Breeding of medicinal solanum and phylanthus amarus.souvick.ppt

Breeding of medicinal solanum and phylanthus amarus

1. BREEDING OF MEDICINAL SOLANUM AND PHYLANTHUS AMARUS Presented By: SOUVICK BANIK 2ND SEMESTER Course Name : BREEDING OF MEDICINAL AND AROMATIC CROPS COURSE NO: PSMA-553 Department of Spices and Plantation Crops FACULTY OF HORTICULTURE Bidhan Chandra Krishi Viswavidyalaya

2. Importance of Medicinal Plants Medicinal plants are the most important source of life saving drugs for the majority of the world’s population. Medicinal plants are those plants which are rich in secondary -metabolites and arc potential source of drugs. These secondary metabolites includes alkaloids, glycosides, coumarins, flavonoids, steroids etc. Medicinal and Aromatic plants form a numerically large group of economically important plants which provide basic raw materials for medicines, perfumes, flavors and cosmetics. These plants and their products not only serve as valuable source of income for small holders and entrepreneurs but also help the country to earn valuable foreign exchange by way of export. Role of MAP in Modern World : According to WHO, 80 per cent of people still rely mainly on traditional remedies such as herbs for their medicines. Income generating role by which collection and cultivation of MAP . MAP playing a key role as natural and alternative to conventional drug and food . MAP have Lesser side effect and it can be safely used in drug, food and cosmetics . Use as phytogenic feed additives . SOURCE :-1. WHO monographs on selected medicinal plants. Vol. 3., 1993 2. Role of Biotechnology in Medicinal Plants. 2003; 2 (2)

3. MEDICINAL SOLANUM ( Solanum nigrum L . ) Family: Solanaceae The plant Solanum nigrum Linn (Solanaceae) commonly called as black night shade in English, Makoi in Hindi, Kachchipandu in Telugu, Munatakali in Tamil, Piludi in Gujarati & Kamuni in Marathi. It is an erect, divaricately branched, unarmed, suffrutescent annual herb. Leaves ovate or oblong, sinuate-toothed or lobed, glabrous ; flowers 3-8 in extra- axcillary drooping subumbellate cymes; fruits purplish black or reddish berries; seeds many, discoid, yellow, minutely. Fig :- Whole plant of Solanum nigrum Fig :- Botanical view of Medicinal solanum

4. ` The main chemical components of citronella oil are solasodin , solasonine , sapogenin , diosgenin, Tigogenin, solanadine , solarmargine , uttrinine - A and uttroside -A . Source : https://www.cropscience.bayer.com/en/crop-compendium/pests-diseases-weeds/weeds/solanum-nigrum

5. USES The leaves are used for joint pain ( muscle pain ), skin diseases, used in the treatment of anti tuberculosis and are said to produce Diaphoresis. Leaves are also used in dropsy, nausea and nervous disorders . The decoction of the berries and flowers are useful in cough, erysipelas (specific, acute, cutaneous inflammatory disease caused by a haemolytic streptococcus and are characterized by red hot). These are remedy for pulmonary tuberclosis and Bronchitis, diuretic. The juice of the berries used as an anti-diarrhoea, opthalmopathy and hydrophobia. It is also used in anasarca and heart disease. Berries are used to posses tonic, diuretic and cathartic properties. Seeds are useful in giddiness and dipsia . They are also useful in inflammations and skin diseases. The roots are useful in otopathy , ophthalmopathy, rhinopathy and hepatitis. Ghosh et. al,2011 reported that the whole plant used as antiseptic, anti-inflammatory , expectorant, cardiotonic, digestive , diuretic, laxative, diaphoretic, sedative, swelling, cough , asthma The plant is also effective in curing cardiopathy, leprosy, haemorrhoids, nephropathy, ophthalmopathy, dropsy and general debility. Decoction of the plant depresses the CNS and reflexes of the spinal cord . References : Ghosh, N; Ghosh, R; Mandal, V; Mandal, SC (Sep 2011). "Recent advances in herbal medicine for treatment of liver diseases". Pharmaceutical biology . 49 (9): 970–88.

6. Breeding Objectives To increase content of solasodine , solasonin , α and β- solanigrine etc. Resistance to major diseases like Powdery Mildew and Coller Rot . Adaptability to various agro -climatic situations Improves response to added nutrients also require emphasis. Resistant to different pest but the plant is sufficiently hardy to resist pest . Fig :-Black nightshade berries

7. Solanum nigrum (2n = 24), belonging to the family Solanaceae. For purposes of hybridization flower buds were emasculated two days prior to their opening by removing the anthers only. All the plants used in crosses were grown in insect proof green house and the flowers were protected with butter paper bags before and after pollination. Pollen was collected on to a clean sterilised slide by tapping the flowers and it was applied by gently rubbing the stigma against the pollen surface of the slide. Hybridization: Source : https://www.cropscience.bayer.com/en/crop-compendium/pests-diseases-weeds/weeds/solanum-nigrum

8. In vitro Clonal Propagation of black nightshade: Fresh shoot segments of black nightshade were used as explants to establish cultures in vitro. These explants were surface sterilized with 0.1% HgCl2 for 3-4 min . These were washed with autoclaved distilled water for 5-6 times , followed by washing with antioxidant solution (ascorbic acid (0.1%) + citric acid (0.05%) for 10-15 min. Surface sterilized explants were in inoculated on MS (Chopra et. al.,1986) media with various concentrations of cytokinins (BAP and Kinetin). The initiated shoots were multiplied on various concentrations and combinations of PGRs. The in vitro cloned shoots were rooted in vitro on various concentrations of IBA and NAA (Naphthalene acetic acid). The nodal shoot segments from unpruned plants showed less response (40-45%), whereas explants from pruned plants showed 100% response on MS + BAP (3μM) with 5-6 shoots initiated per node. Excessive defoliation and yellowing of shoots occurred on MS + BAP (1 μΜ ) + Kin (0.5 μM ) + IAA (0.5μM). The combinations of BAP (2μM) + Kin (1.5μM) proved to be supportive for shoot multiplication. BAP (2μM) was significant for shoot multiplication, as 8-10 shoots were regenerated from each clump. Same effect of cytokinins have been observed in Gymnema (Sharma et. al., 2010) and Leptadenia (Rathore and Shekhawat (2010)) . The in vitro regenerated shoots rooted in vitro with a response of 40-45%. Rooting was observed on IBA (3μM). All the micropropagated shoots rooted on ½ strength of MS media with 10μM IBA. Further investigation will be carried out for ex vitro rooting, hardening and acclimatization. Source : 1.Rathore, M. S. and Shekhawat , N. S. (2010) Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn -An endangered plant. Environmental and Experimental Botany. 2. Sharma, B. and Bansal, Y. K. (2010) In vitro propagation of Gymnema sylvestre Retz. R.Br through apical bud culture. Journal of Medicinal Plants Research. 4 (14): 1473-1476.

9. Salient Features and Achievements : Improved Varieties are RRL-20-2, RRL-SL-6, Glaxo, Arka Sanjivini and Arka Mahima suitable for cultivation . Among them , Arka Sanjivani :- It is diploid variety released by Indian Institute of Horticulture, Bangalore for cultivation . Arka mahima : - It is a tetraploid variety which is generally released by IIHR, Bangalore for Cultivation .

10. PHYLANTHUS AMARUS Family : Phyllanthaceae or Euphorbiaceae Botanical name : Phylanthus amarus L. Erect herbs to 30 cm tall. Leaves 6-8 x 3-4 mm, oblong, base unequal sided, apex obtuse to acute, lower surface glaucous; stipules lanceolate, scarious . Male flowers towards tip of branchlets, solitary, axillary; tepals 5, ovate; stamens 3, exserted ; filaments connate; disc of 5 glands. Female flowers c. 1.5 mm across; tepals 5, oblong; ovary globose; style erect, recurved; pedicel to 2 mm long. Capsule are 2 mm across, globose. Seeds 6, trigonous , vertically muriculate . Fig : Phyllanthus amarus   Economic part: Whole plant

11. Genus: Phyllanthus Family: Phyllanthaceae or Euphorbiaceae Subfamily: Phyllanthoideae Tribe: Phyllantheae Binomial name Phylanthus amarus L.

12. Phyllanthin (0.4-0.5%) Hypophyllanthin L interalin 5 - Demethoxyniranthin D emethilenodioxyniranthin N iranthin N irtetralin H inokinin

13. USES Phyllanthus amarus Schum. & Thonn . belongs to the family Euphorbiaceae is a small herb well known for its medicinal properties and widely used worldwide. P. amarus is an important plant of Indian Ayurvedic system of medicine which is used in the problems of stomach, genitourinary system, liver, kidney and spleen. It is bitter, astringent, stomachic, diuretic, febrifuge and antiseptic. The whole plant is used in gonorrhea , menorrhagia and other genital affections. It is useful in gastropathy, diarrhoea, dysentery, intermittent fevers, ophthalmopathy, scabies, ulcers and wounds. Reference : Chien , CF; Wu, YT; Tsai, TH (Jan 2011). "Biological analysis of herbal medicines used for the treatment of liver diseases". Biomedical Chromatography. 25 (1-2): 21–38

14. Breeding Objectives: 1) Breeding for better quality and high yield as per bio-active chemical compounds . e.g. CIM –Jeevan Phyllanthin ranging between 0.70-0.77% in dry herb, produces hypophyllanthin ranging between 0.32-0.37% in dry herb 2) Developed cultivars resistance to diseases. e.g. Powdery Mildew during it growth period . 3) Breeding for adaptability to wide agro-climatic conditions. 4) Breeding heat tolerant new high yielding cultivars .

15. The present study was aimed at the development of a somoclonal variant and isozyme marker for Phyllanthus amarus Schum & Thonn using inter-nodal segments and the enzyme peroxidase. Maximum callus proliferation was obtained on Murashige and Skoog’s medium supplemented (MS) with 1.0 mg/l of 2,4-Dichlorophenoxyacetic acid. Three weeks-old pale yellowish white semi-friable callus was used for organogenesis ; the maximum percentage of multiple shoot formation (85%) was achieved after 4 weeks when callus was cultured on Murashige and Skoog’s medium fortified with 1.0 mg/l of 6-Benzylaminopurine . The multiple shoot let formations were also achieved in the presence of the same concentration. The maximum formation of rootlets was observed on MS medium augmented with 1.0 mg/l of Kinetin and 0.5 mg/l of Naphthalene acetic acid. The banding pattern and phytochemical constituent differences were observed between mother plants, directly regenerated via nodal segments, calli , and calli mediated plants. The calli mediated somoclonal variation was confirmed through isozyme (peroxidase) and phytochemical analysis. The isoperoxidase banding profile showed a difference in calli and calli mediated plants. The phytochemical study confirmed the presence of more alkaloids, saponins, tannins and others from calli and calli mediated shoots and roots. Hence the isozyme banding patterns can be used as molecular markers in future plant breeding or genetic improvement programmes. Somaclonal Variation of Phyllanthus amarus Source :- 1. http://intranet.cimap.res.in 2. agritech.tnau.ac.in

16. Clonal propagation of Phyllanthus amarus Xaviers et. al, 2012 reported that MS nutrient medium was used for micropropagation studies. The growth regulators at required concentration were added and homogenized. The volume was made up to 1 L after the pH of the medium was adjusted to 5.6–5.8 using 0.1 N HCI or 0.1 N NaOH . The agar 8 g/L was dissolved at the time of boiling with constant stirring. Then, they were sterilized in an autoclave at a temperature of 121°C at a pressure of 15 pounds per inch for 20 min. The explants were collected from plants grown in net house. For micropropagation studies, the leaves were trimmed off and the shoot tips (6 mm long) and nodal segments (single node) were used for initiation. The explants were first washed in running tap water followed by treatment with 20 emulsifier solution (two to three drops in 100 mL distilled water) for 5 min. After that , the explants were taken to the laminar airflow chamber for further sterilization. Shoot tips and nodal segments were initially sterilized in ethyl alcohol (70%) for 30 seconds followed by sterilization in 0.1% mercuric chloride for 5 min. The treated explants were then washed four to five times with sterile distilled water to make them free from sterilants. For shoot elongation, the individual shoots were excised and inoculated in MS medium supplemented with either BAP alone (0.2 mg/L) or BAP (0.2 mg/L) and Indole Acetic Acid (IAA) (2.0 mg/L). After a week, basal callus was seen in all the cultures. Shoot length of around 7 cm was recorded after 20 days of subculturing with an average of eight internodes in the medium supplemented with BAP and IAA. Shoot length of only 5 cm with six internodes was observed in medium with BAP . Reference :Xaviersetal.,2012ClonalpropagationofPhyllanthusamarus:Ahepatoprotector|RequestPDF.221967567_Clonal_propagation_of_Phyllanthus_amarus_A_hepatoprotector [accessed Aug 24 2018]

17. In Vitro culture: Bhattacharya et. al, 2000 reported an efficient tissue culture protocol was developed for the medicinally important plant Phyllanthus amarus using leaf, node and internode explants on Murashige and Skoog’s basal medium with different combination and concentrations of growth regulators. It has been observed that when MS medium was fortified with 2,4-D (0.5 mg/L), the internodal segment produced maximum shoot formation (80%), at the 2.0 mg/L BAP concentration and the internode explants produced maximum percentage of root (60%) when the MS medium contained 3.0 mg/L IAA . The tissue cultured Phyllanthus amarus crude extracts at different concentrations were evaluated for antimicrobial activity against human bacterial pathogen Pseudomonas aeruginosa. Among the different concentrations of crude extracts, 150 µL (17.0%) recorded the maximum percentage of inhibition when compared to the standard antibiotic (3.3%). In the present study, the in vitro developed shoots elongated better in a medium supplemented with BAP with IAA than in a medium with BAP alone. Shoots grew to a length of around 7 cm with an average of eight internodes after 20 days of sub culturing in the medium supplemented with BAP and IAA, whereas shoot length of around 5 cm with approximately six internodes was noticed in the medium with BAP alone. Higher concentrations of BAP were observed to inhibit the formation of shoots, and the shoots so formed were thick and short. The rooted shoots were successfully transplanted (70%) to plastic cups containing garden soil, and the humidity was maintained at approximately 80%. The plants were watered twice every 7 days, and after 3 months they were transferred to larger pots for acclimatization. S ource : http://intranet.cimap.res.in References : -1.Bhattacharyya R, Bhattacharya S. High frequency in vitropropagation of Phyllanthus amarus by shoot tip culture. Indian J Exp Biol 2001;39:1184-7. 2.Katarzyna L, Halina W. In vitro propagation of Cataipa ovata. Plant Cell Tissue Organ Cult 2000;60:171-6. 3. Seeni S, Latha PG. In vitro multiplication and ecohabituation of endangered blue vanda (Vanda coerulea ). Plant Cell Tissue Organ Cult 2001;6:1-8.

18. Salient Achievements :   Navyakrit (CIMAP) - High herbage and active constituents, released by CIMAP, Lucknow. CIM- Jeevan : Released by CIMAP, Lucknow. The present invention relates to a cultivar of  Phyllanthus amarus  ‘CIM-Jeevan ’, producing high amount of herb, phyllanthin and hypophyllanthin, wherein said cultivar is developed through y-irradiation of superior gemplasm , the said plant produces high amount of herbage yield ranging between 1.0-1.15 kg per sqm fresh total plant herb, possesses high vegetative erect growth with a height ranging between 60 to 65 cm, produces phyllanthin ranging between 0.70-0.77% in dry herb, produces hypophyllanthin ranging between 0.32-0.37% in dry herb, and shows seed germination of about 90%.