Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro

Summary The promotive effect of ethylene inhibitors (Els), i.e. AgNO₃ and aminoethoxyvinylglycine (AVG) on de novo shoot regeneration from cultured cotyledonary explants of Brassica campestris ssp. pekinensis cv. Shantung in relation to polyamines (PAs) was investigated. The endogenous levels of free putrescine and spermidine in the explant decreased sharply after 1–3 days of culture, whereas endogenous spermine increased, irrespective of the absence or presence of Els. AgNO₃ at 30 M did not affect endogenous PAs during two weeks of culture. In contrast, explants grown on medium containing 5 M AVG produced higher levels of free putrescine and spermine which increased rapidly after three days and reached a peak at 10 days. An exogenous application of 5 mM putrescine also resulted in a similar surge of endogenous free spermine of the explant. More strikingly, shoot regeneration from explants grown in the presence of 1–20 mM putrescine, 0.1–2.5 mM spermidine, or 0.1–1 mM spermine was enhanced after three weeks of culture. However, exogenous PAs generally did not affect ethylene production, and endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC of the explant. This study shows the PA requirement for shoot regeneration from cotyledons of B. campestris ssp. pekinensis in vitro, and also indicates that the promotive effect of PAs on regeneration may not be due to an inhibition of ethylene biosynthesis.Abbreviations PAs polyamines - AVG aminoethoxyvinylglycine - SAM S-adenosylmethionine - ACC 1-aminocyclopropane-1-carboxylate - Els ethylene inhibitors     

多胺对宁夏枸杞愈伤组织器官发生和体细胞胚发生的影响

利用已建立的宁夏枸杞（Lycium barbarumL.)愈伤组织器官发生和体细胞胚发生体系,对多胺在其离体形态发生中的作用进行研究。通过检测内源多胺含量发现,在所研究的三种多胺中,Put是器官发生途径的主要多胺,而在体细胞胚发生途径Spd含量占优势。Put含量变化在两条途径中相似：在愈伤组织分化的早期迅速积累不仅又下降,随着芽原基和球形胚的形成含量又进一步上升。器官发生中Spd最高含量仅在培养的第一天后,Spd含量才开始上升,到第十天时达到最高值。三种外源多胺的添加均有有效地促进两种离体分化途径的形态建成：Spd(100 μmol/L）能显著增加不定芽数,而体细胞胚发生中Spd(100μmol/L）而Put（100μmol/L）的单独处理能最好地促进体细胞胚形成和进一步发育成苗;尽管Spm在离体形态发生中含量较低,但添加外源Spm也促进了不定芽形成和体细胞胚形成然后成苗。多胺生物合成抑制剂CHA处理阻碍不定芽形成和和体细胞胚的进一步发育;但是MGBG对器官发生途径中的形态建成没有影响,即降低体细胞胚的发生频率及再生苗数。添加Spd(50μmol/L)能部分逆转CHA、MGBG的抑制效应。以上结果表明,多胺对宁夏枸杞器官发生和体细胞胚发生途径的离体形态建成有一定影响。     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

Effects of exogenous polyamines on nitrate tolerance in cucumber

Putrescine (Put), spermidine (Spd), and spermine (Spm) are the major polyamines (PAs) in plant, which are not only involved in the regulation of plant developmental and physiological processes, but also play key roles in modulating the defense response of plants to diverse environmental stresses. In this study, Cucumis sativus L. seedlings were cultivated in nutrient solution and sprayed with three kinds of PAs (Put, Spd, and Spm). The effects of PAs were investigated on excess nitrate stress tolerance of C. sativus by measuring growth and nitrogen (N) metabolism parameters. The contents of NO_3-^?N, NH_4-⁺N, proline and soluble protein in leaves were increased; while plant height, leaf area, shoot fresh and dry weight, root fresh weight were decreased under 140 mM NO₃^? treatment for 7 d. In addition, the activities of nitrate reductase (NR), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) were significantly inhibited under 140 mM NO₃^? treatment for 7 d. With foliar treatment by 1 mM Spd or Spm under stress treatment, the contents of Spm, Put, and Spd in leaves increased significantly, except that Spm content decreased under Spd treatment. The activities of NR, glutamine synthetase (GS), GOGAT and GDH and plant height, leaf area, shoot fresh and dry weights were significantly increased. The contents of proline and soluble protein in leaves were significantly enhanced. In contrast, the accumulation of NO_3-^?N and NH_4-⁺N were significantly decreased. However, there were minor differences in activities of N metabolism enzymes and the content of osmotic adjustment substances under 1 mM Put treatment. These findings suggest that 1 mM exogenous Spm or Spd could enhance the capacity of N metabolism, promote growth and increase resistance to high concentrations of NO₃^?. The ameliorating effect of Spd was the best, and that of Put the worst.     

Do exogenous polyamines have an impact on the response of a salt-sensitive rice cultivar to NaCl?

In order to analyze the putative impact of polyamines (PAs) on the plant response to salt, seedlings from the salt-sensitive rice cultivar I Kong Pao (IKP) were exposed for 5, 12 and 19 days to 0, 50 or 100 mM NaCl in the absence, or in the presence of exogenous PAs (putrescine (Put), spermidine (Spd) or spermine (Spm) 1mM) or inhibitors of PA synthesis (methylglyoxalbis-guanyl hydrazone (MGBG) 1mM, cyclohexylammonium (CHA) 5mM and D-arginine (D-Arg) 5mM). The addition of PAs in nutritive solution reduced plant growth in the absence of NaCl and did not afford protection in the presence of salt. PA-treated plants exhibited a higher K+/Na+ ratio in the shoots, suggesting an improved discrimination among monovalent cations at the root level, especially at the sites of xylem loading. The diamine Put induced a decrease in the shoot water content in the presence of NaCl, while Spd and Spm had no effects on the plant water status. In contrast to Spd, Spm was efficiently translocated to the shoots. Both PAs (Spd and Spm) induced a decrease in cell membrane stability as suggested by a strong increase in malondialdehyde content of PA-treated plants exposed to NaCl. These results are discussed in relation to the putative functions of PAs in stressed plant metabolism.     

Effects of methylglyoxal-bis (guanylhydrazone) and abscisic acid on polyamine metabolism in embryonectomized barley seeds

Incorporation of L-[U-¹⁴C] arginine or L-[U-¹⁴C] ornithine into putrescine (Put), spermidine (Spd) and spermine (Spm) in embryonectomized barley seeds (Hordeum vulgare L. cv. Himalaya) was studied following imbition with methylglyoxal-bis (guanylhydrazone) (MGBG) and abscisic acid (ABA). Both radiolabeled amino acids were incorporated into the amines as a result of active polyamine biosynthesis in the seed during imbibition. In the aleurone layer, the Spd and Spn existed mainly in the free form (acid soluble). However about 50% of Put was recovered in conjugated form(s) (acid insoluble). Imbibition with 5 and 10M ABA for 3 days increased the accumulation of the free form of ¹⁴C-Put, probably as a result of inhibition (70%) of ¹⁴C-Spd accumulation. The ABA treatment showed no significant effect on levels of the conjugated form of Put and Spd. Imbibition with millimolar concentrations of MGBG resulted in (i) abnormal accumulation of the free form of Put and incorporation of ¹⁴C-amino acids into the diamine, (ii) progressive inhibition of the accumulation of the free forms of ¹⁴C-Spd and Spm, and (iii) reduction of the ¹⁴C incorporation into the conjugated forms of Put and Spd. Uptake of ¹⁴C-amino acids was not affected by MGBG treatment. The results indicate that MGBG may inhibit not only the synthesis of Spd and Spm, but the catabolism (e.g. oxidation) of Put in the aleurone layer.This paper is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Enhancement of shoot regeneration from cotyledon explants of Brassica rapa ssp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors

The presence of benzyladenine or naphthaleneacetic acid in seed germination medium markedly enhanced subsequent shoot regeneration from the base of the excised cotyledon explants of Brassica rapa cv. Horizon. Cotyledon explants from younger seedlings (3 or 4-day old) produced more shoots than those from older seedlings. Addition of the ethylene inhibitor aminoethoxyvinylglycine (1.0 M) to the regeneration medium improved shoot regeneration three fold.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - MGBG methylglyoxal-bisguanylhydrazone - MSBN ms (murashige & skoog 1962) medium supplemented with 4.4 m BA & 5.4 m NAA, 2% sucrose - NAA naphthaleneacetic acid     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of leaf tissues

The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2 mM in combination with 0.5 M aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same concentration but in combination with 60 M silver thiosulphate and four-day pulses with 9 M 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos

Organogenesis and somatic embryogenesis were induced from megagametophyte and zygotic embryo explants of 2 cycad species, Ceratozamia hildae and C. mexicana, cultured on modified B5 medium containing kinetin (0–13.9 M) and 2,4-d (0–9.0 M). Organogenesis occurred from megagametophyte explants of both species on the range of media tested. Somatic embryogenesis was largely restricted to zygotic embryo explants. Somatic embryos germinated in vitro: however, rooting of adventitious shoots was unsuccessful.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid R-014515.     

水稻细胞质雄性不育系及其保持系幼穗发育过程中的多胺代谢

在幼穗发育过程中,不育系和保持系幼穗多胺含量先剧降后稳定或略回升,精氨酸脱羧酶活性快速下降,而二胶和多胺氧化酶活性缓慢下降。从雌雄蕊形成期到花粉母细胞形成期,不育系的多胺含量和精氨酸脱羧酶活性明显低于保持系;不过,两系二胺氧化酶和多胺氧化酶活性却差别不大。外施D-Arg抑制两系Put合成,也抑制以Put为前体的Spd的合成;外施MGBG抑制Spd和Spm的合成;同时,D-Arg或MGBG对不育系花粉育性影响不大,但明显降低保持系花粉育性,D-Arg＋MGBG对花粉育性的降低效应更强;Put和pd＋Spm可抵消（或部分抵消）D-Arg和MGBG的降低效应。且Put＋Spd＋Spm能使不育系花粉的育性得以轻度恢复。     

The effects of proline, thioproline and methylglyoxal-bis-(guanylhydrazone) on shoot regeneration frequencies from stem explants of B. napus

Internode segments from aseptic shoot cultures are the most prolific explants for the regeneration of Brassica shoots in vitro. These explants also have the advantage of not being subject to the genotypic variations in regeneration response observed in hypocotyl and cotyledon explants. Despite reports of 80–100% shoot regeneration from stem explants, observed frequencies are typically 50–60%. Three media additives, proline, thioproline and methylglyoxal-bis-(guanylhydrazone) (MGBG), were tested for their efficacy in promoting shoot regeneration from stem explants of two B. napus varieties, Westar and Cobra. The effects of proline and thioproline on both varieties were neutral or deleterious. In Cobra the MGBG treatments caused a uniform reduction in explant regeneration. However, at low concentrations (0.35M) MGBG resulted in a 50% increase, to 92%, in regeneration from Westar. The potential of MGBG in promoting explant regeneration in B. napus is discussed in the light of its interaction with the explant genotype.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MGBG methylglyoxal-bis-(guanylhydrazone) - NAA naphthalene acetic acid - thioproline thiazolidine-4-carboxylic acid     

Changes of free, soluble conjugated and bound polyamine titers of jojoba explants under sodium chloride salinity in vitro

Jojoba (Simmondsia chinensis L.) single node explants were cultured in a basal medium supplemented with 17.8 microM 6-benzyladenine and four levels of sodium chloride concentration (0, 56.41, 112.82 and 169.23 mM). The free, the soluble conjugated and the insoluble bound forms of polyamines (PAs) (putrescine (Put), spermidine (Spd) and spermine (Spm)) were determined monthly during a 3-month proliferation stage. Free Put and Spd were found in higher levels in the control treatment, while Spm content was higher in the salt treatments. All soluble conjugated PAs were found to be in lower concentrations in explants growing on medium supplemented with salt, while the opposite was true for the insoluble bound PAs. It appeared that certain PAs and PAs forms could play a significant role in the adaptation mechanism of jojoba under saline conditions.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na⁺, K⁺ and Cl⁻ concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na⁺ and Cl⁻ accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K⁺ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.     

Efficient adventitious shoot regeneration and somatic embryogenesis in pea

A rapid and simple method for adventitious shoot regeneration and somatic embryogenesis from immature cotyledon explants of pea (Pisum sativum L.) is described. Cotyledon size and the explant orientation to the medium surface were shown to have a clear effect on shoot regeneration. The highest frequency of shoot regeneration was achieved when the distal end of the greenest cotyledons (7–8 mm in size) were placed in contact with the agar surface. Shoots rooted at a frequency of 80–90% and grew into normal fertile plants. Somatic embryos were induced in cultures of immature cotyledons on modified MS medium containing high levels of -naphthaleneacetic acid (27–215 M) and 2,4-dichlorophenoxyacetic acid (23–181 M). A higher frequency of somatic embryos with a normal morphology were induced using -naphthaleneacetic acid.Abbreviations BA 6-benzyladenine - 2,4-d dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Micropropagation of Alnus cordata (Loisel.) Loisel.

Procedures were developed for micropropagation of Alnus cordata through in vitro axillary shoot multiplication of axillary bud explants cultured in Murashige & Skoog (MS) medium. Establishment of cultures from plants grown in the field was very difficult due to bacterial contamination and phenolic oxidation in explants causing severe browning. Explants were first cultured on an MS medium containing 4.4 M 6-benzyladenine and 87.6 mM sucrose (initiation medium) for 7 days and then transferred to an MS medium containing 1.1 M 6-benzyladenine and 333 mM glucose (multiplication medium) for a further 20–25 days. It was necessary to transfer cultures from initiation medium to multiplication medium after 7 days to minimize excessive callus growth, abnormally thick and brittle leaves, inhibition of shoot elongation, and senescence. Shoot multiplication comparable to the above method was achieved by culture of axillary bud explants in MS medium supplemented with 1.1–4.4 M 6-benzyladenine and 333 mM glucose 4–5 weeks after culture. Shoots rooted in MS medium (1/2 x macro-nutrients) supplemented with 1.2–4.9 M indolebutyric acid. Also, 98% rooting was achieved when cultures were treated with 625 mgl^-1 indolebutyric acid for 24 h at the end of the shoot production stage and rooted in vivo as mini-cuttings. Plantlets established well in soil.