DiscussionDiscussionDiscussionDiscussion

CONTENTS

Page No.

5.1. Growth parameters 108

5.2. Biochemical parameters 111

5.3. Yield and quality parameters 117

5.4. Conclusions 120

5.5. Proposal of future work 121

107

Chapter 5

DISCUSSION

Around 1.5 million people die every year of malaria; every 30 seconds a child

dies due to this preventable and curable disease. Most of the affordable

antimalarial drugs have become ineffective because Plasmodium falciparum, the

malarial parasite responsible for the most severe malaria cases and deaths, has

developed resistance to them (WHO 2010). Artemisia annua is a well-known

medicinal plant that has been utilized as one of the most potent plant against

malaria. A component and extract, artemisinin, is the source of other derivatives

which are also suitable for pharmaceutical use, has a very rapid type of action

against the malarial parasite (Wilcox et al. 2004). Only artemisinin-based

combination therapies (ACTs) meets international standards set-up by WHO

and UNICEF for the cure of malaria. As stated earlier, the artificial synthesis of

artemisinin is complex and expensive, its isolation from Artemisia annua is only

dependable alternative. The present demand for artemisinin is far more than

that of supply, therefore, researchers are working round the world towards

improving artemisinin content in the plant by various means.

Radiation, a convenient tool for degradation of polysaccharides, can be

performed at room temperature and the degraded product obtained by this

process can be used without purification. Although sodium alginate can be

depolymerised through enzymatic degradation but plant researchers suggest

the use of radiation processing technology for degrading the polysaccharides as

it is economical and environment-friendly (Hien et al. 2000; Kume et al. 2002;

Lee et al. 2003). Irradiated sodium alginate (ISA) has several novel unique

features that can be useful in agriculture. In fact, polysaccharides, such as

sodium alginate (SA), undergo chain scission by irradiation and it affects the

overall polymer cross-linking process. Consequently, its application influences

the biological properties of the plant cells (El-Rehim 2006). In addition, various

researchers have emphasized that ISA could successfully act as a plant growth

promoter and also a potent enhancer of the activity of various enzymes in the

plants (Tomoda et al. 1994; Akimoto et al. 1999; Hien et al. 2000; Kume et al.

Chapter 5: Discussion

108

2002; Luan et al. 2003; Luan et al. 2005; Kume 2006; Mollah et al. 2009; Idrees

2011; Khan et al. 2011; Sarfaraz et al. 2011).

It is common knowledge that absorption of nutrients continues throughout

the life span of plants. However, their nutrient requirement may increase with

the development of sink. When plants grow old, the absorption process may

become slow. It may be due to weakening of root system or unavailability of

nutrients resulting from leaching, fixation and decomposition. Under such

conditions the adequate supply of nutrients through supplemental top

dressing/foliar spray and/or their efficient absorption and utilization with the

help of some stimulants, is highly desirable to ensure the realization of their

genetic potential. Understandably, this has proved a highly successful technique

for profitable cultivation of a number of crop plants, including medicinal plants

(Singh 2008). There is general agreement that all the nutrient amendments

made to soil, nitrogen and phosphorus fertilizer application has by far, the most

important effects in terms of increasing crop production. They are considered to

be of prime importance as they play several important roles in metabolic

regulatory processes of plants.

Keeping these points in mind, four field experiments were conducted on A.

annua to assess the feasibility of foliar ISA and its combined application with

basal nitrogen and phosphorus. The main aim was to test the realization of the

growth, yield and artemisinin production through newly introduced plant

growth promoter, ISA (as foliar sprays) and adequate, balanced and efficient

utilization of nutritional inputs. The results have been discussed in the light of

knowledge of subject and research work carried out by other workers.

5.1. Growth parameters

The main processes that determine the quality and quantity of plant growth are

cell division, enlargement and differentiation. These processes are affected by

various internal and external factors including supply and absorption of

nutrients, which have critical importance in cell metabolism and involvement of

phytohormones/ plant growth regulators for maintaining a healthy source-sink

relationship (Patel and Golakia, 1988). Plant growth regulating substances get

involved through the modification of transcription, translation and/or

Chapter 5: Discussion

109

differential sensitivity of the tissue. In the present investigation, ISA was used as

plant growth regulating substance.

As observed in Experiment 1 various concentrations of ISA improved

growth attributes (shoot and root lengths per plant as well as shoot fresh and

dry weights per plant) of Artemisia annua significantly (P<0.05) studied at pre-

flowering and flowering stages (Tables 2-5). The maximum values of growth

characteristics were determined at flowering stage as the plants continued to

grow. In conformity with our results, Albersheim and Darvill (1985), Natsume et

al. (1994), Tomoda et al. (1994), and Kume et al. (2002) reported significant

improvement in plant growth attributes by the application of radiation-derived

oligosaccharides of alginate. Luan et al. (2003) suggested a key role gamma

irradiated sodium alginate in enhancing the biological activity of the plants.

Albersheim and Darvill (1985) suggested that biologically active

oligosaccharides act as signal molecules that regulate growth and development

of the plant as well as defense reactions by regulating gene expression. Mollah et

al. (2009) also confirmed the growth promoting effect of irradiated sodium

alginate in Amaranthus cruentus. In fact, polysaccharides, such as sodium

alginate (SA), undergo chain scission by irradiation. The irradiation of SA by

gamma rays affects the overall polymer cross-linking process. Consequently, its

application influences the biological properties of the plant cells (El-Rehim,

2006). Khan et al. (2011) studied the effects of different concentrations of ISA on

Papaver somniferum in green house conditions and found that the growth of the

plants were significantly influence in positive manner. However, the

phenomenon which stimulates the processes related to promotion of plant

growth still needs further investigations.

In Experiment 2 and 3, different levels of soil-applied nitrogen and

phosphorus were given combined with foliar ISA80 (ISA80 came out as the best

concentration in improving overall performance of the crop). Growth attributes

were found to be significantly affected by various nitrogen and phosphorus

doses when applied with ISA80 in the separate experiments (Tables 21-24 and

36-39). Among various doses applied ISA80+N80 and ISA80+P40 proved best in

respective experiments. The involvement of nitrogen in influencing plant growth

Chapter 5: Discussion

110

and development and biomass production is universally acknowledged

(Marschner 2002). The increase in total biomass of the plants may be attributed

to the fact that uptake of nitrogen increases as the nutrient supply increases as it

was found in the present study upto 80 kg ha-1 soil nitrogen application with

foliar ISA80. Further higher nitrogen i.e. 100 kg ha-1, did not increase the growth

and biomass but decreased somewhat because more high N could be a limiting

factor. Enhanced dry matter production induced by elevated plant nitrogen is

extremely well documented and probably driven by increase in photosynthetic

efficiency (Davies et al. 2009). Also, increasing levels of nitrogen application

resulted in higher absorption and utilization, which accumulated the

photosynthetic assimilates for faster growth of the plants (Gautam et al. 2008).

The observed positive effects of soil-applied phosphorus with foliar ISA80 may be

ascribed to the roles of phosphorus various physiological processes. It is

indispensible for all forms of life because of its role in energy transfer via ATP

(Samuel et al. 1993). Moreover, it plays an important role in various metabolic

processes as it is an integral part of several compounds such as co-enzymes,

nucleic acid, nucleotides, phosphoric acid and phosphorylated sugars

(Marschner 2002). Among essential nutrients, P is often limiting due to low

availability, therefore its supply by any means may improve directly or

indirectly cell division, cell enlargement and tissue and organ development as

observed in Experiment 3, in which different doses of phosphorus were supplied

along with foliar ISA80. Beneficial effects of phosphorus either alone or in

combination with other nutrients/growth regulators were reported by many

workers on different crops including A. annua (Singh 2000; Ferreira et al. 2005;

Weathers et al. 2005; Naeem 2007; Singh 2008; Davies et al. 2011).

There were marked differences in growth parameters when nitrogen and

phosphorus was applied in two splits with ISA80 in Experiment 4 compared to

Experiment 2 and 3. ISA80+N40+40+P20+20 proved best over the all other

treatments including control (Tables 47-50). Results indicate that application of

fertilizers in two equal splits were more effective than single application. The

increase in plant height and biomass by applying fertilizer in two splits might be

attributed to the saving it in proper time and maximizing the utilization through

Chapter 5: Discussion

111

minimizing losses of the applied fertilizer compared to single dose. Laughlin and

Chung (1992), as well, mentioned that a partial application of fertilizer before

and after vegetative stage under optimal moisture conditions resulted in an

increase in plant growth. Applying nitrogen and phosphorus as one dose is

expected to cause high loss by continuous leaching. Split application is, however,

minimize losses by leaching, and hence increase the available nitrogen and

phosphorus to the crop. The similar results were reported by Gough et al.

(2004), Finzi et al. (2006) and Maier et al. (2008) on different crops.

5.2. Biochemical parameters

As regards photosynthetic pigments, viz. contents of total chlorophyll and

carotenoids, studied in Experiment 1 (Tables 6 and 7), the application of ISA at

80 mg L-1 (ISA80) proved optimum for total chlorophyll content; while, the ISA

application did not affect the total carotenoids content significantly. As the

oligosaccharides (obtained by degrading the alginate) induce cell signalling that

in turn, leads to stimulation of various physiological processes in various plants

(Farmer et al. 1991; Darvill et al. 1992; John et al. 1997), the application of ISA

might stimulate the improvement in total chlorophyll content in this study.

When different doses of nitrogen and phosphorus were applied along with ISA80,

further improvement in chlorophyll content was observed in Experiments 2 and

3, respectively (Tables 21 and 36). The enhancement in chlorophyll content as a

result of nitrogen and phosphorus application together with ISA80 (Experiments

2 and 3) could possibly be attributed to the increase in number and size of

chloroplast, the amount of chlorophyll per chloroplast and proper granna

development in the chloroplast (Akimoto et al. 1999). This perception is further

strengthened by the correlation studies, wherein leaf-nitrogen and -phosphorus

content was positively correlated with total chlorophyll content (Tables 62-65).

The positive effect of nitrogen supply on the formation of chloroplasts during

leaf growth has been reported by Kutik et al. (1995). In turn, the chloroplast

formation leads to an increase in the lipid content of the leaves and chloroplast

constituents such as chlorophyll. Moreover, phosphorus is an integral part of

many compounds of plant cells, including sugar-phosphates, intermediates of

respiration and photosynthesis and that of phospholipids that constitute the

Chapter 5: Discussion

112

plant membranes (Devlin and Witham, 1986). Therefore, the supplementation

of soil-phosphorus might be helpful in enhancing chlorophyll content directly or

indirectly. As shown by the data of Experiment 4, when phosphorus was applied

in splits together with ISA80, it further increased the chlorophyll content in

comparison to its single dose application (Table 51). In fact, as mentioned above,

when fertilizer is applied in splits, its loss due to leaching is minimized,

rendering it available for the plants and, thus, resulting in improved

physiological, biochemical and other parameters. This is what happened in this

study.

Nitrate reductase (NR) is the rate-limiting enzyme in nitrogen assimilation

and a key enzyme of metabolic regulation. Nitrate reducing power of the plant is

one of the important factors determining the plant growth (Taiz and Zeiger

2006). However, the process of nitrate reduction is directly or indirectly

dependent on the metabolic sensors and/or signal transducers (Campbell

1999). The level of the enzyme is dependent on a number of factors, born within

or outside the plants. One of the major regulatory factors, determining the

activity of NR, is the level of endogenous phytohormones or those added from

outside (Khan et al. 1996). As a result of ISA application, there was significant

improvement in NR activity at both the stages compared with the control in

Experiment 1 (Table 8). The increase in NR activity by ISA might be related to

the increase in the uptake of nitrogen (Tables 14) that might have increased the

concentration of nitrate in the leaves to be acted upon by NR. The nitrate

essentially induces functional NR by producing a nitrate sensing protein of

unknown nature that presumably binds with the regulatory region of NR-genes

and the transcript (NR-mRNA) and other regulator proteins involved in

metabolic response (Scheible et al. 1997; Campbell 1999, 2002). In this

connection, the present results are concordant with those obtained by Khan et

al. (2011) regarding opium poppy and by Sarfaraz et al. (2011) regarding fennel.

As observed in Experiments 2 and 3, NR activity was also increased by nitrogen

and phosphorus application applied together with ISA80, respectively (Table 24

and 38). NR catalyzes one of the most controlled reactions in plants, receiving

inputs from light photosynthesis, CO2, oxygen availability and nutrient status at

Chapter 5: Discussion

113

the transcriptional, post-transcriptional and post-translational levels (von

Wiren et al. 2000). Therefore, enriching soil-nutrient status must certainly have

a positive impact on NR activity as observed in this study (Table 24 and 38).

Depletion of soil-applied fertilizer occurs due to their high demand by the crop

after initial stages of growth. The plants at that time need further addition of

fertilizers to compensate growth. Thus, owing to increasing the availability of N

and P for plants by their split application, this study might have resulted in a

greater level of NR activity compared to single dose application of these

nutrients. Increasing rates of fertilizer incorporation have earlier been reported

to increase the activity of NR in several crops (Khan et al. 1996; Wang et al.

2000).

Carbonic anhydrase (CA) is an enzyme that plays diverse roles in

physiological processes such as ion exchange, acid-base balance, carboxylation/

decarboxylation reactions and inorganic carbon diffusion between the cell and

its environment as well as within the cell (Badger and Price 1994; Georgios et al.

2004). RuBisCO, the key CO2 fixing enzyme in plant systems, constitutes

approximately 70 per cent of the total proteins present in the leaves. As a rate

limiting photosynthetic enzyme, it regulates the synthesis of carbon compounds,

which are transported to the sink organs and are subsequently utilised to

sustain growth and development of plants. There are several other reports

regarding the significant effect of ISA application on CA activity in the case of

fennel, mentha, lemongrass and opium poppy (Idrees 2011; Khan et al. 2011;

Sarfaraz et al. 2011). The ability of ISA to promote RuBisCO activity and,

thereby, to increase the yield has been reported by Idrees (2011) for Mentha

arvensis. The overall carbon fixation rate relates to the relative activities of

carboxylase and oxygenase functions of RuBisCO. Therefore, the observed effect

of ISA on CA activity might be a reflection of the combined effect of a number of

underlying factors associated with ISA-mediated stimuli. In fact, the role of ISA

in the activation of RuBisCO and PEP carboxylase has also been documented

(Tomoda et al. 1994; Akimoto et al. 1999; Hien et al. 2000). In the present

investigation, CA activity was improved over the control as a result of nitrogen

(Experiment 2) and phosphorus (Experiment 3) application, together with ISA80

Chapter 5: Discussion

114

(Tables 24 and 39). Such an increase in RuBisCO activity might be expected in

this study as in the presence of optimum nutrient supply, hydration of CO2 is

catalyzed rapidly that, in turn, increases the supply of RuBisCO at the site of its

action, and probably it might be a strong reason behind the improvement in the

rate of photosynthesis in the treated plants as reported by Siddiqui et al. (2008).

As observed in this study (Table 24), an enhanced level of CA activity has earlier

been observed in other plants (Khan et al. 1998; Shah et al. 2007; Siddiqui et al.

2008), using other growth regulators, such as GA3, in combination with graded

levels of nitrogen.

A greater increase in CA activity due to application of split doses of

nitrogen and phosphorus along with ISA80 in comparison to the single-dose

application of these nutrients applied with ISA80 could be as a result of adequate

and timely availability of the nutrients in Experiment 4 (Table 54). It indicates

that the fertilizers, applied in a single full-dose, do not fully replenish the crop

with the required nutrients and, thus, the application of fertilizers in at least two

splits needs to be carried out in order to offsets the crop hidden hunger for the

required nutrients. The observed positive effects of split-fertilization on CA

activity are in accordance with the findings of Khan et al. (2002), Ashraf et al.

(2006) and Siddiqui et al. (2008) in this regard.

Photosynthetic parameters, viz. net photosynthetic rate, stomatal

conductance and internal CO2 concentration, were progressively improved

stage-wise from pre-flowering to flowering stage in Experiment 1 (Tables 10-

12). Compared to the control, application of ISA80 (80 mg L-1 of ISA) enhanced

the net photosynthetic rate both at pre-flowering and flowering stage. Hein et al.

(2000) also observed significant enhancement in net-photosynthesis and CO2

assimilation using depolymerised SA. Seemingly, the value-enhancement of

previously discussed physiological processes like NR and CA activities, might

have ultimately resulted in an increase in the photosynthetic rate and the

related attributes. The results obtained by Luan et al. (2003) are similar to those

recorded in this investigation, showing positive effect of ISA on photosynthetic

attributes regarding Limonium, Lisianthus and Chrysanthemum. Data presented

in Experiments 2 and 3 show the improvement in photosynthetic parameters,

Chapter 5: Discussion

115

when nitrogen and phosphorus were supplied with ISA80 (Tables 25-27 and 40-

42). A number of compounds involved in photosynthesis, such as chlorophylls,

enzymes and coenzymes, being themselves nitrogenous in nature, not only

depend upon this essential nutrient element for their production but also show

a linear increase to increasing quantities of added nitrogen within limits

(Salisbury and Ross 1992, Mohammad et al. 1997, Taiz and Zeiger 2006). The

improved photosynthetic rates, as observed in this study, might be attributed to

a high level of RuBisCO activity and CO2 fixation together with increased cell

expansion as a result of phosphorus application (Rao and Terry 1989). An

increase in phosphorus-mediated stomatal conductance and internal CO2

concentration in this study could be considered as beneficial effects of

phosphorus nutrition on photosynthesis as reported by Garg et al. (2004) in the

case of Vigna aconitifolia. Moreover, the present research indicates that

photosynthetic rate may be limited by nutrient deficiencies (Rodriguez et al.

1998; Marschner 2002), but additional fertilizer supply after initial growth

phase may be more useful in improving the photosynthetic attributes as

observed in Experiment 4 (Tables 55-58).

Application of ISA at 80 mg L-1 (ISA80) increased the content of H2O2 as

compared to the non-treated plants (control) in Experiment 1 (Table 13),

indicating that ISA could induce the formation of H2O2 in plant cells. Association

of increased formation of H2O2 with other plant growth regulators has, in fact,

been reported by other workers. For example, Wallaart et al. (2000), Ferriera

(2007) and Pu et al. (2009) reported an increase in H2O2 content in consequence

of night frost, soil-K deficiency and due to salicylic acid in A. annua. Additionally,

Guo et al. (2010) and Aftab et al. (2011a) noticed enhanced formation of H2O2 in

A. annua plants treated with methyl jasmonate. From these studies, it is quite

clear that plant growth regulators may promote the formation of ROS up to an

extent, which is useful for the plants too. Therefore, speculations can be made

that ISA, being a plant growth promoter, might somehow be involved in

inducing H2O2 formation. In Experiment 2 and 3 (Tables 28 and 43), formation

of additional H2O2 was noticed when nitrogen and phosphorus was applied with

ISA80; however, no direct evidences are available in literature about the increase

Chapter 5: Discussion

116

of ROS by the N and P nutrients. Nonetheless, there are reports in which

antioxidant enzymes have been shown to be positively regulated by nutrient

supply (Nguyen and Niemeyer 2008; Jalloh et al. 2009). As reported (Quereshi et

al. 2005), antioxidant enzymes are generally increased when ROS are generated;

therefore, it can be presumed that nutrient supply may regulate ROS status

inside the cell.

As per Experiment 1 (Tables 14-16), leaf-nitrogen and -phosphorus

content of A. annua plants was enhanced significantly due to ISA application,

while leaf-potassium content remained unaffected. Thus, ISA appears to

facilitate the efficient absorption and utilization of mineral nutrients as also

observed by Darvill (1998) and Idrees (2011). There are limited reports

regarding the effect of ISA on leaf-N, -P and -K contents (Idrees 2011; Sarfaraz et

al. 2011). It was observed that ISA may increase the membrane permeability like

the common plant growth regulators (Wood and Paleg 1972; Crozier and

Turnbull 1984; Al-Wakeel et al. 1995). An increase in membrane permeability

would, in turn, facilitate the absorption and utilization of mineral nutrients and

the transport of assimilates in plants (Ansari 1996; Khan et al. 1998). This

would also contribute toward enhancing the capacity of biomass production by

plants as reflected by the increase in fresh and dry weights of plants in the

present investigation. The data of Experiments 2 and 3 show that soil-applied

nitrogen and phosphorus together with foliar-ISA80 enhanced the leaf-nitrogen

and -phosphorus contents significantly (Tables 29-31 and 44-46). An adequate

supply of mineral nutrients at initial vegetative stage plays a pivotal role in

growth and development of plants. However, the initial nutrient-dose needs to

be supplemented with another dose of nutrients at peak vegetative stage for the

require results. Owing to the split application of N and P fertilizers in this study,

the plants received an adequate supply of nutrients on the one hand; and on the

other, the growth of plants was promoted by foliar sprays of ISA, leading to

healthy vegetative growth and unhindered translocation of nutrients to the

shoot that might be the reason behind the improved growth attributes together

with enhanced leaf-nutrient contents in the N-P-ISA-treated plants in

Experiment 4 (Tables 59-61). These results are in conformity with the findings

Chapter 5: Discussion

117

of Naeem (2007) and Singh (2008) in the case of coffee senna and ginger,

respectively.

5.3. Yield and quality parameters

The data on yield and quality parameters, viz. dry-leaf yield, artemisinin content

and artemisinin yield were significantly improved by the doses of N and P

nutrients and ISA concentrations (Experiments 1-4; Figs. 9-24).

Dry-leaf yield, calculated at flowering stage, was positively regulated by

different ISA treatments in Experiment 1 (Fig. 9). As mentioned earlier, the

biologically active oligosaccharides, derived from depolymerization of sodium

alginate, may prove as signal molecules in order to regulate the growth,

development and defense reactions in plants, following the expression of genes

concerned (Albersheim and Darvill 1985). As a result, the biological properties

of the plant cells may get stimulated (El-Rehim, 2006), leading to overall

improvement in the agricultural performance of the crop. In the present

investigation, such an improvement in growth might have manifested in

significant promotion of yield and quality of the crop (Experiment 1; Figure 9-

12). Moreover, the absorption of nutrients was increased in ISA-treated plants

ensuring better nutrient status of plants (Tables 14 and 15), which might have

manifested in the efficient nutrient assimilation and improved rate of

photosynthesis, leading to enhancement in growth, yield and quality of crop. As

evident by the work of various researchers, the ISA-mediated enhancement in

values of yield attributes might be ascribed to the positive role of ISA in plant

growth and development in general (Hien et al. 2000; Tham et al. 2001; Kume et

al. 2002; Luan et al. 2003; Luan et al. 2005; Kume 2006; Idrees (2011); Khan et

al. (2011); Sarfaraz et al. 2011).

In accordance with Experiments 2 and 3, the application of nitrogen and

phosphorus, together with that of ISA80 sprays, increased the dry-leaf yield

significantly (Figs. 13 and 17). Increase in dry matter production of A. annua has

also been reported by Singh (2000), Özgüven et al (2008), Davies et al. (2009)

and Jha et al. (2010), using fertilizer dressings. As nitrogen application is known

to increase the levels of cytokinin, leading to cell wall extensibility (Taiz and

Zeiger 2006), it is logical to speculate that nitrogen could be involved directly or

Chapter 5: Discussion

118

indirectly in the enlargement and division of cells and production of new tissues

which, in turn, might have been responsible for improvement in the biomass

yield (Fig. 13). In addition, improved yield due to phosphorus supply may be

attributed to the assured availability and continuous utilization of phosphorus

in the synthesis of carbon skeleton, amino acids and energy rich molecules such

as ATP.

According to Experiment 1 (Figs. 10 and 11), the total content and yield of

artemisinin, recorded at flowering stage, increased with increasing

concentrations of ISA up to 80 mg L-1. Idrees et al. (2011) and Khan et al. (2011)

observed an ISA-mediated improvement in the content and yield of alkaloid in

Catharanthus and Papaver, respectively. It appears that ISA promoted the

artemisinin synthesis by recuperating the level of H2O2, which converts

dihydroartemisinic acid into artemisinin during artemisinin biosynthesis (Pu et

al. 2009; Guo et al. 2010). As per suggestion given by Wallaart et al. (1999),

dihydroartemisinic acid might act as a scavenger of ROS (H2O2 in this case) that

are released in plant cells due to various reasons. During the biochemical

reaction, dihydroartemisinic acid hydroperoxide (DHAA-OOH) is generated that

gets converted to artemisinin (Fig. 25). In this regard, the present results are in

agreement with the observations recorded by Pu et al. (2009), Wallaart et al.

(2000) and Ferreira (2007), who advocated that relatively high levels of ROS

might be generated as a result of K-deficiency in the soil, foliar application of

salicylic acid and occurrence of night frost that, in turn, could result in a greater

conversion of dihydroartemisinic acid to artemisinin. In fact, there has been

noticed a positive relationship between H2O2 and artemisinin contents by Aftab

et al. (2010c and 2011a) in the net-house conditions. The plant may adopt its

metabolic pathway in response to nutrient application. Therefore, nitrogen and

phosphorus might have acted upon the co-regulated compounds, like

artemisinic acid and dihydroartemisinic acid, which may be considered as

promising targets for artemisinin biosynthesis. Since artemisinin was estimated

in dry leaves, the increase in dry-leaf yield and the improvement in artemisinin

content might have resulted into much higher yield of artemisinin compared to

control. Singh (2000), Kapoor et al. (2007), Özgüven et al (2008) and Davies et

Chapter 5: Discussion

119

Fig. 25. Summary of artemisinin biosynthesis in Artemisia annua L. (Olofsson et al. 2011) Cytosol: AACT: acetoacetyl-CoA thiolase; ADS: amorpha-4,11-diene synthase; ALDH1: aldehyde dehydrogenase 1; BAS: β-amyrin synthase; BFS: β-farnesene synthase; CPR: cytochrome P450 reductase; CPS: β-caryophyllene synthase; CYP71AV1: amorphadiene-12-hydroxylase; DBR2: artemisinic aldehyde Δ11(13) reductase; ECS: epi-cedrol synthase; FDS: farnesyl diphosphate synthase; GAS: germacrene A synthase; HMGR: 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; HMGS: 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; IDI: isopentenyl diphosphate isomerase; MVK: mevalonate kinase; PMD: diphosphomevalonate decarboxylase; PMK: phosphomevalonate kinase; RED1: dihydroartemisinic aldehyde reductase; SMO: squalene monooxygenase; SQS: squalene synthase. Plastid: BPS: β-pinene synthase; CMK: 4-cytidine 5’-diphospho-2-C-methyl D-erythritol kinase; DXR: 1-deoxy-Dxylulose-5-phosphate reductoisomerase; DXS: 1-deoxy-D-xylulose-5-phosphate synthase; GGDS: geranylgeranyl diphosphate synthase; GDS: geranyl diphosphate synthase; HDR: hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HDS; hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; IDI: isopentenyl diphosphate isomerase; LS: linalool synthase; MCT: 2-C-methyl-D-erythritol-4-(cytidyl-5-diphosphate) transferase; MCS: 2-C-methyl-D-erythritol-2,4 cyclodiphosphate synthase.

Chapter 5: Discussion

120

al. (2009, 2011) are some of workers, who reported increased content and yield

of artemisinin by the application of nitrogen and phosphorus.

5.4. Conclusions

The following meticulous information emerged from this field-study on

Artemisia annua L. conducted at Aligarh, Western Uttar Pradesh, might be

claimed as the first report in the scientific literature regarding the effect of foliar

sprays of ISA applied alone or in combination with nitrogen and phosphorus:

(i) The optimum concentration of irradiated sodium alginate (ISA) was 80 mg

L-1 (ISA80), the application of which enhanced the growth, photosynthetic

rate, activities of enzymes and content as well as yield of artemisinin in

Artemisia annua plants (Experiments 1).

(ii) The optimum soil-applied dose of nitrogen was 80 kg ha-1, which gave the

best results when applied with foliar ISA80 in terms of growth,

photosynthetic rate, activities of enzymes and content as well as yield of

artemisinin in Artemisia annua plants (Experiments 2).

(iii) The optimum soil-applied dose of phosphorus was 40 kg ha-1, which gave

the best results when applied with foliar ISA80 in terms of growth,

photosynthetic rate, activities of enzymes and content as well as yield of

artemisinin in Artemisia annua plants (Experiments 3).

(iv) Split dressing of nitrogen (N40+40) and phosphorus (P20+20) applied in

combination with foliar ISA80 (i.e. ISA80 + N40+40 + P20+20) proved superior to

all other ISA-N-P treatments, including that comprised of single dose of

nitrogen (N80) and phosphorus (P40) applied in combination with foliar ISA80

(i.e. ISA80 + N80 + P40) (Experiment 4).

Conclusively, ISA80 + N40+40 + P20+20 proved the best treatment combination

for growth, metabolism, yield and quality of field-grown Artemisia annua crop.

Therefore, it may be recommended for the field-cultivation of Artemisia annua in

agro-climatic conditions of Aligarh, Western Uttar Pradesh.

In this investigation, the effect of foliar application of ISA applied alone or

in combination with soil-applied nitrogen and phosphorus fertilizers has been

studied for the first time in order to improve growth, metabolism, yield and

Chapter 5: Discussion

121

quality of A. annua in field conditions. Since malaria is the major problem in

tropical and sub-tropical countries, the detailed study on the plant having

antimalarial activity (Artemisia annua L.) is quite justified.

The author wishes to claim, with all the modesty at his command, that he

has been able to enrich the scientific literature on the effects of ISA alone or that

of ISA in combination with soil-applied nitrogen and phosphorus fertilizers on

Artemisia annua, which is a high-value medicinal plant, having antimalarial

activity.

5.5. Proposal of future work

As evident from the present study, irradiated sodium alginate (ISA) alone and in

combination with nitrogen and phosphorus can stimulate growth,

photosynthetic efficiency, enzyme activities and, most importantly, the content

and yield of artemisinin in Artemisia annua plants. However, the exact

mechanism as to how the ISA acts to improve growth and development of plants

is still unclear; therefore, unfolding the underlying molecular and other

mechanisms, could be the research work to be carried out in future in this

regard.