**5.1 Role of endophytes in the production of podophyllotoxin**

Endophytes are the group of microorganisms that are found to colonize the interior of the plant irrespective of the type of association they have to maintain with the host. Although this group includes a number of species of microbes, it is less explored. They have great potential of application in agriculture, bioremediation, medicine, etc. [38]. A study reported the isolation of an endophytic fungus, *Fusarium solani*, from the roots of *P. hexandrum*, which was found to synthesize podophyllotoxin at a rate of 29.0 μg/g on a dry weight basis [39]. The results were confirmed by HPLC and mass spectroscopy techniques. Upon isolation of the fungal hyphae from the roots of *P. hexandrum*, selection of the fungal species was done by assessing the presence of podophyllotoxin in the three strains that had been isolated, after which the rDNA analysis confirmed the fungus under study to be *Fusarium solani*. Shake flask experiments were performed, the results of which indicated that maximum biomass production was obtained on the 10th day of growth while the maximum yield in podophyllotoxin was found around the 8th day of growth.

#### **5.2 Identification of key transcription factors involved in regulation of biosynthetic pathway for podophyllotoxin production in** *P. hexandrum*

Although the exact pathway of biosynthesis of podophyllotoxin is still not known, information about the regulatory components of this pathway is also unavailable. Considering the importance of transcription factors and their role in upgrading the industrial synthesis of podophyllotoxin, the present study worked to identify different transcription factors that might be involved in regulating the pathway of podophyllotoxin biosynthesis [40]. The study worked to identify various classes of transcription factors via the mining of transcriptomes of *Podophyllum* species and validation of these factors by qRT-PCR analysis coupled with the analysis of podophyllotoxin content from the different tissues of *P. hexandrum*. Extensive survey of available literature revealed that four transcription families (TFs), basic leucine zipper (bZIP), myeloblastosis (MYB), WRKY, and basic helix-loop-helix (bHLH) were involved in the regulation of phenylpropanoid pathway in several species of plants. It was therefore hypothesized that these TFs might also be involved in the regulation of biosynthesis of podophyllotoxin in the species of *Podophyllum*. Two distinctive transcripts were identified which encoded for bZIP and MYB TFs in the rhizomes of *P. hexandrum* and which were associated with podophyllotoxin content. Upon quantifying the content of podophyllotoxin and analyzing the comparative expression between the high (2.51%) versus the low (0.59) content of podophyllotoxin accessions, the results showed a 0.04- to ~16-fold increase in the transcripts of the transcription factors, further supporting the involvement of the identified TFs with the content of podophyllotoxin. For *P. hexandrum*, the highest transcript abundance was observed for bZIP (19.60-fold) in the rhizome showing 2.51% of podophyllotoxin as compared to the shoots which showed only 0.01% of the resin. In silico analysis of putative promoter regions of the genes associated with this pathway in other species of plants have shown the presence of certain sequence elements for MYB and WRKY TFs, which suggested their involvement in regulating the production of podophyllotoxin. Abundance of the transcript was evaluated with respect to the transcription families using fragments per kilobase of transcripts per million mapped reads (FPKM) and qRT-PCRbased transcript by in silico techniques. The values of TFs FPKM (fragments per kilobase of transcripts per million mapped reads) ranged between 0.0014–12.01 and 0.014–1162.01 in the transcriptomes correlating with shoots and rhizomes of *P. hexandrum* and *P. peltatum*, respectively. Gene expression pattern was observed through two different platforms and the results were in synchrony with each [31].

### **6. Conclusion**

From the study, it can be concluded that growth of *Podophyllum hexandrum* is difficult in vitro, but the plant can be grown successfully by somatic embryogenesis provided that media and other culture conditions are optimized in a stepwise manner. The traditional culturing techniques do not tend to increase the podophyllotoxin production, whereas its production can be increased by coculturing of hairy roots of the plant along with *L. flavum*. This process is called is precursor feeding while molecular techniques such as genetic engineering of the plant with the help of *Propagation of* Podophyllum hexandrum *Royale to Enhance Production of Podophyllotoxin DOI: http://dx.doi.org/10.5772/intechopen.93704*

*Agrobacterium tumefaciens* have also showed an increase in podophyllotoxin production. The other methods to increase metabolite production include the growth of hairy root culture and spraying of methyl jasmonate in the plants under greenhouse conditions. There are certain gaps which have not been taken care of like there is very less literature available showing the growth of *Podophyllum hexandrum* using micropropogation techniques. The genetic data about the plant's genes involved in metabolite production are not easily available. Use of bioreactors for the growth and production of podophyllotoxin has not yet been evaluated properly. There is further in-depth study required to be done on *Podophyllum hexandrum* before we can begin the large-scale podophyllotoxin production from the plant.

The formation of State Medicinal Plant Board ensures increased cultivation and conservation of the medicinally important endangered plants by providing appropriate funding to conserve these species [41]. Although the efforts of agrotechnological innovation and biotechnological processes cannot be undermined, only a few of these endemic plant species are under cultivation majorly by the efforts of local farmers and NGOs [3].

## **Acknowledgements**

The authors would like to acknowledge the support and constant guidance of Dr. Hemant Sood whose invaluable input to this review chapter has made it worth a read. The authors would also like to thank the Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, for providing them with the opportunity to work on this project.

The authors also wish to acknowledge the support of Mr. Priyansh Srivastava for the help that he extended during the writing of this chapter.

#### **Conflict of interest**

The authors declare that they have no conflict of interest whatsoever.

#### **Author details**

Utkarsha Srivastava\* and Hemant Sood Jaypee University of Information Technology, Solan, Himachal Pradesh, India

\*Address all correspondence to: utkarsha0587@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
