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Micropropagation and in vitro scaling up protocols for medicinal plant: Scutellaria barbata Terri A. Brearley, Aisha L. Hill, Brajesh N. Vaidya, Nirmal Joshee. College of Agriculture, Family Sciences and Technology,
Fort Valley State University, Fort Valley, GA 31030. E-mail: [email protected]
Abstract Scutellaria barbata is a clinically well researched medicinal plant on various types of cancer cells causing apoptosis and inhibiting tumor growth. TEAC assay suggests high antioxidant capacity of leaf extracts that could be tied to its therapeutic role. Histochemical studies of foliar trichomes reveal presence of flavonoids. S. barbata nodal explants (1–1.2 cm) were examined for adventitious shoot induction in Murashige and Skoog medium using six different cytokinins at 5 µM with 0.1 NAA and were observed and transferred after 7, 14, and 21 days post inoculation. The cultures were transferred to basal MS for elongation and shoot number was recorded. m–Topolin was superior at 14 and 21 dpi. After establishing superiority of m–Topolin, nine carbon sources (0.1 M) with 5 µM m–Topolin and 0.1 NAA were tested for 14 and 21 days for adventitious shoot induction. Glucose and fructose induced highest number of adventitious shoot buds. To study scaling up potential, ten week old multiplying cultures were transferred to Liquid Lab Rocker® boxes containing a napkin and 30 mL of basal MS with 3% sucrose. The cultures in the Liquid Lab Rocker ® boxes will be removed and fresh weight, shoot number, and dry weight will be recorded to analyze biomass production. Microshoots were transferred for rooting into Magenta™ vessels filled with autoclaved potting mixture and a rooting protocol with close to 100% rooting has been established. Prolific in vitro flowering and seed set was seen during rooting stage and it is of value for rare and valuable genotypes. Sterile vessels with microshoots were placed under 16/8 light at 25 °C. Micropropagation experiments using transverse Thin Cell Layer explants are in progress.
Introduction Scutellaria is the second largest genus in Lamiaceae with over 360 species that are geographically widespread around the world (Paton, 1990). Biomedical research employing Scutellaria extracts and individual flavonoids on various types of cancer cells, in vitro and in vivo, have resulted in inducing apoptosis, and inhibiting tumor angiogenesis. Scutellaria barbata has antibacterial properties of apigenin and luteolin, antitumor compound pheophorbide, and other flavones such as scutellarein, scutellarin, carthamidin, isocarthamidin, and wogonin (Chan et al. 2006; Goh et al., 2005; Sato et al. 2000). Majority of plant material for this purpose and supplying to the herbal market is taken from the wild and is subject to loss of genetic material or contamination. Protocols need to be developed to ensure that genetic material is saved for further research and medicinal use. Due to genetic variability between plants a protocol must be adapted as we have done here with S. barbata. Ex-situ conservation protocols need to be developed to assist conservation and scaling up of biomass production in the greenhouse or field.
Materials and Methods
1. Micropropagation (Nodal explant, cytokinins, and incubation period): Murashige and Skoog (1962) (MS) medium was supplemented with 3% sucrose, pH adjusted to 5.8, and 7 g L-1 agar, then autoclaved at 121°C for 15 min. There were six cytokinin treatments: 6-benzylaminopurine (BAP), 6-furfurylaminopurine (Kinetin), 6-(α,α-dimethylallylamino)purine (2iP), 6-(3-hydroxybenzylamino)purine (meta-Topolin - m-T), N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron- TDZ), and 6-(4-Hydroxy-3-methylbut-2-enylamino)purine (Zeatin) (PhytoTechnology Laboratories, Shawnee Mission, KS) that were added as filter sterilized solutions at 5 µM. Filter sterilized auxin 1-naphthylacetic acid (NAA) was added to cooled medium at 0.1 µM. Control received no plant growth regulators (pgrs). Each treatment had three replicates of five culture tubes.
Two month old mother stock of S. barbata was provided from in-house cultures. Under sterile conditions, 10 mm ± 2 mm nodal
explants were excised from shoots and all treatment tubes were inoculated on the same day. All cultures were maintained at 25 oC ± 2 oC with 16/8 hour photoperiod with a light intensity of 40 µmol m-2 s-1.
The incubation time assay used MS basal as elongation medium (no plant growth regulators). All seven treatments had three
replicates of five culture tubes with 15 mL of medium. The time periods in the assay were 7, 14 and 21 days with all three time periods having their own set of explants. One replicate from each cytokinin treatment was randomly picked and the explants were transferred into the elongation medium after 7 days post inoculation (dpi) then repeated at 14 and 21 dpi. All cultures were maintained in the same conditions as above.
Shoot and height counts of the explants started at 21 dpi and continued till 63 dpi. Height measurements were taken from the
surface of medium to longest shoot tip. Transverse Thin Cell Layer cultures: tTCLs are explants that have been excised from part of the plant < 1mm wide. Explants of
less than and greater than 1mm wide were tried on leaves and stems. Two treatments were tested, BAP with IAA and BAP with 2,4-D. The concentrations for BAP were 0.1, 1.0, 5.0, and 10.0 µM, and for IAA and 2,4-D it was 0.01, 0.1, 1.0, and 10.0 µM. The MS medium with the PGRs were tested two different ways, one was semisolid and the other was liquid on Whatman filter paper. Both were in 100 x 15 mm petri dishes stored in same conditions as cultures above, under diffused light.
2. Nodal segment, carbon sources, time experiment: MS medium by supplementing 0.1 M of a carbon source (sugar), m-Topolin at 5
µM and NAA at 0.1 µM, pH adjusted to 5.8, 7 g L-1 of agar, then autoclaved as above. The control had sucrose with no m-Topolin or NAA. The carbon source treatments: Sucrose, D-Maltose, D-Glucose, Fructose, D(+)-Mannose, myo-Inositol, D-Mannitol, and D-Sorbitol (PhytoTechnology Laboratories, Shawnee Mission, KS). All treatments had three replicates of five culture tubes with 15 mL of medium. The time periods were 14 and 21 dpi with the two time periods having their own set of explants. In this experiment elongation medium contained 1 % sucrose. Shoot number and height counts of the explants were initiated at 21 dpi and continued till 49 dpi.
3. Scale up for biomass: Ten week old cultures from carbon source experiment were transferred to sterile Liquid Lab Rocker® boxes (LLR). The boxes contained brown paper as substratum and 30 mL of liquid basal MS medium supplemented with 3% sucrose. There were explants from eight carbon treatments with two-five explants in each box. Liquid MS was added when there was no extra liquid flowing in the box. After three weeks the cultures were recorded for fresh and dry weight, and shoot count.
4. Rooting and Acclimatization: After seven weeks in culture microshoots were transferred to a sterile unit of two Magenta Boxes®
joined by a connector. The bottom box was filled halfway with a 1:1 mixture of potting soil and vermiculite, liquid basal MS with 3% sucrose, and indole-3-butyric acid (IBA) at 5 µM. Cultures were maintained at 25 oC ± 2 oC with 16/8 hour photoperiod. After six weeks the top box was opened slightly to reduce humidity. Two weeks after opening the boxes the plants were transferred to the greenhouse for further growth and to record final survival.
Results and Discussion
Control Fructose Glucose Maltose
Sucrose Myo-Inositol Mannitol Sorbitol
B
A
B
B B
B B
A A
A
A A A
A C
C
B B
A
C
C CD
BC
BC BC
A
D
B
0
1
2
3
4
5
6
7
8
Control 2iP BAP Kinetin m-Topolin TDZ Zeatin
Hei
ght
(cm
) an
d S
ho
ot
Co
un
t
Treatments
S. barbata 7, 14, & 21 dpi
Height(cm)
ShootCount 7dpi
ShootCount 14dpi
ShootCount 21dpi
A
B B B
C C C
B E
A A
B
DE
C
DE D
C
B
A
C
C
C
C
C
0
5
10
15
20
25
Hei
ght
(cm
) an
d S
ho
ot
Co
un
t M
ean
s
Treatments
S. barbata 14 & 21 dpi
Height(cm)
14 dpiShootCount
21 dpiShootCount
Con Fruc Glu Mal Suc Myo–I Mann Sor BAP Kin 2iP m-T TDZ Zeatin Con
0
10
20
30
40
50
60
70
80
90
Co
ntr
ol
Fru
cto
se
Glu
cose
Mal
tose
Man
nit
ol
Myo
-In
osi
tol
Sorb
ito
l
Sucr
ose
Co
ntr
ol
Fru
cto
se
Glu
cose
Mal
tose
Man
nit
ol
Myo
-In
osi
tol
Sorb
ito
l
Sucr
ose
Ave
rage
Sh
oo
ts p
er C
lum
p
14 dpi Treatments 21 dpi
Results and Discussion Cytokinin and regeneration: For the height of the cytokinin treatments, 2iP was significantly different than the other treatments. For 14 and 21 dpi m-Topolin was significantly different from the other treatments. Carbon Source, Shoot induction, and growth: For the height of the carbon source treatments, control was significantly different than the other treatments. Fructose and glucose are significantly different for 14 dpi, and glucose for 21 dpi on shoot count. Mannose is not included in the graph due to total mortality of explants during the experiment. Scaling up Experiment: Carbon source 14 dpi resulted in fructose treatment having highest average shoot number (49.6), whereas highest dry biomass was accumulated in plants with maltose treatment (0.176 g per clump). Similarly, at 21 dpi mannitol had the highest shoot number average (84.75) and had the highest dry biomass accumulated (0.262 g per clump). Rooting and acclimation experiment: Rooting for S. barbata was 97.5 % in the boxes and 80% upon transfer to the greenhouse conditions. This protocol ensures high survival and we are expanding it to other species. Transverse Thin Cell Layer culture experiment: Progress is being made, calluses are being observed for somatic embryogenesis. Cytokinin m-Topolin was superior for adventitious shoot bud induction at 14 and 21 dpi. Carbon sources were tested along with m-Topolin and glucose was the best for 14 and 21 dpi, going with 14 dpi is more cost effective. For scaling up mannitol at 21 dpi is the best option due to almost doubling the shoot count over fructose and increasing biomass by two-thirds.
Biochemistry and physiology of higher plants indicate that species that metabolize mannitol have several advantages over those that exclusively translocate sugars. One advantage is increased tolerance to salt- and osmotic-stress as a result of mannitol's function as a ‘compatible solute’. Another advantage is a possible role in plant responses to pathogen attack— thus mannitol metabolism may play roles in plant responses to both biotic and abiotic stresses.
References Chan, J. Y., P. M. Tang, P. Hon, S. W. Au, S. K. Sui, M. M. Waye, S. Kong, T. C. Mak, and K. Fung. 2006. Planta Medica 72:28-33. Goh, D., E. S. Ong, and Y. H. Lee. 2005. Journal of agricultural and food chemistry 53 (21):8197-8204. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473-497. Paton, A. 1990. A global taxonomic investigation of Scutellaria (Labiatae). Kew Bull 45:399-450. Sato, Y., S. Suzuki, T. Nishikawa, M. Kihara, H. Shibata, and T. Higuti. 2000. Journal Ethnopharmocol 72:483-488. TranTran Thanh Van, K., H. Chlyah and A. Chlyah, 1974. Regulations of organogenesis in thin layers of epidermal and sub epidermal cell. Tissue Culture and Plant Sciences. Academic Press, Pp 101-139.
Acknowledgement 2011-2014. Capacity building USDA NIFA. Title: Germplasm conservation, Anti-adipocytic and anticancer activity and metabolic engineering in the genus Scutellaria. CSREES Award # 2011-38821-30928. P.I.: Nirmal Joshee Thank you to Dr. David I. Shapiro-Ilan of USDA-ARS Byron, GA for SAS guidance. Thanks for time to time help from lab members- Alhassan I Zakaria, and to Vicki Owen for maintaining Scutellaria germplasm in the greenhouse
.
Fig. 1 Micropropagation, rooting, scaling up and acclimation of S. barbata. A. In vitro 3 wks. B. and C. In vitro rooting D. and E. LLR boxes with inset picture showing a single box and a clump from that box F. Carbon source assay G. Cytokinin assay H. In vitro plants acclimated to the greenhouse I. In vitro plants after six weeks in the greenhouse (fructose is flowering) J. In vitro plant flowering, setting seed, and growing plantlets K. tTCLs with callus induction.
A B C D
E F G H
I J K
Fig 2. A. Cytokinin effect on the height and total shoot count for three time periods B. Carbon source assay representing the height measurements and total shoot count for each time period C. Graph representing the percent rooting and survival of acclimated plants in the greenhouse D. Number of shoots per clump in the LLR boxes in scaling up experiment. The GLM Procedure and Student-Newman-Keuls Test was executed on SAS for graphs A and B.
A B C D
