<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of eight species or subspecies of <Emphasis Type="Italic">Turbinicarpus (Cactaceae)</Emphasis>

Summary In vitro propagation systems by means of areole activation were developed for Turbinicarpus laui, T. lophophoroides, T. pseudopectinatus, T. schmiedickeanus subsp. flaviflorus, T. schmiedickeanus subsp. klinkerianus, T. schmiedickeanus subsp. schmiedickeanus, T. subterraneus, and T. valdezianus. In vitro-germinated seedlings were used as a primary source of explants. Multiple shoot formation from areoles was achieved for three explant types (apical, lateral, and transverse), cultured on Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 10 gl⁻¹ agar and several treatments with cytokinins. Efficiencies were in the range from 7.8 shoots per explant in T. valdezianus up to 19.7 shoots per explant in T. pseudopectinatus, using the best treatment for each species and in a single proliferation cycle. Four of the studied species responded best when 6-benzylaminopurine (3.3–8.8μM) was used, while 6-(γ,γ-dimethylallylamino)purine (19.7–24.6μM) showed better results in two species. The two remaining species showed no significant differences in their response to both cytokinins. Regarding explant type, the best results were obtained with transverse cuts for five species, with apical explants for one species, and the two remaining species showed no significant differences among the explants tested. Rooting of the in vitro-generated shoots was achieved most efficiently on half- or full-strength MS basal medium. Rooting frequencies were in the range from 54.2 to 94.2%, and the frequency of survival of the plants once transferred to soil was 91.6% on average.     

Variation in Phytochemical,Morphological, and Ploidy Levels of Iranian Thymus Species

Thymus is one of the most important genera of the Lamiaceae family. This work was performed to assess inter and intra species variation, which is an indispensable prerequisite for the selection and the exploitation of the germplasm, using yield, secondary metabolites, and ploidy level criteria. Nineteen Iranian populations belonging to 11 Thymus species which includes T. vulgaris were used in this study. The results of cytological observations on the 19 populations revealed the three root-tip chromosome numbers of 2n=2x=30, 2n=4x=56 or 60 (diploid and tetraploid). This study also presents the results of a two-year field experiment that evaluates the agronomic and morphology of the 19 populations of Thymus spp. Cluster analysis grouped the populations into six groups and explained the relationships among ploidy levels, morphological traits, and essential oils (EOs). In general, diploid species belonged to the thymol chemotype, whilst carvacrol chemotype consistently dependent on the gene-dosage effect. Thymus migricus, T. daenensis-2, T. serpyllum, and T. trautvetteri populations with diverse thymol background were the best selection as the parents to improve thymol in a breeding program. Moreover, dry and fresh weight criteria can be used to improve EO content in thyme. Achieving this goal would be expected by crossing T. migricus and T. daenensis-2. Finally, providing relevant information on the ploidy level of Thymus species, with emphasis on morphology and EO components variations, may be recommended for the selection of populations or species to improve bioactive components as well as morphological traits in future breeding programs.     

In vitro plant regeneration of Mammillaria elongata normal and cristate forms

In the normal form of Mammillaria elongata, shoots were regenerated in vitro, through callus, from tubercle explants excised from the upper part of the branch and cultured on Murashige and Skoog medium (MS) with 1.07 μM α-napthaleneacetic acid (NAA) and 22.20 μM 6-benzylaminopurine (BA). A high percentage of tubercles explants of the M. elongata cristate form, excised from the tip of the branch and cultured on MS with 0.54 μM NAA and 0.44 μM BA or 1.07 μM NAA, responded by initially forming an inflated cristate shoot, which gave cristate and normal shoots, without callus intervention, when transferred on basal MS. Callus formed on cristate tubercles explants gave both cristate and normal shoots when transferred onto basal MS. Normal and cristate shoots were rooted in vitro on MS with 9.84 μM or 0.98 μM indole-3-butyric acid, respectively, and established ex vitro. In both normal and cristate form, the differential response appeared to be associated with the site of the explant excision. The formation of shoots was influenced by the season of culture; i.e., explants excised in October had a higher shoot formation rate than those excised February. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors affecting the initiation of mini-rhizomes from Trillium erectum and T. grandiflorum tissues in vitro

Leaf and stem explants of Trillium grandiflorum and T. erectum produced mini-rhizomes (MRs) in vitro which gave rise to shoots and roots. The apical portion of the stem and the basal portion of the leaves were the most effective explants from these tissues, while stem tissue was more responsive than leaf tissue. The best response with both species was observed on half-strength MS basal medium supplemented with cytokinin and auxin. T. erectum was more responsive than T. grandiflorum overall, and in some cases produced MRs in the absence of growth regulators. Culture at 21°C appeared to stimulate growth from T. grandiflorum tissues, compared with controls at 27°C, whereas the outgrowth of shoots from MRs was inhibited in both species at 21°C. In vitro production of MRs could provide a more rapid, alternative propagation method for these species than traditional methods.     

Rapid in vitro production of true-to-type plants of Pogostemon heyneaus through dedieferentiated axillary buds

Summary Rapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitro-derived plantlets. At the optimum level of BA (2.22μM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40d. Retaining the culture without subenlture enhanced the number of shoots (>30 shoots). Inereased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60d) initiated 25–30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22μM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40d. Subeulture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 μM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristies identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns.     

Variation of Essential Oil Content and Composition,Phenolics, and Yield Related Traits Using Different Pollination Systems in Populations of Thymus Species

The production of self-pollinated plants could be important for improving medicinal plants secondary metabolites. In this study, 11 Thymus populations from eight species were evaluated to determine the effect of self and open pollination on agro-morphological characteristics, total phenolic content (TPC), essential oil (EO) content, and EO components. Inbreeding led to some positive effects of above mentioned traits in most of the studied populations. Total phenolic content ranged from 7.07 to 52.69 mg tannic acid equivalents (TAE) g⁻¹ dry weight (DW) in open pollinated derived populations, while it varied from 1.2 to 55.03 mg TAE g⁻¹ DW in self-pollinated ones. Under open and self-pollination condition, the highest EO content was obtained in T. trautvetteri (3.37 %) and T. pubescens (1.96 %), respectively. Gas chromatography-mass spectrometry (GC/MS) identified 42 compounds including thymol, carvacrol, linalool, p-cymene, γ-terpinene, terpinen-4-ol, and α-terpineol as the main compounds. In most cases, selfed plants compared to open pollinated ones, revealed higher thymol content. T. daenensis-1 showed a significant increase in thymol content (from 25.22 % to 74.3 %) due to self-pollination. Moreover, self-pollination led to emergence of some new compounds. Carvacrol methyl ether was the constituents of Thymus EO that are being reported in self-pollinated populations. Finally, inbreeding in Thymus might be suggested as a useful tool to increase genetic homogeneity for the selection of superior plants for improving secondary metabolite.     

Clonal propagation and production of cichoric acid in three species of Echinaceae

In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N⁶-benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39–0.73 mg/g dw) were observed in shoots and leaves.     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens– a valuable medicinal plant

A rapid and efficient protocol for the large‐scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15‐day‐old aseptic seedlings is described. Of the three different cytokinins, 6‐benzyladenine (BA), kinetin (Kin) and 2‐isopentenyl adenine (2‐iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half‐strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α‐naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole‐3‐butyric acid (IBA). 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 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.     

Genetic Diversity and Chemical Polymorphism of Some Thymus Species

To ascertain whether there are chemical and genetic relationships among some Thymus species and also to determine correlation between these two sets of data, the essential‐oil composition and genetic variability of six populations of Thymus including: T. daenensis ?elak. (two populations), T. fallax Fisch . & C.A.Mey ., T. fedtschenkoi Ronniger , T. migricus Klokov & Des .‐Shost ., and T. vulgaris L. were analyzed by GC and GC/MS, and also by randomly amplified polymorphic DNA (RAPD). Thus, 27 individuals were analyzed using 16 RAPD primers, which generated 264 polymorphic scorable bands and volatiles isolated by distillation extraction were subjected to GC and GC/MS analyses. The yields of oils ranged from 2.1 to 3.8% (v/w), and 34 components were identified, amounting to a total percentage of 97.8–99.9%. RAPD Markers allowed a perfect distinction between the different species based on their distinctive genetic background. However, they did not show identical clustering with the volatile‐oil profiles.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

Control of hyperhydricity in Eucalyptus axillary shoot cultures grown in liquid medium

Shoots of four clones of two Eucalyptushybrids were successfully grown in a liquid medium containing modified MS basal salts supplemented with 0.01 mg l ^–1 (0.04 M) BAP and various concentrations of a proprietary anti-hyperhydricity agent (sold commercially as anti-vitrification agent EM2). Shoots of a single clone were also grown using the same medium but with the anti-hyperhydricity agent replaced with various concentrations of a commercially available gelling agent containing pectin (M-Gel) or a polysaccharide extract (iota-carrageenan) from seaweed. A range of supplements applied to the media were effective in reducing the hyperhydricity of shoots. The addition of EM2 and M-Gel led to a significant decrease in hyperhydricty, without showing any significant detrimental effect on the numbers of shoots produced for three of the four genotypes tested. At a concentration of 5 g l^–1, for either compound, the percentage rooting and percentage survival of shoots, both in vitro and ex vitro, either equalled or exceeded those of untreated shoots. Addition of iota-carrageenan, although beneficial in reducing hyperhydricity and improving percentage rooting in vitro, was detrimental to shoot production and acclimatisation ex vitro. Common factors of these agents' contributions to their anti-hyperhydric properties are discussed.     

<i>In Vitro</i> Propagation of <i>Agave guiengola</i> Gentry Using Semisolid Medium and Temporary Immersion Bioreactors

Agave guiengolaGentry is an endemic plant from a very small locality in Oaxaca, Mexico. Its conservation status is fragile and can rapidly worsen. Because of its scarcity, this agave has been used solely for ornamental purposes, but it could have other uses if more plants were available. In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium (MS) as well as basal medium supplemented with cytokinins 6-Benzylaminopurine (BA) or 6-(γ,γ-Dimethylallylamino)purine (2iP). The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^–1 BA, obtaining a mean of 3.7 shoots per explant. Other interesting responses were observed, such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid (2,4-D); root induction without Plant Growth Regulators (PGR); and generation of shoot clusters. These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors, obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies. All new plants rooted and survived the transfer to soil. This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation, economical purposes, or to carry out studies in this species to assess its full potential, avoiding exploitation from wild plants.     

Essential Oil Composition of Five Thymus Species from Bulgaria

In this study, the essential oil composition of five Thymus species, belonging to Sect. Hyphodromi (A. Kerner) Halácsy – Thymus atticus Čelak., T. leucotrichus Halácsy, T. striatus Vahl, T. zygioides Griseb. and T. perinicus (Velen.) Jalas. was studied by GC/MS/FID. T. atticus, T. leucotrichus, and T. striatus were characterized by high amounts of sesquiterpenoids (57.7, 78.9 and 79.7 %, respectively) with β-caryophyllene and caryophyllene oxide as the main constituents. Aromatic compounds (61.2 %) were the most abundant group in T. zygioides essential oil, with thymol (51.2 %) as the principal component. The essential oil from the endemic species T. perinicus contained almost equal amounts of monoterpenoids (37.8 %) and aromatic compounds (36.0 %) with borneol (17.9 %) and thymol (20.9 %) as the major components. The obtained results revealed the existence of new chemotypes of T. atticus (caryophyllene oxide/β-caryophyllene), T. leucotrichus (β-caryophyllene/elemol/germacrene D) and T. striatus (β-caryophyllene/germacrene D/caryophyllene oxide). The essential oil content of endemic T. perinicus is reported for the first time. Principal component analysis (PCA) and cluster analysis (CA) were used to investigate the variations in the essential oils of different Thymus species from Sect. Hyphodromi (A. Kerner) Halácsy.     

A rapid in vitro propagation protocol for Piper barberi Gamble, a critically endangered plant

Summary A protocol is described for rapid multiplication of Piper barberi Gamble (Piperaceae) through shoot tip and nodal explant cultures. Nodal explants with a single axillary meristem showed three times better response with respect to shoot proliferation when compared to shoot tip explants. The best shoot proliferation response of nodal explants was observed with a cytokinin combination of N₆-benzyladenine (4.43 μM) and kinetin (2.32 μM), with 88% bud break. The number of shoot initials (2.4) produced per nodal explant was twice the number of shoot initials (1.2) per shoot tip. An average of 6.9±0.58 adventitious shoots were observed from the proximal end of the internodal explants on Mursashige and Skoog (1962) (Ms) basal medium supplemented with N₆-benzyladenine (2.22 μM) and kinetin (0.46 μM). A multiplication rate of 82 shoots per explant could be achieved after 9 wk of subculturing. The in vitro shoots were rooted on one-half and one-quarter MS basal medium. The shoots rooted on one-quarter MS in the dark produced eight roots with an average root length of 3.36 cm and 98% survival. These plants were transferred to the field with a survival rate of 75%.     

贝莱斯芽孢杆菌Bv-303对水稻白叶枯病菌的拮抗活性及其应用

本研究明确了一株新型贝莱斯芽孢杆菌(Bacillus velezensis) Bv-303菌株对黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv. oryzae,Xoo)的拮抗活性及其对水稻白叶枯病(bacterial-blight,BB)的生物防治效果。采用牛津杯法测定了菌株Bv-303发酵上清液(cell-free supernatant, CFS)对白叶枯病菌体外拮抗的活性及其稳定性;通过对接种白叶枯病菌的水稻叶片进行喷雾处理,在水稻体内测试了该菌株发酵液(cell-culture broth,CCB)、发酵上清液及菌悬液(cell-suspension water,CSW)对白叶枯病菌的抑制效果;并统计了该菌株对水稻种子发芽率与幼苗生长的影响。结果表明,在体外,菌株Bv-303发酵上清液对白叶枯病菌的生长抑制率可达85.7%–88.0%,对热、酸、碱、紫外线等具有较好的稳定性;在水稻叶片上,喷施该菌株的发酵液、发酵上清液及菌悬液均能提高植株对白叶枯病的抗性,其中发酵液的效果最佳,抗病性提高率高达62.7%;且发酵液对水稻种子萌发和幼苗生长均没有副作用。因此,菌...     

In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi-an endangered medicinal plant native to Taiwan

Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l⁻¹ BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shool multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l⁻¹ indole-3-butyric acid and 0.1–0.2 mg l⁻¹ α-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2+DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4wk) and ex vitro-acclimated plantlets.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Clonal propagation and hydroxycitric acid production from <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of <Emphasis Type="Italic">Garcinia indica</Emphasis> using root suckers as explant

In vitro shoots from mature tree explants often show poor root initiation response. Garcinia species are known for vegetative propagation through root suckers in natural environment. To establish a protocol for clonal propagation, apical and intercalary buds of the root suckers obtained from a mature elite tree of Garcinia indica were used as explant source. Multiple shoots were initiated in woody plant medium fortified with combinations of 6-benzylaminopurine and 1-phenyl-3-(1,2,3-Thiadiazol-5-YL)-urea. A pulse treatment of indole-3-butyric acid ranging from 4.9 to 19.6 mM for 30 s and 1 min was tried for root induction. The resulting in vitro plants were successfully hardened. The initiated shoots were also used for in vitro synthesis of hydroxycitric acid in shake flask cultures. The effect of basal medium, incubation period and plant growth regulators on production of hydroxycitric acid in vitro was studied.