Modulation of in vitro morphogenesis in nodal segments of Salix tetrasperma Roxb. through the use of TDZ, different media types and culture regimes

A comparative performance of two different media formulations (woody plant medium (WPM) and Murashige and Skoog??s (MS) medium) for their ability to inflict in vitro shoot development in nodal segments of Salix tetrasperma Roxb. has been carried out. Thidiazuron (TDZ) in various concentrations was used as a supplement to the basal media. Media types, TDZ concentrations, exposure duration and culture regimes played an important role in affecting multiple shoot production. WPM supplemented with 2.5???M TDZ for 4?weeks exposure was found to be the best for maximum (4.53?±?0.27) shoots production in vitro. Transfer to a secondary medium consisting of 6-benzyladenine (1.0???M) and ??-naphthalene acetic acid (0.5???M) enhanced the multiplication rate and by the end of 12?weeks, 20.33?±?0.33 shoots with shoot length, 4.70?±?0.26?cm were produced on WPM. Rooting of the regenerated shoots was achieved on half strength basal media (either WPM or MS) containing 0.5???M indole-3-butyric acid. In all the experiments, different growth parameters were scored and WPM was found to be superior to MS medium. The regenerated plantlets were successfully acclimatized in the field with about 81?% survival.     

An Efficient Micropropagation Protocol for High-Frequency Plantlet Regeneration from Liquid Culture of Nodal Tissues in a Medicinal Plant,Turnera ulmifolia L.

A highly efficient, stable, and cost-effective micropropagation protocol for the conservation of a medicinal plant Turnera ulmifolia L. was established from nodal tissues via multiple axillary shoot proliferation on using Murashige and Skoog’s (MS) liquid nutrient medium. To begin with, nodal explants were placed on agar gelled medium amended with 2.0 mg L^?1 6-benzylaminopurine (BAP) and 0.1 mg L^?1 indole-3 acetic acid (IAA) for shoot induction. Subsequently, elongation of regenerated shoots could be possible on liquid MS medium supplemented with 0.5 mg L^?1 BAP and Kin (kinetin) each along with 0.1 mg L^?1 IAA where high frequency of regeneration in terms of number of shoots (47.2 shoots/explant) was achieved. Furthermore, long and healthy shoots (4?5 cm in length) were rooted on agar gelled half-strength of MS medium supplemented with 2.0 mg L^?1 indole-3 butyric acid (IBA). Finally, in vitro regenerated plantlets were gradually acclimatized in the greenhouse and transferred to the field successfully.     

Micropropagation of Vitex negundo L. and Random Amplified Polymorphic DNA Analysis of Micropropagated Plants

A micropropagation protocol was established for a medicinal plant Vitex negundo. Genetic stability of micropropagated plants was investigated. Multiple shoots were induced from nodal explants cultured on Murashige and Skoog (MS) medium with 0.53 μM naphthalene acetic acid (NAA) and 11.0 μM benzyl aminopurine (BAP) along with additives (ascorbic acid, 283.9 μM; citric acid, 130.1 μM; and arginine, 143.6 μM). Shoots were further multiplied by repeated transfer of the mother explant. The shoots were further multiplied on MS medium + 0.57 μM indole-3-acetic acid (IAA) and 6.6 μM BAP. The micropropagated shoots were pulse treated with 122.5 μM indole-3-butyric acid (IBA), in liquid MS medium and then transferred to autoclaved soilrite. These rooted ex vitro. Shoots were also rooted in vitro on a half-strength MS medium + 2.45 μM IBA. The survival rate of in vitro rooted plantlets was poor during hardening compared to ex vitro rooted plantlets. About 95% of the ex vitro rooted, hardened plantlets survived in the field. Genetic stability of micropropagated plants was tested by using 25 random amplified polymorphic DNA primers. The cloned plants exhibited no variation in banding pattern in comparison with the mother plant.     

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA₃). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.This revised version was published online in November 2005 with corrections to the Cover Date.     

Clonal propagation ofGmelina arborea Roxb. byIn vitro culture

In vitro propagation technique ofGmelina arborea multipurpose and a fast growing tree species was studied. Nodal segment including axillary bud was used as a explant. They were cultured on MS media containing various concentrations (0–10 mg/l) of BAP alone or in combination with 0.002 mg/l of IBA. Nodal segments showed axillary bud proliferation in almost all media tested. MS media containing 0.22 mg/l of BAP alone and 2 mg/l of BAP in combination with 0.002 mg/l of IBA were effective for inducing multiple shoots and shoot elongation. MS medium supplemented with 0.02 mg/l of NAA and 1 mg/l of IBA gave the best result for rooting. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the green house. Adventitious shoots production from stem explants that were taken from regenerated plantletin vitro was also discussed. Stem segments were tested for their morphogenetic potential on MS media with various combinations and concentrations of BAP, zeatin and TDZ. Successfull result was obtained on MS media supplemented with 2 mg/l of BAP and 1 mg/l of zeatin or supplemented with 0.5 mg/l of BAP and 0.5 mg/l of TDZ. The shoots obtained on MS media containing 2 mg/l of BAP and 1 mg/l of zeatin rooted on MS media containing 0.02 mg/l of NAA and 1 mg/l of IBA, and plantlets were successfully obtained. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forest Society (1998).     

Multiplication of Elaeagnus angustifolia L. from a Single Shoot in Vitro

1INTRODUCTIONElaeagnusangusifoliaLisahighlytolerantnativewoodyspeciesinsouthernEuropeandAsia.Itmaybeusedinreforestationandsoil-waterconservationandforanexcellentwindbreaktoarrestwind(Bertrand,1985;LiShaozhong,etal,1997),materialsoffragranceandmedicineandnutritivefood(ChangZhaofeng,etal,1994;Goncharova,1997;Rasekh,1999;Ahmadiani2001;JiangFashou,etal,2002).Insomepreviousresearches,BA,2ip,KN(Bertrand,1985,Economou,1988)andBA(Iriondo1995;Mariella,1996)wereusedinshootmultiplicationofE…     

A modified Murashige and Skoog media for efficient multipleshoot induction in G. arborea Roxb.

This paper reports on the effect of various micropropagation factors of Gmelina arborea Roxb. through multiple shoot induction. Factors like the source and age of explants, plant growth regulators （PGRs）, media composition, and carbon source affected multiple shoot-ing in the present study. Among all the explants used, only shoot tips derived from one, two, and three week old seedlings could form multiple shoots. Besides, the formation of multiple shoots depended on the con-centration and combination of PGRs. Among all the PGRs, BAP （6-benzylaminopurine） alone gave the highest regeneration efficiency. Simi-larly, IBA （Indole-3-butyric acid） was the most efficient PGR in inducing root formation in the microshoots. Media composition and carbon source also affected the regeneration efficiency. MS （Murashige and Skoog medium） proved to be the best media for regeneration followed by B5, SH （Schenk and Hilderbrandt medium） and WPM （Woody plant medium） in that order. Similarly, among sugars, only sucrose and glucose sup-ported induction of microshoots. Based on this study we recommend the use of glucose in place of sucrose in MS medium for maximum regenera-tion efficiency.     

Micropropagation of Elaeagnus angustifolia from mature trees

Multiple shoots were obtained from nodal segments of mature trees of Elaeagnus angustifolia L. cultured on MS medium (Murashige and Skoog 1962) supplemented with 0, 0.88 or 2.22 micro M N(6)-benzyladenine. When nodal segments taken from the in vitro proliferated shoots were cultured under the same conditions, additional multiple shoots were obtained. Rooting of the in vitro propagated shoots was achieved on full strength MS medium or on MS supplemented with 2.46 micro M indole-3-butyric acid. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

In vitro plantlet regeneration from Australian Red Cedar (Toona ciliata,Meliaceae)

In vitro regeneration of complete plants from nodal single-bud segments of 2-year-old Australian Cedar (Toona ciliata) trees were obtained under defined nutritional and environmental conditions. Explants were dissected from plants obtained by germination of seeds and growth in pots in a greenhouse. The best medium for shoot regeneration was that of Murashige and Skoog at 1/4 strength with 3% sucrose (1/4 MS), supplemented with 0.1 mg/l IBA and 0.5 mg/l BAP. Rooting of regenerated shoots was observed in MS medium with 0.1 mg/l IBA. Using mature tree material was more difficult. Forced flushing was used to induce shoot development on branches of a 10-year-old tree. Nodal segments of these epicormic shoots formed shoots in vitro on 1/4 MS + 0.01 mg/l IBA + 5 mg/l BAP, but rooting was never observed.     

Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

Preservation of Juniperus thurifera L.:a rare endangered species in Algeria through in vitro regeneration

Juniperus thurifera L.is an endemic Cupres saceae from the Aure`s Mountains of north eastern Algeria and endangered,in part,due to the scarcity of viable seeds It is threatened by other abiotic factors and the lack of an effective management strategy will increase its risk o extinction.The dearth of information on its in vitro regeneration impedes its application in forest managemen programs.We therefore developed a micropropagation protocol using microcuttings with auxiliary buds.Cuttings were grown on different combinations of media supplemented with plant growth regulators at different concentrations.The highest number of shoots and branches regenerated from original shoots was obtained on Woody Plant Medium(WPM)supplemented with 6-benzylaminopurine(BAP)(0.5 mg L^-1)and 2,4-dichlorophe noxyacetic acid(2,4-D)(0.25 mg L^-1).The best elongation of shoots was achieved with WPM supplemented with0.5 mg L^-1of BAP and 0.25 or 1 mg L^-1 of 2,4-D.On the second subculture,shoots had a higher number of branches than those of the first.The highest rooting rate,38.8%,was obtained with shoots cultured in 1/2 Murashige and Skoog(MS)medium supplemented with 5.0 mg L^-1each of indol-3-butyric(IBA)and naphthalene acetic acid(NAA).Similarly,the highest root numbers and lengths were produced on 1/2 MS medium supplemented with IBA and NAA(5.0 mg L^-1each).During transfer to acclimatization,rates of plant losses of 50% occurred.The second part of the experiment showed that the best shoot callusing was on WPM supplemented with BAP and 2,4-D,with either the combination 0.5+0.25 or 0.25+0.25 mg L^-1.The results of this research provide a starting point for further studies on in vitro regeneration of J.thurifera for the sustainable management of its unique ecosystem in the Mediterranean basin.     

爬行卫矛下胚轴高频离体再生体系的建立(英文)

In this paper,a protocol for efficient shoot regeneration was successfully developed from hypocotyl explants of Euonymus fortunei var.radicans.Some factors that influenced shoot regeneration such as different combinations of plant growth regulators,types of medium and inoculation ways were studied in order to establish an efficient plant regeneration for transformation.The results showed that hypocotyl explants wero horizontally cultured on a basic medium composed of MS medium supplemented with 0.5 mg·L-1 BAP and 0.01 mg·L-1 NAA for induction and development of adventidous shoots.Ninety-four percent of regeneration frequency and 5.1 shoots per explants were obtmned after 30 days of culture.Regenerated shootsproliferated efficiently on a shoot multiplication medium consisting of MS medium containing 1.0 mg·L-1 BAP and 0.1 mg·L-1 NAA.Microshoots were rooted on a rooting medium made up of MS medium enriched with O.5 mg·L-1 IBA and O.5 mg·L-1IAA.After hardening,90% of plants were successfully established under greenhouse conditions.Histological observation revealed that shoot primordium originated from subepidermal cells of hypocotyl explants and directly developed into adventitious shoots without caHus formation.     

Micropropagation of Anogeissus latifolia (Roxb. Ex DC.) Wall,ex Guill. & Perr.- A Tree of Fragile Ecosystems

Abstract

A micropropagation process was developed for Anogeissus latifolia-a tree of fragile ecosystems. Multiple shoots were regenerated from cotyledonary node and epicotyl explants on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole-3-acetic acid (IAA) + 1.5 mgl ^-l 6-benzylaminopurine (BAP) + additives (25 mgl ^-l each of adenine sulphate, L-arginine, ascorbic-, citric acids and 1.0 mM L-asparagine) + 200 μM Fe-EDTA (ferric-ethylenediaminetetraacetic acid) salt. The shoots differentiated in vitro were subcultured and repeatedly transferred onto fresh medium but with 1.0 mgl^-1 of BAP to achieve 4-5 fold rate of shoot multiplication. After every 4th week of culture, from each culture bottle 8-10 shoots could be harvested for rooting. The shoots produced in culture were rooted in vitro on half strength MS medium with 1.0 mgl ^-l either of IBA (indolebutyric acid) or NAA (naphthaleneacetic acid). The shoots were pulse treated with combination (100 mgl ^-l each) of IBA and NOA (2-naphthoxy acetic acid) rooted ex vitro. The ex vitro root induction method is highly efficient and plantlets so generated could be acclimatized and pot transferred. The process developed can be used for large scale production of plants of A.     

Direct plant regeneration from nodal explants of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> L. (Del.): a valuable medicinal tree

This report describes in vitro shoot induction and plant regeneration from nodal segments of Balanites aegyptiaca on Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA), thidiazuron (TDZ) and kinetin (Kin) (0.5–20.0 μM). MS medium supplemented with BA (12.5 μM) was the most effective in inducing bud break and growth and also in initiating multiple shoot proliferation. However, the optimal level of TDZ supplementation to the culture medium was 5.0 μM. Shoot cultures were established by repeatedly subculturing the original nodal explants on the same medium. Highest number of shoots (11.5 ± 0.7) and shoot length (5.0 ± 0.2 cm) were achieved when cultures were subcultured on MS medium supplemented with 12.5 μM BA and 1.0 μM α-naphthalene acetic acid (NAA). The shoots regenerated from TDZ supplemented medium when subcultured to hormone free MS basal medium considerably increased the rate of shoot multiplication and shoot length by the end of fifth subculture. Rooting of the shoots was achieved on MS medium augmented with 1.0 μM indole-3-butyric acid (IBA) plus 0.5% activated charcoal followed by their transfer to half strength MS basal medium. The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 70% survival rate. The results of this study provide the first successful report on in vitro direct plant regeneration of B. aegyptiaca.     

Micropropagation of Selected Genotype of Aloe vera L.—An Ancient Plant for Modern Industry

Aloe vera Linn. (Syn. Aloe barbadensis Mill; Gwar-patha in Hindi) belongs to family Liliaceae. The plant, for its medicinal properties, has commercial value. Some of the genotypes of Aloe vera are consumed as a vegetable and processed to make curry and other edible products. We report here on the development of an efficient method for rapid clonal propagation by shoot proliferation from axillary meristem(s) of selected germplasm of Aloe vera. Explants were pretreated with 0.1% aqueous solution of both streptomycin and bavistin separately, each for 15 min. These were surface sterilized with 0.1% aqueous solution of mercuric chloride (HgCl₂) for 4–5 min and washed several times with autoclaved water. These were kept in a chilled, sterile antioxidant (200.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 of citric acid, and 25.0 mg L^?1 of polyvinylpyrrolidone; PVP) solution and cultured on semi-solid Murashige and Skoog's (MS) medium. The bud explants produced multiple (10.3 ± 0.675/explant) shoots on MS medium containing 13.32 μM of 6-Benzylaminopurine (BAP) and 100.0 mg L^?1 of ascorbic acid, 50.0 mg L^?1 each of citric acid and PVP, with 25.0 mg L^?1 each of arginine and adenine sulphate as additives. The shoots were further multiplied by (a) repeated transfer to fresh MS medium with additives + 13.32 μM BAP, and (b) subculturing on MS medium with a lower (4.44 μM) concentration of BAP. On MS medium containing 4.44 μM of BAP and additives, a maximum number (27.8 ± 0.63) of shoots were produced. In liquid MS medium with 4.44 μM of BAP, the rate of shoot multiplication increased and the vigor of the shoots improved. One hundred percent of the cloned shoots rooted under in vitro conditions on hormone-free half-strength MS salts containing 200.0 mg L^?1 of activated charcoal at 32 ± 2°C. The cloned shoots treated with 2.46 mM of indole-3-butyric acid (IBA) or 2.473 mM of β-naphthoxyacetic acid (NOA) for 5 min rooted under ex vitro conditions in the greenhouse. The rooted plants were hardened in the greenhouse and stored under an agro-net house. The cloned plants were transferred under different field conditions at various sites in Western Rajasthan. These plants grew normally. The higher rate of shoot multiplication and easier approach of direct rooting and hardening make this method superior to the methods previously reported on cloning/tissue culture of Aloe species. From a single shoot bud, approximately 5000 plants can be produced within 180 days.     

Establishment of in vitro culture of Populus euphratica Olivier

The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine (BAP) and gibberellic acid (GA) in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingjiang, west China, on selected media with MP2 0.5 mg·L-1 BA 0.1 mg·L-1 NAA. The shoots were elongated on a medium with 0.25 mg·L-1 BAP, 0.1 mg·L-1 NAA and 2 mg·L-1 GA and were then rooted on a medium with 0.2-0.5 mg·L-1 IBA. All the media were incorporated with 30 g·L-1 sucrose and an adjusted pH at 6.3.     

Development of an efficient protocol for plant regeneration from nodal explants of recalcitrant bamboo (Bambusa arundinacea Retz. Willd) and assessment of genetic fidelity by DNA markers

The present study describes an efficient method for in vitro plant regeneration in B. arundinacea through axillary shoot bud proliferation. Nodal explants were excised, cultured on MS medium containing different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN) (0.5–5.0 mg l^?1) alone and/or in combinations with KIN/BAP (0.5 mg l^?1). The highest frequency (91.5 %) of multiple shoot bud induction with maximum number of shoots (85 shoots/explant) was noticed on MS medium + 3.0 mg l^?1 BAP + 0.5 mg l^?1 KIN. The regenerated multiple shoots were elongated on MS medium + 4.0 mg l^?1 KIN + 2.0 mg l^?1 gibberellic acid (GA₃) with maximum shoot length (4.9 cm). The elongated shoots were transferred to MS medium containing indole-3 butyric acid (IBA; 0.5–5.0 mg l^?1) alone and/or in combination with 0.5 mg l^?1 KIN and BAP. Highest frequency of rooting (75 %) was obtained on half-strength MS medium + 2.0 mg l^?1 IBA + 0.5 mg l^?1 KIN. After hardening, the plantlets were shifted to the green house and subsequently established in the field conditions with 90 % survival rate. random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the regenerants. RAPD profiles generated from the regenerated plants were found to be monomorphic, similar to the control. Results confirmed that the regenerated plants were true-to-type in nature and the developed micropropagation protocol could be used for large scale plant production of B. arundinacea.     

In vitro flowering of green and albino Dendrocalamus latiflorus

To propagate Dendrocalamus latiflorus, we used in vivo inflorescences to produce calli on Murashige and Skoog basal (MS) medium supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/l kinetin, 250 mg/l polyvinyl pyrrolidone (PVP), and 1% coconut milk. Multiple shoots were generated on MS medium supplemented with 0.1 mg/l thidiazuron (TDZ). The green plantlets were successfully transferred to soil. Multiple albino shoots also regenerated and were able to proliferate on medium containing cytokinins, especially TDZ. Albino multiple shoots rooted in medium containing α-naphthaleneacetic acid (NAA), and callus formation was observed in the presence of 2,4-D and picloram. Green and albino regenerates flowered after 8 months of subculture. The flowering ratio increased to 44% after three treatments in medium containing 1 mg/l TDZ. Morphological observations revealed that the in vitro green and albino flower organs were normal. However, pollen derived from the in vitro flowers of both the green and albino plants were sterile.     

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L^?1 L-ascorbic acid, 25 mg L^?1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L^?1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.     

Plantlets regeneration from root explants ofRobinia pseudoacacia L. by suspension culture

A rapid and efficient method for the regeneration of plantlets from root explants ofRobinia pseudoacacia L. by suspension culture was established. The roots taken from aseptically grown 15-day-old seedlings were used as explants. It was determined that photoperiodicity was necessary for root proliferation, and that the promotive effect of IAA (3-indoleacetic acid) on root proliferation was better than that of IBA (3-indolebutyric acid). The roots cultured in 1/2 MS liquid medium containing 3 μM IAA and 1% sucrose at 25°C under 16-hour photoperiod with 50 μmol m⁻²s⁻¹ PPFD (photosynthetic photon flux density) shaking at 100 times/min reciprocally showed high efficiency for root proliferation. BAP (6-benzylaminopurine) was found to be essential to induce adventitious shoots from the roots, and the roots cultured in the medium supplied with 3 μM BAP combined with 1–6 μM IAA for 3 weeks under the same conditions as in the root proliferation period were most suitable for adventitious shoot inducement.