Adventitious shoot organogenesis and plant regeneration from cotyledons of Dalbergia sissoo Roxb., a timber yielding tree legume

A procedure is outlined to induce adventitious shoot organogenesis from semi-mature as well as mature cotyledons lacking the embryonic axis of Dalbergia sissoo Roxb., an economically important leguminous tree. Shoot buds were induced in the proximal region of the semi-mature cotyledons on Murashige and Skoog's (MS) medium supplemented with 4.44 M 6-benzyladenine (BA) and 0.26 M -naphthaleneacetic acid (NAA). These buds elongated into shoots following transfer to a similar medium containing half-strength macro-nutrients. Adventitious shoot bud formation was also induced in the mature cotyledons. However, unlike the semi-mature explants, the mature cotyledons exhibited shoot bud differentiation on MS medium containing 22.20 M BA without NAA. Pre-culture of mature cotyledons in liquid MS medium containing 8.88 M BA for a duration of 48 h improved shoot bud regeneration up to six-fold. Regenerated shoots, derived from semi-mature and mature cotyledons, rooted on half-strength MS medium containing 1.23 M and 4.92 M indole-3-butyric acid (IBA), respectively. Plantlets were acclimatized and established in soil.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species

One of the alternative methods adopted in recent years is to use biotechnological approaches for improving the tree species. The synthetic seeds offer several advantages, e.g., easy handling, storability, reduced size of propagules, and transportability. Germplasm can be effectively stored in the form of synthetic seeds. A protocol has been developed for plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb. Nodal segments collected from basal sprouts of mature trees were encapsulated in calcium alginate beads. Inability of nodal segments entrapped in calcium alginate beads to form root was a major problem. To avoid this problem, an appropriate root induction treatment was given to nodal segments for 10 days, prior to encapsulation to allow formation of root primordia. For synthetic seeds production and subsequent conversion into plantlet, nodal segments with root primordia were encapsulated using sodium alginate and calcium chloride as gelling matrix. The best gel complexation was achieved using 3% sodium alginate and 75 mmol/L CaCl2 2H2O. Maximum percentage response (85%) for conversion of encapsulated nodal segments into plantlets was achieved on 1/2-MS medium without plant growth regulators, after 25 days of culture. The frequency of conversion of encapsulated nodal segments into plantlets affected by the concentration of sodium alginate, and the presence or absence of 1/2-MS nutrients in calcium alginate beads. Plantlets with well developed roots and shoots were transferred to pots containing autoclaved mixture of peat moss and soil (1:1). Plants were also established in pots. The conversion of encapsulated nodal segments into plantlets also occurred when calcium alginate beads having entrapped nodal segments were directly sown in autoclaved peat moss moistened with 1/2-MS0 medium. Out of 60 encapsulated nodal segments, in each experiments, stored at 4 degrees C for 30 days, 44 plants developed under in vitro conditions, and 27 on peat moss moistened with 1/2-MS0.     

Intra-fruit seed abortion in a wind dispersed tree,Dalbergia sissoo Roxb: Proximate mechanisms

Dalbergia sissoo, a tropical tree with wind-dispersed pods, exhibits a highly positively skewed distribution of seeds per pod with predominantly only one of the four or five ovules maturing into seed. The abortion cannot be attributed to lack of pollen or resources. This study examines the hypothesis that the abortion is due to an intense rivalry among the developing sibs to gain dispersal advantage. Aqueous extract of the dominant embryos at the stigmatic end that generally develop to maturity significantly inhibited the uptake of labelled sucrose by the young developing (subject) embryos in an in vitro assay system. Extracts of tissues such as subordinate (peduncular embryos), unfertilized ovules and pod coat did not cause such inhibition. Aqueous diffusate of dominant embryos also inhibited the uptake of labelled sucrose by subject embryos. The chemical substance responsible for the inhibition appears to be heat-stable and non-proteinaceous. HPLC analysis indicated the presence of two retention time peaks, different from that of standard indole acetic acid, but with considerable overlap. We hypothesize that the compound could be an indole derivative. We propose that the stigmatic embryos have a head start due to earlier fertilization and produce a chemical that either directly (by metabolically killing) or indirectly (by preventing the uptake of assimilates) kills the proximally placed peduncular embryos.     

In vitro clonal multiplication of mature trees of Dalbergia sissoo Roxb.

Multiple shoots were obtained from nodal explants of 30-year-old trees of Dalbergia sissoo Roxb. on a defined medium, MS (Murashige & Skoog medium) supplemented with auxin-cytokinin combinations. IAA (Indole accetic acid) alone promoted 15% rooted shoot buds. A combination of IAA+Kn (Kinetin) gave 100% rooted shoot buds. A combination of NAA (napthaleneacetic acid) + Kn and NAA+BAP (6-benzylaminopurine) also gave high percentages of rooted shoot buds. Ascorbic acid in the medium prevented the death of callus and plantlets, which followed darkening of the medium.     

A high-frequency in vitro multiplication,micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub

Physiology and Molecular Biology of Plants - An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments...     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation

Guadua angustifolia Kunth was successfully propagated in vitro from axillary buds. Culture initiation, bud sprouting, shoot and plant multiplication, rooting and acclimatization, were evaluated. Best results were obtained using explants from greenhouse-cultivated plants, following a disinfection procedure that comprised the sequential use of an alkaline detergent, a mixture of the fungicide Benomyl and the bactericide Agri-mycin, followed by immersion in sodium hypochlorite (1.5% w/v) for 10 min, and culturing on Murashige and Skoog medium containing 2 ml l^?1 of Plant Preservative Mixture^®. Highest bud sprouting in original explants was observed when 3 mg l^?1 N⁶-benzylaminopurine (BAP) was incorporated into the culture medium. Production of lateral shoots in in vitro growing plants increased with BAP concentration in culture medium, up to 5 mg l^?1, the highest concentration assessed. After six subcultures, clumps of 8–12 axes were obtained, and their division in groups of 3–5 axes allowed multiplication of the plants. Rooting occurred in vitro spontaneously in 100% of the explants that produced lateral shoots. Successful acclimatization of well-rooted clumps of 5–6 axes was achieved in the greenhouse under mist watering in a mixture of soil, sand and rice hulls (1:1:1).     

Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree

Rapid clonal multiplication of Aegle marmelos (L.) Corr. (Rutaceae), a medicinal tree, was achieved by enhanced axillary bud proliferation in young single-node segments of a 25-year-old tree cultured in Murashige and Skoog (MS) nutrient medium. Bud break was dependent on cytokinin supply, but the synergistic combination of 2.5 mg l^–1 6-benzylaminopurine (BAP) and 1.0 mg l^–1 indole-3-acetic acid (IAA) induced the formation of 12.1 shoots of up to 5.2 cm length in 48% of the explants after 7 weeks of culture. Explants of in-vitro-grown shoots – node, whole leaf, shoot tip and internode – were subcultured in the presence of 0.05–2.5 mg l^–1 BAP to produce 11.3, 18.4, 5.3 and 3.2 shoots and shoot buds at a 100%, 70%, 95% and 40% rate respectively, in 7 weeks. Different shoot nodes and leaves were equally regenerative and adventitious organogenesis in the latter was confined to cut petiolar ends. Nodal explants responded most favourably at low BAP (0.05–0.1 mg l^–1) and produced uniform (3.8–5.3 cm) shoots facilitating their simultaneous harvest for rooting. Repeated subculturing through five cycles of nodes and leaves of shoot cultures enabled continuous production of healthy callus-free shoots without any sign of decline. Shoot cuttings (3.0–5.2 cm) were best rooted in half-strength MS medium with 0.5 mg l^–1 IAA (70%) or 10.0 mg l^–1 indole-3-butyric acid (90%). Eighty-eight percent of the rooted plants were established in polybags after hardening. Received: 4 April 1996 / Revision received: 8 September 1997 / Accepted: 20 September 1997     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

Rapid in vitro multiplication of <Emphasis Type="Italic">Melia azedarach</Emphasis> L. (a multipurpose woody tree)

An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l⁻¹ ammonium sulphate, (NH₄)₂SO₄, and 100 mg l⁻¹ K₂SO₄, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About 90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully acclimatized first under culture room conditions, then to green house with 85% survival rate.     

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White’s, Gamborg’s B₅ or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened species.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Enhanced shoot multiplication in Ficus religiosa L. in the presence of adenine sulphate,glutamine and phloroglucinol

Ficus religiosa (Pipal) is a long-lived valuable multipurpose forest tree. The tree is exploited because of its religious, ornamental and medicinal value and the regeneration rate in natural habitat is low. An in vitro propagation protocol has been developed from nodal segments obtained from a 45–50-year old tree. The highest bud break frequency (100 %) followed by maximum number of multiple shoots (13.9) as well as length (2.47 cm) were obtained on Woody Plant medium (WPM) supplemented with 1.0 mg/l BAP along with 0.5 mg/l IAA. Two modifications in this medium resulted in enhanced shoot regeneration-one with 200 mg/l glutamine + 150 mg/l ADS (called as MM-1) giving 32.5 shoots per nodal explant while another modification—with 200 mg/l glutamine + 150 mg/l ADS + 100 mg/l phloroglucinol (called as MM-2) giving 35.65 shoots per explant. These two media were used for sub-culturing of shoots for 4 months. The rate of shoot multiplication was same during the first three sub-cultures on MM-1 and the shoots regenerated were healthy, afterwards shoot multiplication declined. While on MM-2, shoot multiplication declined after first sub-culture and shoots underwent the problem of early leaf fall. Rooting was best induced in micro-shoots excised from proliferated shoot cultures on semi-solid as well as liquid WPM modified with 2.0 mg/l IBA and 0.5 mg/l IAA. The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 25–30 days before transfer to soil conditions, where the established plants showed more than 90 % survival.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

Genetic diversity and population structure of Butea monosperma (Lam.) Taub.- a potential medicinal legume tree

Three molecular marker systems, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) were employed to investigate the genetic structure and diversity among the 14 natural populations of Butea monosperma collected from different geographical regions of India. Detected by 17 RAPD, 15 ISSR and 11 SRAP primer combinations, the proportions of polymorphic bands were 84.2 %, 77.2 % and 91.9 %, respectively, and the mean Nei’s genetic distances among the populations were 0.13, 0.10 and 0.13, respectively. Partitioning of genetic variability by Analysis of molecular variance (AMOVA) revealed that the high genetic diversity was distributed within the populations. AMOVA also revealed that the coefficient of gene differentiation among populations based on F_ST was very high irrespective of markers used. The overall gene flow among populations (Nm) was very low. Cophenetic correlation coefficients of Nei’s distance values and clustering pattern by Mental test were statistically significant for all three marker systems used but poor fit for ISSR data than for RAPD, SRAP and combined data set of all three markers. For all markers, a high similarity in dendrogram topologies was obtained, although some differences were observed with ISSR. The dendrogram obtained by RAPD, SRAP and combined data set of all three markers reflect relationship of most of the populations according to their geographic distribution.     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.