Short-term storage of Solanum trilobatum L. synthetic seeds and evaluation of genetic homogeneity using SCoT markers

다국어 초록 (Multilingual Abstract)

The current study demonstrates an effective protocol for encapsulation of in vitro nodal segments of Solanum trilobatum L. for short-term storage. In a modified Murashige and Skoog medium supplemented with 8.8 µM 6-benzyl adenine (BA), 0.5 µM indole-3-acetic acid (IAA), and 25 mM ammonium nitrate, a gelling matrix of 3% sodium alginate and 80 mM CaCl2.2H2O promoted the highest shoot regeneration frequency (98.4%). The effect of storage temperature, sucrose concentration, and storage time interval on shoot regrowth efficiency was assessed. Synthetic seeds maintained at culture room temperature (25 °C) were shown to be more competent than low-temperature storage (8 °C and 4 °C), with better regeneration efficiency (82.2%). The alginate matrix added with 1.5% sucrose increased the storage potential of synthetic seeds up to 12 weeks with a regeneration frequency of 57.7%. Using start codon-targeted polymorphic (SCoT) markers, the genetic fidelity of plants regrown after 12 weeks of storage was assessed. The SCoT fingerprinting approach has confirmed the post-storage genetic stability of recovered plants with their in vitro source. The present investigation is the first report on the development of synthetic seeds of S. trilobatum L., which would greatly facilitate germplasm conservation and exchange of this invaluable medicinal plant.

참고문헌 (Reference)

1 Anand Y, "Synthetic seeds: a novel approach of in vitro plantlet formation in vasaka (Adhatoda vascia Nees.)" 19 : 159-162, 2002

2 Pintos B, "Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring" 95 : 217-225, 2008

3 Sharma S, "Synseed technology- a complete synthesis" 31 : 186-207, 2013

4 Ray A, "Storage and conversion of Eclipta alba synseeds and RAPD analysis of the converted plantlets" 54 : 547-550, 2010

5 Saikat Gantait, "Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ 3 Vanda coerulea Grifft. ex. Lindl." 한국식물생명공학회 7 (7): 257-266, 2013

6 Xiong FQ, "Start codon-targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (Arachis hypogaea L.) genotypes" 38 : 3487-3494, 2011

7 Bhattacharyya P, "Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl. an endangered medicinal orchid species" 529 : 21-26, 2013

8 Collard BCY, "Start Codon Targeted (SCOT) Polymorphism: a simple novel DNA marker technique for generating gene-targeted markers in plants" 27 : 86-93, 2009

9 Singh AK, "Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species" 163 : 220-223, 2006

10 Standardi A, "Recent perspectives on synthetic seed technology using non embryogenic in vitro-derived explants" 159 : 968-978, 1998

11 Jawahar M, "Rapid proliferation of multiple shoots in Solanum trilobatum L" 14 : 107-112, 2004

12 Alagumanian SV, "Plant regeneration from leaf and stem explants of Solanum trilobatum L" 86 : 1478-1480, 2004

13 Singh SK, "Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species" 31 : 649-653, 2009

14 Singh AK, "Plant regeneration from alginate to encapsulated shoot tips of Phyllanthus amarus Schum and Thonn, a medicinally important plant species" 42 : 109-113, 2006

15 Singh AK, "Plant regeneration from alginate encapsulated somatic embryos of Dalbergia sissoo Roxb" 9 : 319-324, 2010

16 Prattipati Subhashini Devi, "Plant Regeneration from Encapsulated Nodal Segments of Sterculia urens Roxb., an Endangered Gum-Yielding Tree" 한국작물학회 17 (17): 21-25, 2014

17 Naik SK, "Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange" 108 : 247-252, 2006

18 Iram Siddique, M. Anis, "Morphogenic Response of the Alginate Encapsulated Nodal Segment and Antioxidative Enzymes Analysis during Acclimatization of Ocimum basilicum L." 한국작물학회 12 (12): 229-234, 2009

19 Prasad A, "Molecular, chemical and biological stability of plants derived from artificial seeds of Centella asiatica (L.) Urban—an industrially important medicinal herb" 60 : 205-211, 2014

20 Saha S, "Molecular and phytochemical analyses to assess genetic stability in alginate-encapsulated microshoots of Ocimum gratissimum L. following in vitro storage" 57 : 33-43, 2014

21 Faisal M, "Molecular and biochemical characterization in Rauvolfia tetraphylla plantlets grown from synthetic seeds following in vitro cold storage" 169 : 408-417, 2013

22 Agarwal T, "Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers" 120 : 313-323, 2015

23 Nadkarni KM, "Indian Material Medica" Popular Prakasan Pvt Ltd 1153-, 1976

24 Sharma S, "In vitro propagation and synseed production of scarlet salvia (Salvia splendens)" 25 : 359-368, 2014

25 Cheruvathur MK, "In vitro propagation and conservation of Indian sarsaparilla, Hemidesmus indicus L. R. Br. through somatic embryogenesis and synthetic seed production" 35 : 771-779, 2013

26 Verma SK, "In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L" 124 : 517-521, 2010

27 Shilpha J, "Improved in vitro propagation, solasodine accumulation and assessment of clonal fidelity in regenerants of Solanum trilobatum L. by flow cytometry and SPAR methods" 117 : 125-129, 2014

28 Mehrotra S, "ISSR and RAPD based evaluation of genetic stability of encapsulated micro shoots of Glycyrrhiza glabra following 6 months of storage" 52 : 262-268, 2012

29 Yang Linlin, "High-frequency in vitro plantlet regeneration in Lilium davidii var. unicolour Salisb, an important edible and medicinal plant, and confirmation of genetic fidelity of regeneration plantlets using ISSR markers" 한국식물생명공학회 15 (15): 435-446, 2021

30 Yonga CF, "Germplasm genetic diversity of Myrica rubra in Zhejiang Province studied using inter-primer binding site and start codon-targeted polymorphism markers" 170 : 169-175, 2014

31 Rathore NS, "Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis" 36 : 555-559, 2014

32 Bhattacharyya P, "Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid" 2 : 489-504, 2014

33 Thakur J, "Genetic homogeneity revealed using SCoT ISSR and RAPD Markers in micropropagated Pittosporum eriocarpum Royle- An endemic and endangered medicinal plant" 2016

34 Largia MJV, "Genetic fidelity assessment of encapsulated in vitro tissues of Bacopa monnieri after 6 months of storage by using ISSR and RAPD markers" 37 : 1008-1017, 2013

35 Zeng B, "Genetic diversity of orchard grass (Dactylis glomerata L.) germplasms with resistance to rust diseases revealed by Start Codon Targeted (SCoT) markers" 54 : 96-102, 2014

36 Saha S, "Encapsulation, short-term storage, conservation and molecular analysis to assess genetic stability in alginate-encapsulated microshoots of Ocimum kilimandscharicum Guerke" 120 : 519-530, 2014

37 Ahmed MR, "Encapsulation technology for short-term storage and germplasm exchange of Vitex trifolia L" 26 : 133-139, 2015

38 Sharma S, "Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn" 111 : 191-198, 2012

39 Hung CD, "Encapsulation technology for short-term preservation and germplasm distribution of the African mahogany Khaya senegalensis" 107 : 397-405, 2011

40 Redenbaugh K, "Encapsulation of somatic embryos in synthetic seed coats" 22 : 803-809, 1987

41 Rai M, "Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange" 118 : 33-38, 2008

42 Danso KE, "Encapsulation of nodal cuttings and shoot tips for storage and exchange of Cassava germplasm" 21 : 718-725, 2003

43 Perveen S, "Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production" 36 : 2067-2077, 2014

44 Bhattacharyya P, "En-masse production of elite clones of Dendrobium crepidatum: A threatened, medicinal orchid used in Traditional Chinese Medicine (TCM)" 3 : 168-176, 2016

45 Baskaran P, "Embryogenesis and synthetic seed production in Mondia whitei" 121 : 205-214, 2015

46 Saha S, "Effects of plant growth regulators on efficient plant regeneration efficiency and genetic stability analysis from two Ocimum tenuiflorum L. morphotypes" 27 : 609-628, 2016

47 Dhavala VNC, "Effect of explant age, hormones on somatic embryogenesis and production of multiple shoot from cotyledonary leaf explants of Solanum trilobatum L" 8 : 630-634, 2009

48 Tsvetkov I, "Effect of alginate matrix composition on regrowth of in vitro-derived encapsulated apical microcuttings of hybrid aspen" 50 : 722-724, 2006

49 Khilwani B, "Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst" 127 : 433-442, 2016

50 Ahmad N, "Direct plant regeneration from encapsulated nodal segments of Vitex negundo" 54 : 748-752, 2010

51 Joshi C, "Context sequences of translation initiation codon in plants" 35 : 993-1001, 1997

52 Sawant SV, "Conserved nucleotide sequences in highly expressed genes in plants" 78 : 123-131, 1999

53 Hatzilazarou S, "Conservation, regeneration and genetic stability of regenerants from alginate-encapsulated shoot explants of Gardenia jasminoides Ellis" 13 : 1666-, 2021

54 Chaudhuri RK, "Conservation of Swertia chirata through direct shoot multiplication from leaf explants" 2 : 213-218, 2008

55 Adhikari S, "Assessment of genetic stability of Cucumis sativus L. regenerated from encapsulated shoot tips" 170 : 115-122, 2014

56 Mishra J, "Assessment of genetic fidelity of encapsulated microshoots of Picrorhiza kurrooa" 104 : 181-186, 2011

57 Devi SD, "Artificial seed for short-term storage: using nodal buds in Aquilaria malaccensis Lam" 115 : 2103-, 2018

58 Kumar S, "Alginate-encapsulation of shoot tips of jojoba [Simmondsia chinensis (link Schneider] for germplasm exchange and distribution" 16 : 379-382, 2010

59 Singh SK, "Alginate encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)" 32 : 607-610, 2010

60 Murashige T, "A revised medium for rapid growth and bioassays with tobacco tissue culture" 15 : 473-497, 1962

61 Govindan S, "A pilot study on the clinical efficacy of Solanum xanthocarpum and Solanum trilobatum in bronchial asthma" 66 : 205-210, 1999