Stock-plant etiolation causes drifts in total soluble sugars and anthraquinones, and promotes adventitious root formation in teak (Tectona grandis L. f.) coppice shoots

The effects of stock-plant etiolation on coppice-shoot growth, drifts in total soluble sugars and anthraquinones (AQs; C₁₄H₈O₂), and rooting potentiality of shoot cuttings were examined in Tectona grandis L. f. (clone FG1). When seedlings were one-year-old, they were coppiced and maintained in the dark for etiolation, with a parallel set kept under natural light in an open environment. Coppice shoots were made into single-node leafy cuttings (SNCs), which were cultured under intermittent mist for rooting. These SNCs were treated with different concentrations of NAA (0, 2000 and 3000 mg l⁻¹). Etiolation significantly increased the coppice-shoot length, internode length, number of coppice shoots, number of leaves, number of nodes and total soluble sugars. The HPTLC analysis showed qualitative and quantitative differences in AQs in coppice shoots obtained from etiolated and non-etiolated stock plants. The study showed that AQs could be used as a marker for maturity and juvenility in teak. Stock-plant etiolation caused a significant increase in percent rooting and sprouting, shoot length, number of shoots and number of leaves per SNC, but a decrease in callusing at the base of the SNC. NAA at 2000 and 3000 mg l⁻¹ had inhibitory effects on rooting and sprouting of SNCs. The result showed that stock-plant etiolation fostered rooting by rejuvenating the coppice shoots.     

Etiolation and flooding of donor plants enhance the capability of <Emphasis Type="Italic">Arabidopsis</Emphasis> explants to root

Rooting of cuttings depends not only on the rooting treatment and the genotype, but also on the condition of the cuttings at the time of excision. The physiological and developmental conditions of the donor plant may be decisive. We have examined in Arabidopsis the effect of two donor plant pre-treatments, etiolation and flooding, on the capability of flower stem and hypocotyl segments to root. For etiolation, plantlets were kept in the dark, hypocotyls up to 12 days and plantlets for 12 weeks. Flooding was applied as a layer of liquid medium on top of the semi-solid medium. This procedure is also referred to as “double layer”. Both pre-treatments strongly promoted rooting and we examined possible mechanisms. Expression of strigolactone biosynthesis and signaling related genes indicated that promotion by etiolation may be related to enhanced polar auxin transport. Increased rooting after flooding may have been brought about by accumulation of ethylene in the cutting (ethylene has been reported to increase sensitivity to auxin) and by massive formation of secondary phloem (the tissue close to which adventitious roots are induced). Both pre-treatments also strongly lowered the endogenous sucrose level. As low sucrose favors the juvenile state and juvenile tissues have a higher capability to root, the low sucrose levels may also play a role.     

In vitro propagation of Lagerstromia parviflora Roxb. from adult tree

A micropropagation protocol based on axillary bud proliferation has been developed from mature Lagerstromia parviflora adult tree. Nodal segments cultured on woody plant medium supplemented with 5.0 microl. BAP and 0.25 microm IAA gave maximum (86.9%) morphogenetic response. Proliferated shoots (10.7 per explants) were elongated to 3.9 cm within 6 weeks. In vitro produced micro-shoots were subjected to an IBA treatment (500 ppm for 2 min. dip) and placed under misting conditions for rooting. Misting beds were prepared with sand: soil (3:1) for 80.6% rooting and was acclimatized. Shoot length seems to be important to induce adventitious roots. The highest (91.7%) rooting was recorded on shoots ranging a length between 3.1-4.0 cm. Rooted and hardened plants were later transferred to poly bags and maintained in shadenet house. The protocol has the realizes capacity to produce 260 plants from a single explants within 10 months multiplication cycle.     

“艾西丝”南瓜诱导生根过程中过氧化物酶活性及同工酶的研究

以“艾西丝”南瓜组培苗为材料,研究其在诱导不定根形成过程中过氧化物酶活性及同工酶谱的变化。结果表明,当南瓜无根苗从含有较高浓度的ＢＡ培养基转入１／２ＭＳ培养基后,可诱导无根苗茎基部不定根生成,一般在转入生根培养基第３ｄ开始生根,至第６ｄ生根率达７０％以上。在此期间,南瓜茎内过氧化物酶活性由低增高,即在不定根形成之前,过氧化物酶活性处在较低水平,而当不定根形成时,过氧化物酶活性迅速升高,以后一直维持     

Stock plant etiolation and stem banding effect on the auxin dose-response of rooting in stem cuttings of Carpinus betulus L. ‘Fastigiata’

Experiments were undertaken to determine the effect of stock plant etiolation and stem banding, prior to cutting propagation, on the auxin dose-response of rooting in Carpinus betulus L. Fastigiata stem cuttings. Stock plants were forced in a greenhouse, etiolated for 10 days and banded with black, light-tight Velcro for 8 weeks. Indole-3-butyric acid was applied to cuttings at concentrations ranging from 0 to 79 mM. Rooting percentages and numbers increased to a peak reponse at 20 mM in light-grown and 40 mM in etiolated shoots, followed by an inhibition at higher concentrations for all except etiolated and banded shoots. Cuttings prepared from shoots which had been etiolated or banded rooted better than controls at low and optimal IBA concentrations. Cuttings from shoots receiving both etiolation and banding also yielded higher rooting percentages and more roots per rooted cutting. Furthermore, etiolation and banding reduced the sensitivity of cuttings to supra-optimal auxin-induced inhibition of adventitious root initiation.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

PP333对分蘖洋葱试管苗增殖和生根的影响

以MS 0.1 mg·L-1NAA 0.4 mg·L-1 6-BA为增殖培养基,1/2MS 1.5 mg·L-1IBA 0.01 mg·L-1NAA为生根培养基,添加不同浓度PP333的结果表明:增殖培养基中添加1.0 mg·L-1PP333对分蘖洋葱试管苗增殖生长有促进作用,减少超度含水态苗的发生;生根培养基中添加0.1 mg·L-1PP333对分蘖洋葱试管苗生根壮苗有良好效应.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Genetic Transformation of Leaf Explants of Populus tomentosa

The leaf disc method developed by Horsch et al. (1985) has been used for transformation of Populus tomentosa. The strain of Agrobacterium tumefaciens used harbored a reconstructed Ti plasmid which contained gene 4 of T–DNA and the chimeric CAT(chloramphenicol acetyltransferase) gene. Leaf explants from shoot cultures of Populus tomentosa were co-culfivated with the bacterium. On the hormone free medium, teratoma-like shoots developed from the edge of the leaf explants. When the abnormal shoots were excised from the explants and transferred onto rooting medium, a mass of callus formed at the base of shoots, with new shoots developing, but without root formation. The measurement of'endogenous cytokinin showed that the transformed shoots produced 14 times as much iso-pentenyl adenosine as untransformed shoots did. All teratoma-like shoots-tested showed the presence of nopaline, and were able to grow well. on the medium containing 60-100μg/ml chloromycetin, while normal shoots turned white after 40 days. Pretreatment of A. tumefaciens with phenolic compound, salicylic acid, would increase the frequency of transformation significantly.     

Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

云南山茶花茎尖培养中多芽体的形成和生根的研究

本文报道云南山茶花(Camellia reticulataL.)的茎尖培养诱导形成多芽体和生根的条件及影响因素。MS培养基中含有较高浓度(3—5 mgL~(-1))BA和适量(1—1.5 mgL~(-1))IAA或NAA诱导形成多芽体。外植体的母株年龄明显影响多芽体的形成,幼龄种苗外植体的多芽诱导率高于成年树。添加CM(椰乳)、ZT、尿囊素对成年芽条返幼态有明显的促进作用。采用“浸没-纸桥生根法”诱根,以高浓度生长调节剂(0.5gL~(-1)IBA或ABT生根粉)溶液浸芽条基部短时间(20—30 min),生根效果最佳。母树的年龄对芽条的生根也有显著影响,种苗来源的芽条的生根率和根的数目都比来源于成年树的高。生根培养基中蔗糖浓度为15—20 gL~(-1),生根率高。MW和1/2 ER培养基比MS培养基的生根效果好。试管植物移栽入土后生长正常并已开花。     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

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.     

Micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch.-Hams. ex Wall.: a critically endangered medicinal herb

An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA₃ (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.     

Responses of the photosynthetic system and peroxidase activity to the rooting conditions of oak micropropagation

The effects of indole butyric acid (IBA) concentration and the duration of the plant growth regulator (PGR) treatment on the rooting ability, peroxidase level and photosynthetic activity of young Quercus robur L. plants were studied. Four-week-old oak shoots were transplanted to rooting media supplemented with 10 or 20 μM IBA. After 2, 3, 4, 5 or 7 days the shoots were transplanted again to a fresh, PGR-free medium. On the tenth day after transfer to rooting medium, the CO₂ fixation capacity, pigment content and peroxidase activity were measured. The photosynthetic parameters varied as a function of the time spent on medium containing PGR, showing maximum values in plants transplanted on the third to fourth day to PGR-free medium. The rooting percentage of these plants reached its maximum within two weeks. However, peroxidase activity was the highest in plants transferred later to PGR-free medium. The most pronounced stimulating effect on rooting was achieved with the higher initial IBA concentration followed by a transfer to PGR-free medium on the third to fourth day. These plants showed the highest vitality and the best rooting ability. This revised version was published online in June 2006 with corrections to the Cover Date.     

Successful propagation in vitro of apple rootstock MM106 and influence of phloroglucinol.

Successful in vitro propagation of clonal apple rootstock MM106 was achieved by culturing axillary buds on MS basal medium with BAP (1 mg/L), GA3 (0.5 mg/L) and IBA (0.1 mg/L). Use of liquid medium (LM) in initial cultures reduced phenol exudation to a greater extent and gave maximum sprouting percentage when transferred to solid MS medium. Phloroglucinol (PG) did not enhance sprouting of buds but increased the rate of multiplication when added in the medium. Maximum number of shoots were obtained when MS medium was supplemented with BAP (0.5 mg/L), GA3 (1 mg/L), IBA (0.1 mg/L) and PG (100 mg/L). For rooting, in vitro regenerated shoots were placed in IBA (30 mg/L) for 3 hr and transferred to solidified auxin free medium. Rooting was recorded in about 80% of shoots. Inclusion of PG in rooting medium was not beneficial but shoot cultures grown in its presence gave higher rooting percentage. Rooted plantlets showed about 70% survival rate in potting mixture of sand:soil:perlite (1:1:1).     

Influence of indole-butyric acid and electro-pulse on in vitro rooting and development of olive (<Emphasis Type="Italic">Olea europea</Emphasis> L.) microshoots

The effects of indole-butyric acid (IBA) and electro-pulses on rooting and shoot growth were studied in vitro, using olive shoot cultures. Tested shoots were obtained from seedlings belonging to three Spanish cultivars, ‘Arbequina’, ‘Manzanilla de Sevilla’ and ‘Gordal Sevillana’, which have easy-, medium- and difficult-to-root rooting abilities, respectively. The standard two-step rooting method (SRM), consisting of root induction in olive rooting medium supplemented with 0, 0.1 or 1 mg/l IBA followed by root elongation in the same rooting medium without IBA, was compared with a novel one-step method consisting of shoot electro-pulses of 250, 1,250 or 2,500 V in a solution of IBA (0, 0.1 or 1 mg/l) and direct transferral to root elongation medium. The rooting percentage of the seedling-derived shoots obtained with the SRM was 76% for ‘Arbequina’ and ‘Gordal Sevillana’ cultivars and 100% for ‘Manzanilla de Sevilla’ cultivar, whereas with the electro-pulse method, the rooting percentages were 68, 64 and 88%, respectively. IBA dipping without pulse produced 0% rooting in ‘Arbequina’ seedling-derived shoots. The electroporation in IBA not only had an effect on shoot rooting but also on shoot growth and development, with longer shoots and higher axillary shoot sprouting and growth after some of the treatments. These effects were cultivar-dependent. The electro-pulse per se could explain some of these effects on shoot development. I.M.G. Padilla and I. Vidoy contributed equally to this article.     

米老排的组织培养和快速繁殖

以米老排人工林成年优树当年生枝条茎段为外植体,建立了“以芽繁芽”的组织培养快速繁殖体系。丛芽诱导培养最快的无性系,经过3个月的培养,一个外植体可获得17．2个芽。在添加6-BA1．0mg·L-1的Ms培养基上增殖率最高,月增殖率为2．43。促进苗高生长的最有效培养基是Ms＋6-BA0．4mg·L-1＋GA30．4mg·L-1。生根培养基为1／2MS＋IBA0．4nag·L-1＋O．1g．L-1活性炭,生根率达81．8％以上,每株苗生根7．8条,平均苗高为1．2cm。经生根培养20d和目光温室炼苗15d后,试管苗移植入黄泥和泥炭土（4：1,刃功混合基质中,成活率达85％以上。     

圆叶娃儿藤离体组织培养的快速繁殖研究

圆叶娃儿藤实生苗的上胚轴在H+6—BA2毫克/升的培养基上可产生大量不定芽,并发展成无根苗。当无根苗转至1/2 MS+NAA0.25毫克/升的培养基上10天后即可生根获得完整再生植株并移栽成活。通过这一实验程序已可使组织培养这一方法成功地作为圆叶娃儿藤快速繁殖的一种手段。