Albanian j. agric. sci. 2014 (Special edition)

Agricultural University of Tirana

(Open Access)

RESEARCH ARTICLE

Regeneration of eggplants Solanum melongena var.Sukthi through somatic embryogenesis GJOK VUKSANI1*, ANGJELINA VUKSANI2, PASHK LEKAJ1, URAN ABAZI1 1

Agricultural University of Tirana, Albania

2

Agricultural Ministry of Albania

*Corresponding author e-mail: [email protected]

Abstract The experiment is carry out at Humbold University of Berlin, in laboratory of “Cell cultures and invitro”, “Institute of Horticultural Sciences” Germany.Explants obtained from Hipokotili and Kotiledonet and with a size of about 0.3 cm are placed in a substrate multiplication in - vitro MS / 2 to which are added vitamins, Were raised six different variants with treatment with phyto-hormones 2,4 / D and NAA promotes calluses formation with respective concentrations. After 8 weeks (4 weeks after callus) has been experimented formation of organo-genesis of shoot formation to regenerate the calluses cells. Organo-genesis of shoot formation has been experimented fully with MS with an additional 0.25% active carbon and adding vitamins: For each variant were taken from callus explants 20 and 20 of Cotyledon, Hypcotic explants by 4 containers with 25 ml (5 explants for each container). The numbers of variants and origins are the same callus to show that formed calluses are taken with the same concentration of phyto-hormone that also tested the impact of different treatments callus with phyto-hormone in organo-genesis of cuttings. As e resultants we can say that somatic embryos in Solanum melongena Var.”Sukthi ” is successful in shot regeneration from cotyledon with concentration of 0,8mg/l 2,4D 60% and 0,1 mg/l NAA with about 75 %. But the shot regeneration development is better in variant with 0,8 ml/l 2,4D than others variants tested. The shots regeneration from explants with origins from Hypcotic is very poor development with only 40% in variant use 0,8mg/l 2,4D. So as conclusion we can stress that

regeneration of eggplants, Solanum melongena Var.”SUKTHI” through somatic embryogenesis is successful with explants taken from cotyledon with concentration of 0,8mg/l 2,4D and 0,1 mg/l NAA. Keywords: Propogation, explants, somatic embryogenesis, phytohormon, callus

Introduction Eggplant (Solanum melongena ) is a widely cultivated and economically important vegetable crop in many countries but is susceptible to many pests and diseases. Eggplant is highly amenable to morphogenesis in general and somatic embryogenesis (SE) in particular. Different factors such as genotype, explant type and position, growth regulators, polyamines and some other factors have been shown to affect SE. Most of the studies have focussed on induction, development and maturation of SE. Molecular aspects of SE. Somatic embryogenesis is an important pathway for the regeneration of plants from cell culture systems and a method commonly used in large scale production of plants and synthetic seeds [3]. Both somatic embryogenesis and in vitro cultured zygotic embryos are being employed to address long-standing questions concerning the role of growth regulators as well as the limits of influence of maternal tissues and 241

endosperm on zygotic embryos [8]. The analysis of somatic embryo development can also significantly contribute to the understanding of early morphogenetic events in plant embryogenesis, such as the establishment of polarity and the differentiation of tissue systems [11]. The site of somatic embryo induction and their developmental pattern are highly variable according to the species and explant type. Histological analysis performed in several plant species showed that somatic embryo development may occur from epidermal [2], parenchymatous [6] or vascular cells [9]. Eggplant somatic embryogenesis studies using different growth regulators and various explants including leaves [5], cotyledons [7], zygotic embryos [10], and hypocotyls [4], have been reported. In the present work development of somatic embryos in Solanum melongena Var.”Sukthi ” was performed in order to improve the usefulness of this system for such studies.

Vuksani et al

The experiment is carry out at Humbold University of Berlin, in laboratory of “Cell cultures and invitro”, “Institute of Horticultural Sciences” Germany. Material and Method The seeds of Solanum melongena var.Sukthi eggplants are obtained by genetic benches and the experiment is carried out in the Laboratory of "Cell cultures and invitros", "Institute of Horticultural Sciences" of the "Humboldt" in Berlin, Germany. Seeds were sterilized with Calcium hypochlorite. Calcium hypochlorite Sukthi 3% for 10 minutes. After germination of seeds and when the seedlings have reached the age of 7 to 10 days, were taken explants (part 0,3 / 0,5 cm) from cotyledon and hypcotic and are located in the substrate. Eggplant somatic embryogenesis studies using different growth regulators and various explants, [5], cotyledons and hypocotyls [4], have been reported. Explants obtained from hipokotili and kotiledonet and with a size of about 0.3 cm are placed in a substrate multiplication in / vitro MS / 2 to which are added vitamins, MS/2 Macro.+ Thiamin 2,5 ml/l Biotin 0,2 ml/l Pyridoxine 0.2 ml/l Were raised six different variants with treatment with phyto-hormones 2,4 / D and NAA promotes calluses formation with respective concentrations: 1-0,1 mg/l 2,4-D. 2-0.5 mg/l2,4-D 3-0,8 mg/l 2,4-D and 4-0,1 mg/l NAA. 5-0.5 mg/l NAA 6-0,8 mg/l NAA. Both somatic embryogenesis and in vitro cultured zygotic embryos are being employed to address long-standing questions concerning the role of growth regulators …..[8]. For each variant were taken from 20 explants Kotiledonet and Hypcotic. These explants are placed in containers with about 25 ml of 5 explants feeder for each pot by adding active carbon and 0.25% for the coloring of the substrate. After 8 weeks (4 weeks after callus) has been experimented formation of organo-genesis of shoot formation to regenerate the calluses cells.

Organo-genesis of cuttings has been experimented fully with MS with an additional 0.25% active carbon and adding vitamins: Thiamin 2,5 ml/l Biotin 0,2 ml/l Pyridoxine 0.2 ml/l For each variant were taken from callus explants 20 and 20 of Cotyledon, Hypcotic explants by 4 containers with 25 ml (5 explants for each container). The numbers of variants and origins are the same callus to show that formed calluses are taken with the same concentration of phyto-hormone that also tested the impact of different treatments callus with phytohormone in organo-genesis of cuttings. Results and discussion 1-The impact of phyto-hormones 2,4 D and NAA in initiating the cells of callus from explants were received from Cotyledon and Hypcotic.

Figure1. The formed callus after 4 weeks

After 4 weeks, the calluses rsults taken from Cotyledon explants are presented in the following graph: 25 20 15 10 5 0

Zero

Little

Good

Total c. formation

Figure 2: No of Callus formation from Cotyledons 242

Regeneration of eggplants Solanum melongena var.Sukthi through somatic embryogenesis

As seen from the graph, we have a better development of calluses from explants taken from treatments of Cotyledon particularly taken with the 2.4 D concentrations 0.2 mg / l and 0.5 mg / l 2,4 D that reaches 100% to 20 callus explants experimented for each variant. But we can say that the phyto-hormone NAA gives a better result in the initiation of the particular callus concentration 0.2 mg / l NAA with about 90% to 20 explants obtained for this variant despite its development is relatively small.

The following Figure shows strands of shoots that are differentiated by amorphous calluses cells derived from Experiment 1 described above. For each calluses batch cell derived from the division of a single explants are created hundreds of shoots.

25 20 15 10 5 0

Figure 4: Shoot formation for Sukth 0,8 mg/l 2,4D Cotyledon 25 Zero

Little

Good

Total c. formation

20

Figure 3: The results of callus formation

15

from Hypcotic

10

As for the effect of phyto hormones 2,4-D and NAA in initiating callus explants, we received from Hypcotic, as seen from the graph, we have a better development of callus almost in the same manner as for 2,4 D NAA also observed no statistically validated quantitative changes. \While as a conclusion we can say that for explants in callus phase of eggplants Sukth taken from Hypcotic and Cotyledon have a good development and initiate callus cell in the range of 75% to variant 6 with the use of phyto-hormone concentration of 0.8 mg / l. NAA in explants originating from Cotyledon up to 100% in Variant 1 and Variant 2, with 0.2 mg / l and 0.5 mg / l to 2.4 D originating from explants of Cotyledon. Calluscells are easily induced, but the puzzle lies in the difficulty of the way they are presented and organogenesis from these cells callus produced. 2-The impact of phyto-hormones 2,4 D and NAA in the formation of new cuttings and seedlings from cells taken from callus explants of Cotyledon and Hypcotic. After 8 weeks (4 weeks after calluses) has been proved the formation of organogenesis of explants that originate from callus cells. 243

5 0

Zero

Little

Good

Total shoot. formation

Roots form

Figure 5: Number of shoot formation from Cotyledons

As is obvious from the figuregiven above, initiation of organogenesis from cells originating from calluses explants taken from cotyledons varies from 10% to 6 Variant treated with 0.8 mg / l NAA to 30% to Variant 1 treated with 0.2 mg / l 2,4 D achieving better results for version 3 and 4 where are used concentrations respectively 0.5 and 0.8 mg / l 2,4 D by about 60% and the maximum to that concentration used in Variant 4: 0 2 mg / l NAA with about 75%. Also the process of rooting is relatively good and does not create any problem for the production of seedlings using MS / 2. So initiation of organogenesis (development of shoots ) from cells derived from cotyledons calluses is

Vuksani et al

relatively good and gives a satisfactory shoots productivity.

Figure 6: Formation of organo-genesis of new seedlings, 0,2 mg/l NAA, Cotyledon 9 8 7 6 5 4 3 2 1 0 0,2mg/l 0,5/lmg 2,4-D 2,4-D Little

Good

0,8mg 2,4-D

0,2mg/l 0,5mg/l 0,8mg/l NAA NAA NAA

Total shoot formation

wherein the variant is used 0.2 mg / l NAA at 15% to 2 wherein the variant is used 0,5 / 2,4-D LMG with a result of the maximum to 3 wherein the variant is used 0, 8mg 2,4-D and has a shoots formation to the extent of 40% but with a reduced development as observed quite clearly from the figureNo.4. Finally the data from organo-genesis (shoots formation ) are formed from cotyledon compared with those created from hypocotyls. The analysis of variance of the data obtained in experimental means emerges that F is 8.25376 and F crit is 3.6823. We raise the hypothesis: H0 explant factor does not affect; H1 factor affects explants. According to the Fisher test since F (religion calculated) is greater than Fcrit (table F) Null hypothesis falls, then explants factor affects. Similarly, since propability P-Value is less than the probability 0:05 (level of importance, shows that once again falls remains H0 H1, eksplant factor influences. Finally statistically is verified that explants obtained from cotyledon from Albanian eggplants Sukth give a greater number than shoots of explants taken from hypocotyls by the method of Somatic Embio-Genesis. Conclusions

Roots

Figure 7: Number of shoot formation from hypocotyls

Figure 8: Formation of organo-genesis in new seedlings from hypocotyls, 0,8mg 2,4-D

From the results obtained by treatment of calluses cells with hypocotyls to different concentrations of phytohormone indicated that organogenesis (shoots formation) is weak enough to hypocotyls. Shoot formation ranging from 0% to 4 244

Based on experimental data carried out in a modern European laboratory, that of Humbold, University of Berlin Germany, we can conclude the following recommendations: Solanum melongena multiplication eggplants var. Sukth technique through Genesis somatic embryo is successfully implemented and is recommended to be used by specialists for scientific purposes. Callus cells are easily induced by the use of different concentrations of phyto hormones 2,4-D and NAA. Calluses phase explants for the eggplants Sukth taken from Hypcotic, Cotyledon and have a good development and initiate callus cells in the range of 75% to VARIANT 6 with the use of phyto-hormone concentration of 0.8 mg / l NAA to explants originating from Cotyledon to 100% to Variant 1 and Variant 2 with 0.2 mg / l and 0.5 mg / l to 2.4 D to explants of Cotyledon origin. Organogenesis from callus cells originating from cotyledons explants achieve best results than version 3 and 4 that used concentrations respectively 0.5 and 0.8 mg / l 2,4 D by about 60% and the maximum in

Regeneration of eggplants Solanum melongena var.Sukthi through somatic embryogenesis

Variant 4 which used concentration 0.2 mg / l NAA with about 75%. So as a conclusion, it is statistically verified that explants taken by Albanian eggplants Sukth cotyledon provide a greater number than explants taken from hypocotyls by the method of Somatic Embiogenesis.

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