Large-scale Multiplication Research Articles

High frequency shoot regeneration and plant establishment of Rauvolfia serpentina: An endangered medicinal plant

An efficient method was developed for high frequency shoot regeneration and plant establishment from nodal segment explants of Rauvolfia serpentina. Among the three different cytokinins (6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP)) tested as supplements to Murashige and Skoog (MS) medium, BA at 5.0 μM concentration was found effective in inducing multiple shoots. Effects of different medium (Murashige and Skoog (MS), Gamborg medium (B5), Schenk and Hildebrandt (SH) and Woody Plant Medium (WPM)) on in vitro regeneration were also evaluated. The highest frequency of shoot regeneration (90%) and maximum number of shoot (45.4 ± 3.2 per explants) with an average shoot length of 4.7 ± 0.21 cm were standardized on WPM supplemented with 5.0 μM BA along with 1.0 μM α-naphthalene acetic acid (NAA). In vitro regenerated micro-shoots rooted best on WPM supplemented with 1.0 μM NAA. Plantlets with well-developed shoot and roots, following acclimation in the greenhouse, were successfully established in field condition, and 90% of plants were survived. The regenerated plants were morphologically normal and did not show any immediate detectable phenotypic variation. The method developed can be successfully employed for large-scale multiplication and long-term in vitro conservation of R. serpentina. Key words: Nodal explants, plant growth regulators, in vitro, plant regeneration, micro-propagation.

Micropropagation of self-heading Philodendron via direct shoot regeneration

The present study describes a direct shoot regeneration-based micropropagation procedure for the self-heading cultivars of Philodendron. Three types of explant (i.e. leaf lamina, petiole, stem nodal segment) were screened for their shoot induction potential following a three months treatment with 0.5mgl−1 of either 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or both. Results indicated that the leaf laminas were poor candidates for shoot induction whereas the petioles showed potential for adventitious shoot production at frequencies of 2.8–11.1% in two of the cultivars tested. Stem nodal segments were the most responsive among the three as shoots formed directly following the TDZ treatment at frequencies of 16.7–41.7% depending on the cultivar. When comparing the effectiveness of different cytokinins to induce shoot proliferation on stem nodal segments, it was found that the 0.5 and 1mgl−1 of kinetin (Kn) and 6-benzyladenine (BA) treatments resulted in higher shoot formation percentages compared to the 0.5 and 1mgl−1 of TDZ treatments in two of the three cultivars. Furthermore, more shoots were produced on BA than on Kn-supplemented media in all the three cultivars. Shoots derived from the 0.5mgl−1 of BA treatment can be induced to root following one month incubation with 0.1–1mgl−1 of indole-3-butyric-acid (IBA). The rooted shoots showed 100% survival after acclimatization in the greenhouse. The procedure reported in the present study can assist in the large-scale multiplication of elite self-heading cultivars of Philodendron in the future.

Micropropagation, antinociceptive and antioxidant activities of extracts of Verbena litoralis Kunth (Verbenaceae)

This work describes an efficient micropropagation protocol for Verbena litoralis and the study of the antinociceptive and antioxidant activities in extracts of this species. For the establishment in vitro, surface-sterilization procedures and PVPP showed high efficiency in fungal-bacterial contamination and phenol oxidation controls. Nodal segments cultivation in MS medium supplemented with 6-benzyladenine (7.5 µM)/α-naphthaleneacetic acid (NAA; 0.005 µM) induced multiple shoots. Elongated shoots were rooted with IAA (0.2 µM). Acclimatization rates were elevated and the plants showed the typical features of this species. The hexanic fraction (HF) of powdered leaves presented a radical scavenging activity with IC(50) = 169.3 µg mL(-1). HF showed a non-dose dependent analgesic activity in the writhing test; its antinociceptive activity in the hot plate test was restricted to 500 mg kg(-1), which is the highest dose. The results of this study showed the potential of tissue culture on conservation and large scale multiplication and confirmed the traditional folk medicine use of V. litoralis.

In vitro multiple shoot induction through axillary bud of Cocculus hirsutus (L.) Diels: A threatened medicinal plant

In this study, we describe a method for rapid in vitro multiplication for conservation of a threatened medicinal plant, Cocculus hirsutus (Menispermaceae) via enhanced axillary bud proliferation from nodal explants collected from young shoots of a mature elite plant. The physiological effect of different plant growth regulators such as 6-Benzylaminopurine (BAP), kinetin (Kn), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on in vitro multiplication was studied. Multiple shoots were induced in vitro from the nodal segments of stem on Murashige and Skoog’s (MS) medium containing BAP or Kn alone or in combination. Maximum number of shoots (45 ± 0.69 shoots per explant) was observed on the medium containing BAP (0.5 mg/L) along with additives like adenine sulphate (50.0 mg/L) and glutamine (150 mg/L). The shoots were best elongated on medium containing 0.25 mg/L BAP along with additives. Elongated shoots were rooted on ¼ MS medium supplemented with 0.5 mg/L IBA. The rooted in vitro raised plantlets were acclimatized in growth chamber. The method described here can be successfully employed for large-scale multiplication, long term in vitro conservation and for further investigation of medicinally active constituents of C. hirsutus . This is the first report on in vitro multiplication of C. hirsutus. Key words: Cocculus hirsutus , in vitro multiplication, threatened, nodal explants.

In vitro propagation of Etlingera elatior (Jack) (torch ginger)

An efficient protocol for complete plant regeneration from suckers of Etlingera elatior (Jack) has been developed. The addition of N6-benzyl amino-purine (BAP) (0, 13.32, 22.20, 31.08, and 44.40μM) to the culture medium comprising Murashige and Skoog (MS) basal salts, 3% sucrose, 0.4% Gelrite™ did not show any significant effects on percentage of shoot induction and mean number of shoots produced. Various concentrations of BAP (0, 13.32, 22.20, 31.08, and 44.40μM), 6-furfurylaminopurine (kinetin) (0, 13.95, 23.25, 32.55, and 46.50μM) and N6-(2-isopentenyl) adenine (2-iP) (0, 14.76, 24.6, 34.44, and 49.2μM) were tested for shoot multiplication. BAP at all levels were found suitable for the multiplication of shoot. However, the low level of 13.32μM BAP was chosen as the best concentration of BAP due to economic feasibility. The best root proliferation was observed on MS medium without plant growth regulator (PGR). Assessment of various potting media for acclimatization showed medium containing soil:sand:peat moss (1:1:1) produced high survival of plantlets, number of leaves produced per plant and the plant height. This protocol provides a basis for future studies on mutation induction, genetic engineering, and could be applied for large-scale multiplication of this valuable plant.

Shoot regeneration and micropropagation of Panax vietnamensis Ha et Grushv. from ex vitro leaf-derived callus

The methods for leaf-derived callus induction, callus proliferation, adventitious shoot induction and plant regeneration of Vietnamese ginseng ( Panax vietnamensis Ha et Grushv.) were examined. In this study, callus induction was formed on both medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with thidiazuron (TDZ). The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2,4-D and 0.2 mg/l TDZ. The best callus proliferation medium was Schenk and Hildebrandt (SH) supplemented with 0.2 mg/l TDZ and 1.0 mg/l 2,4-D. The maximum callus-derived shoot number (8.2) was obtained on SH medium supplemented with 50 g/l sucrose in combination with 2.0 mg/l 6-benzylaminopurine (BA). The most successful rooting of regenerated adventitious shoots was obtained on SH medium with 1.0 mg/l α-naphthalene acetic acid (NAA). Plantlets were successfully acclimatized without growth chamber facility on Ngoc Linh mountain with a survival rate of 85% after two months. On the other hand, substantial increase of root length was observed. This study describes an efficient method for in vitro regeneration of P. vietnamensis , which could be considered for large-scale multiplication and propagation of this important medicinal plant. Key words : Acclimatization, callus, Panax vietnamensis, regeneration, root, shoot.

Thidiazuron induced high frequency axillary shoot multiplication in Withania somnifera L. Dunal

An efficient and improved shoot regeneration technique for the micropropagation of Withania somnifera L. through in vitro culture of nodal segments with axillary buds using thidiazuron (TDZ) is developed. Murashige and Skoog (MS) medium containing TDZ (0.0 to 10.0 µM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication and subsequent elongation on hormone free MS medium. TDZ at a concentration of 0.5 µM was found to be most effective in bud break and also produced maximum regeneration frequency (98%), number of shoots (23.8 ± 0.33) with shoot length of (4.83 ± 0.66 cm), after 4 weeks of culture. Such proliferating shoots when sub-cultured on MS media devoid of TDZ, showed the highest number (32.4 ± 0.24) of shoots and shoot length (7.66 ± 0.08 cm) at the end of fourth subculture passage. Among the different concentrations of Indole- 3-butyric acid (IBA) (50 to 500 µM) tested, the maximum percentage of rooting 100% in vitro regenerated microshoots was achieved in soilrite when basal portion of the microshoots were treated with 200 µM (IBA) for 15 min, which induced maximum roots (18.3 ± 0.16) with root length of (7.63 ± 0.08 cm) per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. Micropropagated plants grew well, attained maturity and flowered normally. The present regeneration process favoured large-scale multiplication and long-term in vitroconservation of W. somnifera. Key words: Axillary, Ashwagandha, conservation, genotype, nodal explants, regeneration, thidiazuron.

In vitro propagation of Cuphea procumbens Orteg. and Evaluation of genetic fidelity in plantlets using RAPD marker

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cuphea procumbens Orteg. using cotyledonary node explants excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at a concentration of 2.5 μM was effective in inducing multiple shoots. The highest number of multiple shoots (9.33 ± 0.60) and maximum average shoot length (4.16 ± 0.44 cm) was standardized on MS medium supplemented with 2.5 μM BA alongwith 0.5 μM NAA. Addition of 200 mg/l Casein hydrolysate (CH) to the shoot induction medium enhanced the growth of regenerants. Rooting of in vitro regenerated shoots was best achieved on 1/2 strength MS medium. The in vitro raised plantlets with well developed shoots and roots were hardened, successfully established in earthen pots containing garden soil and maintained in greenhouse with 80% survival rate. Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate the genetic stability among in vitro regenerated progenies. All RAPD profiles from the micropropagated plants were monomorphic and similar to control plant. These results suggests that the culture conditions used for the axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with genetic integrity of in vitro regenerated plants. The described method can be successfully employed for large-scale multiplication and in vitro conservation of C. procumbens.

Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis

Jatropha curcas (Euphorbiaceae), a drought resistant non edible oil yielding plant, has acquired significant importance as an alternative renewable energy source. Low and inconsistent yields found in field plantations prompted for identification of high yielding clones and their large scale multiplication by vegetative propagation to obtain true to type plants. In the current investigation plantlets of J. curcas generated by axillary bud proliferation (micropropagation) using nodal segments obtained from selected high yielding genotypes were assessed for their genetic stability using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) analyses. For RAPD analysis, 21 out of 52 arbitrary decamer primers screened gave clear reproducible bands. In the micropropagated plantlets obtained from the 2nd sub-culture, 4 out of a total of 177 bands scored were polymorphic, but in the 8th and 16th sub-cultures (culture cycle) no polymorphisms were detected. AFLP analysis revealed 0.63%, 0% and 0% polymorphism in the 2nd, 8th and 16th generations, respectively. When different genotypes, viz. IC 56557 16, IC 56557 34 and IC 56557 13, were assessed by AFLP, 0%, 0.31% and 0.47% polymorphisms were found, respectively, indicating a difference in genetic stability among the different genotypes. To the best of our knowledge this is the first report on assessment of genetic stability of micropropagated plantlets in J. curcas and suggests that axillary shoot proliferation can safely be used as an efficient micropropagation method for mass propagation of J. curcas.

DATE PALM GENETIC DIVERSITY CONSERVATION FOR SUSTAINABLE PRODUCTION

DATE PALM GENETIC DIVERSITY CONSERVATION FOR SUSTAINABLE PRODUCTION

In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation

This communication describes for the first time an efficient and reproducible protocol for large-scale multiplication of Bambusa nutans. Nodal segments collected from field-grown clumps and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kin) gelled with 0.2% gelrite yielded 80% aseptic cultures with 100% bud-break. The in vitro-formed shoots obtained after bud-break were successfully multiplied in MS liquid medium supplemented with 13.2 μM BA, 2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every 3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-fold. Shoot clusters containing three to five shoots were successfully rooted with 100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA, 2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthaleneacetic acid (NAA), and 3% sucrose. Plantlets grown in vitro were acclimatized and subsequently transferred to the field. Inter-simple sequence repeat analysis has confirmed the genetic uniformity of the tissue-cultured plants up to 27 passages.

Tissue culture of Cecropia glaziovii Sneth (urticaceae): vegetative micropropagation and plant regeneration from callus

Cecropia glaziovii is a tree with used in Brazilian popular medicine. Methods allowing the clonal propagation of this species are of great interest for superior genotype multiplication and perpetuation. For this reason, we examined the effect of different culture media and different types of explants on adventitious shoot regeneration from callus and buds of C. glaziovii. Leaves, petioles and stipules obtained from aseptically grown seedlings or from pre-sterilized plants were used to initiate cultures. Adventitious shoot regeneration was achieved when apical and axillary buds were inoculated on gelled Murashige & Skoog (MS) medium supplemented with 6-benzylaminopurine alone (BAP) (1.0, 5.0 or 10.0 mg L-1) or combined with -naphthalene acetic acid (NAA) (1.0 or 2.0 mg L-1), after 40 days of culture. Best callus production was obtained after 30 days of petioles' culture on gelled MS medium with 2,4 dichlorophenoxyacetic acid (2,4-D) (5.0 mg L-1) combined with BAP (1.0 mg L-1). Successful shoot regeneration from callus was achieved when MS medium supplemented with zeatin (ZEA) (0.1 mg L-1) alone or combined with 2,4-D (1.0 or 5.0 mg L-1) was inoculated with friable callus obtained from petioles. All shoots were rooted by inoculation on MS medium supplemented with indole-3-acetic acid (IAA) (1.0 mg L-1). Rooted plants transferred to potting soil were successfully established. All in vitro regenerated plantlets showed to be normal, without morphological variations, being also identical to the source plant. Our study has shown that C. glaziovii can be propagated by tissue culture methods, allowing large scale multiplication of superior plants for pharmacological purposes.

Adventitious rooting of conifers: influence of physical and chemical factors

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species.

Trichomes, essential oils and in vitro propagation of Lavandula pedunculata (Lamiaceae)

Lavanders ( Lavandula spp.) are aromatic plants with high industrial and commercial value, used in food industry, perfumery and pharmaceutical preparations. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Lavandula pedunculata and to evaluate the potential of these plants for essential oil production with industrial application. In vitro cultures were established on media with different concentrations of benzyladenine. Trichome morphology was examined by SEM and their secretion analyzed by histochemical tests. Essential oils were isolated by hydrodistillation and analyzed by GC and GC-MS. The essential oils and trichomes of in vitro plants were compared with those of field-growing plants. Best propagation rates were achieved with 0.25 mg/l BA. Rooting occurred without treatment with auxins thus preventing callus growth. Trichomes and essential oils of the in vitro propagated plants were similar to those of the parent plants. Two chemotypes were characterized: 1,8-cineole/camphor type and fenchone type. Since trichomes and essential oils of L. pedunculata micropropagated plantlets are identical to those produced by field-growing plants, in vitro cultures can be used for essential oil production without affecting natural resources. Moreover, the quality of the essential oils is assured and this protocol can be further applied to the propagation of selected chemotypes for industrial purposes.

MICROPROPAGATION STUDIES ON IMPORTANT FOREST TREES OF HIGH IMPORTANCE: AN EFFORT FOR LARGE-SCALE COMMERCIAL MULTIPLICATION AND THEIR FIELD PLANTATION

The present project is taken up with the aim to develop micropropagation protocols for large and commercial scale multiplication of highly important forest trees, i.e., Eucalyptus hybrids. Two Eucalyptus hybrids namely as FRI-5 (Eucalyptus cainaldulensis Dehn x Eucalytpus tereticornis Sm.) and FRI-14 (Eucalyptus torelliana F.V. Muell × Eucalyptus citriodora Hook) were selected for the present course of investigation with the aim to develop mass scale micropropagation technique using nodal segments as explants from the mature trees (30-32 years old) of both the hybrids. These Eucalyptus hybrids were produced by Forest Research Institute, Dehradun in the early 1960-70s though controlled crossing are available in very limited number and need multiplication true to type for field-testing to ascertain its superiority under varied agro-climatic conditions. These hybrids showed 3-5 superior in terms of total biomass and wood volume when compared to their parents. Cultures were established and multiplied on MS medium supplemented different concentration of BAP either alone or in with combination of auxins i.e. IBA/NAA. MS medium was found to be effective and suitable for all the experiments during plant production. Eighty-five to 92% rooting was achieved on half strength MS medium supplemented with IBA. Ninety-five to 98% plantlets survival was obtained after hardening and acclimatization prior to field transfer. Parameters like height, DBH, clear bole length and self pruning capability of both the hybrid were recorded up to three years to identify the suitability of these hybrids in different varied climatic zones of Uttarakhand.

IN VITRO CULTURE FOR MUTANT DEVELOPMENT

The success of any in vitro mutagenesis programme depends on the establishment of reproducible in vitro plant regeneration procedures, optimization of mutagenic treatments, and efficient screening of the mutagenized populations for desired variations (Ahloowalia, 1998; van Harten et al., 1998; Jain, 2000, 2006, 2007). Cell and tissue culture techniques can improve effectiveness of mutation induction in several aspects. One of the major advantages of tissue culture is that a large number of regenerated plants can be handled at ease for mutagenic treatments (Jain, 2000; Predrieri, 2001). They offer a wide choice of plant material for mutagen treatment (in vitro axillary buds, organs, tissues, protoplasts, and cells) (Broertjes and van Harten, 1988; Constabel and Shyluk, 1994; Jain and Newton, 1989; Duncan, 1997) that is more suited to mutation induction techniques as compared to in vivo buds (Predrieri, 2001). The tissue structures from which plants originate are either multicellular or of single cell origin (Williams and Maheshwaran, 1986; Ochatt et al., 2005; Kulkarni et al., 2007). Normally chimeras are major problems in regenerated plants by mutagen treatment of multicellular structures such as shoot tips or axillary buds. By in vitro culture chimeras can easily be dissociated by several time subcultures of shoot cultures, say 4 generations M1V4 (Jain, 2000; Predrieri, 2001) This, as compared to in vivo buds, means less risk of obtaining chimera plants and a higher probability for mutated cells to express the mutation in the phenotype (Broertjes and van Harten, 1988). It also facilitates rapid completion of the propagation cycles of subculture aimed to separate mutated from non-mutated sectors (Ahloowalia, 1998). In vitro selection and cloning of selected mutants can also be effective when supported by adequate early evaluation tools (Ball, 1990; Duncan, 1997; Ochatt, 2006; Ochatt et al., 2005) as well large scale multiplication of mutant plants. Micropropagation is an ideal system for true-to-type multiplication of mutant plants by using shoot tips or axillary buds (Ahloowalia, 1998). Furthermore, tissue culture provides high phytosanitary conditions, which is an ideal system for obtaining healthy starting material and is maintained throughout the plant regeneration process (Ochatt et al., 1990; Cassels, 1998; Predrieri, 2001; Kulkarni et al., 2007). Therefore, in vitro mutagenesis should always be carried out on healthy explants. Otherwise endogenous contamination may compromise obtaining mutations, as well as selection and/or the survival of mutants.

A scaled-up system for in vitro multiplication of thidiazuron-induced red raspberry shoots using a bioreactor

SUMMARYA procedure for the mass propagation of adventitious shoots regenerated from leaf explants of red raspberry (Rubus idaeus L.) using a liquid medium-containing bioreactor system combined with gelled medium has been described. Leaf segments of the raspberry cultivars ‘Festival’, ‘Heritage’, and ‘Latham’ produced multiple buds and shoots in 4.5 µM thidiazuron (TDZ)-containing shoot induction medium within 6 – 8 weeks of culture initiation. The raspberry cultivars differed significantly with respect to their shoot regeneration and multiplication rate, with ‘Heritage’ producing the highest number of adventitious shoots per explant.TDZ supported rapid shoot proliferation at low concentrations (1.2 – 2.3 µM) in the bioreactor system, but shoot elongation was improved in medium containing 4.4 µM 6-benzyladenine (BA). BA-induced, elongated shoots rooted in the bioreactor vessel containing the same medium, but without any plant growth regulators. In vitro-derived plantlets were acclimatised and eventually established in a greenhouse and in the field. These results suggest the possibility of large-scale multiplication of raspberry shoots using a bioreactorbased system.

Callus induction and plant regeneration in Dorem ammoniacum D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l−1, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l−1 for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l−1 NAA and 2 mg l−1 BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l−1) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l−1 for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l−1 BA and 0.2 mg l−1 IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l−1 IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.

A Scale-up System for Lowbush Blueberry Micropropagation Using a Bioreactor

In an attempt to improve the micropropagation protocol for lowbush blueberry (Vaccinium angustifolium Ait.), a protocol using a bioreactor system combined with a semisolid gelled medium has been developed. Cultures of cultivar Fundy and two wild clones (‘NB1’ and ‘QB1’) were established in vitro on a gelled modified cranberry basal medium (BM) containing 5 μM zeatin or 10 μM N6-[2-isopentenyl]adenine. Multiple shoots were obtained within 8 weeks by transferring zeatin-induced shoots from the gelled BM to a bioreactor containing liquid BM with 1 to 4 μM zeatin. Genotypes differed significantly with respect to multiplication rate in liquid and gelled BM containing 1 μM zeatin with ‘NB1’ producing 8.5 ± 1.1 and 2.9 ± 0.3 shoots per explant in liquid and gelled media, respectively, after one subculture followed by ‘QB1’ (7.1 ± 0.6 and 2.6 ± 0.4 shoots per explant, respectively) and ‘Fundy’ (5.8 ± 0.4 and 2.0 ± 0.2 shoots per explant, respectively). With subculture, there was an increase of shoot multiplication rate for all genotypes. Bioreactor- and gelled medium-proliferated shoots were treated with 39.4 mm indole-3-butyric acid powder, rooted in a 2 peat:1 perlite (v/v) medium, plantlets acclimatized, and eventually established in the greenhouse with 64% to 74% rooting of microshoots and 90% to 99% survival of rooted shoots. Results obtained suggested the possibility of large-scale multiplication of lowbush blueberry shoots in bioreactors.

English

  An efficient protocol is described for the rapid in vitro plant regeneration of a medicinally important plant, Hygrophila spinosa through direct somatic embryogenesis from nodal explants excised from 4 week old aseptic seedlings. Somatic embryos differentiated directly from nodal explants on Murashige and Skoog (MS) medium supplemented with 1.0 µM 6- benzyladenine (BA) after 1 week of culture. The highest number (110.40 ± 3.55) of somatic embryos were recorded on MS medium supplemented with 1.0 µM BA and 0.5 µM NAA, growth regulator free half strength MS medium was found suitable for maturation and conversion of somatic embryos into complete plantlets. The well developed in vitro regenerated plantlets were successfully established in pots containing garden soil and grown in a greenhouse with 85% survival rate. The regenerants present an appearance similar to the seedlings obtained from normal seeds. The described method can be successfully employed for large scale multiplication and long term in vitroconservation of H. spinosa.   Key words: Acanthaceae, Hygrophila spinosa, somatic embryogenesis, medicinal plant, conservation, micropropagation.