Hormone-free Basal Medium Research Articles

English

Dendrobium primulinum Lindl. (D. Primulinum L.) is one of the important epiphytic orchid species for horticultural and commercial use. It is listed as a rare and critically endangered species of orchid. The present study is intended to conserve the orchid species through the micropropagation technique. The in vitro seed germination and seedling growth was carried out by taking a mature pod of D. Primulinum L. in Murashige and Skoog (MS) media, and the MS media supplemented with a varied concentration (0.5, 1.0, 1.5 and 2 mg/L) of 6-benzylaminopurine (BAP), and a fixed concentration (0.5 mg/L) of α-naphthalene acetic acid (NAA). Sequential phases of seed germination, protocorm formation, and seedling development in the presence of growth regulators were determined in the study. The significance of hormonal effects was determined by using One-Way Analysis of Variance (ANOVA, p≤0.05). The seed germination started after two weeks of culture in the media supplemented with BAP. The maximum seedling growth was obtained in the media supplemented with 1.5 mg/L of BAP. Although the hormone-free basal medium revealed an ideal condition for seed germination and spherules formation, the presence of an appropriate concentration of growth regulators such as 0.5 mg/L NAA or a combined 0.5 mg/L BAP + 0.5 mg/L NAA expressed a synergistic effect to enhance the protocorm formation and seedling development. Key words: In vitro, Dendrobium primulinum, 6-benzylaminopurine (BAP), α-Naphthalene acetic acid (NAA).

In vitro regeneration of Pholidota pallida Lindl. (Orchidaceae)

Orchids exhibit a wide range of diversity in forms, size, colour and texture of flowers beyond the imagination of human mind. Orchids grow in nature through seeds but in the absence of appropriate hosts, they do not germinate in adequate number. This obstacle was overcome by adapting tissue culture technique for appropriate germination of orchids. In vitro techniques have been successfully carried out for the mass propagation of orchid plantlets. Hence, a preliminary study was carried out to develop a suitable protocol for mass multiplication of Pholidota pallida. MS (Murashige and Skoog) medium was found to be suitable for the asymbiotic seed germination of Pholidota pallida. MS medium supplemented with IAA (Indole-3-acetic acid), IBA (Indole-3butyric acid), BAP (6-Benzylaminopurine) and KIN (Kinetin) individually and in combinations induced direct protocorm like bodies. Hormone-free MS basal medium was found suitable for the conversion of PLBs (protocorm-like bodies) into complete plantlets

Genetic fidelity of long-term micropropagated Lavandula officinalis Chaix.: an important aromatic medicinal plant

A protocol for long-term in vitro conservation of multiple shoot cultures of Lavandula officinalis and regeneration of true-to-type plants from them is described here. Multiple shoots were developed from apical bud explants on a modified vitamin-enriched Murashige and Skoog’s basal medium supplemented with 2.5 mg/l Kinetin. The cultures were subsequently conserved in vitro for 6 years under slow growth conditions imposed by lowering the sucrose level from 3 to 2.0 % and increasing the agar concentration from 0.8 to 1.2 % in this medium. Complete plant regeneration from in vitro conserved shoots was achieved through axillary shoot proliferation on original 2.5 mg/l Kinetin containing medium followed by rooting of individual shoots in a hormone-free half strength MS basal medium. The genetic fidelity of the regenerated plants was tested by random amplified polymorphic DNA analysis. Twenty one arbitrary decamer primers produced a total of 64 scorable bands (1–6 bands/primer) in the size range of 100–5,148 bp. All DNA fingerprints with these primers displayed monomorphic band profiles indicating homogeneity among the regenerated progeny and their uniformity with the donor parent. Head-space gas chromatographic analysis of the leaf tissue excised from mother as well as regenerated plants also revealed a similar qualitative and quantitative profile of volatile terpenes. The utility of the developed protocol for long-term ex situ conservation and quality plant production for cultivation of this high value aromatic herb is discussed.

Micropropagation of Swertia chirata Buch.-Hams. ex Wall.: a critically endangered medicinal herb

An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA3 (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.

Exogenous Hormones Affecting Morphology and Biosynthetic Potential of Hairy Root Line (LYR2i) of Linum album

A hairy root line of Linum album LYR2i was obtained via the genetic transformation of a cotyledon segment of the plant by cocultivating it with Agrobacterium rhizogenes and culturing it in hormone-free B5 Gamborg liquid medium. Characteristic changes in root morphology associated with variations in biosynthetic capabilities were achieved by adding different combinations of phytohormones to the basal medium. A reversible system between hairy roots and its cultures with a diversified morphology was established by including and excluding phytohormones in the basal medium. The medium containing indole 3 acetic acid (IAA) at a concentration of 3 mg/l (MI3) induced thicker root tips and that containing 2,4-dichlorophenoxy acetic acid (2,4-D) at a concentration of 1 mg/l (MD1) induced compact green callus. Podophyllotoxin and 6-methoxypodophyllotoxin content increased by 1.86-fold and 1.45-fold as a result of adding IAA (MI3) and 2,4-D (MD1) to the basal medium, respectively. After regeneration, the hairy roots regained their original morphology and biosynthetic capability on hormone-free basal medium. The transformation status of the regenerated hairy roots was confirmed by PCR analysis.

Efficient production of transgenic potato ( S. tuberosum L. ssp. andigena) plants via Agrobacterium tumefaciens-mediated transformation

Potato is an important target crop for biotechnological applications and is a valuable model system for studying signaling processes. Efficient transformation is critical for rapid genetic analyses. The production of transgenic potato shoots within 4 weeks from the time of initial inoculation of leaf explants by Agrobacterium tumefaciens has been established with the Solanum tuberosum subspecies andigena. Vigorous stock plants, the precise, uniform wounding of the midrib on the leaf explant, and the composition of the regeneration media play key roles in the development of this efficient shoot regeneration protocol. To produce callus from leaf explants in 7 days, basal medium was supplemented with optimized concentrations of benzyl-aminopurine and napthalene acetic acid. Incubation on basal medium supplemented with a combination of zeatin riboside, napthalene acetic acid, and gibberellic acid induced shoot formation from callus after 28 days of incubation. In this improved protocol, the commonly used pre-culture of explants with nutrient medium was eliminated and the Agrobacterium inoculation medium was not supplemented with any phytohormones. Induction of roots from putative transformed shoots was achieved in hormone-free basal medium supplemented with kanamycin. Normal, healthy root formation was observed within 5 days and 91% of the selected shoots rooted on kanamycin. By using RT-PCR analysis with gene specific primers, all rooted shoots out of 20 selected from five different lines exhibited expression of the full-length StBEL5 transgene driven by the CaMV 35S promoter. The protocol described here is simple, efficient, and produces transgenic shoots in just 4 weeks after inoculation with Agrobacterium.

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids

Stem nodal explants of Paphiopedilum philippinense hybrids (hybrid PH59 and PH60) directly formed shoots when cultured on a modified half-strength Murashige and Skoog (1962) basal medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D: 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45 and 4.54 μM) for 6 mo. On hormone-free basal medium, the percentages of explants with shoots were 33.3% and 0% and the shoot numbers per explant were 1 and 0 in hybrid PH59 and hybrid PH60, respectively. In hybrid PH59, 4.52 μM 2,4-D plus 0.45μM TDZ induced a higher percentage of explants with shoots and shoot number per explant than did the hormone-free treatment. In hybrid PH60, although 4.52 μM 2,4-D and 0.45 μM TDZ promoted shoot formation, the highest shoot number was found with 4.52 μM 2,4-D alone. Plantlets, each having several roots, were obtained from regenerated shoots after transferring onto hormone-free basal medium for 3 mo. The plantlets were potted in sphagnum moss, and acclimatized well in a greenhouse.

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet ( Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N6 medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.

Effects of auxins and cytokinins on direct somatic embryogenesison leaf explants of Oncidium 'Gower Ramsey’

Four auxins (IAA, IBA, NAA and 2,4-D) and five cytokinins (2iP, zeatin,kinetin,BA and TDZ) were examined for their effects on direct somatic embryogenesis onleaf explantsof a sympodial orchid Oncidium 'Gower Ramsey’.On a hormone-freebasal medium, the percentages of embryo formation were 40%, 20%,5% and0% on leaf tips, adaxial sides, wound surfaces and abaxial sides of theleaf explants, respectively, and the average number of embryos per explant was5.6. Embryo formation on leaf explants was retarded by all four auxins tested,but promoted by all the cytokinins. The percentages of embryo formation werereduced to 20%, 5–10%, 0% and 0%,respectively, in the same parts of leaf explants when supplemented with lowdogsages of IAA (0.3–3 mg/l) on the basal medium.Furthermore, embryo formation was totally inhibited by 3 mg/l NAA,0.3–3 mg/l IBA and 2,4-D. The sequence of embryo formationonvarious location of leaf explants was altered by 0.3–1 mg/l2iP and 3 mg/l zeatin, and embryo formation on adaxial sides> leaf tips > woundsurfaces > abaxial sides. The highest percentage of embryoformation on leaf tips, adaxial sides and wound surfaces of explants were75%, 50% and 20% when supplemented with 1mg/lTDZ, 1 mg/l 2iP and 0.3 mg/l kinetin, respectively.The highest average number of embryo per explant (10.7) was found on a basalmedium containing 1 mg/l TDZ.

Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae)

An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid ( Oncidium ‘Gower Ramsey’). Compact and yellow–white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1–3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3–10 mg/l) and peptone (1 g/l) for 4–7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2–4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12–14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3×3 mm 2) was found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse.

Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium Sweet Sugar

Segments taken from flower-stalk internodes of Oncidium Sweet Sugar formed somatic embryos and shoot buds directly from wound surfaces or via nodular masses proliferation within 1.5 months, when cultured on a Gelrite-gelled 1/2-MS basal medium supplemented with thidiazuron (0.1–3 mg l−1) in darkness. In light, when subcultured, these nodular masses proliferated into green compact callus, and produced somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1–1 mg l−1 NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures. Somatic embryos that directly formed from wound surfaces of flower stalk explants usually developed into abnormal structures, but the callus-derived embryos could germinate into PLBs and eventually developed to normal plantlets on a hormone-free basal medium for 3–4 weeks. Both the embryo-and shoot bud-derived regenerants developed into healthly plantlets when potted in sphagnum moss and acclimatized in the greenhouse.

Micropropagation of Plumbago zeylanica Linn

ABSTRACT A rapid and highly effective method for plant micro-propagation from vegetative shoot buds was established for the medicinal plant Plumbago zeylanica Linn. Multiple shoots were proliferated from nodal explants cultured on Murashige and Skoog basal medium supplemented with kinetin or N6-benzyl adenine (0.25–1.0 mg/l). Excised shootlets cultured on hormone-free basal medium rooted within 4 to 7 days. Successful transfer of the rooted shoots to potting soil has been accomplished with 100 percent survival.

Organogenesis and somatic embryogenesis in Codiaeum variegatum (L.) Blume cv. “Corazón de Oro”

Adventive organogenesis and somatic embryogenesis were induced from leaf explants taken from in vitro or in vivo plants of Codiaeum variegatum cv. “Corazon de Oro.” Shoot multiplication occurred with N6-benzyladenine (BA) alone, where the simultaneous production of adventitious buds and somatic embryos occurred at the fourth subculture, and on leaves not in contact with the medium. A medium with BA and 2,4 dichlorophenoxy acetic acid (2,4-D) produced the largest organogenic response, for both in vivo- and in vitro-produced explants. Somatic embryogenesis was only induced when such explants were transferred to a medium lacking 2,4-D. Thus, a medium with BA only produced the largest percentage of explants with shoots and embryos. Replacing BA with thidiazuron induced up to 100% bud regeneration on in vitro-produced explants by 60 d, but was slower for in vitro-grown explants. Both types of embryos exhibited growth arrest that was partially overcome by transfer to hormone-free basal medium with activated charcoal. Rooted plants from all explants were successfully obtained on a medium with indole-butyric acid (IBA).

In vitro propagation of Achillea asplenifolia VENT. through multiple shoot regeneration

A method for the micropropagation of Achillea asplenifolia VENT. (Asteraceae) is described. Axillary shoots regenerated from nodal explants of adult plants could be stimulated to multiple shoot formation on the basal medium of Murashige and Skoog (1962), supplemented with different cytokinins. The best proliferation (5.33 shoots per culture), together with a healthy appearance of the cultures, was achieved with N-benzyl-9-(2-tetrahydropyranyl)adenine at a concentration of 1 mg/l. Shoots could be easily rooted on hormone-free MS basal medium and subsequently acclimatized to greenhouse and field conditions with 100% survival rate. DNA-fingerprinting, chromosome counts, the proazulene pattern as well as the essential oil analysis proved the true to type character of the micropropagated plants.

Roots as a source of explants for the successful micropropagation of carambola ( Averrhoa carambola L.)

A technique for the clonal micropropagation of carambola is described. Multiple adventitious shoots were produced on root explants placed on woody plant medium or Murashige and Skoog medium supplemented with 2.0 mg l −1 6-benzylaminopourine and 0.2 mg l −1 naphthalene acetic acid. When these shoots were excised from the roots and placed on the same medium, a multiplication rate of 2.1 per month was achieved. Shoots left on the multiplication medium for more than 7 weeks became chlorotic and eventually died. Chlorosis was avoided if the shoots were transferred to a hormone-free basal medium for a 10 day period. Shoots were successfully rooted on a basal medium containing 1% activated charcoal and were successfully transferred to soil and planted in the glasshouse. The technique provides a commercially practical technique for the clonal propagation of carambola.

Infection of Somatic Embroys of Tall Fescue with Acremonium coenophialum

The availability of a tall fescue (Festuca arundinacea Schreb.) clone whose members are infected with different strains of its endophytic fungus, Acremonium coenophialum Morgan‐Jones & Gams, would help identify the individual contributions of the endophyte and tall fescue toward traits normally associated with endophyte‐infected tall fescue plants. As embryos and very young seedlings are the only stages of the life cycle during which infection by the endophyte naturally occurs in planta, attempts were made to duplicate this phenomenon in vitro. Callus cultures of tall fescue were induced using leaf basal tissue as the explant source, and Murashige and Skoog (MS) basal medium supplemented with the synthetic auxins 2,4‐D [(2,4‐dichlorophenoxy)acetic acid], 2,4,5‐T [(2,4,5‐trichlorophenoxy)acetic acid], or picloram (4‐amino‐3,5,6‐trichloropicolinic acid). Two tall fescue genotypes were exposed to four concentrations (15, 30, 45, or 60 μM) of each auxin. Calli were transferred to hormone‐free basal medium to permit the development of somatic embryos. Elongating shoots were counted to deter mine the effectiveness of each treatment and transferred to fresh medium to permit their development into plants. The most effective and consistent auxin concentration to stimulate somatic embryogenesis was 2,4‐D at 30 μM, producing almost 14 embryos per callus. Next, 60 explants of one fescue genotype were placed on MS medium supplemented with 30 μM 2,4‐D. Two isolates of the endophytic fungus were used to infect the explants and developing call! by inoculating at weekly intervals. The highest infection rate was obtained by inoculating somatic embryos with the fungus at the time the callus was transferred to hormone‐free medium, the stage at which somatic embryos begin to elongate and germinate. This stage corresponds with the stage at which infection is thought to occur in planta. This method will permit further studies of the interactions between tall fescue and its endophyte.

Maturation and conversion ofLiriodendron tulipifera somatic embryos

Embryogenic suspension cultures of the hardwood forest tree yellow-poplar (Liriodendron tulipifera) have the potential to produce millions of plantlets. However, low conversion frequencies limit the realization of this potential. Using 4 embryogenic yellow-poplar lines, we first tested the ability of somatic embryos, selected for their similarity to mature zygotic embryos, to convert to plantlets, then tested physical and chemical treatments for their effects on promoting maturation of somatic embryos and subsequent plantlet production. Embryos selected based on resemblance to mature zygotic embryos and transferred to a hormone-free basal medium without casein hydrolysate (CH) produced plantlets at a frequency of 63%. Populations of synchronized somatic embryos were obtained by repeated fractionation of liquid medium-cultured proembryogenic masses (PEMs) on stainless steel sieves. These fractionated embryos failed to mature properly when cultured in liquid basal medium, however. Development of embryos cultured in basal medium supplemented with 5×10−7 M abscisic acid (ABA) was slowed and embryos appeared to mature properly, with separated cotyledons and little precocious germination. However, ABA-treated embryos only rarely converted to plantlets, possibly due to residual effects of the ABA. PEMs fractionated on sieves, transferred to filter paper and placed on solidified basal medium gave a 60–70% synchronous population of mature embryos 10–12 days following plating. Mature embryos transferred to basal medium without CH converted at a frequency of 72%. The percentage of all embryos differentiating from PEMs on filter paper that formed plantlets was 32%.

Maturation and conversion ofLiriodendron tulipifera somatic embryos

Embryogenic suspension cultures of the hardwood forest tree yellow-poplar (Liriodendron tulipifera) have the potential to produce millions of plantlets. However, low conversion frequencies limit the realization of this potential. Using 4 embryogenic yellow-poplar lines, we first tested the ability of somatic embryos, selected for their similarity to mature zygotic embryos, to convert to plantlets, then tested physical and chemical treatments for their effects on promoting maturation of somatic embryos and subsequent plantlet production. Embryos selected based on resemblance to mature zygotic embryos and transferred to a hormone-free basal medium without casein hydrolysate (CH) produced plantlets at a frequency of 63%. Populations of synchronized somatic embryos were obtained by repeated fractionation of liquid medium-cultured proembryogenic masses (PEMs) on stainless steel sieves. These fractionated embryos failed to mature properly when cultured in liquid basal medium, however. Development of embryos cultured in basal medium supplemented with 5×10−7 M abscisic acid (ABA) was slowed and embryos appeared to mature properly, with separated cotyledons and little precocious germination. However, ABA-treated embryos only rarely converted to plantlets, possibly due to residual effects of the ABA. PEMs fractionated on sieves, transferred to filter paper and placed on solidified basal medium gave a 60–70% synchronous population of mature embryos 10–12 days following plating. Mature embryos transferred to basal medium without CH converted at a frequency of 72%. The percentage of all embryos differentiating from PEMs on filter paper that formed plantlets was 32%.

Temporal Competence of Embryonic Pinus ponderosa Cotyledons to form Multiple Buds in Vitro

To investigate temporal changes occurring during tissue maturation and loss of organogenic competence, embryos of Pinus ponderosa Laws, were excised from seed and placed horizontally on medium in sterile culture. On a hormone-free basal medium, cotyledons differentiated and failed to form multiple buds. When placed on a benzyladenine (BA)-containing medium, the lower cotyledons in contact with the medium formed multiple buds while those elevated above the medium did not. Cotyledons became incompetent to form buds when embryos were initially placed on basal medium for 2 days before being transferred to the BA-supplemented medium. In order to initiate buds, cotyledons of newly excised embryos had to be exposed to BA for at least 3 days. Exposure to BA for longer than 7 days did not significantly increase the number of cotyledonary buds. Cotyledons elevated above the BA-supplemented medium retained organogenic competence to form buds for up to 14 days but did not form buds unless the cotyledons were placed in contact with the medium. The presence of BA is required to retard cotyledon maturation and sustain tissue competence to initiate buds.