Satish Manohar Nalawade

Studies on the Production of Some Important Secondary Metabolites from Medicinal Plants by Plant Tissue Cultures

Plants are a tremendous source for the discovery of new products of medicinal value for drug development. Today several distinct chemicals derived from plants are important drugs currently used in one or more countries in the world. Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell culture technologies were introduced at the end of the 1960s as a possible tool for both studying and producing plant secondary metabolites. Different strategies, using an in vitro system, have been extensively studied to improve the production of plant chemicals. The focus of the present review is the application of tissue culture technology for the production of some important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.

ASYMBIOTIC GERMINATION OF IMMATURE SEEDS, PLANTLET DEVELOPMENT AND EX VITRO ESTABLISHMENT OF PLANTS OF DENDROBIUM TOSAENSE MAKINO - A MEDICINALLY IMPORTANT ORCHID

SummaryShi-hu (Dendrobium spp. or Dendrobii Herba) is one of the important traditional Chinese medicines. The commercially available crude drug in the traditional medicine market is composed mainly of three species: Dendrobium tosaense, D. nobile, and D. moniliforme. An efficient method of propagation has been developed via asymbiotic germination of seeds in vitro for the medicinally important D. tosaense. Seeds from capsules of D. tosaense collected 8–14 wk after artificial pollination germinated after being cultured on full-strength or half-strength Murashige and Skoog (MS) medium devoid of plant growth regulators and with 3% sucrose. Germination of seeds varied with the medium type and seed maturity. Germinated seedlings after transfer to MS medium with 1.5% sucrose and 8% banana homogenate or potato juice or coconut water and 20 wk of incubation developed into healthy plantlets. Well-developed plantlets were transplanted to moss or moss and tree fern or tree fern as substrates in plastic trays and transferred to a greenhouse for hardening. All plants survived, attained maturity, and developed normal flower and capsule after one and a half years. This protocol of successful plant regeneration by asymbiotic seed germination should permit rapid propagation and conservation of this medicinally important Dendrobium species.

IN VITRO PROPAGATION OF SOME IMPORTANT CHINESE MEDICINAL PLANTS AND THEIR SUSTAINABLE USAGE

SummaryMedicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the demand for the erude drugs, the plants are being overexploited, threatening the survival of many rate species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An overview of tissue culture studies on important Chinese medicinal plants and related species is presented.

IN VITRO PROPAGATION OF THE CHINESE MEDICINAL PLANT, DENDROBIUM CANDIDUM WALL. EX LINDL., FROM AXENIC NODAL SEGMENTS

SummaryDendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l−1 sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l−1 HYPONeX, 80 g l−1 potato homogenate and 2 g l−1 activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l−1 benzyladenine (BA) and 0.1 mg l−1 naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l−1 NAA and the plantlets were successfully acclimatized in soil.

INFLUENCE OF EXPLANTS, GENOTYPES AND CULTURE VESSELS ON SPROUTING AND PROLIFERATION OF PRE-EXISTING MERISTEMS OF COTTON (GOSSYPIUM HIRSUTUM L. AND GOSSYPIUM ARBOREUM L.)

SummaryA simple and efficient method for multiple shoot induction and proliferation was achieved in six Indian cotton cultivars from the pre-existing meristems of 21-d-old in vitro-grown seedlings. Combinations of naphthalene acetic acid (0.3–10.7 μM) and 6-benzylaminopurine (BA; 2.2 or 4.4 μM) were used for induction of shoots. The shoots proliferated and were maintained on MS (Murashige and Skoog) medium supplemented with 4.4 μM BA. Simultaneous elongation of shoots was obtained in the same medium. Optimum multiplication was observed in cv. LRK-516 (19.7±4.6), in cotyledonary nodes isolated from the adjoining node and cultured individually in 250 ml flasks. This indicates lateral inhibition of adjoining meristems. A positive influence of culture flasks as opposed to test tubes on the proliferation of multiple shoots was observed in all six cultivars tested. The morphogenic response varied with genotype and the nature of explants. Rooting of elongated shoots was achieved on MS medium devoid of growth regulators. The plantlets were transferred to the field after hardening in the greenhouse. All plants flowered and formed bolls on maturity.

Propagation of Haemaria discolor via in vitro seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm−3 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm−3 α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of β-glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoogs medium supplemented with B5 vitamins, 0.1 mg dm−3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm−3 α-naphthaleneacetic acid, 3 % glucose + 50 mg dm−3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.

A Rapid and Simple Method for in vitro Plant Regeneration from Split Embryo Axes of Six Cultivars of Cotton

Plant regeneration was achieved from the seed derived decotyledonated split embryo axes of six Indian cultivars of cotton (Gossypium hirsutum L.). Explants were cultured on Murashige and Skoogs basal medium supplemented with 2 % sucrose and 0.65 % agar. Incorporation of 0.25 % charcoal in the medium and incubation of the cultures at 30 ± 2 °C had synergistic effect on the frequency of shoot and root formation. The method employed is genotype independent, simple and rapid.

Induction of multiple shoots and plant regeneration from 'accessory buds' of nodal segments from field-grown mature cotton plants (Gossypium hirsutum L.)

SummarySprouting of a single shoot was obtained from nodal segments of field-grown cotton plants (Gossypium hirsutum L. cv. DCH-32 and NHH-44) when cultured on Murashige and Skoogs (MS) basal medium devoid of plant growth regulators. Pruning of the sprouted shoot followed by re-culturing of the explant on MS basal medium supplemented with 2.22 μM benzylaminopurine triggered the dormant ‘accessory buds’ present in the node to proliferate and differentiate into multiple shoots. The shoots elongated on the same medium and rooted on MS basal medium devoid of growth regulators. Plantlets were hardened under greenhouse conditions and transferred to the field. Flowering and boll formation were observed in these plants at maturity. This technique for successful clonal propagation from mature cotton tissues should permit the rapid multiplication of plants selected based on specific phenotypic or genotypic characteristics.

Multiple Shoot Induction and Plant Regeneration from Embryo Axes of Six Cultivars of Gossypium hirsutum

The report describes in vitro plant regeneration from embryo axis explants of six cultivars of cotton. Induction of a maximum number of multiple shoots in all six cultivars could be achieved on Murashige and Skoogs (MS) salts and Gamborgs (B5) vitamins supplemented with 0.4 μM benzyladenine (BA) and 0.1 μM napthaleneacetic acid (NAA). Elongated shoots could be rooted on half strength medium supplemented with 0.5 μM NAA. Rooted shoots survived (92 %) after hardening in the greenhouse and grew to maturity (100 %) after transfer to field.