PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS IN ...

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this study is the induction of somatic embryogenesis and plantlet formation from the ... Key words: Somatic embryogenesis, Plant regeneration, Tomato cv S-22.
Journal of Cell and Tissue Research Vol. 11(1) 2521 - 2528 (2011) ISSN: 0974 - 0910 (Available online at www.tcrjournals.com)

Original Article

PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS IN CULTIVATED TOMATO (Solanum lycopersicum L.) ?

GODISHALA, V.,1 MANGAMOORI, L.,1 AND NANNA, R.

Department of Biotechnology, Kakatiya University, Warangal 506 009; 1Center for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad 500 072. E. mail: [email protected] Received: January 25, 2011; February 19, 2011 Abstract: An efficient protocol has been developed for inducing somatic embryogenesis and plantlet formation from cotyledon cultures of cultivated tomato (Solanum lycopersicum L.) cv S -22. Cotyledon explants from 10-days old invitro seedlings were cultured on MS medium supplemented with αnapthalene acetic acid (NAA)/2,4–dichlorophenoxy aceticacid ( 2,4-D) / Indole -3-butyric acid (IBA) / Indole acetic acid (IAA) + 6- Benzyl aminopurine (BAP). Cotyledon explants produced embryogenic callus with different stages of embryoids, when cultured on Murshige and Skoog’s (MS) medium supplemented with 0.2/0.5mg/L IAA + 2.0 – 4.0 mg/L BAP. The explants with primary callus developed on NAA / 2,4-D / IBA and IAA were subsequently cultured on MS medium containing IAA (0.1mg/L) + 2.0-4.0 mg/L BAP triggered shoot induction. Treatment with 0.5mg/L IAA + 3.5 mg/L BAP resulted in the induction of maximum frequency of somatic embryogenesis with highest number of somatic embryos directly from the explant. Somatic embryo induction and maturation took place up to bipolar / torpedo shaped on the same medium. Where as bipolar/torpedo shaped somatic embryos developed into whole plantlets on GA3 + BAP containing medium. The distinct feature of this study is the induction of somatic embryogenesis and plantlet formation from the cotyledon explants of cultivated tomato cv S-22 . Key words: Somatic embryogenesis, Plant regeneration, Tomato cv S-22.

INTRODUCTION Tomato (Solanum lycopersicum L.) is an important commercial crop world wide. The tomato crop is very versatile and is grown either for fresh market or processing .Tomato is rich in vitamins A and C and fiber and is also cholesterol free [1]. In vitro regeneration of cultivated tomato has been a subject of research because of the commercial value of the crop and its amenability for further improvement via genetic manipulation through genetic transformation [2]. Development of novel germplasm through plant tissue culture and gene transfer holds great potential for improving the quality, resistance to diseases and agronomic characters of tomato. A prerequisite for genetic transformation of a crop system is the availability of a good protocol for in vitro plant

regeneration. There have been several reports of plant regeneration via organogenesis in tomato using different explants such as cotyledon, leaf and hypocotyls [3-6]. Attempts to regenerate plants via somatic embryogenesis are limited, although somatic embryos were developed from different explants [710]. In this communication we report high frequency plant regeneration via somatic embryogenesis in cultivated tomato cv S-22. This protocol is simple, reproducible and may be suitable for agrobacterium mediated genetic transformation. MATERIALS AND METHODS Plant Material: The seeds of cultivated tomato cv S-22 were obtained from Max Agri-Genetics Pvt Ltd, Hyderabad. These were washed under running tap

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J. Cell Tissue Research 3 weeks

3 – 4 weeks

Step - I MS + 0.2/0.5mg/L IAA + 2.0 -4.0mg/L BAP

Step - II

Embryogenesis with Callus

Step - I Cotyledon explant

MS+0.1 mg/L IAA+ BAP

Bipolar Embryos on GA3+BAP

Organogenesis

Germination of Somatic embryos

Rooting on MS + 0.2 mg/L IAA

Plantlet

3 weeks MS+NAA+BAP MS+2,4-D+BAP MS+IBA+BAP MS+IAA+BAP (0.2/0.5mg/L) + (0.51.5mg/L)

Step - I

Step - II Callusing

IAA+BAP (0.1 mg/L) + (24mg/L)

Organogenesis

3 weeks 3 – 4 weeks

Fig 1 : Schematic representation of the methodology adopted and results obtained during organogenesis and somatic embryogenesis from cotyledon

water for 15 minutes followed by Tween-20 and 0.1 % ( w/v) mercuric chloride for 5 minutes. The seeds were then rinsed three times with sterile distilled water. The sterilized seeds were inoculated on halfstrength Murashighe and Skoog’s (MS) medium [11] and were kept in dark for 24 hrs.The seeds were germinated aseptically. Culture media and culture conditions: Cotyledon explants (10 days old) (0.8-1.0 cm2) from axenic seedlings were inoculated on MS medium supplemented with different concentrations of IAA/ NAA/2,4-D/IBA (0.2/0.5 mg/L) + BAP (0.5-1.5mg /L)/2.0 – 4.0 mg/L BAP and 0.1 mg/L IAA + 2.0 – 4.0 mg/L BAP containing sucrose (30 g/L) and mesoinositol (100 mg/L). After 3 weeks of culture on step I media explants with torpedo/bipolar embryos were transferred on to MS media ( step II ) containing ½ strength MSO, MSO and different concentrations of GA3 (0.1- 0.5) + BAP(0.1-0.5mg/L) (Fig.1) All the media were supplemented with 3% (w/v) sucrose and solidified with 0.8% (w/v) agar (Difco Bacto-Agar). After adding the growth regulators, the pH of the media was adjusted to 5.8 ± 1 either with 0.1 N HCl or 0.1 N NaOH and autoclaved at 121 oC and 1.06 kg/cm2 for 15 – 20 minutes. Explants

cultured singly in culture tubes (25 X 150 mm) and maintained at 25 ± 1 oC under cool white-fluorescent lights with light intensity of 50 µmol m-2 s-1 under 16h of photoperiod for 4 – 6 weeks. The cultures were transferred to fresh medium after an interval of 4 weeks. There were 4 blocks with 20 cultures per treatment and each experiment was repeated twice. Data on somatic embryogenesis and germination were statistically analyzed using mean and standard error. Histological study: To ascertain the embryogenic nature of differentiating structures, cultured tissues were subjected to a histological study. Tissue bearing somatic embryos at different developmental stages was fixed in acetic acid : alcohol (1:3) then dehydrated in ethanol-xylol series, embedded in paraffin wax, sectioned at 10m thickness and stained with haematoxylin and basic fuschin. Total number of embryos and the percentage of globular, heart and torpedo/bipolar shaped were scored. Acclimatization of plants: The regenerated plants were taken out and washed with sterile distilled water under aseptic conditions to remove remains of agar medium. Later these were shifted to plastic pots containing sterile vermiculite: soil (1:1) covered with polythene bags inorder to maintain 80% RH and kept

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Godishala et al.

Fig. 2 a

Fig. 2 b

Fig. 2 c

Fig. 2 d

Fig. 2 e

Fig. 2 f

Fig. 2 g

Fig. 2 h

Fig 2: (a-h) Plantlet formation through somatic embryogenesis in cultivated tomato cv S-22. a- Group of somatic embryos (globular); b-Heart shaped embryo (note the notch); c- Different stages of embryoids ; d - Torpedo shaped embryo ; e- A group of cotyledonary stage embryos; f- Well developed cotyledonary stage embryo; g- Cotyledonary young and mature embryos ; h – Plantlet formation.

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J. Cell Tissue Research Table 1: Effect of IAA + BAP on somatic embryogenesis in cotyledon explants of cultivated tomato cv S-22. * Mean ± Standard error

Concentration of Plant growth regulators (mg/L) IAA + BAP 0.2 + 0.5 0.2 + 1.0 0.2 + 1.5 0.2 + 2.0 0.2 + 2.5 0.2 + 3.0 0.2 + 3.5 0.2 + 4.0 0.5 + 0.5 0.5 + 1.0 0.5 + 1.5 0.5 + 2.0 0.5 + 2.5 0.5 + 3.0 0.5 + 3.5 0.5 + 4.0

Percentage of response for somatic embryo formation Morphogenesis 42 47 50 57 70 80 85 92 50 55 58 65 75 90 98 80

Brown callus Callus Callus Somatic embryos Somatic embryos Somatic embryos Somatic embryos Somatic embryos Brown callus Callus Callus Somatic embryos Somatic embryos Somatic embryos Somatic embryos Somatic embryos

in culture room for 3 weeks. Later, they were shifted to earthenware pots containing garden soil and maintained in the research field.

Cotyledon explants produced embryogenic callus on 0.2/0.5 mg/L IAA + 2.0-4.0 mg/L BAP with globular, heart - shaped and bipolar somatic embryos after four weeks of culture (Fig 2). The highest frequency of somatic embryos development from the initial globular stage to bipolar/torpedo shaped occurred in the presence of 0.5mg/L IAA + 3.5mg/L BAP (Figs. 3-4 ). While 0.5-1.5 mg/L BAP showed no effect on embryo induction, a dramatic increase in somatic

10 ± 0.73 12 ± 0.76 14 ± 0.79 18 ± 0.46 19 ± 0.92

04 ± 0.49 05 ± 0.38 08 ± 0.49 07 ± 0.62 04 ± 0.45

03 ± 0.52 03 ± 0.49 06 ± 0.68 05 ± 0.49 04 ± 0.62

15 ± 0.96 06 ± 0.49 18 ± 0.80 06 ± 0.48 24 ± 0.98 10 ± 0.62 32 ± 0.83 12 ± 0.49 20 ± 0.60 05 ± 0.56

04 ± 0.49 05 ± 0.56 06 ± 0.73 10 ± 0.49 05 ± 0.31

embryogenesis occurred in the presence of 3.5 mg/ L BAP (Table-1). All the stages upto torpedo-shaped were observed on 0.2/0.5mg/L IAA + 2.0-4.0 mg/L BAP (Fig.1).A low concentration of BAP (2.0 mg/L) produced the somatic embryos with less number and percentage of somatic embryogenesis whereas higher concentrations supported the maximum percentage of somatic embryogenesis followed by more number of all the stages of somatic embryos (Table 1). The explants containing somatic embryos were transferred to the same fresh media for further maturation. But no further development was observed even after two subcultures. Development of somatic embryos appeared to be asynchronous with a wide range of varied sizes and structures. As a result, various stages of somatic embryos development was observed in the same cluster of embryos originally from the explant.

RESULTS Cotyledon explants of tomato cv S-22 produced callus on step-I media. Callus produced on each of the two media was morphologically distinct. The media containing 2,4-D + BAP produced nodular callus, whereas NAA + BAP / IBA+BAP / IAA+BAP induced friable callus. However, these types of callus did not differentiate into shoots or embryos after 2-3 subcultures on the same media (Fig.1). After two weeks, the explants with small primary calli were transferred to step II media containing 0.1mg/L IAA+BAP (2-4 mg/L) and cultured for 3-4 weeks on this medium triggered shoots development from meristematic centres originated on the friable/nodular callus. Cotyledon explants produced shoots on step I media containing MS medium fortified with 0.1 mg/ L IAA + BAP (2.0 – 4.0 mg /L) and rooted on 0.2 mg/L IAA (Fig.1).

Average number of Somatic embryos per explant (±SE)* Globular Heart haped Bipolar

Table 2: Effect of GA3 + BAP on germination of somatic embryos in cultivated tomato cv S-22. * Mean ± Standard error, ** Callusing

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Medium + Growth regulators

Germination frequency (Mean ± SE)*

1/2 MSO MSO MS + GA3 (0.1) + BAP (3.0) MS + GA3 (0.2) + BAP (3.0) MS + GA3 (0.3) + BAP (3.0)

33.7 ± 0.702 93.8 ± 0.990 71.2 ± 0.916

MS + GA3 (0.4) + BAP (3.0)**

28.5 ± 0.73

MS + GA3 (0.5) + BAP (3.0)**

9.0 ± 0.76

Godishala et al.

Fig. 2 i

Fig. 2 j

Fig. 2 k

Fig. 2 l

Fig 2: (i – m) Histological studies of somatic embryogenesis in cultivated tomato cv S- 22. i- globular ; j- heart shaped (with clear notch ) k- cotyledonary stage embryo; l – enlarged view of the same (note leaf primordia & apical meristem); m- a group of cotyledonary stage embryos.

Fig. 2 m

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J. Cell Tissue Research Globular

Heart

Bipolar

100 80 60 40 20 0

Avarage number of somatic embryos per explant

% of response for somatic embryo formation

Percentage of Response

20 15 10 5

0. 2+ 0. 5 0. 2+ 1. 0 0. 2+ 1. 5 0. 2+ 2. 0 0. 2+ 2. 5 0. 2+ 3. 0 0. 2+ 3. 5 0. 2+ 4. 0

0

Fig.3: Effect of IAA + BAP on somatic embryogenesis in cotyledon explants of cultivated tomato cv s-22

Concentration of plant growth regulators (IAA + BAP) mg/L Heart

120 100 80 60 40 20 0

Bipolar

35 30 25 20 15 10 5 0

Avarage number of somatic embryos per explant

Globular

Fig.4: Effect of IAA + BAP on somatic embryogenesis in cotyledon explants of cultivated tomato cv s- 22

0. 5+ 0. 5 0. 5+ 1. 0 0. 5+ 1. 5 0. 5+ 2. 0 0. 5+ 2. 5 0. 5+ 3. 0 0. 5+ 3. 5 0. 5+ 4. 0

% of response for somatic embryo formation

Percentage of Res ponse

Conce ntration of plant grow th regula tors ( IAA + BAP ) mg/L

Germination frequency %

Germination frequency 100

Fig.5: FEffect of GA3 + BAP on germination of somatic embryos in cult iv at ed tomato cv s-22

80 60 40 20 0 1/2 MSO

MSO

3.0 + 0.1

3.0 + 0.2

3.0 + 0.3

3.0 + 0.4

3.0 + 0.5

Concentration of BAP + GA3(mg/L)

Somatic embryo germination and plant let formation: The torpedo-shaped/bipolar embryos when transferred to ½ strength and full strength MS basal media didn’t germinate. Whereas the somatic embryos cultured on MS medium supplemented with 0.1-0.5 mg/L GA3+3mg/L BAP for further maturation and complete plants with well developed roots were obtained within 3-4 weeks of culture (Fig. 2eh). Maximum frequency of somatic embryos germination was observed at 0.2 mg/L GA3 in comb-ination with 3.0 mg/L BAP followed by 0.5 mg/L GA3 (Table 2). At high concentration of GA3 less germination frequency was observed (Fig.5) with callusing.

After 4 weeks of somatic embryo germination, the plantlets were transferred to plastic pots containing sterile vermiculite: soil (1:1) mixture. Later, they were shifted to earthenware pots after hardening in the culture room and maintained in the research field under shady conditions. The survival rate of these plants was found to be 68 %. The plants were normal, and morphological and floral characters were similar to donor plants. Light microscopic observations of the sections of different stages of embryo development revealed globular embryo with a suspensor and subsequently

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Godishala et al. the embryo differentiated into heart-shaped and bipolar structures (Fig.2. i-m). Thus the embryos resembled zygotic embryos. During the present studies, proliferation of somatic embryos occurred in two ways: i) Formation of primary somatic embryos from the explants. ii) Proliferation of secondary embryos from primary embryos when somatic embryos maintained for a period of 2-3 months on the induction medium through repetitive embryogenesis. DISCUSSION Regeneration through somatic embryogenesis from cotyledon explants has not been reported so far. Here we report a simple method for inducing somatic embryos from cotyledon explants directly from embryogenic callus. The induction, development and germination was contr olled by plant growth regulators. Auxins (2, 4-D, IAA, NAA, IBA) when supplemented alone produced callus without any meristamatic centres. However IAA in combination with BAP (2.0-4.0 mg/L BAP) induced embryogenic callus tissue which on further culturing, produced somatic embryos. An exogenous supply of BAP has been found to improve somatic embryo development and germination in Solanum surattense [12] and Psoralea corylifolia [13].The supplementation of cytokinins during the histodifferentation phase can compensate for the detrimental effects of auxins on meristem development [14].

In the present investigation, GA3 appeared to promote further maturation and plantlet development. The addition of GA3 to the culture media was required for germination of somatic embryos and elongation; in its absence only torpedo-shaped embryos that usually did not develop further were formed on the callus surface [17]. Thus, somatic embryogenesis is an alternative and efficient method for plant propagation over regeneration via organogenesis [12]. The repetitive somatic embryogenesis found in tomato cv S-22 has great potential for its mass propagation and genetic transformation as it was observed in Solanum surattense [12]. In conclusion, this is the first report of a successful procedure to regenerate cultivated tomato cv S-22 via somatic embryogenesis. The protocol developed by us is simple, r eproducible and genotype independent. The cotyledon based regeneration protocol is expected to be very useful for the genetic improvement of cultivated tomato cv S-22 through recombinant DNA technology .The cotyledon is preferred explant for genetic transformation studies since nutrients readily penetrate the tissues and thus facilitates the antibiotic selection and effective suppression of non transformed cells and also readily availability of explants through out the year. ACKNOWLEDGEMENTS Mr. G. Vikram is grateful to Sri. Ch. Devender Reddy, Secretary cum correspondent, Vaagdevi Institutions and Mr. A. Sheshachalam Principal for their encouragement during his Ph.D work. REFERENCES

The effects of plant growth regulators on the ratio of globular to bipolar/ torpedo-shaped embryos indicated that they interfere with normal distribution of cell division and cell expansion during early embryogenesis. The transition of globular to heart – shaped to bipolar is critical step in somatic embryogenesis. Ammirato [15] pointed that normal development of somatic embryos required a fine temporal and spatial regulation of cell division, elongation and differentiation. Growth regulators may exert multiple effects in these processes depending on concentration or on the embryo stage at the time of application. In the present study asynchronous development of somatic embryo was noticed as reported in Solanum surattense and potato [12,16].

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J. Cell Tissue Research [8] Gill, R., Malik, K.A., Sanago, M.H.M. and Saxena, P.K.: J. Plant Physiol., 147: 273-276 (1995 ). [9] Philip, O.N., Sankaran, K.R. and Praveen, K.S.: Inter. J. Plant Sci., 157: 554-560 (1996). [10] Kaparakis, G. and Alderson, P.G.: J. Hort. Sci. Biotechnol., 77: 186-190 (2002). [11] Murashige, T. and Skoog, F.: Physiol. Plant., 15: 473 493 (1962). [12] Rama Swamy, N., Ugandhar, T., Praveen, M., Venkataiah, P., Rambabu, M., Upender, M. and Subhash, K.: Indian J. Biotech., 4: 414-418 (2005). [13] Sahrawat, A.K. and Chand, S.: Curr. Sci.,81: 13281331 (2001). [14] Merkle, S.A.: Somatic embryogenesis in Magnoliaceae In: Somatic embryogenesis and synthetic seed I. Biotechnology in agriculture and forestery, Vol 30 (Bajaj, Y.P.S. eds), Springer Varlag, Berlin, pp 388-403 (1995). [15] Ammirato, P.V.: Organizational events during somatic embryogenesis. In: Plant Tissue and Cell culture (Green, C.E., Somers, D.A., Hackett, W.P. and Biesboer, D.D. eds), AR Liss, Newyork, pp 57-81 (1987). [16] De Garcia, E., Martinez, S. and De Garcia, E.: J. Plant Physiol., 145: 526-530 (1995). [17] Roest, S. and Bokelmann, G.S. : Potato Res., 19: 173178 (1976).

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