Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

白及假鳞茎薄片诱导不定芽及快繁体系的构建

以白及(Bletilla striata)假鳞茎为外植体,根据上、中、下部位切取薄片,探索假鳞茎不同部位BA、NAA和TDZ对假鳞茎薄片诱导不定芽的影响,比较假鳞茎薄片不同厚度对褐化率和出芽数的影响,采用正交试验,研究了BA和NAA对不定芽增殖的效果,并对组培苗进行壮苗、生根和移栽。结果表明:假鳞茎的部位对诱导不定芽作用极显著,下部的出芽率显著高于上部和中部,BA和TDZ对诱导不定芽作用显著,NAA对诱导不定芽作用不显著。最佳诱导不定芽的方式为假鳞茎下部薄片在基本培养基+2.0 mg·L^-1BA+1.0 mg·L^-1TDZ的培养基上培养4周,出芽率为93.3%,出芽数为15个,厚度为1.6~2.0 mm的假鳞茎薄片其褐化率最低。最佳的增殖培养基为基本培养基+1.5 mg·L^-1BA+0.3 mg·L^-1NAA,增殖系数达4.3,平均苗高为7.8 cm。本研究成功建立了白及假鳞茎薄片诱导芽为关键技术的快繁技术体系,为白及种质资源创新奠定了基础。     

In vitro micropropagation of an endemic fruit tree Syzygium alternifolium (Wight) walp

Shoot proliferation was obtained from shoot tip and nodal bud explants ofSyzygium alternifolium (Wight) walp on modified Murashige and Skoogs medium (MS) supplemented with either BA, KN or AD alone or BA in combination with either IAA, NAA or IBA. A combination of BA and auxins produced more shoots from both types of explants than on the medium containing only cytokinins. The highest multiplication rate was achieved with nodal bud explants in presence of 17.7M BA and 2.6M NAA. Excised shoots were rooted on half-strength MS medium with either IAA or IBA. The regenerated plantlets have been successfully acclimatized and transferred to soil. About 70% of plantlets have survived underex vitro conditions.Abbreviations BA-N⁶ Benzyladenine - KN Kinetin - AD Adenine - IAA Indole-3-aceticacid - IBA Indole-3-butyricacid - NAA-1 Naphthalene aceticacid - MS Murashige and Skoogs medium (1962)     

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 M of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.Abbreviations BA 6-benzyladenine - GD Gresshoff and Doy Medium - IBA indole-3-butyric acid     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Antihepatotoxic agent in micropropagated plantlets of Eclipta alba

A protocol for in vitro micropropagation of Eclipta alba (L.) Hassk from nodal segment explants has been established. The maximum number of shoots was obtained after 60 days of culture in Murashige & Skoog (MS) medium supplemented with 4.4 M benzyladenine. Multiple rooting was achieved using MS medium with 2.4 M 2-isopentyladenine. Wedelolactone was present in shoots cultured in media containing cytokinins.Abbreviations BA benzyladenine - 2iP 2-isopentenyladenine     

Micropropagation of Campanula isophylla Moretti

A method for micropropagation of Campanula isophylla Moretti is described. The method is based on division of the basal parts of shoot clusters into sections, each with four 3 mm stem stubs. Shoots from the shoot clusters are easy to root and give plants without apparent phenotypic aberrations. It is thus possible to propagate the stock and produce rooted plantlets in the same process. Basal sections of shoot clusters formed more shoots than shoot tips or single nodes. The medium used for propagation was MS with 4.4 M benzyladenine (BA). Addition of naphthaleneacetic acid or raising the concentration of BA did not improve the results significantly. As primary explants 2 mm stem segments with an axillary or apical bud were used; smaller explants often failed to grow. For rooting the concentration of macronutrients was reduced to one-half, and BA was omitted. The cultures received an irradiance of 20 mol m^-2 s^-1 fluorescent light; dry weight of shoots decreased if the irradiance was reduced. The method was used for propagation of 113 genotypes; shoot numbers and days to first root differed significantly among genotypes.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

Multiple shoot formation from normal and malformed somatic embryo explants of Eastern redbud (Cercis canadensis L.)

A procedure for multiple shoot formation from somatic embryo explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. TDZ in combination with BA produced more shoots than either treatment alone. The highest number of shoots (3.3 to 3.4 shoots per explant) was obtained from partially desiccated and wounded explants treated with a combination of 5 or 10 M BA and 0.5 or 1.0 M TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 1.5 to 3.2 shoots per explant by cutting through the cotyledonary node prior to culture. In addition, the frequency of explants forming shoots was increased by desiccation of somatic embryo explants to approximately 50% moisture and by using somatic embryos with two well formed cotyledons as explants.Abbreviations ABA abscisic acid - BA benzyladenine - CRD Completely randomized design - DKW Driver and Kuniyuki medium - LSD Least significant differences - TDZ thidiazuron     

Rapid Axillary Bud Proliferation and ex vitro Rooting of Eupatorium triplinerve

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 M benzylaminopurine (BAP) and 2.46 M indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 M IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.     

Production of clonal plantlets of Grevillea robusta in in vitro culture via axillary bud activation

A micropropagation method for Grevillea robusta A. Cunn. was developed using explants from mature trees cultured on Woody Plant Medium plus 4.4 M benzyladenine and 0.27 M naphthaleneactic acid (NAA) for shoot proliferation. Each nodal explant produced three to five shoots within 10 to 12 weeks in three successive transfers. Rooting was obtained by dipping the shoots in 0.54 mM NAA solution. Rooted shoots were established well in soil.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneactic acid     

In vitro propagation of Cleome spinosa (Capparaceae) using explants from nursery-grown seedlings and axenic plants

Summary Two independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.     

Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

In vitro propagation of Artocarpus altilis (park.) fosberg (breadfruit) from mature plant material

Summary A micropropagation protocol for Artocarpus altilis (breadfruit, yellow cultivar) using shoot tip explants taken from mature trees is outlined. Cultures were initiated from shoot tip explants (1–2 mm in length) on either N5K or N15K (Margara, 1978) macronutrient formulation, MS (Murashige and Skoog, 1962) micronutrients and vitamins and 6-benzyladenine (BA, 4.4 μM). Single-node explants obtained from shoots formed at the initiation stage were used at the multiplication stage. Multiplication elongation and maintenance were possible on N30NH₄ (Margara, 1978) macronutrients, MS micronutrients and vitamins and zeatin 2.2 μM. Shoot elongation was not enhanced by the inclusion of gibberelic acid at 1.4 μM, 2.8 μM or 14.0 μM, along with zeatin at 2.2 μM, when compared with those shoots cultured on zeatin only. N30NH₄ was found to be superior (with respect to shoot quality) to MS macronutrient formulations at the multiplication and maintenance stages, because the shoots with development were more vigorous than those formed on the other macronutrient formulations mentioned. Zeatin at 2.2 μM yielded 50% more in vitro-formed shoots when compared with BA at 4.4 μM for the same time period. Shoots rooted on hormone-free medium with a success rate of 60%. Regenerated plantlets were hardened with about a 40% success rate.     

Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source

Shoot cultures established from mature trees of hazelnut (Corylus avellana L.) cvs. Nonpareil and Tonda Gentile Romana were used to determine the effects of basal media, carbon sources and concentrations, pH and cytokinins on shoot multiplication. All factors except pH affected the multiplication rate. Shoot multiplication was the best on a modified Driver and Kuniyuki medium for Paradox walnut (DKW) supplemented with 6-benzylaminopurine (BA) (1.5–3 mg/l). Plants grown on 3% glucose or fructose medium produced more and longer shoots than those on sucrose. The general appearance and growth habit of shoots were better on medium with glucose than fructose. Nonpareil shoots elongated better than those of Tonda Gentile Romana. Changes in medium pH from 4.7 to 5.7 did not significantly affect the multiplication rate. More than 10 genotypes propagated well on modified DKW medium with glucose. This is the first report of the effect of carbon sources on shoot multiplication of hazelnut and provides a basis for further research in the improvement of hazelnut micropropagation.Abbreviations MWPMC modified woody plant medium for chestnut (Yang et al. 1986) - DKW Driver and Kuniyuki (1984) medium for Paradox walnut - BA 6-benzylaminopurine - Z zeatin - 2iP N6-(2-isopentenyl) adenine - K kinetin - IBA indole-3-butyric acid