum concentration. Purthermore, to determine the chlorophyll contents of the in vitro propagated plants' leaves. During the proliferation, the highest number of shoots were observed in the case of using BA + IBA, and on the medium containing 0.75 mg/I BA + 0.05 mg/1 IBA 8.93 shoots were found. The addition of KIN + IBA decreased the number of shoots and increased the sizes of leaves — the widest (11.2 mm) and longest (17.8 mm) leaves were obtained on the medium containing 1.00 mg/I KIN + 0.05 mg/1 IBA. The longest shoots (36.46 mm) were found in the case of applying 0.75 mg/1 BAR + 0.05 mg/I IBA. The BA + KIN + IBA combination resulted the shortest shoots. Sometimes not only shoot regeneration but spontaneous rooting was observed during the multiplication. The highest chlorophyll content (1.569 mg/g total chlorophyll, 1.132 mg/g chlorophyll-a, 0.437 mg/g chlorophyll-b) was obtained in the presence of 1.0 mg/I KIN + 0.05 mg/1 IBA.

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the PHYB Gene from Arabidopsis

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.

Floret primordia differentiation from in vitro cultured sunflower capitula

In vitro culture has become a useful tool for studies concerning the assessment of physiological and biochemical factors controlling floral morphogenesis. For sunflower, in vitro techniques starting from different explants yield good results on culture initiation and plant regeneration. Although inflorescence generation on cultured shoots is sometimes achieved there are no reports about in vitro florets developed from cultured young capitula. This work describes a protocol for the in vitro culture of sunflower reproductive meristems where floral organogenesis can be expressed. Experiments were carried out using undifferentiated sunflower capitula excised from plants grown under greenhouse conditions. White (W) and Linsmaier and Skoog (LS) culture media were compared. LS medium induced a comparatively large capitula receptacle area expansion, reduced the explant hyperhydration and calli formation and was chosen for further experiments. The presence of casein hydrolysate (CH) in LS culture media was tested resulting in reduced surgical stress and recovery of explants. The addition of Kinetin to LS medium plus CH proved to induce differentiation of new primordia at the capitulum generative front. Explants cultured with 0.1 mg l-1 Kinetin only differentiated mother bract primordia. Explants cultured with 1 mg l-1 Kinetin showed larger expansion of the receptacle area and new floret primordia did not differentiate their mother bract. Intermediate levels of kinetin (0.5 and 0.8 mg l-1) were tested in addition to two levels of IAA (1.3 and 2.5 mg l-1). The combination 0.8 mg l-1 Kinetin and 2.5 mg l-1 IAA was capable of differentiating floret primordia that closely resembled those formed in the capitula developed in vivo.

MICROPROPAGATION OF THE APPLE ROOTSTOCK M26 BY TEMPORARY IMMERSION SYSTEM (TIS)

In order to develop an efficient and economic micropropagation technique for fruit trees and ornamental plants, a liquid culturing system in the RITA containers using temporary immersion principle was examined on the apple rootstock M26. Several plant growth regulators were tested in the multiplication and elongation phase on two different types of explants. The results showed that the multiplication rate increased with an increase in the concentration of BAP and IBA in the multiplication medium, but the shoot length decreased at the same time. Hyperhydricity increased with a rise in concentration of BAP and IBA in the multiplication medium. Stem explants produced more shoots than shoot tips. The rooting results showed that shoots from all treatments rooted more than 90%. The higher concentrations of BAP and IBA in the multiplication medium did not affect the rooting percentage, root number and root length, nor did the addition of kinetin in the elongation medium. The plantlets were easily acclimatized in the greenhouse.

Tratamentos pré-germinativos em sementes de <i>Didymopanax morototoni</i> (Aubl.) Dcne. et Planch.

Didymopanax morototoni (Aubl.) Dcne. et Planch, (caixeta), é uma espécie nativa com vasta distribuição natural, rápido crescimento, grande porte e ainda pela sua rusticidade, é considerada uma espécie pioneira de potencial florestal para o Brasil. Porém, as suas sementes apresentam baixa germinabilidade. O objetivo deste trabalho foi avaliar o efeito de tratamentos pré-germinativos que pudessem maximizar o processo de germinação desta espécie. Foram testados os seguintes tratamentos: pré- -embebição em H20 por 8 h; escarificação química (HCl e H2SO4 a 70%); escarificação mecânica (por picotes no tegumento e com lixamento); lavagem com água destilada e lavagem em água + álcool (1:1) por 15, 30 e 45 min. As sementes foram colocadas para germinar em diferentes substratos. Realizou-se ainda teste de germinação in vitro. Sementes foram inoculadas em meio WPM, solidificado com ágar na presença de cinetina e ácido giberélico nas concentrações (0; 0,1; 0,5; 1,0 mg/l), e caseína hidrolisada na concentração 250 mg/l. A incubação foi a 25°C 1°C em câmara de germinação sob fotoperíodo de 16 horas luz, numa intensidade de 14,3 μE.m-².s-¹ ou no escuro. A germinação foi favorecida pelos pré-tratamentos de lavagens com água e água + álcool, sendo o tempo mais eficaz o de 45 min. A adição de cinetina e ácido giberélico provocou um aumento na germinabilidade de 40 a 70 %. Estes resultados confirmam a dormência tegumentar parcial e indicam a presença provável de substancias inibidoras nos tegumentos, removidas pelas lavagens.

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon.

LITHOSPERMUM ERYTHRORHIZON: , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and 1 H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks.

Role of kinetin in alleviation of copper and zinc toxicity in Lupinus termis plants

The effect of exogenous kinetin application on the growth and some physiological processes of Lupinus termis plants growing in metal containing solutions with excess concentrations of Cu and Zn ion were studied. Generally, plants growing in these solutions had a lower chlorophyll (Chl.) content, leaf relative water content (RWC) and produced less biomass than the control plants. Proline content was higher in metal-treated plants than in untreated controls. Chromatography of cell-free-extracts of roots and shoots indicated three main protein peaks with molecular weights about 170, 75--70 and 5--45 kDa. These peaks were coincident with Cu or Zn maxima. Addition of kinetin reduced the decline in Chl. content in metal-treated plants, improved water status of the plants and enhanced growth of the shoots and roots. The Cu or Zn content expressed on a per mg protein basis was raised when kinetin was applied to the growing shoots. Kinetin (Kin), Cu and Zn, singly and in the presence of kinetin (Cu × Kin and Zn × Kin), significantly affected the parameters tested. Only the effects of Cu × Kin and Zn × Kin interactions on shoot fresh weight and Cu × Kin on root length were statistically insignificant. Based on the calculated coefficient of determination (η 2) the roles of Cu and Zn in affecting Chl. content and growth were dominant in comparison to kinetin. Kinetin effect was dominant for root length and proline content, but the role of the interaction was subdominant. The results of this study indicate that kinetin can alleviate the harmful effects of Cu and Zn on the growth of lupin plants through stimulation of Cu and Zn incorporation into metal-binding proteins.

Kinetin-induced caffeic acid O-methyltransferases in cell suspension cultures of Vanilla planifolia Andr. and isolation of caffeic acid O-methyltransferase cDNAs

Two different S-adenosyl-L-methionine:caffeic acid methyltransferases are induced in kinetin-treated cell cultures of Vanilla planifolia. Upon addition of kinetin to the culture, maximum caffeic acid 4-O-methyltransferase and caffeic acid 3-O-methyltransferase activity is obtained after around 2 and 40 h, respectively. The former enzyme is involved in the biosynthesis of vanillic acid and the latter enzyme is co-induced with other enzymes of the general phenylpropanoid pathway and is involved in the biosynthesis of lignin precursors. A number of cDNA clones encoding S-adenosyl-L-methionine:caffeic acid-methyltransferases were isolated by heterologous probe screening of a λZapII cDNA library constructed from mRNA of a kinetin-treated cell suspension culture of V. planifolia. A full-length cDNA clone, Vpomt35, contains a 1 089-bp open reading frame coding for 363 amino acid residues, a 25-bp 5′-end sequence and a 214-bp 3′-end non-coding sequence. The deduced amino acid sequence of Vpomt35 revealed 56 to 80 % sequence identity when compared to those of other plant caffeic acid O-methyltransferases. Detection of methyltransferase activity in extracts of Escherichia coli XL-1 Blue cells transformed with a pBluescript phagemid construct containing Vpomt35 confirmed that this cDNA clone encodes a caffeic acid 3-O-methyltransferase. The enzyme also exhibits a low caffeic acid 4-O-methyltransferase activity (2 % of the caffeic acid 3-O-methyltransferase activity). A number of other cDNA clones encoding caffeic acid O-methyltransferases was also sequenced. Comparison of nucleotide and deduced amino acid sequences of these cDNAs indicated two types of caffeic acid O-methyltransferase cDNAs which showed 94 % sequence identity in the coding region, but only 55 % homology with several gaps in the 3′ untranslated end. Results from Southern blot analysis suggest that the caffeic acid O-methyltransferase gene is organized as a small family with at least two genes, which are expressed in Vanilla cell suspension cultures.

Micropropagation of the medicinal plant, Scilla natalensis Planch.

Primary bulb explants of Scilla natalensis were cultured in vitro on modified MS medium. Some of these explants initiated shoots, which provided a sterile source of secondary leaf and bulb explants. The secondary explants responded similarly to various combinations of plant growth regulators. Shoots were initiated spontaneously on medium containing no plant growth regulators. The number of shoots initiated was increased by the addition of kinetin or thidiazuron (TDZ) alone, but was reduced by the addition of indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) alone. Optimal shoot initiation occurred on medium containing 1 to 2 mg l−1 kinetin and 1 to 2 mg l−1 IAA. These shoots were rooted on medium containing 1 mg l−1 IAA. The plantlets were successfully acclimatised in the misthouse/shadehouse.

In Vitro Multiplication of Two Genotypes of Asparagus (Asparagus officinalis L.)

Asparagus is a vegetable that presents an increase in yield when propagated by meristem culture. On the order hand, the rooting phase in asparagus is greatly affected by the previous phase, i.e,. multiplication. This species presents a better rooting performance when callus is formed at the shoot base. So, the aim of this work was to evaluate treatments during the multiplication phase, which also leads to callus formation at the shoot base. The initial explants came from shoots being cultivated in vitro. It was tested kinetin at: (0.0, 0.5, 1.0, 1.5, and 2.0) μM; ancymidol at (0.0 and 0.5) μM and NAA at (0.0 and 0.5) μM for both genotypes, which were cultured in a MS medium added to sucrose (30 g·L–1), agar (6.0 g·L–1) and myo-inositol (100.0 m g·L–1). Shoots bearing two buds were inoculated in 10-ml test tubes and placed in a growth room for 30 days when they were evaluated. The addition of kinetin significantly improved the number of buds and at 1.3 μM this growth substance presented the best results as number of shoots is concerned. NAA application promoted a negative effect on spear bearing. The addition of ancymidol in this phase did not improve the bud multiplication. It was shown that clone M14 performed better than the hybrid cv. Deco as multiplication is concerned.