Shyamal K. Nandi

6-Benzylaminopurine and its glycosides as naturally occurring cytokinins

Abstract Primary crown gall tumours induced on stems on tomato plants by Agrobacterium tumefaciens yielded extracts with pronounced cytokinin activity. By mass spectrometry and other methods, 6-benzylaminopurine and its 9-β-ribofuranoside were identified as cytokinins in the extracts which also contained a 9-hexoside of 6-benzylaminopurine.

Taxol content in the bark of Himalayan Yew in relation to tree age and sex

Taxol content in the bark of Taxus baccata trees growing in a homogenous (uniform) environment at Jageshwar, District Almora in Central Himalaya has been quantified. The average taxol concentration in the bark of sampled trees was 0.0558+/-0.008% (of dry wt.) and was about 64% higher for male plants (averaged across tree age) in comparison to female trees. Maximum taxol content was recorded in the bark samples collected from trees of >110 yrs age. ANOVA indicates a significant difference in the taxol content of bark from trees of different ages, however, differences were not significant between sexes. Taxol was quantified by HPLC using a standard curve prepared with authentic taxol; the identification of bark taxol was confirmed by UV and mass spectrometry. The total taxol content of the bark of Taxus trees across an age series was found to range between 0.064 to 8.032 g/tree, and a tree of about 100 yrs age can yield 5.74 kg dry bark.

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA3)/ indole-3-butyric acid (IBA). BA (22.19 μM) was effective for induction of multiple shoots and addition of GA3 to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 μM). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 μM). However, this was associated with basal callus formation. Treatment with IBA (25–100 μM) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 μM for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 μmol m−2s−1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 μmol m−2s−1 in Q. leucotrichophora and around 1500 μmol m−2s−1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 μmol m−2s−1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current seasons growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (≥70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (≥90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.

An Overview of Cytokinin Biosynthesis

A critical element in understanding the physiological role of cytokinins is the knowledge of how plants control their cytokinin levels. Therefore, we need to know the pathway(s) and site(s) of cytokinin synthesis, the metabolic fate of cytokinins, and the biochemical mechanisms controlling their biosynthesis and degradation.

In vitro propagation of Aconitum balfourii Stapf.: an important aconite of the Himalayan alpines

Summary Small leaf segments, obtained from in vitro sprouted axillary buds, have been used to develop an ef®cient micropropagation protocol for Aconitum balfourii Stapf., an important medicinal herb of the Indian Himalayan alpines. Following the induction of callus from cultured leaf segments on Murashige and Skoog (1962; MS) medium containing 4.5.mM 6-benzyladenine (BA) and 26.9.mM a-naphthaleneacetic acid (NAA), the highest values for shoot induction were obtained on the same medium supplemented with the same concentration (4.5.mM) of BA and a lower concentration of NAA (1.4.mM). These individual shoots could be further multiplied easily, with maximum shoot proliferation obtained on MS medium containing 1.1.mM BA only. The shoots obtained either directly from the callus or after multiplication on the above medium, were found to root ef®ciently on MS medium supplemented with 12.3.mM indole-3-butyric acid (IBA). After in vitro hardening in the culture room, the plantlets were transplanted to a greenhouse where the growth of plants was normal. Comparison of mitotic chromosome numbers of root tip cells, protein pro®le and alkaloid content of tubers of in vitro raised and seed grown plants of comparable age showed equal number of chromosomes, identical protein pro®le and similar amounts of diterpenoid alkaloids (aconitine and pseudoaconitine). This paper is the ®rst report describing a complete regeneration protocol, along with quantitative data, for A. balfourii using in vitro methods.

Cytokinin biosynthesis in plant tumour tissues

A range of endogenous cytokinins have been identified inDatura crown-gall tissue by GC-MS. Incorporation of [3H]adenine into zeatin riboside, zeatin and its nucleotide(s) is also shown. Metabolism studies usingcis- andtrans-isomers of zeatin riboside indicate that interconversion of the two isomers does not occur in this tissue. Data on the identity of major endogenous cytokinins in a genetic tumour line of tobacco is also provided.

Adventitious Root Formation in Young Shoots of Cedrus Deodara

The effect of auxins [indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA)], phenolics (phloroglucinol and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin, containing 50% carbendazim) on adventitious root formation in stem cuttings (current seasons growth) of Cedrus deodara L. during winter and monsoon (rainy) seasons has been examined. Significant stimulation of rooting due to treatments was observed in cuttings planted in winter in the following order: 0.25 mM IBA (87.5% rooting) > 5 mM coumarin (70.8%) > IBA (0.5 mM) + coumarin (5 mM) (50.0%). In cuttings planted in monsoon only 0.05% Bavistin was found to be effective in inducing rooting (83.3%). Other treatments were ineffective and in some treatments drying of cuttings was noticed.

Effect of pre-germination treatments on seed physiology and germination of central Himalayan oaks?

The continuous decline in regeneration of two important species of central Himalayan oak, namely Quercus glauca and Q. leucotrichophora, is of great concern. A study was therefore, carried out to improve germination ability of these species using various presoaking treatments. Seeds of both the species lost viability following storage; tetrazolium staining pattern and germination capacity of seeds following different period of storage at 4 °C and 20 °C indicated retainment of viability for a period of 12 months at 4 °C. Of the various physical, chemical and plant growth regulator treatments examined to improve seed germination, only KNO3 1.0 % was found to be effective. Seeds scarified at the chalazal end exhibited significant improvement in germination in both the species (94.4 % compared to 56.7 % in control in Q. glauca and 82.7 % compared to 64.0 % in control in Q. leucotrichophora). The results of this study impart simple methods to improve seed germination for developing nurseries for commercial purposes.

GA3 induced flowering in Podophyllum hexandrum Royle: A rare alpine medicinal herb

Podophyllum hexandrum Royle, an important alpine herb, is the source of highly valued podophyllotoxin. The effect of some plant growth substances (GA3, BAP & ABA), uniconazole (an inhibitor of GA biosynthesis), and a combination of GA3 and uniconazole were examined in respect to influence on sprouting in rhizomes of P. hexandrum and on induction of flowering at a lower altitude. Amongst the various chemicals tested, GA3 had a marked effect resulting in uniform sprouting and also induced flowering in about half of the treated rhizomes. While BAP also promoted early sprouting, delayed sprouting was seen in rhizomes treated with ABA. Uniconazole treatment, either alone or with GA3 was found to inhibit flowering and also resulted in reduced plant height. GA3 treatment of rhizomes from plants that was maintained for up to 30 months at a lower altitude also induced flowering thus replacing the normal chilling requirement of plants. These results suggest that treatment of GA3 could be effectively used for inducing uniform sprouting and flowering in rhizomes of P. hexandrum grown at lower altitudes.