Sumita Jha

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the TL T-DNA, and 90% of them carried the TR T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the TL and the TR-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the TR T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the TL T-DNA genes, while all the callusing lines induced by strain A4 contained the TL DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.

In vitro propagation of cashewnut

In vitro plant propagation was developed for seedling shoot tips, leaf axils, and cotyledonary nodes of cashew, Anacardium occidentale. Factors affecting multiplication rate included age of explant source, explant type, medium composition, light requirements, and transfer frequency. Cotyledonary nodes produced more buds than other explant types. Nodes had a 90% viability when transferred daily to fresh medium containing activated charcoal for 7 d while exposed to continuous dark. Cultures were then exposed to low light illumination with weekly transfers. The phytohormone composition producing the most buds was 2.32 μM kinetin, 9.12 μM zeatin and 4.40 μM BA. The highest frequency of rooted shoots was obtained by treating shoots with the bacterium, Agrobacterium rhizogenes. Plants also were recovered by induction of roots using auxin treatment on propagated shoots.

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: Changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes.

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.

In vitro regeneration from bulb explants of Indian squill, Urginea indica Kunth

Callus cultures were established from bulb explants of diploid Urginea indica Kunth (Indian squill) on a modified basal medium of Murashige and Skoog (1962) supplemented with either 2 mg/l-1 2,4-D+15% (v/v) CM or 4 mg/l-1 2,4-D+2 mg/l-1 NAA+2 mg/l-1 KN+1 g/l-1 YE. Shoot primordia developed after 2–3 subcultures in that medium. Increased growth of shoot primordia was obtained in media containing less auxins and vitamins. Rooted bulbous plantlets obtained were maintained in MS medium with 0.5% sucrose.Adventitious shoots were induced from adaxial epidermal cells of outer scales of regenerated bulbs used as secondary expiants in presence of 1 mg/l-1 of 2,4-D with slightly higher concentration of the three vitamins of MS medium. From each scale leaf, approximately 400 bulblets were produced in 18 weeks in liquid culture. 90% of the plants transferred to potted soil have survived.

Micropropagation of Cephaelis ipecacuanha rich.

Shoot cultures of ipecac, Cephaelis ipecacuanha Rich. were established by inoculating seedling nodal explants onto modified Murashige and Skoogs medium supplemented with 8 mg/l kinetin, 0.05 mg/l NAA and 200 mg/l adenine. Upto 12 new axillary shoots per explant were induced after 12 weeks incubation. Shoot cultures were also established by placing shoot tips on medium containing 0.1–0.25 mg/l NAA with 8 mg/l kinetin for 4 weeks and then to shoot multiplication medium for 8 weeks. The multiplication was maintained over several passages. Shoots were rooted using 2 mg/l IBA and normal plants were re-established.

Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors

Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.

Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens

Abstract Transformed organ cultures of Withania somnifera were established following infection with wild type nopaline and octopine strains of Agrobacterium tumefaciens. The oncogenic strains had different levels of virulence on two genotypes studied, the main difference was found in the nature of the galls formed and in their subsequent morphological competence. Ten percent of the galls obtained following infection with nopaline strain N2/73 spontaneously developed shooty teratomas of altered phenotype. The shooty teratomas grew in unsupplemented basal medium and were able to synthesize both the major withanolides of the parent plants. Withanolide synthesis in shooty teratomas was much higher (0.07–0.1% withaferin A and 0.085–0.025% withanolide D) than in non-transformed shoot cultures.

Somatic embryogenesis from immature cotyledons of an elite Darjeeling tea clone

Abstract Cotyledons from immature embryos of Camellia sinensis O. Kuntze var. T-78, an elite Darjeeling tea clone, were exposed to various levels of 2,4-D, NAA, IBA, BAP and Kin (1–10 mg/1). While no embryos were formed in MS basal medium without growth regulators, the auxins tried were also inefficient in stimulating primary embryogenesis. Somatic embryos were induced directly on the cotyledonary explants in MS medium with 10 mg/1 BAP alone. Somatic embryo formation could be enhanced by addition of 0.5 mg/1 IBA and 80 mg/1 adenine to 10 mg/1 BAP. Embryo conversion was 2–3% in MS medium with or without growth regulators. Almost 20% of somatic embryos converted into whole plants following transfer to B5 medium containing 3 mg/1 BAP and 2 mg/1 IBA. Embryogenic potential was maintained for over 30 months by secondary embryogenesis through successive generation of embryos. Somatic embryo derived plants were successfully transferred to potted soil at 70% frequency under green house conditions. Morphologically and cytologically (2n = 30) plants are true to type.

Agrobacterium rhizogenes -Mediated Transformation in Medicinal Plants: Prospects and Challenges

A large number of plant species from several families have been successfully transformed by Agrobacterium rhizogenes and established in culture. In contrast to normal roots, the Ri-transformed root cultures are fast growing, and do not require exogenous supply of plant growth regulators in the medium to support growth. In addition, they are robust, plagiotropic, and produce the full range of secondary products characteristics of roots derived from the parent plant species. Ri transformed roots of some species synthesize novel compounds and screening and selection procedure can also be employed to improve production levels. The most interesting applications of hairy root cultures in a continuous culture program, are with secondary product pathways exclusively active in roots, and from which the product would be secreted into the medium. Large-scale secondary metabolite production from hairy roots can be achieved by using different types of bioreactors. In this review, we provide an overview of synthesis and accumulation of medicinally important secondary metabolites in Ri transformed root cultures and in Ri-transformed plants.

Genetic transformation of Artemisia annua by Agrobacterium tumefaciens and artemisinin synthesis in transformed cultures

Abstract Transformed organ cultures of Artemisia annua were established following infection with two wild type nopaline strains of Agrobacterium tumefaciens . Parameters like explant type, strain type, age of the plant source for explants, affected the tumorigenesis frequency significantly. Crown galls were formed both on in vivo and in vitro plants: 2–3% of the in vitro galls regenerated spontaneously to produce shooty teratoma of altered phenotype. Artemisinin contents were measured in all transformed as well as non-transformed clones. While shooty teratomas synthesized 0.063 ± 0.002 g/100 g DW artemisinin, non-transformed shoots were found to synthesize only 0.0179 ± 0.002 g/100 g DW of the compound.