Micropropagation

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 9 J.regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the sequential use of 0.4 and 0.2 mM phloroglucinol during shoot multiplication, but not at later stages, so as to maximize shoot growth without inhibiting root formation. Moreover, substituting FeEDTA by FeEDDHA diminished chlorotic symptoms and significantly improved the rooting ability of all genotypes, with up to 90 % microshoots developing viable roots at 6.81 mg/L Fe3+. The addition of 83.2 mM glucose during the root expression phase was particularly efficient at promoting plant survival during acclimatization, compared to equimolar amounts of the alternative sugars sucrose and fructose. At the proposed working concentrations, the aforementioned compounds did not cause any deleterious effects on the nine genotypes studied. Microscopic analysis revealed the physical continuity between adventitious roots and stem cambial tissue. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5800 ramets) have been established by applying this protocol.

The methods for leaf-derived callus induction, callus proliferation, adventitious shoot induction and plant regeneration of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) were examined. In this study, callus induction was formed on both medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with thidiazuron (TDZ). The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2,4-D and 0.2 mg/l TDZ. The best callus proliferation medium was Schenk and Hildebrandt (SH) supplemented with 0.2 mg/l TDZ and 1.0 mg/l 2,4-D. The maximum callus-derived shoot number (8.2) was obtained on SH medium supplemented with 50 g/l sucrose in combination with 2.0 mg/l 6-benzylaminopurine (BA). The most successful rooting of regenerated adventitious shoots was obtained on SH medium with 1.0 mg/l α-naphthalene acetic acid (NAA). Plantlets were successfully acclimatized without growth chamber facility on Ngoc Linh mountain with a survival rate of 85% after two months. On the other hand, substantial increase of root length was observed. This study describes an efficient method for in vitro regeneration of P. vietnamensis, which could be considered for large-scale multiplication and propagation of this important medicinal plant.

The plant tissue culture refers to the cultivation of different part of the plant, I both single cell organ tissue and under sterile circumstances at the media and industrial expansion in several controlled circumstances. The method of the plant tissue culture plays a pivotal role in the second green revolution that gene modification and biotechnology can be used for improving the crop harvest and also superiority. Through using plant tissue culture methods plants can be attained from several explants over direct or indirect morphogenesis and by somatic embryogenesis. Direct morphogenesis refers to the manufacture of shoots from explants deprived of passing over callus (unorganised tissue) stage while indirect morphogenesis relates to the generation of shoots over the callus stage. The culture in which an organized form of growing may be incessantly got is mentioned to as an organ culture. The most significant types of organ cultures used for micropropagation are meristem cultures, ...

Thidiazuron (TDZ) is an active cytokinin that was shown to induce increased shoot proliferation and habituation in black walnut, Phaseolus lunatus and evergreen azalea, which are tree species but has not been widely investigated in bananas. Unlike other cytokines commonly in use that are adeninebased, TDZ is a urea based cytokinin and therefore is non-degradable by cytokinin-oxidase enzymes in plant tissues. This quality causes TDZ to be persistent in tissues hence transforming them from cytokinin dependence to cytokinin autonomy. This therefore makes use of TDZ cost effective but there is lack of information on this quality in banana micropropagation. A study was therefore conducted to investigate the carry over effect of varying concentrations of TDZ and 22.2 M benzylaminopurine (BAP) as control on proliferation of five banana cultivars on a hormone free medium under various incubation conditions. The results showed that TDZ had a carry-over effect that enabled shoots to continue proliferating on a hormone free medium as the culture cycles increased and that this effect was significantly (P<0.05) higher than that of BAP. Accumulation of TDZ to high levels resulted in suppression of shoot proliferation but on exposing such tissues to a cytokinin-free medium in subsequent subcultures would result in increased shoot proliferation and elongation. The results further showed dark conditions enhanced higher proliferation rates than light conditions in some cultivars suggesting that banana in vitro proliferation is a photomorphogenically responsive process that is enhanced under dark conditions.

Pecan, [Carya illinoinensis (Wangenheim) C. Koch] is a nut crop of growing worldwide interest and importance. In this research, a micropropagation protocol was developed during 2015–2016 using five pecan cultivars (‘GraTex’, ‘Wichita’, ‘Choctaw’, ‘10 J’ and ‘GraKing’). In the establishment phase, nodal segments of mature pecan trees were collected 9, 13, 17, 21, and 25 days after bud-break and maintained at 4°C for 0, 24, 48, or 72 hours. Next, they were soaked in 2.5% sodium hypochlorite for 1, 2, 3, 4, 5, 6, or 7 minutes. Each explant was cultured on DKW medium individually. For proliferation phase, the best established shoots were cut into nodal segments and cultured on DKW medium supplemented with different BA concentrations (4.44, 13.30, 22.20, and 44.40 µM). Proliferating cultures were established and microshoots were rooted on MS medium containing IBA (4.9, 14.7, 24.5, and 49.0 µM) for 6 days in the dark. Date of cutting field-grown shoots for explanting had a significant effect on disinfection and culture establishment, with optimum success 11–15 days after bud break. Preliminary chilling treatment (4°C for 48 h) prior to cutting into nodal segments improved explant establishment. The best proliferation rate was obtained using 22.2 µM BA. Root induction using 24.5 µM IBA gave the highest rooting percentage. Rooted microshoots were successfully transferred to pots. Although the ‘Wichita’ and ‘10 J’ cultivars had the highest success for acclimatization (89% and 84% respectively) the difference between cultivars was not significant and plantlets of five pecan cultivars were successfully acclimated. The results of this research can be applied by the authors in commercial level.

The effects of potato, papaya, and tomato organic extracts, prepared at various concentrations (0, 5, 10, 20 and 30%) were examined for the successful proliferation of in vitro Vanda Kasem's Delight (VKD) orchid's protocorm-like bodies (PLBs). Results obtained indicated that the growth of PLBs treated with organic extracts was significantly different compared to that of the control which lacked both carbon source and plant growth regulators. The use of Vacin and Went (VW) medium supplemented with coconut water and organic extracts appreciably enhanced the proliferation of PLBs of VKD, although no significant differences were detected among the treatments. It was concluded that VW medium, when supplemented with coconut water and 20% tomato extract, produced the highest proliferation rate for PLBs of VKD. Observations from this treatment included the production of healthy, green and freshlooking PLBs, and a reduction in the occurrence of culture browning. Therefore, this treatment has successfully resolved problems involving the long reproductive cycle and slower proliferation nature of VKD orchid plant.

Stephania japonica (Thunb). (Syn S.
harnendifolia) is a species under the genus of climbers
belonging to family Menispermeceae. It is used in
traditional medicinal practices and is locally known as
Tubuki lota or Goldua. Having high medicinal value, roots
of Stephania japonica are used for treatment of fever,
diarrhea, dyspepsia and urinary diseases. The alkaloid
akanidine shows significant anti-spasmodic activity on
uterine spasms. As this highly valued medicinal plant is
facing rarity, its micropropagation and conservation was
felt necessary. Stephania japonica explants collected
during dry spells (January) in dormant stage showed higher
rate of regeneration. Murashige and Skoog medium
showed good response compared to others. Among the
various hormones used singly or in combinations, best
results were obtained in BAP (4.0 mg/l) + Kn (0.1 mg/l)
after 15 days of inoculation. Among the various
combinations, BAP+Kn showed best results in BAP (4.0
mg/l) + Kn (0.1 mg/l). Best rooting i.e longest roots of 14
mm and maximum regeneration of 17.1 roots were observed
in IBA (1.0 mg/l) in 14 days. For hardening, shoots with
profused rooting were transferred to ½ strength MS liquid
medium with filter paper raft support for two weeks.

The aim of this work was to study the initiation and establishment of in vitro cultures of ‘Rocha’ and ‘Williams’ pear cultivars, as well as the in vitro rooting of micropropagated shoots and of stem discs from micropropagated shoots of ‘Rocha’.
For initiation and establishment, uninodal cuttings from growing shoots of adult trees in the field were used. The effects of salt medium, BAP concentration and ‘Rocha’ clone were studied. The percent contamination was less than 7% and 26% in ‘Rocha’ and ‘Williams’, respectively. The medium containing QL mineral salts increased the fresh and dry weights of the shoots developed from ‘Rocha’ explants, as well as the number of shoots per explant of ‘Williams’, as compared to MS salt medium. The highest BAP concentration (2 mg/ L) yielded the highest number of shoots per explant in both cultivars. ‘Rocha’ clone had a significant effect on the number of shoots per explant and on the fresh and dry weights of those shoots.
For in vitro rooting of micropropagated shoots, either juvenile shoots (Experiment 1) or shoots of adult origin (Experiment 2) were used. In these experiments different conditions of exposure to IBA were assayed which were defined by the duration of the darkness period and/or of the period during which the shoots were kept on an induction medium containing 10 µM IBA. In the experiments with stem discs, only discs from shoots of juvenile origin were used. The above conditions of exposure to IBA were assayed (Experiment 4) as well as the effect of increased IBA concentrations (from 2,5 to 20 µM) in the induction medium (Experiment 3). 
A maximum of 50% rooted shoots and 12% rooted stem discs was achieved in the in vitro rooting assays. Treatment 5D (5 days with IBA in the dark) resulted in the highest shoot development and the least callus formation but gave a lower rooting percent than treatments 5D+5D (5 days with IBA in the dark + 5 days with IBA under a normal photoperiod) and 42D (six weeks with IBA, the first 5 days in darkness and the rest under normal photoperiod). The increase in the dark period duration from 5 to 10 days (treatment 10D) was detrimental to shoot and root development. In the experiments with stem discs the highest rooting percent was achieved using 10 µM IBA in the induction medium. An increase in IBA concentration increased the frequency of callus formation while the exposure treatment affected the type of callus formed (‘smooth’ or ‘filamentous’).
Key words: in vitro culture; ‘Rocha’ pear; in vitro establishment; in vitro rooting; stem discs.

ABSTRACT: Macadamia is categorized into a group of minor crop but can exhibit wide climatic adaptability. It has quick expanding industry demands for its high economic and nutritional values. Graftings, cuttings and micropropagations are the three major propagation methods of macadamia that can be utilized in a combination manner as seedlings with successful graftage can be encouraged to elongate and provide more scion woods for further multiplication such as supporting materials for cuttings. Cuttings can be sprouted to provide explant materials for micropropagation. Therefore, micropropagations are being used for commercial production of macadamia. Micropropagations are comparatively easy and convenient methods that can faster the introduction of any novel varieties into a new region, production of biotic stress tolerant plants, production of plants with greater uniformity and superior branching. Hence, this crop plant might hold an example for the other plants in the next few decades regarding much better application of advance propagation technologies and other genomic approaches.

In this study successful propagation of mature trees of Sorbus torminalis (L) Crantz was obtained by in vitro methods. The efficiency of shoot regeneration was tested in MS medium and in the multiplication phase. A complete bifactorial design between 6-benzylaminopurine (BAP) and indol-3-butyric acid (IBA) with five concentrations each, was applied. The type of hormone, concentration and their interaction significantly influenced multiplication parameters. The presence of BAP is crucial for shoot proliferation and development. The best multiplication rate, between 6.3 and 6.9, was recorded with 1 or 2 mg/L BAP with the lowest IBA concentrations. Elongation of shoots was also significantly affected by the range of hormone concentration, with a positive relation between elongation and IBA concentration in every BAP level. The length of the longest shoot, between 20.8 and 22.4 mm, was recorded in a medium containing 1 or 0.5 mg/L BAP, respectively, with 1 mg/L IBA. Different IBA concentrations in different induction times were tested for the rooting phase. The best results were obtained with the in vitro regenerated shoots immersed in MS agar medium, supplemented with 50 mg/L IBA, for 72 hours, followed by direct peat:perlit soil moisture transfer, with 77% of rooting and almost 100% of acclimatization success. The plantlets transplanted to soil showed normal growth the following spring and did not show any morphological variation.

The plant tissue culture refers to the cultivation of different part of the plant, I both single cell organ tissue and under sterile circumstances at the media and industrial expansion in several controlled circumstances. The method of the plant tissue culture plays a pivotal role in the second green revolution that gene modification and biotechnology can be used for improving the crop harvest and also superiority. Through using plant tissue culture methods plants can be attained from several explants over direct or indirect morphogenesis and by somatic embryogenesis. Direct morphogenesis refers to the manufacture of shoots from explants deprived of passing over callus (unorganised tissue) stage while indirect morphogenesis relates to the generation of shoots over the callus stage. The culture in which an organized form of growing may be incessantly got is mentioned to as an organ culture. The most significant types of organ cultures used for micropropagation are meristem cultures, shoot cultures, embryo cultures and isolated root cultures. Callus cultures, suspension or cell cultures, protoplast cultures or other cultures are clustered as unorganised tissue cultures. The method that was used was the plant tissue culture in the circulation of several plants, that is also the most significant way for strawberry production, to get the plants disease-free viral and harvest plants like the mother plant or the manufacture of new kinds with good potentials that help the turnout for the cultivation of this plant, that is considered by its high fruits nutritional value. Most plants are affected by the micropropagation strawberry essential basics of the media, like hormones, vitamins, amino acids and physical state and the power source to the media. This paper is a summary of the classic and new technology plant tissue culture and the effect of some features, like the media and development managers (hormone plant), vitamins, amino acids and the physical case of the media. It frequently affects most of these features in the circulation of plants using the tissue culture over the effect in the creation of callus or embryos or vegetative emergence and development of roots, as well as in the fraction development of branches and roots as well as the development highlighted in the influence of these influences on the micropropagation of plants. Tissue culture and some of the factors affecting them and the micropropagation of strawberry. Life Sci J 2014;11(8):484-493]. (ISSN:1097-8135). http://www.lifesciencesite.com. 63

Melissa officinalis L. is widely cultivated in the world and Iran as well, due to its useful application in medicine. In order to establish a stable and high frequency of regeneration system with 4 landrace collected from different climate in Iran, major parameters such as regeneration rate, rooting percentage, shooting and proliferation rate were investigated. Statistical analysis of results showed that BAP in combination with NAA had the highest regeneration in shoot tips explants. NAA in combination with IAA and Kinetin had the best response to callus induction. Also 1 mgl/l NAA had the higher response to rooting than other's used auxins. Explants derived from Hamedan landrace had the highest potential for regeneration. This is the first report on high frequency in vitro regeneration in four diverse landraces of M. officinalis selected from various climates regions in Iran.

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species.

A B S T R A C T RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32-33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA 3 (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale. ß

The present study was undertaken to study the in vitro responses of different clones of Dalbergia sissoo. For this study, three clones: one from Uttarakhand (clone No.09), Rajasthan (clone No.59) and Haryana (clone No.79) were investigated. These clones were selected on the phenotypic characters of candidate plus trees namely: higher volume of wood, clean bole length and stem form, etc. These clones were established and maintained at the Vegetative Multiplication Garden of Forest Research Institute, Dehradun. In this study, a standard procedure was developed for rapid multiplication of Dalbergia sissoo through axillary bud proliferation. Nodal explants were collected from different clones and were sterilized with 0.1% mercuric chloride for 15 min. Explants were collected for a year and the effect of season was adjudged. In all the three clones, explants collected from March to May respond at higher frequency and induced more shoots at optimum requirement of each clone. In clone No.9, maximum response of 92.35% was recorded, while in clone No. 59 and clone No.79, it was 89.31 and 91.63% respectively. For in vitro shoot multiplication in clone No.9 BAP at 2.5 µM was found superior with multiplication rate of 2.41, while in clone No.59 and clone No.79 BAP at 5 µM was found superior with 2.35 and 2.37 multiplication rates respectively. In vitro roots were induced on IBA supplemented medium. Effective IBA concentration was found to be 5 µM in clones No.9 and No.79 where 65.12 and 62.74% of root induction was obtained, while 7.5 µM IBA gave maximum of 60.50% of root induction in clone No.59. Plants were hardened and acclimatized before field transfer in all the three clones.

Khalafalla, M. M., Abdellatef, E., Mohameed Ahmed, M. M. and Osman, M. G. 2007. Micropropagation of Cactus (Opuntia ficus-indica) as Strategic Tool to Combat Desertification in Arid and Semi Arid Regions. Int. J. Sustain. Crop Prod. 2(4):1-8

The project was designed to facilitate the development of commercial pharmaceutical products based on indigenous plants of the Caribbean. Countrywide information on medicinal plants was collected and many local plants were investigated for their biologically active compounds for eventual production of nutraceutical and/or pharmaceutical products. Genebanks were established for the main species and tissue culture was used — often after developing species-specific protocols in the laboratory — for conservation and propagation purposes. Hot pepper was chosen as a suitable crop for promotion within the small island States of the Caribbean as even 0.005 hectare has proven to be an economic unit. This crop is priced well in the market and is growing in demand both as a spice and for its therapeutic properties. Neem (Azadirachta indica) was also chosen as a flagship plant for development owing to its potential applications in both medicine and agriculture. Neem oil was tested for its antimicrobial activity and agriculturally useful formulations (biopesticides and a disinfectant) were tested. This project was innovative mainly because it was multifaceted and aimed at strengthening existing or newly established biotechnology-related research teams throughout the Caribbean that could then sustain the initiative beyond the duration of the project.

The effect of N 6 (3-hydroxybenzyl)adenine (meta-Topolin-mT) was compared with that of N 6-benzylaminopurine (BAP) and zeatin at the proliferation stage of micropropagation of red-fleshed Actinidia chinensis var. chinensis 'Zes006' in two separate experiments. Shoot number, shoot weight, leaf number and leaf area were significantly higher in mT-supplemented media compared with BAP or zeatin. When transferred to rooting media, plantlets that were propagated in mT-supplemented media readily produced roots, enabling easy acclimation to the greenhouse, whereas none of the plantlets propagated in BAP-or zeatin-supplemented media produced roots. Using 12 pairs of Simple Sequence Repeat primers designed for A. chinensis var. chinensis, a very low rate of somaclonal variation was detected at some loci in plantlets produced in zeatin-(1.04%), BAP-(0.4%) as well as in mT-(0.2%) supplemented media. Overall, mT in equimolar concentrations was the better cytokinin for tissue culture of 'Zes006' kiwifruit and may well be applicable to many other kiwifruit genotypes. Key message Supplementation of media with meta-Topolin for in vitro propagation of the red-fleshed kiwifruit cultivar 'Zes006' enhanced better shoot proliferation, giving healthy plantlets that were easier to acclimatize to the greenhouse environment compared with media supplemented with 6-benzylaminopurine or zeatin. It also induced a lower rate of somaclonal variation, as detected by SSR markers.

This book is a result of the project ‘Second generation of planted hardwood forests in the EU — Woodnat’, financed by the European Commission, carried out by a companies’ temporal consortium integrating Seistag Innovacion SL (Spain), Industrial Plants OOD (Bulgaria), ECM Ingenieria Ambiental SL (Spain), WAF Estructuras Digitales SL (Spain), Losan Romania SRL (Romania), Asimov Efficiency SL (Spain), Bosques Naturales SA(Spain), Consiglio per la ricerca In agricoltura e l'analisi dell'economia agraria (Italy) and Wale SAS (France).
The objective of this guide is to be a silvicultural manual analyzing the considered as the better practices to be applied for hybrid walnut (Juglans x intermedia) plantation management oriented to timber production, taking into account scientific literature and the experience in the sector of the companies who compound the Woodnat consortium.
This guide tentatively answers the questions any forest manager would have about walnut planted forests. Applied with common sense, combined with new information as available and modified when needed, these guidelines should be useful tools for the management of walnut planted forests under different cultivation systems and circumstances.

Date Palm (Phoenix dactylifera L.) is a salt and drought tolerant fruit crop mainly cultivated in Arabian countries. Shoot
tip explants of offshoots were used traditionally for various micropropagation protocols on research or commercial levels.
However, its main disadvantage was the scarification of the entire plant. Subsequently, this hinders the micropropagation
of male and female recalcitrant individuals with no offshoots or the interesting cultivars with a limited population.
Consequently, current study is investigating the factors affecting commercial production of tissue cultured palms with
cost-effectively and short-production cycle, which may also strongly enhance the transformation protocols. The innovative
way by which the 15 cm long immature inflorescence was excised before emergence between fronds is reported herewith
this study for the first time. Established spikelet explants were able within 2-3 months only without any callus phase
to produce shining globular structures instead of immature florets. These embryogenic structures couldn’t develop
further without maturation process for 1-2 months under full darkness as well. Subsequently, well-matured embryogenic
structures were shifted to the subsequent differentiation medium under illumination conditions. After then, green shoots
and multiple somatic embryos have been subjected to the multiplication stage, then rooting and eventually successfully
transplanted in the greenhouse. All used nutrient media and their sequential usage is reported in this study by which
it became possible from one inflorescence to produce 10000 plantlets in rooting stage right now of Gulistan Pakistani
cultivar without any bad consequences on the mother tree.

Paulownia is a genus of fast-growing and multipurpose tree species that is native to China. Due to their rapid growth and value in the timber market, many Paulownia species are cultivated in several temperate zones worldwide. Economic importance of Paulownia is increasing as new uses and related products are developed. It is also suitable as a lignocellulosic feedstock crop for the bioethanol industry in the Southeastern USA. A number of Paulownia species are valuable sources of secondary metabolites including flavonoids with high antioxidant activities. A high demand for planting material in domestic and international markets for afforestation and bioenergy production has necessitated the development of efficient micropropagation protocols for rapid and mass propagation of Paulownia. Over the past several decades, research on Paulownia species has been conducted to develop micropropagation, somatic embryogenesis and genetic transformation protocols for use in agroforestry and reforestation programs. Given the economic importance and current and potential future uses of Paulownia, this paper reviews the development of biotechnological approaches for plant propagation and genetic improvement, and antioxidant potential of secondary metabolites occurring in species.

Inflorescence-based micropropagation holds great potential for the multiplication of recalcitrant male and female date palm individual trees and culti-vars of commercial interests with limited populations. This can be accomplished in a short time with minimal effort as compared to the traditional practice of using shoot-tip explants. The aim of this technique is to pave the way to use inflorescence explants to micropropagate date palm by direct formation of organs (somatic embryos or shoots) and avoiding most of the constraints that face the shoot tip like high percentage of contamination in the establishment stage, heavy browning, a long time for first cluster initiation, using a considerable number of offshoots and inability to micropropagate the elite palms in case no offshoots are available. The way to excise the immature inflorescence without damage to the mother tree, composition of the nutrient medium for direct organ initiation have remained hindrances to this technique over recent decades, in addition to the technicalities for proper handling of cultures inside the laboratory and successful shifting of plants to the greenhouse. We report for the first time an innovative method used to excise the immature inflo-rescence at a suitable stage for successful culture initiation. Spikelet explants are induced to produce shining globular structures without a callus phase. Also, explants were exceptionally able to develop direct shoots. Two types of organs developed on the differentiation medium, green shoots and intact somatic embryos. Only green shoots and multiple somatic embryos were subjected to proliferation at the multiplication stage. Well-rooted plantlets were hardened and successfully established in soil.

Successful shoot regeneration of Dendrobium nobile was achieved using thin shoot tip sections and triacontanol (TRIA) for the first time. Protocorm-like bodies (PLBs) or proliferating shoot buds were observed when thin shoot tip sections were cultured on the basal medium of Mitra et al. (Indian J. Exp. Biol. 14 (1976) 350) supplemented with 4.0 mg L À1 TRIA. The highest percentage of explants (93%) produced PLBs or proliferating shoot buds (21) at 4.0 mg L À1 TRIA-supplemented basal medium. All the newly formed PLBs or proliferating shoot buds survived and ultimately produced healthy shoots with 2–3 leaves. Shoots produced roots when cultured on basal medium supplemented with 2.0 mg L À1 TRIA. The well-rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1), and a 92% survival rate was achieved. This work reveals that TRIA can be used as an effective growth regulator in the micropropagation and conservation of D. nobile.

Conservation of plant biodiversity, a potential resource for health care, is essential for the very survival of the human race. Increasing importance to traditional medicine in the recent years has threatened the survival of rare species. Conventional plant propagation methods are affected by variable environmental factors and time consuming. In vitro propagation, an ex situ conservation strategy, provides new means for conservation and mass propagation of economically important plants. In addition, in vitro plant systems serve as an alternative approach for the production of bioactive compounds from medicinal plants. The present review is to focus on application of in vitro propagation via organogenesis, somatic embryogenesis and cell suspension culture for plant conservation.

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species.

Phalaenopsis species are much sought after in the orchid flower industry due to their long-flowering periods and their large-sized flowers. There is a need to hasten the micropropagation process and to improve the quality of the Phalaenopsis plantlets through optimization of medium components. Three experiments were carried out in this research to test the following parameters: direct shoot regeneration from nodal explants of Phalaenopsis violacea flower stalks on half-strength MS medium added with different cytokinins (1benzylaminopurine or BAP, thidiazuron or TDZ, and zeatin) under various concentrations; the effects of different cytokinin concentrations on protocorm-like body (PLB) induction from leaf segments of the orchid; and the effects of different cytokinin concentrations on the orchid's PLB proliferation rates. Among the three cytokinins tested, BAP produced the highest percentages of induced shoots from the nodal explants of the flower stalks, after 8 weeks of culture. Both 0.8 µM and 1.0 µM BAP resulted in the conversion of 100% nodal explants to shoots, with each corresponding to the formation of 3.7 and 2.9 shoots per nodal explant respectively. Half-strength semi-solid MS media containing 0.6 µM, 0.8 µM and 1.0 µM BAP induced PLBs in 60%, 70% and 50% of the leaf segments respectively, with 0.6 µM also producing the highest PLB proliferation rate at 75% after 12 weeks of culture. Among the TDZ concentration series, 0.6 µM induced 60% of the explants into shoot formation, producing 2.6 shoots per explants, with both 0.6 µM and 0.8 µM producing PLBs from 40% of the leaf segments. Zeatin produced low percentages of nodal explant conversions and shoot formation with the lowest PLB induction and proliferation rates.

Efficient shoot regeneration of Eria dalzelli (Dalz.) Lindl. was achieved for the first time using shoot tip transverse thin cell layers (tTCLs) and thidiazuron (TDZ). Protocorm-like bodies (PLBs) or proliferating shoot buds was observed when tTCLs of shoot tip sections were cultured on Mitra et al. (1976) basal medium supplemented with 9.08 μM TDZ. The highest percentage of PLB survival was 96%, producing healthy shoots with 2-3 leaves. Shoots rooted when cultured on the same basal medium supplemented with 11.42 μM IAA. Regenerated plantlets grew normally with a 90% survival rate. This simple protocol will be useful for large-scale propagation of E. dalzelli.

Reliable and reproducible protocols to get healthy and well formed plants from nodal explants of the pomegranate (Punica granatum L) cv 'Bhagava' has been developed. Nodal segments were cultured on two different media at full strength Murashige and Skoog (MS) and Woody Plant Medium (WPM). The media was prepared as a basal medium supplemented with 0.2 to 2 mg/L 6-benzylaminopurine (BAP), 0.1 to 1 mg/L 1-naphthalacetic acid (NAA), 0.5 to 2.5 mg/L silver nitrate (AgNO 3 ) and 10 to 50 mg/L adenine sulphate for establishment stage. For proliferation stage, 0.1 to 0.5 mg/L BAP and NAA was tested. For rooting stage 0.0, 0.25 and 0.5 mg/L 3-Indolebutyric acid (IBA) and NAA on MS and WPM medium were tested. The nodal explants grown on MS medium containing 1.8 mg/L BAP, 0.9 mg/L NAA, 1 mg/L silver nitrate and 30 mg/L adenine sulphate had the highest proliferation rate (10 to 15 shoots/explants) in establishment stage. The same trend was found concerning the maximum leaves numbers (15 to 20 leaves/explants) on proliferation medium containing 0.4 mg/L BAP and 0.3 mg/L NAA. The plantlets grown on MS medium were found to have better survival compared to WPM medium. 0.5 mg/L NAA and 0.5 mg/L IBA showed equal rooting response in both the medium, whereas thick root formation was observed in the medium containing IBA.

Pyrethrum (Chrysanthemum cinerariifolium (Trev.) Vis. = Tanacetum cineariaefolium (Trev.) Schultz-Bip.) is a perennial herbaceous plant belonging to the family Asteraceae, native to Albania and the area of former Yugoslavia. It is the only species in the genus Tanacetum having an agronomic importance, although the genus consists of several species producing similar types of bioactive metabolites. The species is grown in order to obtain the insecticidal compounds collectively termed pyrethrins, which are found primarily in the flower heads. In this work we discuss the results found from a worldwide literature review about the micropropagation techniques followed on pyrethrum, the in vitro culture conditions, and the ex vitro establishment trials under Mediterranean environmental conditions. Many technical problems concerning the propagation of the species seem to have been solved, and detailed protocols are available for an easy and fast propagation by seeds, vegetative splits, stem ...

An efficient protocol for in vitro micropropagation of Bambusa nutans Wall. ex. Munro has been described. Nodal explants obtained from 1½-year-old field-grown culms of B. nutans produced up to 7.0 multiple shoots per explant on Murashige and Skoog (MS) basal medium supplemented with 6benzylaminopurine (BAP, 1.0 mg/L). Continuous shoot proliferation up to 11.33 shoots was achieved by sub-culturing shoot clumps (4 shoots/cluster) in BAP (0.5 mg/L) and 0.1 mg/l α-naphthalene acetic acid (NAA) fortified medium every 4 weeks. 85% rooting was recorded on 2.0 mg/L NAA supplemented medium after 30 to 35 days of culture period. Micropropagated plantlets of B. nutans showed 70% survivability during the hardening stage. After hardening, rooted plantlets were successfully transferred to the soil and exhibited 80% survivability and normal growth. Plantlets cultivated in field condition achieved 95% survivability. Seed explants were also used for in vitro culture establishment of B. nutans on different combination of MS medium.

Effect of MS basal medium supplemented with BA, Kin and IAA, both individually and in combinations. Effects of basal media on the regeneration potential of in vitro nodal explants encapsulated with plant growth regulators. Conclusion The encapsulated in vitro nodal explants of A. calva exhibited differential response when treated with various concentrations and combinations of growth regulators. An average maximum of 9 shoots were induced within 10 days from the encapsulated in vitro nodal explants on MS medium supplemented with BA (2.22 µM). The encapsulated in vitro nodal explants failed to regenerate on Kin (9.29 µM) supplemented MS medium. 80% of the in vitro nodal explants encapsulated with BA (2.22 µM) produced shoots (6.9 cm) and roots on SH basal medium (12.5 cm). Incorporation of IAA (2.9 µM) along with cytokinin in the encapsulation matrix resulted in the decrease of overall frequency, number and length of shoots and roots.

Walnuts are species belonging to the Juglandaceae family, widely distributed throughout the temperate and subtropical zones of the planet. They are prized since antiquity for the quality of their fruit and wood. The domestication degree of walnuts is relatively low compared to food crops, and even compared to other tree species. Although the market of wood from walnut often move large amounts of money, its production is mainly based on extensive farms, growing under low-tech systems. Some attempts have been made in the US and Europe to obtain walnut varieties for wood production, however, once walnuts have long life cycles, the characters on which have to be made selection have a high genetic complexity, the limited experience in improvement programs or limitations of commercial protocols available for asexual reproduction, the fact is that there are not available genotypes for that purpose. To develop the ability to reproduce asexually the selected genotypes is the necessary complement of improvement programs. Also the basis of any intensive production system. Traditional systems of vegetative propagation in Juglandaceae family, as well as being ineffective for production of high volumes of plants involve great complexity in its execution and its results are often unpredictable. Micropropagation is seen as the best alternative to overcome the difficulties of these techniques. However, walnuts are considered highly recalcitrant to tissue culture, causing only very few genotypes are propagated commercially. Various stages of micropropagation of walnuts are especially problematic. The control of microbial contaminants, along with the releasing of phenolics and the decay of cultures difficult the in vitro establishment of the vast majority of genotypes. The re-emergence of microorganisms during proliferation is an important source of losses that can also lead to failure of micropropagation. Low rates of rooting and higher mortality during acclimatization, joint to genetic factors, limited the commercial use of this technology to a few genotypes. In order to develop a pre-commercial protocol for micropropagation of a walnut hybrid, attention was paid on the solution of the main problems that hinder the definition of this as a workable technology for the production of clones of selected ortets. So, a methodology for in vitro establishment which reduce the complexity of this phase and improves the success rate for the introduction is presented. A tool for sanitizing contaminated material that serves for the control of microbial re-emergence during proliferation is proposed. Key elements to ensure obtaining rootable microshoots, potentially capable of supporting the transition to ex vitro conditions are analyzed. The replacement of FeEDTA by FeEDDHA as iron source, the introduction of phloroglucinol in the proliferation culture medium and determining a suitable carbon source for the formation of the roots, with a proper management of the stage prior to radical pre-induction, were key aspects for the micropropagation of up to 14 different genotypes of the hybrid walnut. Additionally, performing a detailed analysis of the root system allowed verify that the proposed methodology favored the production of roots that are connected with stalk. Moreover, the presence of morphologically normal stomata, could reduce losses and promotes the hardening process. Complementing the improvement program and cloning, the development of a tool to identify and differentiate the ortets was discussed. Seeking to combine the simplicity of the technique with the maximum discriminative power, the use of genetic markers SSR type was chosen. To a first approximation, some primers previously published (Woeste et al. 2002) and used in several species of the family Juglandaceae (Dangl et al. 2005, Victory et al. 2006, Ross-Davis and Woeste 2008 among others) were evaluated. In spite of that some of these markers yielded interpretable amplification products, their capacity for joint classification was very small and their use was affected by the high kinship degree of progeny. For this reason it was necessary to develop a set of microsatellite markers designed specifically for the hybrid walnut. From 700 sequenced regions, 24 primers showed to be functional and sufficiently polymorphic as to discriminate between siblings. 10 of these markers, along with 2 from the library developed by Woeste et al. (2002), were used in a different population, showing that it was possible to genotyping with a PID of the order of 10-11, which opens the possibility of using this set of markers within the family Juglandaceae.

The rapid clonal propagation of Cymbidium bicolor was achieved by induction of protocorm-like-bodies (PLBs) using transverse thin cell layers of shoot tips when cultured on 24-epibrassinolide (24-epiBL)-supplemented Mitra et al. (1976) basal medium. The highest percentage of explants (86.0%) producing PLBs (65.0 ± 3.9) was recorded when 3.0 μM 24-epiBL was used. All the newly formed PLBs survived and after nearly 12 weeks, small bud-like structures formed healthy shoots. Shoots produced roots when cultured on the same basal medium supplemented with 2.0 μM triacontanol. The well-rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1) and 100% survival rate was achieved. This is only the second report of the use of 24-epiBL, a plant steroid lactone, in the micropropagation of orchids.

Lindl. is one of the horticulturally important orchids of Nepal due to its beautiful yellowish flower and medicinal properties. The present study was carried out for plant regeneration from shoot tip explants of D. densiflorum by tissue culture technique. The shoot tip explants of this species, obtained from in vitro grown seedlings were cultured on Murashige and Skoog (MS) media alone and MS medium supplemented with different combination and concentration of 6benzylaminopurine (BAP) and napthalenacetic acid (NAA). The maximum number of healthy shoot was observed on MS+ BAP (2 mg/l) + NAA (0.5 mg/l) (4 shoots/ culture). The shoot multiplication started after three weeks of culture.

An efficient micropropagation protocol has been developed successfully for Morinda coreia Buch.-Ham. by culturing nodal segments. The explants were washed, sterilized with HgCl 2 and inoculated on semi-solid Murashige and Skoog (MS) medium containing various concentrations and combinations of plant growth regulators (PGRs). Shoot bud initiation was observed after one week and 8.6 ± 0.32 shoots (per explant) harvested after five weeks on MS medium with 4.0 mg l −1 concentration of 6-benzylaminopurine (BAP). The regenerated shoots were further multiplied on semi-solid MS medium augmented with 2.0 mg l − 1 BAP + 1.0 mg l − 1 Kinetin (Kin). Maximum 24.5 ± 0.34 shoots per explant was obtained after five weeks on this media combination. The long (4-5 cm) and healthy shoots were rooted in vitro with 100% success rate on half strength MS medium + 1.0 mg l − 1 indole-3 butyric acid (IBA). Rooting and acclimatization were achieved simultaneously by ex vitro rooting method using 200 mg l − 1 IBA for 5 min with very good success rate (28.67 ± 05.51 roots per shoot with 100% response). The rooted shoots were transferred to the greenhouse for acclimatization for 4-5 weeks. The hardened plantlets were finally shifted to the field for further growth in the natural conditions after another five weeks. This is the first report on micropropagation of M. coreia, which can be successfully used for the large-scale multiplication and conservation of germplasm of this important medicinal plant.

In vitro seedlings were used as explants for protocorm like bodies (PLBs) production which in turn were used for regeneration purpose. PLBs were induced from the base of seedlings (1.0-1.5 cm in size) in MS + BAP (8.88 µM). After 90 days of inoculation, PLBs production rate started declining and most of the PLBs turned into plantlets. Preculture of seedlings in 1.0 µM thidiazuron (TDZ) for 7 days and transfer to BAP supplemented medium resulted in production of 16 PLBs per seedling within 90 days of culture. Increase of TDZ concentration to 2.5 µM and preculture time 15 days, resulted in induction of highest number of PLBs (19 PLBs per seedling) in the basal medium. The results emphasized the importance of thidiazuron (TDZ) concentration and preculture time for PLBs proliferation from the base of seedlings. The PLBs thus produced were used for regeneration studies. Irrespective of single, segmented or clumps of PLBs, the regeneration response was 100% in 2,4-D (4.52 µM) and KN (4.64 µM) but when KN was replaced by BAP (8.88 µM), response was observed only in clumps of PLBs, whereas in single and segmented ones it was 99 and 97%, respectively. Regenerants developed stout root system in half strength M medium supplemented with 2.84 µM of IAA and transferred to greenhouse with 90% survival. The present study holds tremendous potential as the mother plant is not destroyed and PLBs are produced as a continuous system.