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1 Tissue Culture ofMedicinal Plants

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1.4 Tissue Culturing ofVarious Medicinal Plants

Different plants have different requirements when they are cultured in vitro. Concentration of plant growth hormones and essential additional components vary from species to species.

1.4.1 Micropropagation ofNeem (Azadirachta indica L.)

A. indica is one of the major therapeutic plants that contains a plethora of compounds with therapeutic uses. Neem also has anticancerous, anti-tumor, and antiinammatory effects. Many researchers have investigated the antioxidant and anti-cancerous properties of neem. Cervical cancer patients, when treated with neem, showed apoptosis of cancerous cells due to the anti-oxidant properties of neem. The enhanced activities of caspase 3, 8, and 9, IFN-γ levels and a reduction in TNF-α in monocytes were observed in these patients [46]. Azadirachtin is a secondary metabolite with a complex structure found in neem seeds. It is a valuable pesticide and anticancerous compound[47]. Table1.2 shows compounds present in neem [48]. Meliatetraolenone is a new tetranortriterpenoid compound separated

Table 1.2 Medicinal properties of neem plant

Active component Activity Effects Reference

Azadirachtin Anti-oxidant Activity in cervical cancer

Azadirachtin Salannin deacetylgedunin

Azadirachtin Antibacterial Against Staphylococcus

Nimbolide Cytotoxic effect On broblasts [63] Nimbolide Anti-tumor effects Against human

Gedunin Anti-breast-cancer activity Inhibition of a protein Hsp90 [65] Azadirachtin and

nimbolide Nimbolide

azadirachtin Gedunin Anticancer activity against

Quercetin Anti-inammatory and

Quercetin Antimetastatic agent Effective in cervical cancer [70] Quercetin Diminished multiplication of

Insecticide Can kill the larvae of A.

Cytotoxic activity Decreased HeLa cell viability [66]

Anticancerous effect Induction of cell apoptosis [67]

ovarian, colon, and prostate cancer cells

anti-cancerous

ovarian cancer cells

patients

stephensi

aureus and MRSA

choriocarcinoma

Intervention with principal signaling pathways

Represses the viability of HeLa cells in dose dependent manner

Increased action of caspase-3 and caspase-9

[46]

[61]

[62]

[64]

[50, 68]

[69]

[115,

116]

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I. Fatima etal.

•Antifeedant

•Pesticide

•Anticancer

•Antibacterial

Azadirachtin

Meliatetraolenone

Nimbolide

Quercetin

Insecticidal

Fig. 1.15 Pharmaceutically important compounds of neem with their activities

Anticancer

from the fresh neem leaves. It shows insecticidal action [49]. Gedunin, an extract of the neem plant, shows antiproliferative activity that has been veried by many researchers [50–52]. The neem leaf extract is also advantageous in the antiproliferation of breast cancer cell lines [47, 53–56]. Figure1.15 diagram shows the effects of different components of neem extract.

The micropropagation of neem shoot tips is more likely to develop into a callus. The presence of cotyledons affects the developmental stage [57]. The highest concentration for shoot elongation was observed with 0.5µMBAP for young calli [58]. Quraishi and colleagues used the crown and basal-sprout explants. They controlled the leaching of phenol growth inhibitors by supplementing the nutrient medium with 12.5μM PVP-40. They found that Driver Kuniyuki Walnut (DWK) medium supplemented with 0.22μM benzyl adenine was better than the MS medium for shoot propagation. 100% of basal-sprout and seedling explants grew in half-strength DWK medium containing 4.9μM IBA and root formation. Plantlets developed from both explant types showed a 90% survival rate after acclimatization [59]. When the immature ower was used as an explant for neem micropropagation, the best callus formation response was observed in the M9 medium (MS medium containing 3.0% sucrose, 1.0mg/L 2,4-D, 1.0mg/L BAP and 0.2mg/L NAA). Researchers reported that the extent of callus formation was improved when the same explants were rst cultured on MS medium containing 2,4dichlorophenoxyacetic acid (2,4-D)

1.0mg/L, BAP1.0mg/L and 1Naphthaleneacetic acid (NAA) (0.2mg/L) along with 10% sucrose for 15days and then sub-cultured on a same medium supplemented with 3.0% sucrose [60].

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1.4.2 Tissue Culturing ofPinus roxburghii Sarg

Pinus roxburghii Sarg. (common name Chir Pine) is also a pharmaceutically important plant found in the Himalayan region in Kashmir, Tibet, Bhutan, Sikkim, Nepal, and North India. Extract of P. roxburghii Sarg. shows high analgesic activities and is reported to inhibit carrageenan-induced edema in mice by inhibiting cyclooxygenase synthesis. Thus it can act as a nonsteroidal anti-inammatory drug like indomethacin because polyphenolic compounds, bioavonoids, quercetin, and rutin are present in the extract [71] (Fig.1.16). Various chemical constituents that are present in turpentine oil of P. roxburghii Sarg. (Fig.1.17) include α-pinene, β ene, turpine longifolene hydrocarbons (d- and l-pinene), resin acids, camphene, fenchene, dipentene, and polymeric terpenes [72].

The maximum number and length of shoots was shown by the medium supplemented with 10μM BAP at 5.8 pH.BAP(10 μM) showed the best axillary bud induction and proliferation as compared to Kn and Bisphenol A (BPA) with the addition of0.5 and 0.25μM α-NAA, respectively. Best rooting hormone for this plant was α-NAA at 2.5μM [73].

anti-inflammatory

Pinus roxburghii

Sarg

analgesic

carrageenan-induced

-pinene, car-3-

inhibited

edema in mice

useful in eye, ear,

and pharynx

diseases

Fig. 1.16 Medicinal properties of Pinus roxburghii Sarg

α-pinene, β-pinene

car-3-ene

camphene

Fig. 1.17 Showing chief chemical components of Pinus roxburghii Sarg

dipenten

chief chemical

constituents of

turpentine oil from

Pinus roxburghii Sarg

fenchene

polymeric terpenes

longifolene

hydrocarbons (d- and

l-pinene)

resin acids

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I. Fatima etal.

1.4.3 Tissue Culture ofZiziphora tenuior

Ziziphora tenuior L. belongs to family Lamiaceae is an annual scented herb whose extract shows antifungal and antibacterial properties. Pulegone is the main component of its volatile oil. In addition, Z. tenuior has been used to cure fever, dysentery, diarrhea, gut inammation and cough [74–77]. For micropropagation of Z. tenuior, the best concentration for callus initiation from leaf explants was 0.5 mg/L NAA.The maximum shoot number was obtained at 2mg/L BAP.Moreover, 1mg/L Kin and 0.1mg/L α-NAA provided the best shoot multiplication and length [78].

1.4.4 Micropropagation ofAjuga bracteosa

It is a healing herb for the treatment of gout, rheumatism, palsy, and amenorrhoea. A. bracteosa is used to treat headaches, pimples, measles, stomach acidity, burns, boils, jaundice, hypertension, coughand sore throat by the local population [79]. Researchers reported anticancerous and anti-inammatory properties of this herb. The leavesof A. bracteosa are reported to have anti-malarial properties. Thus,can also be used as a substitute for quinine [80, 81]. Kuria and colleagues reported that two components of this herb (ajugarin-1 and ergosterol-5,8-endoperoxide) have antibiotic properties in contradiction of chloroquine-sensitive Plasmodium falci- parum [80].

Fast callusing of A. bracteosa was obtained on MS medium supplemented with IAA (2mg/L) and BA (5mg/L) after 10days of culture. Medium supplemented with IAA 2mg/L and BA 5mg/L encouraged the maximum number of shoots. Best well-differentiated roots were obtained on the medium having 0.5mg/L IBA [20].

1.4.5 Tissue Culture ofPongamia pinnata

P. pinnataplant contains various derivatives of avonoids such as avans, avones, chalcones, and other compounds, including steroids, terpenes, and fatty acids. This plant shows antimicrobial, antioxidant, anti-diabetic, and anti-inammatory activities. Extracts of this plantare reported for low toxicity towards mammalian cells, therefore, can be used as a potential medicinal agent [82]. Tan and colleagues used Woody Plant Medium [83] as a basal medium solidied with 0.28% (w/v) Gelrite at

5.7 pH.Maximum shoot formation was shown in the medium having thidiazuron (TDZ). Longer shoots of P. pinnata were obtained when supplemented with 2IP, zeatin and BAP.The highest multiple shoot bud initiation was reached using Woody Plant Medium at 5μM TDZ.The highest frequency of rooting in plants was observed with Woody Plant Medium encompassing 20μM Indole-3-Butyric Acid (IBA) and 200μM silver thiosulphate (STS) [84].

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1.4.6 Tissue Culture Linum usitatissimum

Table 1.3 shows the therapeutic effects of L. usitatissimum.

The highest root induction from hypocotyl explants of L. usitatissimum is reported when incubated in MS medium covering a combination of 0.5 mg/L TDZ+0.5mg/L kinetin. TDZ (0.5mg/L) alone is effective in producing maximum shoot induction. Success in shoot induction (69%) from nodal explants on medium contained 2mg/L BAP along with 0.5mg/L NAA is recorded [85]. Another study showed that TDZ was better for shoot regeneration than other plant growth regulators [86].

Stem explants of L. usitatissimum cultured in a medium holding 2.0mg/L TDZ combined with 0.1mg/L NAA resulted in considerable quantity of shoot regeneration [87]. Medium encompassing 2,4-D (2mg/L) and BAP 1mg/L exhibited a considerable shoot regeneration [88]. For rooting, IBA (0.1mg/mL) with half strength MS medium showed the best results [89]. Anjum and colleagues observed that the callus cultures established from the leaf explant grown on TDZ (2.0mg/L) showed the highest antioxidant activity [90]. A higher amount of pharmacologically active lignan secoisolariciresinol was observed in cultures grown in a medium containing TDZ+Kin at 0.5mg/L for each [89].

1.4.7 Micropropagation ofMountain Mulberry

Mulberry is a valuable tree with many applications in the pharmaceutical, food, and construction industries [97]. Mulberry silkworms (Bombyx mori) feed on these plants. In traditional medicine, products of the mulberry plant are used to treat

Table 1.3 Therapeutic effects of Linum usitatissimum

Class of compound Example Therapeutic effects References

Lignans Secoisolariciresinol

Other activity Phytoestrogens Regulate estrogen level in the

Polyunsaturated fatty acids

Water and Fat-soluble vitamins

Diglucoside (SDG) Matairesinol (MAT)

Secoisolariciresinol (SECO)

Lariciresinol diglucoside (LDG)

Omega-6-fatty acid Omega-3-fatty acid linoleic acid

Vitamin E Treatment of cardiovascular

Treatement of breast, colon, and prostate cancer

human body Treatment of eart diseases, stroke,

high blood pressure, type-2 diabetes, Alzheimer’s disease, and rheumatoid arthritis

diseases, Alzheimer’s disease

[91, 92]

[93, 89]

[94]

[95, 96]

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ailments and diseases like throat inammations, dysentery, helminthiasis, constipation, and diabetes [97, 98].

Dubey and colleagues found that the rate of bud break and longest shoot development was maximum in MS medium having BAP (1.0mg/L) and NAA (0.5mg/L). Maximum shoot multiplication was reported in the medium supplemented with BAP (1.0mg/L), NAA (0.5mg/L) and 10% coconut water. For root formation, better results were obtained when 20% activated charcoal was added to half-strength MS medium with the highest mean number of roots [99].

I. Fatima etal.

1.4.8 Micropropagation ofHoslundia opposita Vahl

This plant is effective against parasites like ticks, a common problem in Ghana [100]. The active component extracted from the leaves of this plant is ursolic acid, a triterpene. The crude methanolic extract exhibits high anti-larval action, followed by the ethyl acetate segment. For Hoslundia oppsita, best callus induction was observed in MS medium containing 30g/L sucrose, 4.4μM BA solidied with 0.2% gelrite [101].

1.4.9 Micropropagation ofAloe species

Aloe peglerae, a endangered Aloe specie [102] is used in the treatment of infections, blisters, wound curing and laxative [103, 104]. Figure1.18 shows different uses of Aloe sp. [105–108].

For Aloe vera, the highest shooting formation was observed with 0.5mg/L BAP along with 0.5 mg/L NAA [109]. To obtain more shoots, a mixture of 4.0mg/L BAP and 0.2 mg/L NAA showed the best results in A. vera. Different genotypes respond differently to in vitro culture [110, 111]. For Aloe trichosantha, best callus induction was reported when 0.5mg/L BAP and 0.5 mg/L NAA were used. For A. percrassa Todaro, 0.20mg/L BAP and 0.20mg/L NAA were effective [112]. Other researchers found 0.10BAP and 0.50mg/L NAA for the best callus induction in A. vera explants [113, 114]. Maximum shoot numbers were observed in medium combined with 2.0mg/L of BAP.Rooting with the better number was only observed in MS medium at 0.25mg/L to 1.5mg/L NAA (Hailu etal. 2020). For Aloe peglerae, the highest number of shoots were observed in MS medium with 2.5 μM mTR.Hlatshwayo and colleagues ranked the effectiveness of cytokinins in shoot proliferation as follows mTR>mT>Kinetin>BA [117]. For Aloespecies,thebest shooting response was reported in the medium enriched with 1.0 mg/L BAP+0.5mg/L NAA.Enhanced rooting was observed with IBA at greater concentrations (1.0 and 1.5mg/L), but similar results were also obtained with lesser concentrations (0.5 and 1.0mg/L) of NAA [118].

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enhancing

blood flow

anesthetizing

tissues

antifungal,

antiviral, and

antibacterial

activity

healing

wounds and

burns

anti-

inflammatory

Uses of

Aloe gels

& latexes

anti-

helminthic

antiaging

anti-

protozoal

Fig. 1.18 Therapeutic properties of Aloe vera

1.5 Conclusion

The demand for therapeutic compounds derived from plants has increased with an escalating human population. This demand cannot be fullled by growing whole plants owing to the challenges associated with the growth, space, and time requirements. Micropropagation is a reasonable solution to this problem.Micropropagation techniques not only help to overcome space and time issues related to plant growth but alsooffer bulk production of required metabolites quickly.

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I. Fatima etal.

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I. Fatima etal.

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