In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 µM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 µM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.

Callogenesis in leaves of Kalanchoe pinnata Lam. by 2, 4-D and BA action / Calogênese em folhas de Kalanchoe pinnata Lam. pela ação de 2, 4-D e BAP

The Kalanchoe pinnata Lam. is a bush species of the Crassulaceae that is distinguished by its important medicinal properties. Its leaves are used as cataplasm to treat headaches and wounds. There is evidence for a hypotensive and anti-inflammatory effect. Techniques of plant tissue culture have been applied to plant species that produce substances likely to be explored in pharmacology, cell suspension being the main technique. At the industrial level, this method utilizes bioreactors in order to produce secondary metabolites on a large scale. The objective of this study was to evaluate the effects of in vitro combinations of 2,4-dichlorophenoxiacetic acid (2,4-D) and benzylaminopurine (BA) on callus induction in leaf explants of K. pinnata. Leaf fragments were inoculated in MS medium supplemented with 3.0% sucrose, 0.8% agar and factorial combinations of 2,4-D (0.00, 4.52, 9.06, 18.12 µM) and BA (0.00, 4.44, 8.88, 17.76 µM). The cultures were kept in the darkness at 24±2ºC for 50 days. The percentage of callus induction and the area of explants covered by callus cells were evaluated. In the absence of growth regulators, callus induction did not occur, with necrosis of all explants. The highest percentage of callus induction was 100%, obtained with the combination of 9.06 µM 2,4-D and 8.88 µM BA, but the calluses covered only 25% of the leaf area. The most efficient combination was 4.52 µM 2,4-D and 8.88 µM BA, resulting in 91% callus induction with 50 to 100% of the explants being covered by callus cells.

Kalanchoe pinnata Lam. é uma espécie arbustiva da família Crassulaceae que apresenta interessantes propriedades medicinais. Suas folhas são utilizadas em cataplasma para tratar enxaqueca e ferimentos. Há evidência de seu efeito como hipotensiva e anti-inflamatória. Técnicas de cultura de tecidos vegetais têm sido aplicadas para espécies que possuem substâncias passíveis de exploração na farmacologia, sendo a suspensão celular a principal técnica utilizada. A nível industrial, este método utiliza biorreatores para produzir metabólitos secundários em larga escala. Este estudo teve como objetivo avaliar os efeitos in vitro de combinações do ácido 2,4-diclorofenoxiacético (2,4-D) e de benzilaminopurina (BAP) na indução de calos em explantes foliares de K. pinnata. Fragmentos foliares foram inoculados em meio MS contendo 3% de sacarose, 0,8% de ágar e combinações fatoriais de 2,4-D (0,00; 4,52; 9,06 e 18,12 µM) e BAP (0,00; 4,44; 8,88 e 17,76 µM). Os cultivos foram mantidos no escuro, a 24±2ºC por 50 dias. A porcentagem de indução de calos e a área dos explantes coberta por células de calos foram avaliadas. Na ausência de reguladores de crescimento não ocorreu indução de calos, com necrose de todos os explantes. A porcentagem mais alta de indução de calos foi de 100%, obtida com a combinação de 9,06 µM de 2,4-D e 8,88 µM de BAP, mas estes calos cobriram apenas 25% da área foliar. A combinação mais eficiente foi de 9,06 µM de 2,4-D e 8,88 µM de BAP, que resultou em 91% de indução e 50 a 100% da área dos explantes coberta por células de calos.

Somatic embryo-like structures of strawberry regenerated in vitro on media supplemented with 2,4-D and BAP.

Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of strawberry (Fragaria × ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations of 2,4‑dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2 - 4 mg L-1 2,4-D in combination with 0.5 - 1 mg L-1 BAP and 50 g L-1 sucrose. Histological examination of SELS revealed the absence of a root pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of strawberries via somatic embryogenesis is discussed.

Role of PGrs and inhibitors in induction and control of somatic embryogenesis in Themeda quadrivalvis.

Somatic embryogenesis could be achieved in Themeda quadrivalvis (Linn.) O. Ktze -fodder grass species on MS medium supplemented with 2,4-D. Incorporation of putrescine in the medium stimulated embryogenesis, however its lower concentration stimulated production of non-regenerative callus. Other polyamines such as spermine and spermidine could not evoke similar response. Ascorbic acid used as antioxidant could not prevent browning in embryogenic cultures, however it stimulated embryogenesis. Inhibition of auxin polar transport by use of TIBA and HFCA reduced the embryogenic response significantly and produced distorted or abnormal embryos. Antiethylene substances such as AgNO3 and CoCl2 added in the medium adversely affected the process of embryogenesis and counteracting the stimulatory role of ethylene.

Efficient in vitro plant regeneration via leaf base segments of indica rice (Oryza sativa L.).

A reliable and reproducible protocol has been developed for high frequency plant regeneration from 4-5 mm long leaf base segments of 4 days old in vitro germinated seedlings of indica rice (Oryza sativa) cultivar Rasi. The effect of age of seedlings, position of leaf base segments and optimum concentration of 2,4-D on callus induction frequency was investigated with a future aim to use leaf bases for biolistic and Agrobacterium-mediated transformation experiments. Friable, nodular and white to pale yellow embryogenic callus cultures (206 mg fresh weight /explant) were obtained from the first basal segments of rice seedlings on Linsmaier and Skoog (LS) medium supplemented with 2,4-D (11.3 microM) and 3.0 microM thiamine-HCL. Plant regeneration was achieved after the transfer of 54 days old embryogenic callus cultures to Murashige and Skoog (MS) medium supplemented with BAP (2.2 microM) and NAA (0.27 microM). In vitro regenerated plants with multiple shoots and roots transferred to sterile soil in growth chamber and maintained in greenhouse exhibited normal growth and were phenotypically similar to plants germinated from seeds.

Reduction of vitrification in in vitro raised shoots of Chlorophytum borivilianum Sant. & Fernand., a rare potent medicinal herb.

Reduction of vitrification in in vitro raised shoots derived from shoot bases and immature floral buds along with inflorescence axis used as explants of C. borivilianum, a rare medicinal herb is described. Shoot multiplication was obtained on MS medium with 2 mg l(-1) benzylaminopurine (BAP) + 0.1 mg l(-1) indole-3-butyric acid (IBA) and MS medium with 2 mg l(-1) kinetin (Kin) + 0.1 mg l(-1) 2,4-dichlorophenoxy acetic acid (2,4-D) from shoot bases and inflorescence axis respectively. Best multiplication rates were obtained from both the explants on MS medium with 2 mg l(-1) BAP. Vitrification of shoots in cultures appeared during the multiplication stage. Culture bottles with aerated caps reduced the vitrification to 80%. Reduction of BAP concentration from 2 mg l(-1) to zero during subsequent subcultures also minimized vitrification. Use of 0.5-2 mg l(-1) Kin produced healthy shoots when compared to BAP. In vitro raised shoots rooted on Knop salts containing iron and vitamins of MS medium, 2 mg l(-1) IBA and 0.1% activated charcoal. About 80% plantlets survived upon soil transfer. Scanning electron microscopic and image analyzer studies reveal the morphological structural differences between the leaves of normal and vitrified plantlets.

Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats.

Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins. Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production. Berberine was synthesized and released continuously into the liquid medium. Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis. On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth. Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D. IAA and IBA were not favourable for cell growth and berberine synthesis.

In vitro propagation of three commercial cut flower cultivars of Anthurium andraeanum Hort.

In vitro propagation of Anthurium andraeanum Hort. cut flower cultivars viz. Lima White, Tropical White and Tropical Red through organogenesis using mature plant derived leaf explants was established on Murashige and Skoog (MS) medium fortified with different growth regulators. Cultivar, stage and different regions of the source leaf, and type of growth regulators significantly influenced callus induction. Explants from folded brown leaves were superior in induction of callus. Half strength MS medium fortified with 0.88 microM of benzyiadenine (BA), 0.9 microM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.46 microM of kinetin (Kn) at pH 5.5 was most effective for callus induction. Transfer of callus to medium with 0.54 microM of NAA in place of 2,4-D induced higher number of shoots. Subsequent cultures displayed enhanced rate of shoot initiation and multiplication. Transfer of shoots onto half strength MS medium supplemented with 0.54 microM of NAA favoured rooting of shoots. Cultivar Tropical White was superior in callus, shoot and root induction compared to Lima White and Tropical Red. Plantlets after acclimation in greenhouse were transferred to net-house, that exhibited ninety seven per cent survival. Plants flowered normally between 12 and 15 months and were morphologically similar to that of the mother plants.

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble).

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).

Propagation of Coriandrum sativum L. through somatic embryogenesis.

A highly embryogenic callus was obtained from hypocotyl segments of Coriandrum sativum L. when cultured in the medium consisting of MS + H vitamins (MSH). Induction of somatic embryos required 2,4-dichlorophenoxyacetic acid or napthalene acetic acid. Germination of fully developed embryos was accomplished by subculture on half strength MSH medium containing benzylamino purine 0.05 mg/L. Plantlets developed from somatic embryos were transferred to soil and were successfully flowered.

Somatic embryogenesis in Vigna radiata (L.) Wilczek.

Somatic embryogenesis was achieved from immature cotyledon derived callus of mungbean, V.radiata (L.) Wilczek in MS liquid medium. Embryogenic callus was induced on MS medium with NAA (5 mg/L). Differentiation of somatic embryos was observed when embryogenic callus was transferred to MS liquid medium containing 2,4-D (1.5 mg/L) and L-proline (50 mg/L). The torpedo shaped embryos were transferred to MS liquid medium with BAP and ABA (1 mg/L each) for maturation and germination. Fifty per cent of torpedo shaped embryos were converted into tiny plants (8-9 plants out of 17) after one week of culture. The germinated embryos were isolated and transferred to MS half strength basal (solid) medium for further development.

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots.

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.

2, 4-dichloro phenoxy acetic acid alters monoamine levels, acetylcholinesterase activity & operant learning in rats.

2, 4-Dichloro phenoxy acetic acid (2, 4-D) was given at 100 mg/kg body weight per day by oral intubation from postnatal days 2 to 25 to assess its effect on the levels of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in olfactory bulb (OB), hippocampus (HI), visual cortex (VC), cerebellum (CB) and brainstem (BS). NE levels were increased in OB, HI, VC and BS at 10 days of age. However, by 25 days, NE levels were decreased in OB, HI and VC. DA levels were also increased in OB, HI, VC and CB at 10 days of age and again decreased by 25 days in OB, HI and VC. 5-HT levels were increased in HI, CB and BS at 10 days and in VC and CB at 25 days of age. Reduced acetylcholinesterase (AChE) activity in OB and HI and monoamines in different brain regions at 25 days of age might be responsible for the observed deficits in both acquisition and rate of pedal press response when animals were exposed chronically to 2, 4-D during postnatal brain development.

Effect of 2,4-dichlorophenoxy acetic acid on rabbit duodenum.

2,4-dichlorophenoxy acetic acid (2,4-D) produces myotonia in healthy animals. The action of this drug was studied on smooth muscles in vitro using isolated strips of rabbit's duodenum. The drug was found to have a stimulant action on the smooth muscle. The action seems to be a direct one.