Harsh Pal Bais

Role of polyamines in the ontogeny of plants and their biotechnological applications

Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to be involved in a wide range of plant processes, however their exact role is not completely understood. In this review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been focussed.

Putrescine and silver nitrate influences shoot multiplication, in vitro flowering and endogenous titers of polyamines in Cichorium intybus L. cv. Lucknow local.

A bstractThe influence of putrescine (Put) and AgNO3 on shoot multiplication, in vitro flowering and endogenous titers of polyamines in Cichorium intybus L. cv. Lucknow local was investigated. Exogenous administration of Put at a concentration of 40 mM resulted in maximum tissue response in terms of shoot numbers (34.6 ± 2.61) and shoot lengths (7.6 ± 0.57 cm) on MS media supplemented with 2-iP (2.0 mg L−1) and GA3 (0.5 mg L−1) as observed on the 35th day. Exogenous application of 40 μM AgNO3 resulted in maximum shoot number (36.8 ± 2.63) and shoot lengths (7.9 ± 0.76 cm) on day 35 on the same media. Endogenous titers of conjugated spermidine decreased sharply from day 7–21, whereas endogenous conjugated spermine levels peaked on day 28 (1265 ± 94.9 nmoles g−1 FW), after treatment with 40 mM Put. Whereas, AgNO3 (40 μM) fed samples resulted in higher titers of endogenous conjugated spermine (1405 ± 105.6 nmoles g−1 FW, 3.62 fold over control) on day 14. All other treatments showed decreasing endogenous levels during the whole culture period. Both Put (40 mM) and AgNO3 (40 μM) resulted in floral initiation and floral development on day 28 and 14 (3.76 ± 0.16, 4.2 ± 0.21 flowers per shoot apices), respectively. To investigate the role of Put (40 mM) and AgNO3 (40 μM) on morphogenetic response and endogenous conjugated polyamine titers in shoots of chicory, polyamine inhibitors (DFMA and DFMO) were used. The morphogenetic response and the endogenous conjugated pool of polyamines were diminished in DFMA and DFMO treatments, but could be restored by addition of Put (40 mM) and AgNO3 (40 μM). Under exogenous Put feeding, ethylene production was reduced in shoot cultures of chicory. This study shows for the first time the influence of polyamines on multiple shoot initiation from axillary buds of C. intybus L. cv. Lucknow local and also indicates the promotive effect of Put and AgNO3 on autoregulation of polyamine biosynthesis, thereby regulating in vitro flowering, the endogenous pool of polyamines and shoot multiplication.

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

SummaryThe effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l−1 level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.

Enhancement of growth and coumarin production in hairy root cultures of witloof chicory (Cichorium intybus L.cv.Lucknow local) under the influence of fungal elicitors

Studies were conducted to determine the possible production of coumarins in hairy root cultures of Cichorium intybus L.cv. Lucknow local under the influence of microbial agents. Pythium aphanidermatum and Phytopthora parasitica var. nicotiana were cultured and their mycelial and medium filtrate were used for the elicitation of coumarin production. The media filtrate of P. parasitica at 1.0% v/v added to the MS basal medium led to the maximum growth of hairy roots of C. intybus, which was 1.57-fold higher than the untreated control on the 28th day of culture, along with maximum esculin and esculetin yields which were 4.06- and 3.71-fold higher than the control on the 28th day. The yields of esculin and esculetin in hairy root cultures of chicory strongly correlated with growth. To check the effect of these fungal elicitors on endogenous polyamine metabolism, titers of total endogenous polyamines were analyzed. It was confirmed that the media filtrate of P. parasitica at 1.0% v/v concentration resulted in maximum accumulation of total endogenous polyamines, wherein endogenous spermine titers were found to be maximum as compared to endogenous spermidine and putrescine titers on the 28th day. Total endogenous spermine in the case of 1.0% MF v/v P. parasitica was 1.3-fold higher than that of the control on the 28th day of culture. Maximum growth index with greater length of primary root (17.61+/-0.18 cm) and greater number of secondary and tertiary roots was recorded for the medium filtrate of P. parasitica at 1.0% v/v concentration. This study also provided an insight into the morphological changes in terms of branching patterns, occurring in roots under the influence of these fungal elicitors.

Influence of Polyamines on Growth of Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local) and Formation of Coumarins.

Abstract. The effect of polyamines (putrescine, spermidine, and spermine) was examined for growth and production of two coumarins, esculetin and esculin, in the hairy roots of chicory (Cichorium intybus L. cv. Lucknow local). Of the polyamines administered, 1.5 mm putrescine alone resulted in a 2.3-fold higher increase in the growth of hairy roots as well as in the production of esculetin and esculin, which was 3.37 times more than that of the control on day 21. The endogenous level of conjugated putrescine was more than fivefold that of free putrescine levels in untreated samples. The production of esculetin and esculin in hairy root cultures strictly correlated with growth in all of the treatments. Putrescine at 1.5 mm resulted in a greater length of primary root (18.29 ± 1.37 cm) compared with the control (10.96 ± 0.82 cm) and more secondary and tertiary roots. This study also provides insight into the morphogenetic changes that occur in roots in response to the external supply of polyamines.

Biotechnological Production of Plant-Based Insecticides

ABSTRACT:  The demand for natural and nonpersistent insecticides is increasing day by day. Plant cell cultures could be an alternative to conventional methods of production of insecticides from field-grown plants. In vitro cultured plant cells produce a wide array of insecticides as a part of their secondary metabolism. Their ability to synthesize key enzymes and the manipulation of these could lead to the enhanced production of many insecticides of industrial importance. The development of a high-yielding hairy root culture system for thiophenes, nicotine, and phytoecdysones is of considerable interest. In this article, the current literature on various factors that influence the growth, production, and secretion of six insecticidal compounds, namely, pyrethrins, azadirachtin, thiophenes, nicotine, rotenoids, and phytoecdysones which have been prospects for the scale-up of cell cultures, genetic engineering to obtain transgenic plants, and metabolically engineered plants for increased production of bio-molecules, has been discussed. Environmental safety clearance and the future prospects of application of bio-molecules for plant-derived insecticides are presented.

Putrescine Influences Growth and Production of Coumarins in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local)

Abstract. The effect of putrescine (Put) on the growth and production of two coumarins, esculin and esculetin, in hairy roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of Put on growth and production of coumarins, polyamine inhibitors, namely α-dl-difluromethylornithine and α-dl-difluromethylarginine were used at 1 mM concentration. Put treatment at 1.5 mM produced a 1.9-fold increase in the growth of hairy roots, as well as the production of esculin and esculetin. The treatments with polyamine (PA) inhibitors resulted in much lower growth and production of coumarins compared with both 1.5-mM Put treatment and the control. Both free and conjugated PAs were studied over the whole culture period, and conjugates of all three PAs, namely Put, spermidine, and spermine, were higher than free PAs throughout the culture period. The treatments with PA inhibitors showed lower levels of endogenous PAs compared with Put-treated samples. The treatment with 1.5 mM Put showed maximum accumulation of endogenous conjugated Put (2,098 ± 157 nmoles gm−1 fresh weight). The production of esculin and esculetin was strictly correlated with growth in all treatments. Put at 1.5 mM resulted in greater length of primary root (18.3 ± 1.4 cm) as compared with the control (11 ± 0.9 cm) and larger numbers of secondary and tertiary roots.

Influence of polyamines on growth and formation of secondary metabolites in hairy root cultures of Beta vulgaris and Tagetes patula

Growth of hairy roots of Beta vulgaris, which produces betalaines, and of Tagetes patula, which produces thiophenes, was studied under the influence of externally treated polyamines. Of the three polyamines, viz. putrescine, spermidine and spermine, administered singly at 1.5 mM concentration, putrescine and spermidine at 0.75 mM concentration influenced increase in the accumulation of biomass of B. vulgaris and T. patula hairy roots by 1.42 and 1.30 fold over the control. Whereas, the treatment of spermine (1.5 mM) alone resulted in decrease in the biomass in both the systems. Combined administration of putrescine (0.75 mM) and spermidine (0.75 mM) enhanced growth in both B. vulgaris and T. patula than that observed in individual treatments. Polyamines administered alone or in combination did alter production of betalaine and thiophene content. Dose response experiments showed that, when putrescine and spermidine was administered at 0.75 mM concentration, it resulted in maximum biomass and production of beta-laine and thiophene in B. vulgaris and T. patula respectively as compared to the control and the media treated with double and triple strength of nitrates and in combination with putrescine and spermidine at equimolar concentration. In B. vulgaris and T. patula hairy root cultures, endogenous spermine titers were maximum in putrescine and spermidine 0.75 mM each treated, cultures, which was 1.63 and 2.0 fold higher than in control on 28th and 35th days respectively.

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

SummaryA transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d−1) and esculin content (18.5gl−1), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.

Synergistic effect of auxins and polyamines in hairy roots of Cichorium intybus L. during growth, coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.