Leaf Responses of Micropropagated Apple Plants to Water Stress:Changes in Endogenous Hormones and Their Influence on Carbohydrate Metabolism

The changes in the concentrations of endogenous hormones and their influence on carbohydrate metabolism in leaves of micropropagated Fuji apple plants were studied under water deficiency stress. The results showed that water stress induced a rapid increase in the concentration of abscisic acid （ABA） and led to a decrease in concentrations of both zeatin and gibberellins （GAs）. The concentration of indole-3-acetic acid （IAA） changed in an independent manner, which was not correlated with the different levels of water stress. With regard to the carbohydrates, the contents of sorbitol and sucrose increased, whereas the content of starch decreased. The increase in the concentration of ABA was significantly correlated with both the increase in the activity of aldose-6-phosphate reductase （A6PR） and the decrease in the activity of sorbitol dehydrogenase （SDH）, indicating that ABA played a regulatory role in sorbitol metabolism. The concentration of ABA was positively correlated to the activity of sucrose-phosphate synthase （SPS） but negatively correlated to the activities of acid invertase （AI） and ADP-glucose-pyrophosphorylase （ADPGppase） in water-stressed plants, which indicated that ABA promoted sucrose synthesis and inhibited sucrose degradation and starch synthesis at the same time. Under conditions of water stress, the decrease in the level of zeatin was accompanied by a decrease in the activities of SDH and ADPGPPase. GAs concentration showed positive correlation with ADPGPPase activity. IAA showed no significant correlation with any of the enzymes tested in this study. The results of this study suggested that ABA might be one of the key factors regulating the distribution of carbohydrates under water stress. The metabolism of sorbitol and starch under conditions of water stress might be regulated by the combined action of many plant hormones.

Effects of Growth Regulators on Biomass and the Production of Secondary Metabolites in Peppermint (<i>Mentha pi-perita</i>) Micropropagated <i>in Vitro</i>

The effects of plant growth regulators on peppermint (Mentha piperita) cultured in vitro were studied for the purpose of maximizing growth and essential oil production in micropropagated plants. The basal medium was experimentally supplemented with the auxin 4-indol-3-ylbutyric acid (IBA) and the cytokinin 6-benzylaminopurine (BAP) individually and in combination. Supplementation with BAP alone resulted in the highest values for root length, root dry weight, shoot length, and numbers of nodes, leaves, and ramifications. Treatment with IBA alone or with IBA + BAP resulted in a ~50% increase in shoot fresh weight. The production of secondary metabolites was affected only by the addition of cytokinin, which resulted in a ~40% increase in the total yield of essential oils (EOs). Similar trends were observed for yields of the major EO components (menthone, menthol, pulegone, and menthofuran). Our findings demonstrate that the application of growth regulators increases EO production and biomass concomitantly in an herbaceous species rich in commercially valuable terpenes.

A Phenotyping Protocol for Detailed Evaluation of Apple Root Resistance Responses Utilizing Tissue Culture Micropropagated Apple Plants

In the post-genomics era, reliable phenotypes are considered the bottleneck for unraveling the genetic control over the biology of interest. Phenotyping resistance response of roots to infection by soilborne pathogen is more challenging compared to that of plant aerial parts. In additional to the hidden nature and small stature of fine roots where infection occurs, extra obstacles exist for rosaceae tree crops such as apple. Due to self-incompatible reproduction and high-level heterozygosity of apple genome, genetically identical apple plants cannot be produced through apple seed germination. Here we report an established phenotyping protocol which includes a streamlined tissue culture procedure for micropropagation of uniform apple plants, standardized inoculation procedure using Pythium ultimum, and multilayered evaluating methods on apple root resistance traits. Because of the implementation of tissue culture based micropropagation procedure, constant availability of the uniform plants with defined genetic background, equivalent age and non-contaminated roots overcame a longstanding barrier of systematic and detailed phenotypic characterization of apple root resistance traits. Repeated infection assays by root-dipping inoculation demonstrated the reproducible and wide-range plant survival rates, from single-digit to over 90% survived plants for a given genotype. Genotype-specific values due to P. ultimum inoculation on shoot and root biomass reduction, maximum root lengths, leaf number and cumulative leaf areas were quantified between mock-inoculated and P. ultimum infected plants. Use of a glass-box container offered enhanced accessibility and minimized invasiveness for continuous and non-disruptive observation on the necrosis progression patterns along inoculated roots. With the assistance of a dissecting microscope, the genotype-specific resistance responses along the infected apple roots were captured and analyzed in detail. This reported phenotyping protocol represents a major development and should be easily adopted for other rosacea tree fruit crops with minor modifications.

Comparative micromorphological study of wild and micropropagated Dioscorea bulbifera Linn

Objective:To study the leaf epidermis of wild and micropropagated Dioscorea bulbifera Linn.(D.bulbifera)in order to document useful diagnostic features that may be employed for correct crude drug identification and to clear any taxonomic uncertainties in the micropropagated medicinal plant.Methods:Growth responses of micropropagated D.bulbifera were observed on Murashige Skoog medium supplemented with 6-benzylamino purine(1.0 mg/L)+α-naphthaleneacetic acid(0.2 mg/L)+cysteine(20 mg/L)using nodal segments as explants.Leaves of the wild and micropropagated plants were studied microscopically.Results:More than 80%shoot regeneration and formation of 10%-30%whitish-brown callus were observed within 3 weeks.The highest root proliferation was obtained from Murashige Skoog medium of 6-benzylamino purine(0.05 mg/L)andα-naphthaleneacetic acid(0.01 mg/L)with mean root length of(27.00±1.25)mm and elongated single shoot of mean length(38.00±11.09)mm.Leaf epidermal features that revealed similarities between the wild and micropropagated plants included amphistomatic condition,presence of mucilage,glandular unicellular trichome with multicellular head,polygonal cells with smooth walls,stomata type and shape.Slight variations included thick cuticular wall with closed stomata in wild plant compared to thin walled opened stomata in the in vitro plant.Opening of stomata accounted for larger average stomata sizes of(7.68±0.38)μm and(6.14±0.46)μm on the adaxial and abaxial surfaces,respectively of the micropropagated plant compared to the wild.Conclusions:The diagnostic features obtained in the study could serve as a basis for proper identification for quality control for standardization of the medicinal plant.

Growth of Micropropagated Solanum tuberosum L.Plantlets under Artificial Solar Spectrum and Different Mono-and Polychromatic LED Lights

In agriculture,LED light sources have increasingly replaced the standard luminescent lamps and have acquired an important role in plant micropropagation.We studied the effect of different light sources such as narrow-band LEDs(bright blue,blue,green,yellow,deep red,and red)and wide-band LEDs(cold white,white,warm white,full spectrum,and an artificial solar spectrum sun box constructed by us)on development of potato plantlets in vitro.White luminescent lamps were used as a control.The light intensity of 49μmol·m^(-2)·s^(-1)was provided in all light treatments.We showed that the long-wave narrow-band light treatments were inapplicable for potato micropropagation,because plantlets were weak with small leaves,inhibited roots,and significantly elongated stems.Blue lights provided growth of shortened plantlets with large leaves,well-growing roots,and abundant green mass.The chlorophyll content was lower under blue and bright blue light and was at the same level in the remained treatments.Significant differences in the stomatal apparatus development were observed depending on the light source.These differences were not always reflected in the plantlet phenotype:e.g.,plantlets under blue and bright blue lights showed no differences in any characteristics except stomatal density and size of stomatal guard cells.We found no significant effect of blue light portion in the white lights and full spectrum on plantlet growth.An artificial solar spectrum sun box was the most suitable for potato micropropagation,because it supported the development of plantlets with good fitness,uniform internodes length,abundant roots and green mass accumulation.

Early evaluation of micropropagated black locust (Robinia pseudoacacia L.) clones in Hungary

In Hungary, black locust （Robinia pseudoacacia L.） is one of the most important exotic species, forming entire stands. Its importance is increasing in many other countries. As a result of a new selection program eight black lo- cust clones have been improved for establishing clone trials and a seed orchard. In this study juvenile growth and stem quality of micropropagated clones of black locust were evaluated on a marginal site in central Hungary. At ten years old, the clones ＇MB17D3/4＇ and ＇PV201E2/4＇ appeared to be especially promising for high quality wood production under arid hydrological conditions. A tissue culture method can be considered a suitable tool of propagating superior individual trees and offers new prospects for the rapid cloning of selected genotypes.

Molecular Analysis of Genetic Fidelity in Micropropagated Plants of <i>Stevia rebaudiana</i>Bert. Using ISSR Marker

Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of? fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

Biotechnological Potential of Endophytic Bacteria to Improve the Micropropagated Seedling of Variety RB92579 Sugarcane(Saccharum officinarum L.)

Endophytic bacteria may influence agricultural production in several ways, including promoting plant growth. Two experiments were conducted in order to evaluate the combination of endophytic bacteria from the Brazilian Northeast region aims at the commercial introduction of the inoculation of these bacteria in micropropagated sugarcane plants using a temporary immersion bioreactor. One experiment was done in tubes with sterile commercial substrate, and the other was done in pots with soil;both were installed in a greenhouse. A mixed inoculation was performed in six inoculated endophytic diazotrophic bacteria in micropropagated sugarcane plants, variety RB92579. In the experiment with soil, the mixed inoculation significantly increased the shoot dry matter of plants without the addition of nitrogen fertilizer. However, the accumulation of total-N in the tissues showed no significant differences between treatments with and without nitrogen fertilization. The evaluation of micropropagated seedlings showed no increases in the parameters tested. The results showed that the response of inoculation in temporary immersion bioreactor micropropagation is possible, and that the application of homologous strains may have contributed to a better response by the interaction of endophytic bacteria with sugarcane RB92579. Further studies should be conducted to improve the methodology, which indicates a great potential to optimize this process on a commercial scale.

Phenolic Profile and Volatiles of in vitro Propagated Lavandula angustifolia Mill.Seedlings

An effective in vitro propagation protocol was designed for Lavandula angustifolia Miller,a medicinal aromatic plant that is a prominent source of volatile organic compounds(VOCs).Murashige and Skoog media were supplemented with various concentrations of Plant Growth Regulators(PGRs),and the growth parameters of the nodal segments were examined.Nodal explants formed callus when they were supplemented with 2 mg/L of 6-Benzylaminopurine(BAP).The superior hormonal concentration of Murashige and Skoog(MS)media for the proliferation of shoots from callus cultures(39.33%)was 5 mg/L of 2-Isopentenyl adenine(2iP),and the favorable media for the growth of L.angustifolia callus cultures was 1 and 2 mg/L of BAP,with a 98%for-mation rate in each case.The callus cultures and in vitro propagated L.angustifolia seedlings obtained from var-ious PGR concentrations of MS media were exposed to qualitative and quantitative analysis in terms of phenolic profiles,flavonoids,High-performance liquid chromatography(HPLC)analysis of phenolic acids,and headspace-SPME analysis for volatiles.Such analysis revealed that micropropagated seedlings grown in media containing 1 mg/L of 6-Furfurylaminopurine(KIN)accumulated the highest yield(11.95±0.01 mg GAE/g)of phenolic acids.In contrast,the lowest concentration(2.17±0.04 mg GAE/g)was detected in 0.5 mg/L of BAP+0.5 mg/L of Naphthaleneacetic acid(NAA)media.The plantlets grown in 0.5 mg/L of BAP+0.5 mg/L of NAA media showed the highestflavonoid yield(31.67±0.06μg/g QE/g).In contrast,callus samples exhibited the lowest yield(11.59±0.02μg/g QE/g)offlavonoids in MS media supplemented with a concentration of 0.5 mg/L of BAP.HPLC analysis revealed the variability of phenolic acid contents within the callus cultures as well as plantlets,with gallic acid,4-OH benzoic acid,chlorogenic acid,vanillic acid,caffeic acid,cinnamic acid,and rosmarinic acid being the prominent constituents.The presence of twenty-two chemicals was revealed by headspace-SPME analysis.Eucalyptol,nonanal,borneol,carvone,andβ-caryophyllene were the most abundant.This study demonstrated that micropropagation of L.angustifolia may be an effective method to produce large numbers of genetically identical plantlets for the production of high-value bio compounds.

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Different Explants of Lilium lancifolium Have Different Potential to Differentiate and Regenerate in Tissue Culture

[Objective] The aim was to investigate differences in differentiation and regeneration of the explants from different parts of Lilium lancifolium（Yixing Lily） in tissue culture.[Method] The different parts of scale,leaf and root of Yixing Lily were cultured as explants on MS basic medium supplemented with different concentrations of plant growth regulators,so as to compare their capacity to differentiate and regenerate.[Result] The explants had different potential to differentiate（scale root leaf）.The capacity of different scale parts to differentiate was the lower part middle partupper part;the capacity of different leaf parts to differentiate was the leaf base middle part leaf tip;the capacity of different root parts to differentiate was the root base root tip middle part.[Conclusion] Tissue culture could be well applied in propagation of Yixing Lily.

Optimizing Efficient Micropropagation System of Rhododendron Brachycarpum D. Don by Uniform Design Method

[Objective] The study was conducted to establish a high efficient micropropagation system for Rhododendron Brachycarpum D. Don and realize the high efficiency in vitro micropropagation of R. brachycarpum. [ Method ] Young stems of R. brachycarpum were used as explants, suitable medium compositions for axillary bud growing and rooting were screened through uniform design exPeriments. [ Result] MS （modified） ＋ IAA 0,15 mg/ L ＋ IBA 0.30 mg/L ＋ GA3 3.00 mg/L was the most suitable medium with the regeneration rate of 92%. Stems each with one node were cut from regenerated shoots and cultured for propagation, the proliferative multiple was over 45 within one culture period of 35 d. [ Conclusion] High efficient micropropagation system of R. brachycarpum has been successfully established, which provides some basis for development and utilization and industrial seedling growth of the alpine rhododendron in Changbai Mountain northeast China.

In vitro callus induction and plantlet regeneration of Achyranthes aspera L.,a high value medicinal plant

Objective:To study callus induction from different explants(internode,leaf,root)and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.Methods:Sterilized explants were prepared by uning 0.1%HgCl_2 and 0.5%Bavistin and callus was obtained when cultured onto Murashige Skoog's(MS)medium by using different concentrations and combination of 2,4-D.NAA.BAP,IAA,IBA with 3%sucrose and 0.8%agar.Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.Results:Sterilization treatment of 0.1%HgCl_2.for 2-3 min and Bavistin 0.5%for 10-12 min showed the highest percentage of asepsis and survival rate.Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf.Highest shootlets number(4.83±0.l7)and length(3.8±0.16)cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L.Concerted efforts of BAP 10 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number(6.77±0.94).In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations.Experimentally,3.0 mg/L IBA was enabled to induce maximum rootlets number(10.0±9.82)on full strength MS medium.Afterwards,regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized.The survived plantlets showed 66.67%survival frequency without any morphological abnormality.Conclusions:The results demonstrated that different explants were good source of callus induction,morphology analysis as well as indirect plantlets regeneration.

Rapid in vitro propagation of medicinally important Aquilaria agallocha

Aquilaria agallocha can produce fragrant agarwood used for incense, traditional medicine and other products. An efficient plant regeneration system was established via organogenesis from shoots developed from seedlings of Aquilaria agallocha. Shoots generated many buds on MS medium supplemented with 1.3 μmol/L BA （6-benzylaminopurine） in the first 7 weeks, and the buds elongated on MS medium with 1.3 μmol/L BA＋0.5 μmol/L NAA （naphthaleneacetic acid） in another 7 weeks, 2.3 shoots 2 cm in length per explant were obtained within 14 weeks. Plantlets were rooted on l/2 MS medium after being immersed in 5 μmol/L NAA for 48 h, 96.7% of the roots grew up two weeks later. All plantlets that survived acclimatization grew well in the pots.

A Review of <i>Paulownia</i>Biotechnology: A Short Rotation, Fast Growing Multipurpose Bioenergy 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.

Shoot multiplication and plant regeneration in Caragana fruticosa (Pall.) Besser

Different nutrient media can affect in vitro culturing proto- cols, and experimentation under varied growth conditions is valuable in plants where in vitro methods are in preliminary stages. We carried out the first in vitro propagation studies for the endangered species Caragana fruticosa （Fabaceae）. We evaluated various nulrient media for their im- pact on shoot elongation and axillary bud proliferation using different concentrations of 6-benzylaminopurine （BA） and n-naphthaleneacetic acid （NAA）. Shoot elongation was evaluated based on adventitious shoot primary culture and subculture regeneration from Caragana seedlings. Our goal was to improve both micropropagation and regeneration in C. fruticosa. MS nutrient media was superior to 1/2MS macronutrients, DKW, QL, and WPM for shoot elongation and axillary shoot prolifera- tion. Shoots grown on 1/2MS and WPM exhibited some chlorosis, and shoots on QL produced larger leavers than plants growing on normal medium. The shoot proliferation coefficient on MS media supplemented with 2.22 μM BA and 0.44μM BA ＋ 2.69 μM NAA was significantly higher than that with other treatments in the primary culture. Shoots on 2.22 μM BA showed a higher proliferation coefficient （3.17） than others in the subculture. Shoots were rooted on 1/2MS medium with the addition of different concentrations of NAA. The optimal concenWation for rooting was 0.27 μM NAA （74%）. Roots exhibited many stout and long root hairs. Survivl of established plentlets was 82% at 30 days after transfer to soil. Plants established in the green house showed normal growth and displayed no apparent morphological differences compared to stock plants.

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.

Efficient Micropropagation of Chestnut Hybrids(Castanea spp.)Using Modified Woody Plant Medium and Zeatin Riboside

The chestnuts genus(Castanea spp.)is comprised of economically important trees native to the Northern hemisphere that are used as food and hardwood timber.Here,a very efficient method for micropropagation of European×Japanese chestnut hybrids(Castanea sativa×C.crenata)is described.Woody Plant Medium was used as the basal medium.In vitro shoots of four rootstock cultivars were micropropagated without shoot-tip necrosis on multiplication medium containing 5.7 or 11.4μmol·L^(−1)zeatin riboside,and were rooted on rooting medium containing 2.46μmol·L^(−1)indolebutyric acid.Monthly shootmultiplication rates for each cultivarwere 2–5 folds.In vitro rooting percentages for four cultivars were 87%for‘Maraval’,67%for‘Marigoule’,93%for‘Marsol’,and 97%for‘Précoce Migoule’.Within a 5 week period,80%–95%of rooted shoots were successfully acclimated under high humidity conditions after they were planted in either soil or rockwool.

Factors affecting somatic embryogenesis in conifers

This review seeks to examine the extreme response of isolated somatic plant cells of apical meristematic tissues of mature conifer trees towards specific stress conditions in vitro resulting in somatic embryo- genesis. Signal molecules regulating embryo development have been described in angiosperms, but very little is known about somatic rejuve- nation in conifers. Recent studies on cloning of mature conifers provide new perspectives on signal molecules on cellular dedifferentiation into the embryogenic pathway. Our recent studies show that signal molecules such as butenolide, calcium ions, salicylic acid, antioxidants, amino acids triacontanol and 24-epibrassinolide all play an important role in the con- version of somatic cells into an embryogenic pathway in many recalcitrant pines. This constitutes a major breakthrough in forest biotechnology with many practical applications in clonal forestry.

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.