Adventitious Shoot Induction Research Articles

Tehlike Altındaki Endemik Bitki Centaurea zeybekii Wagenitz ‘nin Kallus İndüksiyonu

In this paper we reported the effect of different cytokinins on callus induction and adventitious shoot regeneration from leaf segments of in vitro grown Centaurea zeybekii Wagenitz seedlings. Callus was not induced when the explants were cultured on MS medium devoid of any growth regulators. Callus formation was observed in MS media containing TDZ, BA and KIN. The highest callusing response was observed in the media containing 0.005 mg/Land 0.01 mg/LTDZ (100%). In adventitious shoot induction experiments, well developed shoots were observed only MS media supplemented with KIN at the end of sixth week. The highest number of shoot per explant was obtained from the MS medium containing 1 mg/LKIN (6.2 shoots/explant). The highest average of maximum shoot length was also observed in this medium (4.17 cm). There was no difference between MS and ½ MS media in rooting but it was very low and only 15 % of total shoots could rooted in medium containing 0.5 mg/LIBA.

DNA METHYLATION ANALYSIS DURING THE OPTIMIZATION OF Agrobacterium-MEDIATED TRANSFORMATION OF SOYBEAN.

Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (Decrease in DNA methylation 1 and DNA methyltransferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.

The Effects of Hormone Treatment on Epigenetic Marks During Organogenesis in Pinus radiata D. Don Embryos

Adventitious organogenesis from zygotic embryos is one of the most reliable techniques to propagate clonal Pinus radiata individuals. Regulation of gene expression has an important role to generate a correct caulogenic pattern, and genomic DNA methylation is amongst the main studied regulatory mechanisms in animals and plants. The aim of this work is to evaluate the role of DNA methylation and histone H4 acetylation during different cell reprogramming processes. Embryo macromorphological responses to each growth regulator were correlated to DNA methylation and acetylated H4 histone content over time. Immunodetection of 5-mdC and AcH4 in whole embryos revealed differences in the tissue depending on the growth regulator. Adventitious shoot induction by benzyladenine is associated with a DNA hypomethylation. The distribution along the tissue of the methylation signal suggests a possible inhibition of growth and elongation in the embryos. However, a hypomethylation in the cotyledons during adventitious shoot induction could indicate an active gene expression during the organogenic process. On the other hand, alteration of embryo growth induced by indole butyric acid and 2,4-dichlorophenoxyacetic acid occurs along a wide change in DNA methylation and H4 histone acetylation content and distribution. These results show that the action of the different growth regulators is epigenetically regulated in both apical meristems.

In vitro propagation and preliminary results of Agrobacterium-mediated genetic transformation of Cordyline fruticosa

A method for in vitro propagation through axillary shoot proliferation and induction of adventitious shoots as well as Agrobacterium-mediated transformation of Cordyline fruticosa is presented. In vitro Cordyline shoots were cultured on MS medium supplemented 0–4mgl−1 thidiazuron (TDZ) for their multiplication. MS medium supplemented with 0.1mgl−1 TDZ proved to be the most effective in which 14 shoots per explants were obtained. Leaf segments were excised from in vitro axillary shoots and cultured on MS medium supplemented with TDZ (0–5mgl−1) for callus induction. Following induction, the non-embryogenic calluses were proliferated on MS medium without plant growth regulators. A rapid and efficient method for producing transgenic plants has been developed for Cordyline via Agrobacterium-mediated transformation. Leaf segments from in vitro shoots were co-cultivated with Agrobacterium tumefaciens EHA105, which harbored the binary vector carrying the bar and β-glucuronidase (GUS) genes in the T-DNA region. The results showed that the number of plants expressing GUS gene first increased with inoculation time and bacterial density (OD600), and then dramatically decreased with the increment of both factors. The highest percentage of transformation efficiency (85%) was obtained when the leaf segment explants were inoculated with Agrobacterium for up to 40min at OD600 of 1.0 followed by 80 and 75% at OD600 of 0.8 and 1.2, respectively. The transgenes were confirmed in transgenic plants using GUS primers and gave the expected band size of 750bp. The results suggested that infection time and Agrobacterium density could have some effects on the transformation efficiency. Southern blot analysis was used for definitive confirmation of tDNA integration in Cordyline and confirmed the results of histochemical GUS assay and PCR amplifications. Regenerated plantlets were acclimatized in greenhouse.

Root explant produces multiple shoot from pericycle in Psoralea corylifolia – a leprosy destroyer medicinal plant

Babchi (Psoralea corylifolia L.) is a rare medicinal plant. It has been considered to be efficacious in leprosy and hence given the name ‘Kushtanashini’ (leprosy destroyer) in Ayurveda. The high death rate in wild along with aimless and unlawful gathering and destruction of habitat put the plant in the category of endangered list. In this study a highly reproducible in vitro regeneration system in P. corylifolia using root segments was developed. The effect of different PGRs (Kin BA, and NAA) on the percentage of adventitious shoot induction and the number of shoot buds per root explant was assessed. The maximum response (65.57%) was observed in the media supplemented with 2.22μM BAP and 6.98μM Kin. The treatment had in average 39.67 number of shoot buds and 3.37 elongated shoots. Elongation response was best (92.0%) in MS+2.22μM BAP+6.98μM Kin media fortified with 2.89μM GA3 and 39.7μMPG, producing a mean of 9.3 elongated shoots with an average of 4.6cm length. An anatomical study showed that the origin of shoot buds were directly from pericycle of root. Elongated shoots were carefully excised and rooted on basal MS+4.92μM IBA+158.6μMPG, with 92 per cent rooting. Rooted explants survived in soil: vermicompost: vermiculite (1:1:1) with 100% success.

Vegetative propagation of Metabriggsia ovalifolia W. T. Wang using leaf cuttings and petiole segments

SUMMARYMetabriggsia ovalifolia W. T. Wang. is an endangered species indigenous to China. Leaf cuttings and petiole segments derived from 1-year-old potted plants, originally derived from in vitro cultures, were used to study the effects of various plant growth regulators (PGRs) and orientation (only for leaf cuttings) on the induction of adventitious shoots and roots. Leaf cuttings and petiole segments dipped in 20.0 |iM 6-benzyladenine (BA) for 30 min induced the highest number of shoots per explant (6.8 and 9.8, respectively) among the PGRs tested, and 1–20 iM indole-3-butyric acid (IBA) induced more roots than indole-3-acetic acid (IAA) or a-naphthaleneacetic acid (NAA). Leaf cuttings placed abaxial side down induced significantly more adventitious shoots (5.0 per cutting) than leaf cuttings placed abaxial side up (0.6 per cutting). More than 96% of plantlets survived when transferred to square plastic trays filled with a 1:1 (v/v) mix of sand and perlite. This study provides a simple, quick and efficient method of propagation from leaf cuttings or petiole segments, allowing this endangered ornamental species to be maintained sustainably.

In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property ofScrophularia kakudensisFranch Tissue Extracts

The current study deals with in vitro propagation, antioxidant property estimation, and assessment of acacetin content in Scrophularia kakudensis Franch. Adventitious shoot induction was achieved from the nodal explant with the highest number of adventitious shoots per explant (17.4) on Murashige and Skoog's (MS) medium fortified with 2.0 mg·L−1 6-benzyladenine (BA) and 0.5 mg L−1 indole-3-acetic acid (IAA). Maximum number of roots per plant (16.5) was noted in half strength MS medium supplemented with 0.5 mg·L−1 IAA. The regenerated plants displayed successful survival ratio (95%) in the greenhouse. The highest content of acacetin, a pharmaceutically important flavonoid, was observed in the shoot extracts (in vitro: 32.83 µg·g−1 FW; in vivo: 30.05 µg·g−1 FW) followed by root extracts. Total phenol and flavonoid contents along with free radical scavenging assays revealed the occurrence of larger amount of antioxidants in shoot extract in comparison with callus and root extracts of S. kakudensis. Thus, the outcome of the present study can be highly beneficial for the germplasm conservation and commercial cultivation of S. kakudensis for therapeutic purposes.

In vitro callus and shoot organogenesis from leaf and stem explants of Chamaedaphne calyculata

Leatherleaf Chamaedaphne calyculata (L) Moench is a relict, rare and endangered species in Poland. There are no reports on the micropropagation of Chamaedaphne calyculata in the literature. Therefore, the aim of this study was to propose a propagation protocol for leatherleaf via indirect organogenesis using leaves and stems (internodal segments) derived from mature plants growing in a natural stand and from plants grown in vitro as explants. The medium developed by Anthony et al. (2004) with 100%, 50% and 25% salt concentrations, supplemented with IAA (5 and 10µM) and TDZ (5 and 10µM), was used for cal- lus development and the induction of adventitious shoots. The media developed by Anthony et al. (2004) and Anderson (1980), both containing 10µM TDZ and 5µM IAA or 2.28µM zeatin, were used for adven- titious shoot elongation. Secondary explants proved to be the most effective starting material for callus induction, the regeneration and elongation of adventitious shoots. The most supportive medium for callus induction and growth and the induction of adventitious shoots was the full medium proposed by Anthony et al. (2004) containing 5µM IAA and 10µM TDZ. Anderson's (1980) medium containing 2.28µM zeatin delivered optimal results in the elongation of adventitious shoots of Ch. calyculata. Roots were cultivated on Anderson's (1980) phytohormone-free medium. Approximately 65% of the plantlets survived after transfer to the sphagnum-peat and perlite mixture (3:1). The plants grew normally without any signs of morphological variation. This study makes the first ever attempt to propose an effective micropropagation protocol for Ch. calyculata.

<i>In Vitro</i> Induction and Identification of Tetraploid in Watermelon [<i>Citrullus lanatus</i> (Thunb.) Matsum and Nakai

This research induced tetraploid watermelon through tissue culture. The cotyledons of a diploid mini-watermelon (A7) were treated with different concentrations of colchicine on medium for different time. The autotetraploid plants were identified basing on morphology, determination of the number of chloroplasts in stomatal guard cells and flow cytometry analysis. A stable autotetraploid material was observed. The results showed that tetraploid watermelons could be obtained under different treatments, and the highest tetraploid induction rate was up to 25 %. The most effective way was cutting the proximal cotyledons at the 7th day after sowing, then explants were cultivated on MS medium with 0.1 %(w/v) colchicine for 72 h, the adventitious shoot induction rate was 62.5 %, and multiplication coefficient was 3.6.

Shoot organogenesis from leaf explants of Dayaoshania cotinifolia W. T. Wang

Dayaoshania cotinifolia W. T. Wang is a rare and endangered member of the Gesneriaceae family which is endemic to China. To conserve this species, an efficient in vitro propagation and regeneration system via shoot organogenesis was established from young leaf explants. Adventitious shoot induction was possible within 50–60 d on basal Murashige and Skoog medium supplemented with 1–3 μM 6-benzyladenine, although 5 μM 6-benzyladenine induced hyperhydricity. Basal medium containing 1–5 μM thidiazuron induced fewer shoots, while 1–5 μM α-naphthaleneacetic acid induced numerous adventitious roots and a few adventitious shoots. However, when thidiazuron and α-naphthaleneacetic acid were combined, both the induction percentage and number of shoots increased. Leaf explants cultured on induction medium supplemented with 1–5 μM 2,4-dichlorophenoxyacetic acid become necrotic and died. Induction medium supplemented with 1 μM α-naphthaleneacetic acid and 1–3 μM 6-benzyladenine was optimal for inducing adventitious shoots as was the combination of 1–3 μM thidiazuron and 1 μM α-naphthaleneacetic acid. Induction medium containing 2.0 μM 6-benzyladenine and 0.5 μM indole-3-acetic acid was optimal for the multiplication of adventitious shoots. Rooting was achieved on half-strength MS medium supplemented with 3.0 μM indole-3-acetic acid or α-naphthaleneacetic acid and 0.1% activated charcoal. Plantlets were transplanted to a mixture of sand, vermiculite, and humus (1:1:1); 92% survived. This protocol is a unique and effective means to micropropagate this rare and important plant and could serve as a solution for in vitro and ex vitro conservation.

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05mg/l NAA plus 2mg/l BA (96.3%) and MS medium supplemented with 0.5mg/l IAA plus 2mg/l BA (88.3%). Root induction was obtained on MS medium supplemented with 0.5mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93%) and ATCC15834 (89%) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.

MORPHOLOGICAL AND HISTOLOGICAL EVENTS IN THE PRELIMINARY TISSUE CULTURE OF HAPLOID AND DIPLOID PELARGONIUM ZONALE VAR . 'KLEINER LIEBLING'

Pelargonium species are know and popular as the ornamental plants valued for their colorful, showy flowers and attractive leaf shape. Furthermore, some of these species are used in the pharmacy, aromatherapy, perfumery and cosmetic industry as well (Moyo et al. 2012). Most of approximately 280 species of the genus Pelargonium (fam. Geraniaceae) are native for South Africa (Mithila et al. 2001). Some of them such as P. zonale (L.) L'Her. ex Aiton were brought to Europe as early as in 1609. P. zonale var. ‘Kleiner Liebling’ is one of the pelargonium dwarf varieties with chromosomes number nine, and it is true haploid (monoploid) (Daker 1966), which makes it an interesting model plant.The aim of our study was the observation of in vitro culture of haploid and diploid plants of P. zonale var. ‘Kleiner Liebling’. It seems that this is the first attempt to establish an efficient protocol for regeneration in tissue culture conditions for these plants. These studies may serve as a start point for further studies of concerning plants at different ploidy levels.To obtain the plant regeneration, in the tissue culture conditions, we used explants both from mature haploid and diploid. Fragments of stems, parts of leaf petioles and leaf lamina were maintained on MS (Murashige and Skoog) basal media supplemented by several plants growth regulators. The first fodder contained 1 µM 2,4-D and 1 µM KIN; the second – 18,2 µM TDZ and 0,57 µM IAA; the third – 0,44 µM BAP and 0,54 µM NAA; and the fourth – 5,4 µM NAA and 0,44 µM BAP.The morphological and histological examinations showed the differences in morphogenetic response of haploids and diploids in applied tissue culture conditions. The response time for haploid and diploid P. zonale var. ‘Kleiner Liebling’ plants was also different. First of all, the callus formation on diploid stem explants was observed on eleventh day of culture on the medium with NAA and BAP combination. Two types of callus were observed on both haploid and diploid explants: endogenous and exogenous. The efficiency of callus production varied depending on type of explants used. The most frequent callus formation was found on fragments of stems and leaf blades (they responded in 100%), while leaf petioles were less efficient, 70% in diploids and 50 % for haploids.Organogenesis (mainly direct) was achieved in three types of explants; however, the response was different for haploids and diploids. Shoots were observed in most cases on haploid petiole leafs, whereas direct organogenesis in diploid explants was observed on stems and leaf blades.The optimal combination of plant hormones for the induction of adventitious shoots were TDZ and IAA (second fodder), while in the medium supplemented with 2,4‑D and KIN organogenesis was not recorded at all. Regenerated adventitious shoots were transferred to solid 1/2 MS medium, where their multiplication and successful development were observed.Direct rhizogenesis was induced only on stem explants. For haploids, roots were obtained on the medium containing TDZ with IAA and 2,4-D with KIN (the first and the second fodders), for diploids third and fourth media (supplemented with NAA and BAP in different concentrations) were suitable.The histological analysis confirmed the direct organogenesis and rhizogenesis, as well as heterogeneous origin of callus in diploid and haploid plants.In conclusion the crucial factor for organogenesis induction seems to be TDZ with combination with IAA application, what correlates with other experimental reports, where TDZ has been reported to induce adventitious shoot buds in number of plant species (Acharjee et al. 2012)In our experiments the somatic embryogenesis induction was not observed, although it is described in Pelargonium (Wojtania et al. 2004). However the SEM analysis showed the ECM (extracellular matrix) presence in diploid and haploid explants of leaf petioles, which may suggest the embryogenic potential of this experimental system. Future investigations are necessary for improving the culture protocols to obtain somatic embryos, plant regeneration and improvement of shoots formation.

멸종위기종 둥근잎꿩의비름 (Hylotelephium ussuriense (Kom.) H. Ohba)의 기 내 증식

멸종위기수종인 둥근잎꿩의비름 기내 부정아유도에 미치는 식물생장조절물질의 종류 및 배양부위의 영향과 기내생육 및 엽록소함량에 미치는 배지, sucrose 농도 및 배양용기에 따른 환기효과를 연구하였다. 부정아 유도는 3.0 m g/L의 BA와 0.01 mg/L의 IBA가 첨가된 배지에 액아포함 줄기를 배양하였을 때 가장 효과적이었다. 기내 부정아의 줄기와 뿌리 신장에 MS배지의 농도와 sucrose의 농도는 영향을 주지 않았다. 환기처리가 기내 건전 식물체 생산에 미치는 영향을 조사한 결과, 기내 환기처리구는 줄기신장에 효과적이었고, 뿌리의 신장은 유의적 차이는 없이 모든 실험구에서 10 cm 이상 증식하였다. 엽록소의 함량은 환기처리 시 총엽록소 함량이 3.12 mg/g으로 높게 나타남을 확인하였다. 둥근잎꿩의비름의 부정아 유도에 농도와 배양부위가 중요한 요인으로 작용하고 기내식물체 신장에 미치는 배지 및 sucrose농도의 영향은 크지 않은 것으로 나타났다. 기내 건전식물체의 생산은 환기가 용이한 배양용기하에서 양호한 것으로 나타났다. To establish the system of in vitro plant regeneration, the different explants (stem with axillary bud and stem without axillary bud) of Hylotelephium ussuriense were cultured on the Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). The adventitious shoot induction was more effective in the stem with axillary bud explants than the stem without axillary bud explants, and was the best on MS medium containing 3.0 mg/L BA and 0.01 mg/L IBA. Frequency of plantlet growth was not significantly treated on MS and sucrose. Total chlorophyll contents under ventilation treatment were higher than those in control (non-ventilation). This in vitro propagation protocol will be useful for conservation and mass propagation of this endangered plant.

Adventitious shoot induction from leaf segments in Anthurium andreanum is affected by age of explant, leaf orientation and plant growth regulator

Anthurium is one of the main potted plants on the flower market, and micropropagation is the most used technique for clonal and large scale propagation of this species. Calli, adventitious shoots and regenerated plantlets were obtained from leaf segments of Anthurium andraeanum. The influence of genotype, explant orientation, plant growth regulator, and age of donor plants were tested. We obtained regenerated plants in three (‘Red One’, ‘Red Dark’, and ‘Snow White’) out of four different cultivars. The ‘White Beauty’, however, was considered to be recalcitrant under these conditions. Therefore, the second study was conducted only with this recalcitrant cultivar, and the use of juvenile rather than adult leaf explants enhanced plantlet regeneration at the frequency of 0.3-2.8 plantlets per leaf segment. The use of 1.0 mg·L−1 6-benzylaminopurine (BAP) and no addition of 2,4-D in the culture medium resulted in direct plantlet regeneration of Anthurium. Plantlets obtained from ‘Red One’ and ‘White Beauty’ were successfully micropropagated and acclimatized. In ex vitro phases, leaf-derived plants of these two genotypes maintained their most important horticultural and ornamental characteristics.

English

A plant regeneration system was established from cotyledon explants of chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How). To obtain optimal conditions of adventitious shoot induction, the cotyledon explants were excised from seedlings of different genotypes as well as seed germination conditions, and then cultured on media containing different concentrations of 6-benzylaminopurine (6-BA)/1-naphthaleneactic acid (NAA). Among the eight genotypes, the highest rate of shoot regeneration was obtained from the cotyledons of inbred line A39. The adaxial portion of cotyledons of seedlings cultured for 3 days in darkness and 1 day in light was the appropriate explants for adventitious shoot organogenesis. The highest frequency of adventitious shoot organogenesis (52.2%) and mean number of shoots per explant (4.2) were achieved on Murashige and Skoog (MS) medium supplemented with 6 mg l-1 6-BA and 0.2 mg l-1 NAA. Adventitious shoots were observed to regenerate directly from cotyledons rather than from calli. A medium supplemented with AgNO3 was not beneficial for shoot induction. Adventitious shoots were elongated in MS medium supplemented with 3 mg l-1 6-BA and 0.2 mg l-1 NAA. Elongated shoots were rooted in ½ MS medium with 0.5 mg l-1 indole-3-acetic acid (IAA). Regenerated plantlets were transferred to a greenhouse for about 1 month, and subsequently transplanted into an open field. Key words: Benincasa hispida, chieh-qua, adventitious shoot, genotypes, plant regeneration.

Plant regeneration from callus of Iris odaesanensis Y. N. Lee native to Korea via organogenesis

Abstract Iris odaesanensis Y. N. Lee. is an important endangered and native plant belonging to the family Iridaceae in Korea. This study describes a method for rapid micropropagation of this species via from leaf, rhizome and root explants derived calli. Leaf, rhizome and root explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) for callus induction. Rhizome explants yielded calli at a frequency of 72% when cultured at 1.0 mg/l 2,4-D. Calli were maintained at 1.0 mg/l 2,4-D. These calli were transferred to MS medium supplemented with 0, 0.5, 1.0, and 2.0 mg/l 2,4-D in combination with 0, 0.5, 1.0, and 3.0 mg/l BA for adventitious shoot induction. The highest number of adventitious shoot (228.9 per petri-dish) were formed at 1.0 mg/l 2,4-D and 1.0 mg/l BA. WPM medium was the best to convert calli into plantlets, where up to 98.2% of calli were regenerated into plantlets. This in vitro propagation protocol should be useful for conservation of this endangered plant.

English

The objective of this study was to establish an efficient and reproducible in vitroplant regeneration for Citrullus lanatus cv. Zaojia. To achieve optimal conditions for adventitious shoot induction, five explants (entire cotyledons, distal cotyledons, proximal cotyledons, cotyledonary node A and cotyledonary node B) were tested on MS medium supplemented with different concentrations and combinations of growth regulators (0 to 0.2 mg/L IAA and 1.0 to 5.0 mg/L BA), the results showed that entire cotyledons cultured in MS + BA (2.0mg/L) + IAA(0.2mg/L) achieved the highest regenerated rate (89.67%) and the optimal protocol screened in this experiment had 7.69 ± 0.10 shoots per explants. Adventitious shoots were able to elongate both on MS medium with 0.2 mg/L KT and 0.2 mg/L NAA; IBA 0.3mg/L was found to be effective in the production of root. Acclimatized plantlets transferredto pot resumed growth, and their stems and leaves elongated and expanded in one month.   Key words: Watermelon (Citrullus lanatus Thumb.), optimized system, regeneration, cotyledon explants, cotyledonary node.

Direct Shoot Organogenesis in Murraya paniculata (L.) Jack: A Prerequisite for Genetic Transformation

An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

Regeneration of Plants from Fraxinus nigra Marsh. Hypocotyls

Fraxinus nigra Marsh. (black ash) is a native North American hardwood tree species that is ecologically important and has ethnobotanical significance to American Indian communities of the eastern United States. Black ash has immature embryos at seed set, combined with complex stratification requirements, making natural regeneration difficult. This, combined with the threat and devastation being caused by the emerald ash borer, makes an in vitro adventitious shoot regeneration and rooting protocol imperative for mass propagation, conservation, and genetic improvement of this species. Hypocotyls were cultured for 4 weeks on a modified Murashige and Skoog (MS) medium containing 13.3 μM 6-benzylaminopurine (BA) and 4.5 μM thidiazuron for adventitious shoot induction. Shoots continued to regenerate when explants were then cultured on MS medium supplemented with Gamborg B5 vitamins plus 0.2 g·L−1 glycine (B5G), 6.7 μM BA, 1 μM indole-3-butryic acid (IBA), and 0.29 μM gibberellic acid (GA3) for 4 weeks, followed by transfer to MSB5G medium with 13.3 μM BA, 1 μM IBA, and 0.29 μM GA3 for shoot elongation. Once elongated, the microshoots were continuously micropropagated through nodal sectioning, and cultured on MSB5G medium supplemented with 13.3 μM BA, 1 μM IBA, 0.29 μM GA3, and 0.2 g·L−1 casein hydrolysate. Rooting of elongated microshoots was successful using woody plant medium supplemented with 4.9 μM IBA and 5.7 μM indole-3-acetic acid with a 10-day initial dark culture for root induction followed by culture under a 16-h photoperiod. Rooted plantlets were successfully acclimatized to the greenhouse with 100% survival.

Development of an efficient micropropagation procedure for Aglaonema ^|^#8216;Lady Valentine^|^#8217; through adventitious shoot induction and proliferation

Development of an efficient micropropagation procedure for Aglaonema ^|^#8216;Lady Valentine^|^#8217; through adventitious shoot induction and proliferation