1. Research regarding in vitro propagation of some Lycium barbarum and Lycium chinense hybrids
Authors names:
Cătălina Nicolae
Adrian Peticilă
PhD. student Oana Venat
Assoc.prof.dr. Adrian Asănică
Popescu Dan
Coordinator Professor:
Assoc.prof.dr. Adrian Peticilă
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

2. Goji berry – a superfruit
Lycium barbarum and Lycium chinense are spinous shrubs in the nightshade Solanaceae family.
Their fruits have been used in traditional chinese medicine for over years.
Some of the health benefits include: antioxidant, anti-aging, regulator of the immune system, inhibition of tumoral growth and anti-diabetes effect.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

3. Goji berry – a superfruit
Goji are considered superfruits for their high content of nutrients
Polysaccharides
B Vitamins
Thiamine
Riboflavin
Piridoxine
5-8%
xylose, glucose, arabinose,
rhamnose, mannose, galactose
Vitamin C
42 mg/100 g
Carotenoids
The most abundant - zeaxanthin
18 aminoacids
Of which 8 are essential
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

4. Propagation of Lycium barbarum
In Romania, studies on propagation of Lycium barbarum mainly regarded seeds and cuttings, but also in vitro cultures.
Generative propagation offers a diversity of biological material, therefore fruits may differ in terms of size, color, shape, and biochemical composition.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

5. Micropropagation of Lycium barbarum
Micropropagation tehniques allow the production of large number of plants in relatively short period of time.
Micropropagation of Lycium barbarum has been studied by numerous authors and explant sources used include shoot tips, nodal segments, stem axillary buds, leaf segments and root segments, on different media types with wide range results. 1 2 3 4 5 6
Lycium barbarum – photos taken during in vitro stages: 1- root development, 2,3 – callus mass 3,4 – in vitro seedlings, 5 – root buds

6. Materials and Methods Culture initiation:
Seeds from 15 hybrids of Lycium barbarum and Lycium chinense were germinated aseptically.
Prior to sterilization, seeds were immersed in 45°C water (warm water).
Nr.
HYBRID
Medium
Seeds nr. 1
E I
MS (-) 12 6
C I 9 11
D I 5
B5(-) 8
soil
sol 2
E I -1 7
C I - 1
D I - 1 3
A I 4
C II - R7P1 13
G I - 1
B I
C II - R7P3 14
G II
B I liber 10
C II - R7P5 15
F II
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

7. Materials and Methods Culture initiation: Seed sterilization:
0,4 % Flint fungicide for 10 minutes;
3 rinses in sterile bidistilled water, 5 minutes each;
12 % Domestos commercial solution (sodium hypoclorite 4,5 %) 15 minutes;
3 rinses in sterile bidistilled water, 5 minutes each.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

8. Materials and Methods Culture conditions
a. IN VITRO
Seeds were taken from a range of hybrid collection and were inoculated for on two different culture mediums: Murashige et. Skoog (MS) and Gamborg (B5), both without growing hormones. Number of seeds/berry was not standardized, the condition of growth, ripening and number of berries for each hybrid has varied.
Seeds were incubated in the growth room, at 22 °C, 50 % humidity and under dark conditions until germination.
After germination, seedlings were kept at 16 hours photoperiod and lx light intensity. Germination observations were made every two days during a period of 30 days and measurements were made every 2 weeks. Data shown was germination rate, seedlings growth: lenght, leaves and internodes.
After a complete in vitro micro propagation cycle, most of hybrids were encapsulated in artificial seeds for testing their resistance after dormancy period in cool environment at 3-5 °C in dry/liquid medium (data not shown) for storing.
b. SOIL
For each hybrid, a number of seeds were germinated nonaseptically, in seedlings substrate and kept at room temperature, in lab environment, without particular conditions.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

9. Materials and Methods Culture conditions a. IN VITRO b. SOIL
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

10. Results and discussions
I. Germination percentage
In vitro
In vitro: average germination percentage was 68,82 %. GI-1 ( 100%), CIIR7P5 (90%) and CI (88,23%) had the highest germination percentage. Only 3 hybrids - BI, BI liber and DI presented less 50% germination percentage.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

11. Results and discussions
I. Germination percentage
soil
Soil germination: Average germination percentage was 33,52 %. CI (83,3 %) and FII (66,6 %) had the best germination rate. 11 hybrids- EI, AI, BI, BI liber, CI- 1, CI I- R7P1, CII - R7P3, CII - R7P5, DI- 1, GI- 1 and G II had a germination percentage under 50 %. DI had 0 % germination.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

12. Results and discussions
I. Germination percentage
Comparison between in vitro and soil propagation
all seeds germinated in vitro conditions showed a higher germination percentage than the ones germinated in soil
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

13. Results and discussions
I. Germination percentage
MS (-) Culture medium
The average germination percentage on MS (-) medium was 61,28 %. EI-1, CII-R7P5 and GI-1 report a 100% of germination percentage on MS medium and 66,67% of hybrids had a germination percentage over 50 %.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

14. Results and discussions
I. Germination percentage
B5 (-) Culture medium
The average germination percentage on Gamborg B5 (-) medium was 76,36 %.
AI, CI, DI-1, GI-1 and GII reported a 100% of germination on B5 (-) medium and 86,7% of hybrids had a germination percentage over 50%.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

15. Results and discussions
I. Germination percentage
MS(-) and B5 (-) Culture medium
Comparation between mediums reveals that B5(-) medium provided a superior germination rate and promoted a much better growth environment for seeds.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

16. Results and discussions
I. Germination percentage on soil and culture medium
MS and B5 medium
E I, A I, B I liber, C I, D I – 1, G II, F II showed a higher germination percentage on B5 medium;
E I- 1, C II R7P1, C II R7P3, C II R7P5 showed a higher germination percentage on MS medium;
B I, C I – 1, D I, GI – 1 – recorded no differences on the two growing mediums.
Nr.
HYBRID
MS (-)
B5 (-)
Soil 1
E I 75
83,3 50 2
E I - 1
100 60 57 3 AI 16 4 BI 20 5
BI Liber 6 CI
77,7 7
CI - 1 8
C II R7P1 80 9
C II R7P3 70 30 10
C II R7P5 11 DI 12
DI - 1 13
G I - 1 40 14
G II
66,6 15
F II
88,8
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

17. Results and discussions
II. Seedlings growth on MS(-) culture medium (average results)
One month after inoculation, 5 hybrids did not have growth to report (BI liber, CII- R7P3, CII-R7P5, DI, DI-1); the best result in terms of growing is shown by GI- 1 with an average of 2,17 cm; from the hybrids that had a maximum germination rate on MS(-) - EI-1, CII-R7P5 and GI-1- only GI-1 reported a better growth; the average growth rate is 1,10 cm.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

18. Results and discussions
II. Seedlings growth on B5(-) culture medium (average results)
All hybrids reported growth on Gamborg B5 medium. The highest growth was recorded by CI – 1 (2,9 cm), CII R7P3 (2,6 cm) and E I (2,5 cm). The average seedling growth was 1,73 cm on Gamborg B5 Medium.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

19. Results and discussions
II. Seedlings length growth on MS(-) and B5(-)
During the first month after inoculation, all hybrids recorded better growth in terms of length on B5 medium, with an 1,73 cm average of growing, while on MS medium, the average growth was 1, 10 cm.
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

20. Results and discussions
II. Seedlings growth
Differences between the two growing medium on two hybrids, after one month from germination.
E I
C I
On left – MS medium;
On right – B5 medium
On left – B5 medium;
On right – MS medium
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

21. Results and discussions
III. Seedlings foliage growth on MS(-) and B5(-)
Five (5) hybrids did not have foliage growth on MS (-) to report (BI liber, CII R7P3, C II R7P5, DI, DI – 1) . Foliage growth on B5(-) for AII, CI-1, CII R7P1 was double than MS (-). Four hybrids have shown no difference on the two growing mediums (EI, EI- 1, CI, GI-1).; F II was the only hybrid to show a better foliage growth on MS (-)
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

22. Conclusions and recommendations
In vitro conditions provided to be superior in terms of germination percentage.
On average, comparing the two growing mediums, seeds inoculated on Gamborg B5 (-) medium shown a higher germination percentage, higher seedling and foliar growth compared to MS (-)
CI-1, CII R7P3, EI-1, GI-1 responded overall better then other hybrids to micropropagation
The rate of germination and the potential of explants depends in a high percentage on seed maturation and pollination (data not tracking)(tbd)
The potential of in vitro propagation is higher on this species because of one particular aspect of it biology: there is a considerable activity on explant to generate new plants form dormant buds on the roots (genetic behavior).
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania

23. References
Amalgase H., Farnsworth N.R., 2011 A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of Lycium barbarum fruit (Goji). Food research International 44,
Asănică A., 2017 – Vertical training of berry shrubs, Total Promotion Publishing House.
Asănică A., Tudor V., Teodorescu R.I., Iacob A., Zolotoi V., Tudor A., Results on hardwood cuttings propagation of some Lycium sp. Genotypes. Frut Growing Research, Vol XXXII.
Cho-Wing Sze S., Song JX., Wong RN., Feng YB, Ng TB, Tong Y., Zhang KY., Application of SCAR (sequence characterized amplified region) analysis to authenticate Lycium barbarum (wolfberry) and its adulterants. Biotechnol. Appl. Biochem., 51,
Dănăilă Guidea SM, RV Dobrinoiu, L. Vişan, R.C. Toma, Protocol for efficient in vitro multiplication of Lycium barbarum L. by direct organogenesis. Scientific Bulletin. Series F. Biotechnologies, Vol. XIX
Fira A., N. Joshee, V. Cristea, M. Simu, M. Hârţa, D. Pamfil, D. Clapa, Optimization of micropropagation protocol for Goji Berry (Lycium barbarum L.). Bulletin USAMVM Horticulture 73 (2).
Kocyigit E., Sanlier N., A review of Composition and Health Effects of Lycium barbarum. International Journal of Chinese Medicine. 1 (1): 1-9.
Potterat O., Goji (Lycium barbarum and Lycium chinense): Phytochemistry, pharmacology and safety in the perspective of traditioal uses and recent popularity. Planta Med. 76 (1): 7-9.
Redgwell, R. J., Curti, D., Wang, J., Dobruchowska, J. M., Gerwig, G. J., Kamerling, J. P., & Bucheli, P. (2011). Cell wall polysaccharides of Chinese Wolfberry (Lycium barbarum): Part 1. Characterisation of soluble and insoluble polymer fractions. Carbohydrate Polymers, 84(4),
Tudor V., Asănică A., Teodorescu I.R., Gîdea M., Tănăsescu Constantin, Tudor A.D., Ţiu J.V., Germination capacity of some Lycium barbarum L. and Lycium chinense Mill. biotypes seeds. Romanian Biotechnological Letters. Vol. 22, No. 1
Images
International Student Symposium "Hortus Academicus“ April 27, Bucharest, Romania