Axillary Bud Explants Research Articles

Rapid in vitro propagation by liquid culture system and genetic homogeneity assessment of Plectranthus bourneae Gamble, an endemic plant species to South India

The present investigation describes the in vitro propagation of Plectranthus bourneae via liquid culture system from axillary bud and shoot tip explants. Multiple shoots were initiated from axillary bud and shoot tip explants on Murashige and Skoog (MS) medium supplemented with different concentrations (0.1–2.0 mg/l) and combinations of cytokinins [BA (6-benzyladenine), KN (kinetin), and TDZ (thidizauron)] along with (0.5–2.0 mg/l) auxins [NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid)]. Maximum shoot multiplication (12.14) was achieved from axillary bud explant followed by shoot tip explant (8.85) at 0.5 mg/l TDZ with 1.0 mg/l NAA. A maximum of 9.71 roots/shoot was observed at 0.7 mg/l IBA. The rooted plantlets successfully hardened and transferred to greenhouse condition with 83% survival. Inter-simple sequence repeat markers exhibited genetic fidelity with 100% monomorphism in regenerants. The authenticated protocol supports rapid production of true-to-type plants by liquid culture in vitro and therefore could supply a valuable target material for genetic transformation study.

In vitro propagation of Xao tam phan (Paramignya trimera (Oliv.) Guill.)

Paramignya trimera (Oliv.) Guill., a woody climber commonly known as "Xao tam phan", has been used in Vietnamese folk for the treatment of numerous cancers. Due to word of mouth about the anticancer properties of this plant, its stems and roots have been overexploited leading to the serious decline of this species in Phu Yen, Khanh Hoa and Ninh Thuan provinces. The aim of the study was to establish an in vitro propagation protocol for the conservation of P. trimera. In this research shoot clusters (5–8 shoots/cluster) were regenerated from axillary bud explants of 1–3 year-old trees after 3 months of cultures on the WPM (woody plant medium) supplemented with STS 3 and BA 5–7 mg/L. STS (silver thiosulfate) was used to prevent the leaf abscission. These shoot clusters grew slowly and reached 1–3 cm in heights after 4 months of the cultures. These shoot clusters did not form any roots after 2 months of culture on the rooting media with IBA and/or NAA 1–5 mg/L. However, there was 51 % of the treated shoot clusters acclimatized and produced new stem and leaves after 2 months growing in greenhouse. WPM supplemented with STS 3, BA 5 and IBA 5 mg/L showed the best response for callus induction in leaf explants after 3 months of cultures. Among the callus types, the milky white compact calli were induced at the cut surface of leaf explants after 3 months of the cultures and became the compact and nodulated calli within 4 weeks later.

Ethnobotany and In vitro regeneration of Acorus calamus L. (Acoraceae): a high valued medicinal and economic plant

Acorus calamus L. is a species of enormous medicinal and economic importance. In vitro propagation of this plant was achieved using axillary bud explant. In the present investigation, naturally grown axillary bud and rhizome explants were cultured on standard MS and B5 medium supplemented with different concentration and combination of cytokinines and auxines. The best shoot proliferation was observed in MS medium containing Kn (1.0mg/l) +IBA (0.5mg/l) with 3.33±0.58 nos. of Shoots, 7.33±0.58 nos. of roots and 15.33±0.58 nos. leaves. In B5 medium best results found in Kn (1.5mg/) + NAA (1.0mg/l) with 2.67±0.58 nos. shoots, 3.67±0.58 nos. of roots and11.67±0.58 nos. of leaves. They were then transplanted in soil: sand: cow dung mixture (1:1:2) and kept in shade for 4 to 5 weeks and then transferred to field for one month. Survival rate was found 80 % in MS medium and 100 % in B5 medium. The present investigation was carried out with a view to standardize an in vitro culture technique for mass propagation of this important medicinal plant species and was found successful.

Genetic Stability of Cassava Plants Regenerated Through Organogenesis Using Microsatellite Markers

Tissue culture technology of cassava (Manihot esculenta Crantz) is a viable alternative to currently adopted techniques for mass propagation, germplasm conservation and genetic improvement. However, somaclonal variation is a common phenomenon in tissue culture which makes it mandatory to monitor the genetic stability of plants. Therefore, the objective of this study was to evaluate the genetic stability of cassava plants regenerated from axillary bud explants through direct organogenesis using simple sequence repeat (SSR) markers. High shoot regeneration (81.2 – 90.0%) occurred in MS medium supplemented with 10 mg/L 6-bnzylaminopurine (BAP) and multiple shoots (2 – 4 shoots per from axillary bud explant) were formed for all the three cultivars (TME14, TMS60444 and Kibandameno) tested. High frequency of rooting (100%) was obtained after transferring the plantlets to cassava basic medium (CBM) and the rooted plants were successfully hardened and acclimatized in the glasshouse with 100% survival rate. Three-month old plants exhibited normal morphological characters comparing with the mother plant. A total of 10 SSR markers were used to validate the genetic homogeneity amongst five randomly selected plants along with the donor mother plants. DNA fingerprints of axillary bud regenerated plants displayed monomorphic bands similar to mother plant, indicating homogeneity among the regenerated plants with donor mother plant. The effect of subculture frequency on genetic stability of axillary bud-derived regenerants and micropropagated plants was also assessed using SSR markers. All the SSR profiles from axillary bud regenerants and micropropagated plants were monomorphic and comparable to mother plants from 1st to 5th subculture, confirming the genetic stability among clones and mother plants. At the 6th subculture, similarity indicators between the progenies and the mother plants ranged from 0.95 to 1.0 and such a similarity indicated a very low polymorphism. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis of the 6th subculture revealed 96% similarity amongst axillary bud regenerants and micropropagated plants with donor mother plant. This low polymorphism ratio between mother plants, axillary bud regenerants and micro-propagated plants indicates the little effect of somaclonal variations, the high genetic similarity between mother plants and progenies and demonstrates the reliability of this propagation system for cassava. These results suggest that direct organogenesis from the axillary buds is the safest method for regeneration of true-to-type plants and this system can be used for clonal mass propagation, germplasm conservation and genetic transformation of cassava.

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

ABSTRACTSuccessful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.

Effect of silver nitrate on leaf abscission in culture during establishment of explants of custard apple (Annona squamosa l.) cv. Balanagar

The present investigation was carried out on “Micropropagation in custard apple (Annona squamosa L.)” with objective to develop commercially feasible and economically viable protocol for micropropagation technique of custard apple. While standardization of micropropagation in custard apple, Effect of silver nitrate on leaf abscission in culture during establishment of explants of custard apple cv. Balanagar were examined. In order to control leaf abscission silver nitrate at concentrations of 0, 2.5, 5 and 10 mg/l was tested to medium MS + 0.5 mg/l BAP + 0.5 mg/l KIN + 100mg/l CH. The results revealed that Silver nitrate significantly reduced leaf abscission in both shoot tip and axillary bud explants. The least leaf abscission was reported in treatment N3 (5 mg/l) followed by treatment N4 (10 mg/l) and N1 (2.5mg/l) in both shoot tip and axillary bud explants. Whereas, in treatment N1 (without silver nitrate) more than 80% leaf abscission was observed. The number of leaves produced by the explants was maximum in treatment N3 (5 mg/l) in both shoot tip (7.5) and axillary bud (8.5) explants. Whereas, when silver nitrate was applied at 10 mg/l (Treatment N4) this was phytotoxic and inhibited leaf production.

Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

Dioscorea rotundata, commonly known as white yam, is an important staple food crop widely cultivated in West Africa and provides food security to millions of people. Genetic improvements of this crop using the advanced biotechnology tools have been hampered hitherto by the recalcitrant nature of regeneration through somatic embryogenesis. Therefore, we have developed an efficient and reproducible system for plant regeneration via somatic embryogenesis. Explants of different types (immature leaf, node, stem internode, root segment, petiole, and axillary bud) of D. rotundata accession TDr 2436 were tested for their embryogenic potentials on Murashige and Skoog (MS) medium supplemented with various auxins (2,4-D, NAA, and picloram). Among all explants tested, axillary bud explants cultured on MS medium supplemented with picloram (0.5–12 mg/l) favored the production of calli. Maximum proliferation of calli (526 mg fresh weight/explant) was achieved on MS medium supplemented with picloram (0.5 mg/l), casein hydrolysate (600 mg/l), and proline (1 g/l). Histology analysis confirmed that the embryogenic calli produced on this medium were mixed with non-embryogenic calli. Transfer of calli on MS basal medium supplemented with activated charcoal (1 %) changed the color of calli to purple and promoted the production of somatic embryos (87 embryos/callus) as well as adventitious shoot buds. Furthermore, upon transfer to MS medium supplemented with BAP (0.4 mg/l), the embryos continued their differentiation and maturation and germinated into complete plantlets. The adventitious shoot buds produced multiple shoots on MS medium supplemented with BAP (0.4 mg/l). Well-developed germinated plantlets were acclimatized in the screen house with 90 % survivability. Histology studies confirmed that the regeneration of D. rotundata reported here followed dual regeneration pathways. The embryogenic calli regenerated through development of somatic embryos and germinated into complete plantlets, however non-embryogenic calli regenerated through organogenesis and developed multiple shoots. The developed protocol has potential for somatic hybridization, mass clonal propagation, and genetic transformation applications.

Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development.

In vitro Propagation of Plectranthus bourneae Gamble? An Endemic Red Listed Plant

An efficient protocol of in vitro propagation of Plectranthus bourneae Gamble (Lamiaceae), a valuable medicinal important and endemic Red listed plant of Western Ghats, (Tamil Nadu, India) was standardized by improved shoot multiplication from axillary bud explant. An in vitro propagation system has been reconnoitered on MS with the effective concentration BA (0.7 mg/l) followed by a combination of BA (0.7 mg/l) and TDZ (1.0 mg/l) which promoted high number of shoots. The multiple shoot rate was enhanced further by adding AdS (50 mg/l). Beneficial shoot length was achieved when cultured on MS containing GA3 (0.5 mg/l). Rooting was increased on MS augmented with IBA (1.5 mg/l). Micropropagated plants were acclimatized and the survival rate was 80%. Acclimatized P. bourneae plants can be used as substitute alternative to natural populations. Using this protocol the propagated plants can be used for conservation strategies.Plant Tissue Cult. &amp; Biotech. 25(2): 273-284, 2015 (December)

Role of Silver Nitrate on &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Flowering and Shoot Regeneration of &amp;lt;i&amp;gt;Solanum nigrum&amp;lt;/i&amp;gt; (L.)—An Important Multipurpose Medicinal Plant

Silver nitrate induced MS media enhanced multiple shoot regeneration and in vitro flowering from axillary bud and leaf explants of Solanum nigrum (L.) an important antiulcer medicinal plant. Healthy axillary bud and leaf explants were inoculated on MS medium supplemented with BAP/Kn (1.0 - 2.0 mg/l) in combination with IAA/NAA (0.5 mg/l) and AgNO3 (0.1 - 1.0 mg/l). The explants were responded effectively and good regeneration frequencies were observed in all the combinations of silver nitrate tested when compared to control. Maximum number of multiple shoots (34.3) was found in leaf explants cultured on MS media supplemented with BAP (2.0 mg/l), NAA (0.5 mg/l) and AgNO3 (0.4 mg/l). These regenerated shoots were sub-cultured on to the flowering media. Maximum number of in vitro flowers (12) was obtained from axillary bud explants in BAP (2.0 mg/l), Kn (1.0 mg/l), IAA (0.5 mg/l) and AgNO3 (6.0 mg/l) supplemented media when compared to leaf (4) and control. All the in vitro raised shoots were transferred to rooting medium supplemented with NAA, IBA (1.0 - 2.0 mg/l) and AgNO3 (0.1 - 0.6 mg/l). The best rooting response (24.6) was observed in 2.0 mg/l IBA + 0.4 mg/l AgNO3. The well rooted plantlets were transferred to polybags containing soil + vermiculate in 1:1 ratio for hardening and finally the hardened plantlets were transferred to field conditions.

Effect of explant’s position and culture method on shoot proliferation and micro-cuttings for a rare and endangered species, Abeliophyllum distichum Nakai

Using either the apical or axillary bud of the endangered species Abeliophyllum distichum Nakai, we tested the effect of bud position and culture method on shoot proliferation and rooting. In shoot proliferation, the axillary bud explant was more effective than the apical bud and the effect was fostered by BA treatment, whereas no differences were observed in shoot elongation by the explant position. Spontaneous rooting was observed in the MS basal medium and resulted in conspicuous differences in the explant position : more than 80% in apical bud explant and 28% in axillary bud explant was achieved, respectively. The positional effects were also observed in BA pre-treatments: generally vertical culture method appeared to be better in shoot proliferation, growth, and rooting than that of the horizontal culture method regardless of the BA pre-treatment duration. The highest shoot multiplication was achieved through the vertical culture method with axillary bud explant, whereas the best shoot elongation and rooting was obtained using the vertical culture method with the apical bud explant. Apical bud explant was superior to axillary bud explant in ex vitro micro-cuttings and revealed a significant difference in shoot growth and root development. The above results suggest that explant position and culture method influence the efficiency of micropropagation for a rare and endangered plant Abeliophyllum distichum.

Induction of callus and somatic embryogenesis from explants of Parkiabiglobosa (Jacq.)Benth and ParkiaroxburghiiG.Don.

Legumes are very important species including herbs, shrubs and trees, they are cultivated worldwide for different economic uses.Parkiais genus belongs to the Legumefamily, Fabaceae. The most common known species are P. biglobosaBenth (African locust bean) and P. roxburghii (Tree Bean)which were introduced to Egypt since 1890. The population of the tree is rapidly declining as result conservation efforts is needed to prevent it from extinction. In an attempt to conserve this genetic resource, tissue culture studies were carried toestablish a callus culture of both plants from different explants and regeneration of the developed callus and to study the effect of explant type, media composition on the callus production and the regeneration. Apical bud,axillary bud and root explants from in vitro germinated seedlings of P. biglobosaBenthwere cultured on MS and B5 medium supplemented with (0.4., 0.8, 1.2, 1.6 or 2.0 mg l-1 NAA) + Glutamine 0.5 mg l-1 + Proline 0.5 mg l-1 + Casein 0.3 mg l-1 + 2,4D 2.5 mg l-1. Each medium was supplemented with 30 g l-1 sucrose, 3 g l-1 agar and PH 5.8.for callus induction .and (0.4.,0.8,1.2,1.6 or 2.0 mg l-1 NAA) + Glutamine 0.5 mg l-1 + Proline 0.5 mg l-1 + Casein 0.3 mg l-1 + 6-BAP 2 mg l-1. Each medium was supplemented with 30 g l-1 sucrose, 3 g l-1 agar and PH 5.8.for regeneration.The study presented here demonstrates the successful attempt at regeneration of plantlets of P. biglobosaBenth via indirect organogenesis.It can be concluded that the medium composition has a significant effect on the calli production together with the type of the explant which showed relatively higher significance. Seeds germination stage apical bud explants media 8 followed by media 10 showed the most favorable mean performances values for the studied characters, Number of days to callus initiation (day) , Callus weight (mg), Dead calli percentage (%),Albino plants percentage (%),Green plants percentage (%) and Root percentage (%). Moreover, axillary bud, showed remarkable increase in the mean performance of average. However, the overall response of MS medium was found to be superior to that of B5 medium. Explants cultured on MS medium fortified with combinations of 2,4-D and BAP induced rapidly proliferating calli that turned more friable and nodular.

In Vitro Propagation of Ceropegia anjanerica Malpure et al.: A Rare Endemic Plant from Maharashtra

An in vitro micropropagation system has been developed for the Ceropegia anjanerica Malpure et al., a rare endemic plant species having ornamental as well as medicinal potential but a limited reproductive capacity. Axillary bud explants were cultured on Murashige and Skoog medium supplemented with 6-Benzylaminopurine, Thidiazuron and 6-Furfurylaminopurine along with combination of Indole-3-Butyric Acid. Although all concentrations showed multiple shoot formation, the best results (8.0 ± 0.5) were achieved with MS + 6-Benzylaminopurine (8.87 µM) + 6-Furfurylaminopurine (18.58 µM). Half strength Murashige and Skoog medium fortified with Indole-3-Acetic Acid (8.56 µM) showed good rooting response (4.0 ± 0.7). Well grown plantlets hardened, acclimatized and established in greenhouse exhibited 78 % survival.

희귀 수종 미선나무(Abeliophyllum distichum Nakai.)의 기내 증식 및 발근에 미치는 LED (light emitting diode) 효과

희귀 멸종위기 식물인 미선나무를 재료로 기내 증식 및 발근에 미치는 광질 및 절편체(정아지 및 액아지) 효과를 조사하였다. 광질은 냉백색 형광등(F), 100% 청색광 LED(B), 혼합광(BR; 50% blue LED+50% red LED) 및 100% 적색광(R)을 사용하였다. 줄기의 증식은 절편체 종류에 따른 뚜렷한 차이를 보이지 않았으나 광질에 따른 차이를 보였다. 전반적으로 줄기의 증식은 청색광 및 혼합광에서 촉진되는 것으로 나타났으며, 특히 청색광에서는 일부 절편에서 발근이 이루어 졌다. 줄기의 생장 역시 광질에 따른 현저한 차이가 없었다. 적색광에서 줄기 신장이 촉진된 반면 잎의 발달은 저조하였다. 발근은 전반적으로 광질에 관계없이 정아지가 액아지보다 양호하였고, 뿌리수도 증가하였으나 혼합광에서는 발근이 억제되었다. 한편 적색광에서는 뿌리의 발달이 매우 부진하여 다른 광질과 유의적인 차이를 보였다. 발근유도 시 줄기는 주로 단일 줄기로 자랐다. 이상의 결과를 볼 때 LED를 이용한 미선나무의 기내번식은 정아지를 절편으로 청색광 및 혼합광 LED (BR) 하에서 배양함이 좋은 것으로 나타났다. This study was conducted to elucidate the effect of light sources and explant types on in vitro shoot multiplication and rooting of a rare and endangered plant Abeliophyllum distichum. Both apical buds and axillary buds were used as explants under 4 different light sources, cool white florescent light (F), 100% blue light-emitting diode (LED) (B), 50% blue and 50% red LED mixture (BR), and 100% red LED (R). Clear difference was observed in terms of shoot proliferation by light sources types but not by position-dependent explant types. Multiple shoot induction rates were enhanced under both B and BR light sources. Spontaneous rooting was induced in shoot induction medium under B light source. Both the rates of rooting and numbers of roots per explant were higher in apical bud explants compared to axillary bud explants. Interestingly R light source stimulated shoot elongation but inhibited root development. Therefore, our results suggest that the use of apical bud explants under B or BR light sources is suitable for in vitro micropropagation of a rare and endangered plant species, Abeliophyllum distichum.

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.

Optimization of factors influencingin vitroflowering of gherkin (Cucumis anguriaL.)

This study investigated the factors influencing in vitro flowering of gherkin (Cucumis anguria L.). Multiple shoots were efficiently regenerated from cotyledonary node and axillary bud explants of C. anguria within 15 days on MSB5 medium containing 3% sucrose and supplemented with 1.5 mg l-1 6-benzyladinine (BA). The elongated shoots were excised and transferred to MSB5 medium containing 4% sucrose supplemented with 0.5 mg l(-1) gibberellic acid (GA(3)) and 1.0 mg l(-1) indole-3-butyric acid (IBA) induced maximum number of flowers (9.5 flowers/plant) and root induction (16.5 roots/plant). Factors that influence the in vitro flowering were optimizing pH, photoperiod and temperature. In vitro flowering was significantly early and higher number of flowers produced at pH (5.8), photoperiod (12/12 h) and room temperature (28 °C). In vitro developed flowers were less viable (80 ± 1.0%) compared to control plants (90 ± 2.0%). Our in vitro flower induction procedures provide an extremely effective method for further research on flowering regulation mechanisms in C. anguria. These plantlets were successfully transferred to the soil where they grew well for 3 to 5 weeks with 90% survivability. Plants grew normally and produced flowers with viable pollen and fertile seeds.

In Vitro Flower Induction from Shoots Regenerated from Cultured Axillary Buds of Endangered Medicinal Herb Swertia chirayita H. Karst.

In vitro flowering and effective micropropagation protocol were studied in Swertia chirayita, an important medicinal plant using axillary bud explants. The Murashige and Skoog's medium (MS) supplemented with benzyl amino purine (BAP) 1.0 mg L−1 and adenine sulfate 70.0 mg L−1 was found optimum for production of multiple shoots. In the present study, incubation of flowering cultures on BAP supplemented medium (during shoot multiplication) was found necessary for flowering (6 weeks). However, concentrations of auxins-like IBA (0–2.0 mg/L) were ineffective to form reproductive buds. Subculture duration, photoperiod, and carbon source type do have influence on the in vitro flowering. The mature purple flowers were observed when the cultures were maintained in the same medium. This is the very first report that describes in vitro flowering system to overcome problems associated with flower growth and development as well as lay foundation for fruit and seed production in vitro in Swertia chirayita.

Influence of Bavistin, Cefotoxime, Kanamycin and Silver Thiosulphate on Plant Regeneration of &amp;lt;i&amp;gt;Solanum viarum&amp;lt;/i&amp;gt; (Dunal)—An Important Anticancer Medicinal Plant

The effects of bavistin, cefotoxime and kanamycin along with the ethylene inhibitor silver thiosulphate on in vitro regeneration using the axillary buds of Solanum viarum (D.) have been investigated. Axillary bud explants when cultured on MS medium supplemented with bavistin (100 mg/l) maximum number of shoots (4.21 ± 0.83) were observed. In a separate experiment, bavistin along with growth regulators such as BAP and TDZ was added to MS medium containing sucrose (3%). Maximum number of shoots was obtained (5.55 ± 0.28) in MS medium supplemented with bavistin (100 mg/l) and BAP (2.0 mg/l). Cefotoxime and kanamycin (10-150 μM/L) were added to MS medium supplemented with BAP (2.0 mg/l) with sucrose (3%) having shown the maximum shoot regeneration (4.38 ± 0.45 and 3.4 ± 0.48) at 80 μM/L and 50 μM/L respectively. Similarly the regeneration medium was supplemented with (5.0-100 μM/L) of STS. Maximum number of shoots (4.75 ± 0.43) was seen with 40 μM/L. These plantlets were further maintained for root emergence on a rooting medium supplemented with growth regulators such as (0.5-2.0 mg/l) IAA, IBA and NAA. In MS medium supplemented with IBA (1.0 mg/l), maximum number of roots (24.3 ± 0.31) was seen with 100% regeneration. The rooted plants were acclimatized and transferred to field plots with (95%) of plant surviving in the field.

Impact of Different Carbohydrates and Their Concentrations on &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Regeneration of &amp;lt;i&amp;gt;Solanum viarum&amp;lt;/i&amp;gt; (Dunal)—An Important Anticancer Medicinal Plant

In this present study, the effect of various carbon sources such as sucrose, glucose, fructose and maltose was investigated on in vitro shoot regeneration of Solanum viarum using axillary bud explants. The frequency, growth and multiplication rate were highly influenced by the type and concentration of carbon source used. Among the different concentrations (1%-6%) of carbohydrates studied, the maximum number of shoots (22.6 ± 0.50) and shoot length (5.92 ± 0.13 cm) was obtained on MS medium supplemented with 4% (w/v) fructose. The least number of shoots (1.5 ± 0.32) was obtained on 6% maltose and the least shoot length (1.2 ± 0.23) was observed on 6% glucose. Among the four types of carbon sources that were employed in the present study, fructose at 4% proved to be better choice for multiple shoot regeneration followed by sucrose, glucose and maltose from axillary bud explants of S. viarum.