Quantitation of endogenous levels of IAA,IAAsp and IBA in micro-propagated shoots of hybrid chestnut pre-treated with IBA

Endogenous levels of indole-3-acetic acid (IAA), indole-3-acetylaspartic acid (IAAsp) and indole-3-butyric acid (IBA) were measured during the first 8 d of in vitro rooting of rootstock from the chestnut ‘M3’ hybrid by high performance liquid chromatography (HPLC). Rooting was induced either by dipping the basal ends of the shoots into a 4.92-mM IBA solution for 1 min or by sub-culturing the shoots on solid rooting medium supplemented with 14.8-μM IBA for 5 d. For root development, the induced shoots were transferred to auxin-free solid medium. Auxins were measured in the apical and basal parts of the shoots by means of HPLC. Endogenous levels of IAA and IAAsp were found to be greater in IBA-treated shoots than in control shoots. In extracts of the basal parts of the shoots, the concentration of free IAA showed a significant peak 2 d after either root inductive method and a subsequent gradual decrease for the remainder of the time course. The concentration of IAAsp peaked at day 6 in extracts of the basal parts of shoots induced with 14.8-μM IBA for 5 d, whereas shoots induced by dipping showed an initial increase until day 2 and then remained stable. In extracts from basal shoot portions induced by dipping, IBA concentration showed a transient peak at day 1 and a plateau between day 2 and 4, in contrast to the profile of shoots induced on auxin-containing medium, which showed a significant reduction between 4 and 6 d after transferred to auxin-free medium. All quantified auxins remained at a relatively low level, virtually constant, in extracts from apical shoot portions, as well as in extracts from control non-rooting shoots. In conclusion, the natural auxin IAA is the signal responsible for root induction, although it is driven by exogenous IBA independently of the adding conditions.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.     

Changes in peroxidase activity,auxin level and ethylene production during root formation by poplar shoots raised in vitro

Shoots of poplar (Populus tremula × P. tremuloïdes) were multiplied in vitro and rooted on a rooting medium in the presence of NAA. No rooting occurred in the absence of exogenous auxin. A peak of soluble peroxidase activity, which corresponded to a decrease in the free IAA level in the shoots, preceded rooting These events were considered as corresponding to the initiative phase of rooting. They are preceded by a peak in free IAA activity which might initiate the inductive phase of the rooting process. A burst of ethylene production was measured in both rooting and non-rooting shoots, but the ethylene peak from rooting shoots appeared earlier and was higher. The use of ACC indicated that the exogenous auxin might have enhanced ACC-synthetase activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - NAA naphthaleneacetic acid - IAA indole-3-acetic acid - 2-iP 2-isopentenyladenine - IAAsp indole-3-acetylaspartic acid - IBA indole-3-butyric acid - GC gas-chromatography     

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out duringadventitious root formation in avocado microcuttings. Both auxinand conjugate were monitored in control cuttings (rooted inthe absence of auxin) as well as in cuttings treated with arooting promotor (IBA) or an auxin transport inhibitor (TIBA).Additionally, a histological study to follow root differentiationwas carried out. In control cuttings IAA levels remained constantthroughout the rooting process, however, in IB A-treated cuttingsIAA levels increased 2-fold during the first 6 d. Addition of200 µM TIBA induced a slight decrease of IAA levels andinhibited root formation. As for IAAsp levels, both control and IBA-treated cuttings showeda big increase before root differentiation occurred and as theprocess went on, a progressive decrease took place. However,in TIBA-treated cuttings IAAsp levels not only did not increasebut diminished progressively during the process. The role ofauxin conjugates during the rooting process of avocado is discussed. Key words: Avocado, IAA, IAAsp, rooting     

Putrescine and polyamine inhibitors in culture medium alter in vitro rooting response of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &amp; Arn.

The influence of polyamine putrescine (PUT), and polyamine inhibitors were tested for in vitro rooting response from micro shoots that initially established on Murashige and Skoog (MS) medium comprising 2.7 µM α-Naphthaleneacetic acid (NAA) and 8.9 µM 6-Benzylaminopurine (BA) by using nodal explants of Decalepis hamiltonii. Incorporation of putrescine alone in rooting medium devoid of auxins supported the best response for in vitro rooting qualitatively and quantitatively. Incorporation of putrescine at 50 µM able to induce 8.62?±?1.93 roots with a maximum root length of 9.10?±?1.65 cm wherein, the root fresh weight was also found to be high compared to all other treatments (5.248?±?1.71 g). Addition of putrescine inhibitor cyclohexylamine (CHA) in medium curtailed rooting response from microshoots. Among the three polyamine inhibitors, CHA in presence of 9.8 µM Indole-3-butyric acid (IBA) outperformed α-DL-difluromethylarginine (DFMA) and α-DL-difluoromethylornithine (DFMO) combination with 9.8?µM IBA. The least response for root number (1.55?±?0.72), root length (1.96?±?0.45 cm), and root weight (1.94?±?0.35 g) was found for IBA?+?PUT?+?DFMA and the best response was noted for IBA?+?PUT?+?CHA (2.6?±?1.1, 2.92?±?0.73 cm, 3.03?±?0.75 g) respectively. Endogenous content of putrescine, spermidine and spermine supported the rooting response from in vitro shoots. These results have clearly demonstrated that putrescine plays a crucial role in rooting of D. hamiltonii. Plantlets were transferred to micro-pots for a short acclimatization stage in greenhouse where they survived at 90?%. This highly reproducible procedure can be adopted for large scale swallow root propagation. Overall, supplementing putrescine in the rooting medium enhances the quantity and quality of roots in D. hamiltonii, thus confirming its role.     

Root Development Enhanced by Using Indole-3-butyric Acid and Naphthalene Acetic Acid and Associated Biochemical Changes of In Vitro Azalea Microshoots

Based on the importance of producing in vitro adventitious roots, this study was carried out to investigate the effects of indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) at a concentration of 2 mg L^?1 on the formation of adventitious roots of azalea and their impact on biochemical changes and endogenous hormones. The rooting percentage, root number, and root length were increased in the microshoots of both studied cultivars (‘Mingchao’ and ‘Zihudie’) when the growth medium was supplemented with IBA. Additionally, peroxidase, indole acetic acid oxidase, hydrogen peroxide, and soluble protein contents were improved in both cultivars by auxin treatments especially during the first 7 days of the rooting period. However, application of IBA and NAA increased catalase and polyphenol oxidase in both cultivars during the first 14 and 28 days of culture. The increase in endogenous indole acetic acid (IAA) levels was accompanied by low activity of IAAO during most periods of root induction of microshoots in all treatments. Endogenous gibberellic acid levels were increased after 7 days of culture and then increased again after 28 days of culture. In contrast, the levels of endogenous zeatin riboside and isopentenyl adenosine were decreased with auxin treatments in the first period of the rooting process and then increased after 21 and 28 days of culture. The present study demonstrated that IBA at a concentration of 2 mg L^?1 has a strong effect on azalea rooting. Moreover, the efficiency of IBA and NAA effects on biochemical changes during adventitious root induction was investigated, which may provide new horizons of in vitro rooting production and provide valuable information for the micropropagation of Rhododendron plants.     

Auxin metabolism and rooting in young and mature clones of Sequoia sempervirens

The capacity of young and mature Sequoia sempervirens clones to produce roots in vitro was studied after wounding and indole-3-butyric acid (IBA) treatments. Rooting was not observed in mature or in young cuttings cultivated for 30 days in medium without IBA. The presence of 25 μ M IBA in the medium resulted in the appearance of roots at the base of the cuttings. More roots appeared and grew faster on cuttings of the young than on the mature clone. This difference in rooting capacity between young and mature cuttings may be related to differences in the hormone levels at the base of the 5 mm long cuttings during the first 4 days of the root inductive period. After HPLC fractionation. IAA. IBA and related compounds, including indole-3-aspartic acid (IAAsp) and IBA-glucose ester (IBA-GE), were determined by MS and MS-MS and their levels measured by ELISA. Another immunoreactive compound was also found and determined to be N,N-dimethyltryptophan (DMT), a compound previously reported to inhibit auxin-enhanced ethylene production. Wounding of the stem without IBA treatment revealed a transient increase in IAA, IAAsp and DMT levels in young cuttings while a dramatic increase in the levels of DMT was observed in mature cuttings. Following IBA treatment. IAA levels increased in both clones, but higher levels were measured in the young than in the mature clone. IBA and IBA-GE were also found but in higher levels in the mature clone. Thus, the difficult-to-root mature clone differs from the young clone in its auxin metabolism.     

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut ( Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non-rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.     

Auxin stability and accumulation during in vitro shoot morphogenesis influences subsequent root induction and development in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Recent results showed that after 16 months in the field, micropropagated eucalyptus plants have an inferior root system to cuttings. Such differences may be due to the plant growth regulators supplied during the culture stages of standard protocols, which are targeted at optimising plantlet yields and not root quality. This study investigated such a proposal, focusing on auxins in an easy-to-root clone. Initial results showed that the auxin provided in the standard protocol (NAA for multiplication and IBA for elongation) enabled 100% rooting in auxin-free medium, where rooting was faster than on IBA-rooting media. When auxin supply was omitted from multiplication and restricted to NAA or IAA during elongation, rooting in an auxin-free medium was reduced to 68 and 31%, respectively, reflecting the stabilities of these auxins in plant tissues. Additionally, 15% of shoots from the NAA-medium and 65% from the IAA-medium produced roots with altered graviperception. GC–MS analysis of these shoots revealed a relationship between free IAA-availability and altered graviperception. This was further tested by adding the IAA-specific transport inhibitor 2,3,5-triiodobenzoic acid to rooting media with IBA, IAA or NAA, which resulted in 100, 70.9 and 20.6% rooting, respectively. At least 40% of the sampled root tips had atypical starch grain deposition and abnormal graviperception. It is proposed that, at least in this clone, while IBA and NAA can be used for in vitro root induction, IAA is necessary for development of graviresponse.     

Effect of Applied and Endogenous Auxin on Callus and Root Formation of In Vitro Shoots of the Apple: Rootstocks M26 and A2

The influence of exogenous IBA (indol-3yl-butyric acid) on rootand callus formation was studied in shoots of the apple rootstocksA2 and M26. The shoots grown in vitro were derived originallyfrom meristems of both juvenile and adult trees. Endogenousindol-3yl-acetic acid (IAA) concentrations in leaves and stemswere correlated with the responses to applied IAA. After 30 subcultures shoots from A2 and M26 rooted easily, butA2 did so more readily and even without IBA. Treatment withIBA improved percentage rooting and number of roots in bothrootstocks. Ex-adult and ex-juvenile shoots of A2 formed rootsto the same extent. However, ex-adult shoots of A2 showed ahigher IBA optimum for root number than ex-juvenile A2 and werealso less sensitive to supra-optimal IBA concentrations. Incontrast, in M26, there were no differences between ex-adultand ex-juvenile shoots. The results imply that rooting ability is associated more withdifferences between cultivars than with the origin of the explants.The best rooting occurred in ex-adult shoots of A2 which hadthe lowest endogenous IAA concentration, while callus formationwas correlated with high endogenous auxin concentration. Ex-adultA2 produced almost no callus even after exposure to high IBAconcentrations (25µM) whereas ex-adult M26 formed muchmore callus at 1/10 of the IBA concentration. Malus sylvestris (L.) Mill. var. domestica Borkh., Malus pumila Mill., apple rootstocks A2 and M26, in vitro culture, root and callus formation, HPLC analyses of IAA     

Azospirillum brasilense inoculation,auxin induction and culture medium composition modify the profile of antioxidant enzymes during in vitro rhizogenesis of pink lapacho

Optimal in vitro plant growth can be stimulated by selecting specific nutritional and environmental conditions. However, the culture conditions, dissection, and disinfection of plant material are stressful and may induce disruption of the plant physiological homeostasis. This can be modified by inoculation with rhizobacteria as Azospirillum brasilense, by the culture medium type, and by auxin induction. Here, we performed rooting experiments in two auxin-free culture media with ‘pink lapacho’ (Handroanthus impetiginosus) shoots previously induced with 0, 10, 30, or 50 μM indole butyric acid (IBA) for 3 days and inoculated with A. brasilense Cd and Az39. Peroxidase (PO), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) activities were determined on days 0, 3, 6, 9, 12, and 15. Also, weekly absolute rooting percentage was evaluated. All enzymatic activities were higher in A. brasilense-inoculated shoots, linked to early and high rooting percentage. The culture medium type and IBA concentration also affected enzymatic activities. The positive correlation between PO and PAL activities on day 9 and successful final in vitro rooting of H. impetiginosus allows using these activities as early markers of rhizogenesis reducing the selection time of easy-to-root plants. The changes in enzymatic levels performed here are discussed on the basis of their role in rooting and in vitro stress and contribute to the knowledge of the physiology of trees and their interaction with rhizobacteria.     

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L.

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.     

Temperature-Sensitive Plant Cells with Shunted Indole-3-Acetic Acid Conjugation

Cells of henbane (Hyoscyamus muticus L.) grow indefinitely in culture without exogenous auxin. Cells of its temperature-sensitive variant XIIB2 grow like the wild type at 26[deg]C but die rapidly at 33[deg]C unless auxin is added to the medium. Despite this temperature-sensitive auxin auxotrophy, XIIB2 produces wild-type amounts of indole-3-acetic acid (IAA). IAA is the predominant auxin and is important for plant growth and development. Since the IAA production of the variant is functional, we investigated whether the synthesis or degradation of IAA metabolites, possibly active auxins themselves, is altered. The IAA metabolites were IAA-aspartate (IAAsp) and IAA-glucose. The wild type converted IAA mainly to IAAsp, whereas the variant produced mainly IAA-glucose. Exogenous auxin corrected the shunted IAA metabolism of the variant. The half-life of labeled IAAsp in the variant was reduced 21-fold, but in the presence of exogenous auxin it was not different from the wild type. The temperature sensitivity of XIIB2 was also corrected by supplying IAAsp. Pulse-chase experiments revealed that henbane rapidly metabolizes IAAsp to compounds not identical to IAA. The data show that the variant XIIB2 is a useful tool to study the function of IAA conjugates to challenge the popular hypothesis that IAA conjugates are merely slow-release storage forms of IAA.     

Auxins and polyamines in relation to differential in vitro root induction on microcuttings of two pear cultivars

The internal levels of indole-3-acetic acid (IAA) and polyamines (PAs) and the metabolism of indole-3-butyric acid (IBA) were studied in relation to the in vitro rooting process of two pear cultivars, the easy-to-root Conference and the difficult-to-root Doyenne d'Hiver. Doyenne d'Hiver required about a 10 times higher concentration of IBA to achieve a rooting percentage similar to that of Conference. One- or two-day exposures to IBA were sufficient to stimulate rooting but with different efficiency for each cultivar. Longer exposure to auxin strongly increased the root number in Conference, whereas root elongation was inhibited in both cultivars. The metabolism of IBA in both cultivars was not significantly different when IBA was used at a high concentration to stimulate maximal rooting in Doyenne d'Hiver. IBA was mainly conjugated into IBA glucose, which was accumulated, and a small amount was converted into free IAA in both cultivars. However, in Doyenne d'Hiver this metabolic pathway appears to be active only at a higher exogenous IBA concentration. At a high IBA concentration more callus was formed by Doyenne d'Hiver, indicating that the cells of Doyenne d'Hiver are not capable of responding to the hormone in the same manner as Conference cells. Anatomic observations indicated that the capacity to induce initial dividing cells was more efficient in Doyenne d'Hiver, but subsequently the number of root primordia formed and root development were much reduced relative to Conference. A possible correlation between these processes and an early increase followed by a decrease of free IAA was seen in Conference. By day 4, a significant increase in IAA conjugates and free putrescine was observed in Doyenne d'Hiver. This higher putrescine content may be related to the lower amount of root development. Together with previous studies these results indicate that differences in the uptake and metabolism of applied auxins may affect rooting ability and the subsequent development of adventitious roots in microcuttings of pear.Abbreviations IBA indole-3-butyric acid - IAA indole-3-acetic acid - PA(s) polyamine(s) - HPLC high pressure liquid chromatography - GC-MS gas chromatography-mass spectrometry - TCA trichloroacetic acid dansyl, 1-dimethylaminonaphthalene-5-sulfonyl - TLC thin layer chromatography - TBA terbutilic alcohol - IBAGluc IBA glucose - IAAGluc IAA glucose - IAAsp IAA aspartate     

Morpho-anatomical and biochemical changes associated with rooting of micropropagated ninebark cuttings

Ninebark (Physocarpus opulifolius) is an attractive ornamental shrub with poor rooting characteristics in some cultivars, which is a limiting factor in commercial production This study was designed to optimize rooting conditions of ninebark cuttings and to observe the effect of exogenous auxin IBA on some morpho-anatomical and biochemical changes associated with rhizogenesis in the in vitro conditions. Both auxins under study: the indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) gave comparable effects but the combination of ½ MS?+?1 mg·L^?1 IBA was the most cost effective for all rooting parameters. Anatomical changes at the cuttings’ bases during root formation were typical for woody plants and they were accelerated by auxin in the culture medium. High levels of the endogenous indole acid and hydrogen peroxide were temporarily associated with intensive cell divisions in cuttings, and the polyphenolic acid contents kept increasing during rooting above the initial levels and those in controls.

     

Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport

Indole-3-butyric acid (IBA) was recently identified by GC/MS analysis as an endogenous constituent of various plants. Plant tissues contained 9 ng g^?1 fresh weight of free IBA and 37 ng g^?1 fresh weight of total IBA, compared to 26 ng g^?1 and 52 ng g^?1 fresh weight of free and total indole-3-acetic acid (IAA), respectively. IBA level was found to increase during plant development, but never reached the level of IAA. It is generally assumed that the greater ability of IBA as compared with IAA to promote rooting is due to its relatively higher stability. Indeed, the concentrations of IAA and IBA in autoclaved medium were reduced by 40% and 20%, respectively, compared with filter sterilized controls. In liquid medium, IAA was more sensitive than IBA to non-biological degradation. However, in all plant tissues tested, both auxins were found to be metabolized rapidly and conjugated at the same rate with amino acids or sugar. Studies of auxin transport showed that IAA was transported faster than IBA. The velocities of some of the auxins tested were 7. 5 mm h^?1 for IAA, 6. 7 mm h^?1 for naphthaleneacetic acid (NAA) and only 3. 2 mm h^?1 for IBA. Like IAA, IBA was transported predominantly in a basipetal direction (polar transport). After application of ³H-IBA to cuttings of various plants, most of the label remained in the bases of the cuttings. Easy-to-root cultivars were found to absorb more of the auxin and transport more of it to the leaves. It has been postulated that easy-to-root, as opposed to the difficult-to-root cultivars, have the ability to hydrolyze auxin conjugates at the appropriate time to release free auxin which may promote root initiation. This theory is supported by reports on increased levels of free auxin in the bases of cuttings prior to rooting. The auxin conjugate probably acts as a ‘slow-release’ hormone in the tissues. Easy-to-root cultivars were also able to convert IBA to IAA which accumulated in the cutting bases prior to rooting. IAA conjugates, but not IBA conjugates, were subject to oxidation, and thus deactivation. The efficiency of the two auxins in root induction therefore seems to depend on the stability of their conjugates. The higher rooting promotion of IBA was also ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. IAA was converted to IBA by seedlings of corn and Arabidopsis. The K_m value for IBA formation was low (approximately 20 μM), indicating high affinity for the substrate. That means that small amounts of IAA (only a fraction of the total IAA in the plant tissues) can be converted to IBA. It was suggested that IBA is formed by the acetylation of IAA with acetyl-CoA in the carboxyl position via a biosynthetic pathway analogous to the primary steps of fatty acid biosynthesis, where acetyl moieties are transferred to an acceptor molecule. Incubation of the soluble enzyme fraction from Arabidopsis with ³H-IBA, IBA and UDP-glucose resulted in a product that was identified tentatively as IBA glucose (IBGIc). IBGIc was detected only during the first 30 min of incubation, showing that it might be converted rapidly to another conjugate.     

An efficient protocol for micropropagation of lemon (<Emphasis Type="Italic">Citrus limon</Emphasis>) from mature nodal segments

The influence of the basal medium and different plant growth regulators on micropropagation of nodal explants from mature trees of lemon cultivars was investigated. Although the basal medium did not affect any of the variables, explants on DKW medium were greener. Several combinations of 6-benzyladenine (BA) and gibberellic acid (GA) were used to optimise the proliferation phase. The number of shoots was dependent on the BA and GA concentrations and the best results were obtained with 2 mg l⁻¹ BA and 1 or 2 mg l⁻¹ GA. Explants length was shorter with the higher BA concentrations and, in all genotypes, shoot length was greater with 2 mg l⁻¹ GA. The best results for productivity (number of shoots × the average shoot length) were obtained with 2 mg l⁻¹ BA and 2 mg l⁻¹ GA, although explants with chlorosis and narrow leaves were observed. The presence of BA and GA in the proliferation medium was essential for the explant multiplication but GA had a greater influence. The transfer of in vitro shoots to rooting media, containing different concentrations of indole butyric acid (IBA) and indole acetic acid (IAA) produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The highest rooting percentages were obtained on media containing 3 mg l⁻¹ IBA alone or IBA in combination with 1 mg l⁻¹ IAA and on these media the highest numbers of roots were produced. The average root length was affected significantly by the IBA and IAA concentrations. Root length was greater when only 3 mg l⁻¹ IBA was used, and in this rooting medium explants had a better appearance, with greener and larger leaves. The success during the acclimatisation was close to 100% and the plantlets exhibited normal growth in soil under greenhouse conditions.     

Restart of Lignification in Micropropagated Walnut Shoots Coincides with Rooting Induction

The lignin content of walnut shoots did not change during in vitro shoot multiplication. Lignin content started to increase as soon as shoots were passed to a rooting medium with auxin. Exogenous auxin (applied for rooting) caused a transient elevation of the endogenous free indoleacetic acid (IAA) content with a simultaneous decrease of peroxidase activity. These events typically marked the completion of the rooting inductive phase (before any visible histological event, that is before the cell divisions beginning the rooting initiation phase). This meant that either the given exogenous auxin or the endogenous IAA has served as signal for the stimulation of lignification. Continued increase of lignification in the shoots required completion of root formation; this increase indeed was slown down when root emergence did not occur. It was further shown that lignification varied conversely to the content of the soluble phenol content, itself apparently being related to the activity of phenylalanine ammonia-lyase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.