Establishment of somatic embryogenesis of some mango cultivars (Mangifera indica L.) grown in Indonesia

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1 Establishment of somatic embryogenesis of some mango cultivars (Mangifera indica L.) grown in Indonesia Tri Muji Ermayanti*, Deritha Ellfy Rantau Research Centre for Biotechnology-Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor Km. 46 Cibinong 16911, Bogor, Indonesia Abstract Induction of somatic embryogenesis was carried out in several mango cultivars (Mangifera indica L.) grown in Indonesia. Immature fruits collected from Bogor, Cibinong, Serpong, Indramayu and Pasuruan areas were used as explants. Different compositions of basal medium, addition of 2,4-D ( mg/l) in the medium and the use of Phytagel at g/l were also tested in order to enhance somatic embryo developments. Cultures were treated with kinetin, GA 3 and TDZ to stimulate embryo germination and plantlet development. The results showed that in 3M medium containing 2.5 g/l Phytagel, formation of embryogenic callus was affected by length of explants and mango genotype, 23 out of 24 cultivars tested formed embryogenic callus. Depending on the cultivars tested, basal medium gave different effect on the maturation of somatic embryos. Addition of 0.5 mg/l 2,4-D was the best for somatic embryos development of Kensington Pride cultivar. Phytagel at concentration of 3 g/l was the best for embryo maturation of Lalijiwo, Gadung 21 and Bapang compared to those grown in medium with addition of 2.5, 3.5 and 4 g/l Phytagel, however, Phytagel at 4 g/l was the best for Gedong 115, and that at 2.5 g/l was the best for Irwin cultivar. Addition of GA 3 alone or in combination with TDZ enhanced embryo germination and plantlet development of Kensington Pride compared to the use of medium containing 2,4-D in combination with kinetin. Keywords: Mango, Somatic embryogenesis, Maturation, Germination INTRODUCTION Mango (Mangifera indica L.) is one of the famous tropical fruits, which have typical taste, and various fruit shapes. This fruit has great economic impor- *Correspondence to: Dr. Tri Muji Ermayanti Tel ; Fax tmermayanti@hotmail.com tance. In Indonesia more than 224 hundred indigenous and introduced cultivars grown in many areas. The central collection areas of mango cultivars in Indonesia are in Pasuruan (East Java) and Indramayu (West Java). Mango trees are highly heterozygous, and sexually-produced seedlings are therefore very variable (IBPGR, 1986). Usually they are propagated mainly from seeds or vegetatively by grafting and budding. Rooting of cuttings and layers is also possible for propagation but these techniques are not applied for commercial purposes (PROSEA, 1992). Tissue culture is an alternative method for micropropagation especially for specific purposes such as for in vitro conservation of superior cultivars and genetic manipulation. Many research showed that somatic embryogenesis using nucellus as explants was the most successful methods for in vitro propagation on mango. This technique has been applied on some Indian, Australian and American mango cultivars (Litz, 1984; Litz et al., 1998; Thomas, 1999). Many investigation had been done using these cultivars with most results reported that somatic embryogenesis induction and development was very much genotype-dependent. Production of somatic embryos had been investigated by addition of various combination of plant growth regulators (Litz et al., 1993; Pliego- Alfaro et al, 1996) and the use of semi-solid culture (Monsalud et al., 1995). Germination and plantlet regeneration from encapsulated somatic embryos (Ara et al., 1999) as well as plant regeneration from protoplast using Indian cultivars (Ara et al., 2000) had also been carried out. However, the applications of such technique had not been reported in mango cultivars grown in Indonesia. Induction of somatic embryos was initiated in Manalagi cultivar, but very limited results has been reported (Husen et al., 2001; Ermayanti et al., 2003). The aims of the research were to establish the induction of somatic embryogenesis on some mango cultivars grown in Indonesia, to stimulate maturation Establishment of somatic embryogenesis of some mango cultivars (Mangifera indica L.) grown in Indonesia 1

2 of somatic embryos well as to germinate the somatic embryos to plantlets. MATERIALS AND METHODS Immature mango fruitlets were collected from both mono- and polyembryonic cultivars grown in Bogor, Cibinong, Serpong, Indramayu and Pasuruan areas. Fruitlet length ranges from 15 to 57 mm were used as explants. Fruitlets were washed with tap water containing 1% liquid detergent and left in the running water for about 30 min followed by surface sterilization using 96% ethanol for 2 min. Explants were then soaked in 1% sodium hypochlorite for 30 min, and rinsed with 3 changes of sterile distilled water. Before dissection, the fruitlet length was measured. Fruitlets were then bisected longitudinally to produce symmetrically equal halves. The seeds were separated from the seed coat and zygotic embryos were cut and discarded, only nucellar tissues attached with ovules used to induce the formation of embryogenic callus. Explants were then placed in a modified 3M medium (Litz & Yurgalevich, 1997) containing 2.5 g/l Phytagel as gelling agent with the nucellar tissues touched the medium for induction of embryogenic callus. The cultures were incubated in a culture room at 26 o C in darkness for callus induction. For embryo maturation, embryogenic callus cultures were maintained in an incubation room with low light intensity ( lux), and in higher light intensity ( lux) for germina-tion of embryos and plantlet development. Subculturing into a fresh medium was done frequently every 1-2 weeks when the medium had turned to brown due to the production of the phenolic compounds. Medium with different nutrient compositions (the use of half and full strength of B5 macro nutrients, and elimination of (NH4) 2 SO 4 ) and level of 2,4-D ranged from 0.5 to 2.0 mg/l were used to develop somatic embryo maturation, whilst medium containing various growth regulators, such as 2,4-D, kinetin, GA 3 or TDZ were utilized to enhance embryo germination. The growth regulator utilized in this experiment referred to the best treatment for Indian mango cultivars investigated by Thomas (1999). Phytagel at concentration of g/l were also added to 3M medium for embryo maturation. The medium ph was adjusted to 5.8 prior to autoclaving at 1 atm, 120 o C for 20 min. RESULTS AND DISCUSSION The results showed that 23 out of 24 mango cultivars formed somatic embryos and developed into proembryonic masses. The highest embryo-genesis was obtained from Gadung 21 collected from Pasuruan, and the lowest level of embryo-genesis found in Piit cultivar (Table 1) both cultivars are polyembryonic. The different number of initial culture was merely due to the limited number of fruitless collected in the field. Some fruits were infected by pests and diseases, therefore, only fresh fruitlets used for this experiment. Embryogenic callus was formed 4-6 weeks after culturing explants to 3M medium. In 3 days, cultures turned to brown, but this did not affect callus formation. Cultures were transferred into the fresh medium 5-6 weeks after planting. Table 1. Somatic embryogenesis of 24 mango cultivars after 2 months cultured in 3M medium. Cultivar Number of explants cultured Length of fruitlets producing somatic embryos (mm) Number of explants producing somatic embryos (%) Apel (24.0) Arumanis & 36 2 (14.3) Arumanis (33.3) Bapang 7 47 & 52 2 (28.6) Cengkir (31.2) Daging (17.4) Delima & 36 2 (14.3) Gadung (50.0) Gadung (66.7) Gedong (33.3) Gedong Gincu (22.2) Golek (9.1) Golek & 43 3 (33.3) Irwin (28.6) Kensington Pride (20.1) Madu anggur (37.5) Malgova (38.5) Manalagi (30.8) Lalijiwo (36.1) Nam doc mai 11-0 Piit 156 * 11 (7.1) Saigon (25.0) Swarnarika (44.4) Yampulu (28.6) Note: *length for explants was not recorded Induction of somatic embryogenesis was affected by the length of immmature fruits used as explants, and this response was strongly cultivar-dependent. Besides browning, contamination was another problem which need to be eliminated. In the previous experiment, contamination could be eliminated in Malgova, Swarnarika, Gadung and Golek 31 cultivars (Ermayanti et al., 2003). In Indian and American mango cultivars, cultures forming callus was also affected by the size of immature fruits, length of discarded zygote and the presence of 2,4-D in the culture medium (Litz, 1984). In our experiment, 3M medium was also countaining 2,4-D at 1.0 mg/l. Development of Ermayanti and Rantau 2

3 embryonic callus was very slow in some cultivars tested, therefore, only a few cultivars which had better growth used for embryos maturation and germination experiments. Growth and development of somatic embryos was also influenced by basal medium compositions. Medium with full strength of B5 (Gamborg et al., 1968) macro nutrient was the best for embryo maturation of Lalijiwo, Kensington Pride, Irwin, Swarnarika, Gedong Gincu, and Gedong 115 cultivars, whilst the medium with half strength of B5 macro nutrients was the best for both Malgova and Piit cultivars. Elimination of (NH4) 2 SO 4 from the medium reduced somatic embryos formation and their development in Gadung 21, Gedong 115, Gedong Gincu, Lalijiwo, Malgova and Piit cultivars. Gamborg s B5 and Rugini Olive (RO) media enhanced formation of embryogenic callus on Indian Arka Anmol cultivar compared to MS medium. In this experiment intact half ovule nucellus cultures gave higher callusing response compared to excised nucellus used as explants (Thomas, 1999). Gamborg s B5 medium with full strength of macro nutrients was also best for germination and plantlet development of monoembryonic Amprapali cultivar compared to that with quarter and half strength of macro nutrients (Ara et al., 1999). Table 2. Effects of medium compositions on production of somatic embryos of some mango cultivars. Medium C = 3M medium; medium D = medium C with full strength of B5 macro nutrients; medium E = medium C with no (NH4) 2SO 4. Cultivar Number of somatic embryos on the medium C D E Gadung Gedong Gedong Gincu Irwin Kensington Pride Lalijiwo Madu Anggur Malgova Piit Swarnarika The level of 2,4-D also influenced development of somatic embryos in polyembryonic Kengsington Pride cultivars investigated. The development from embryogenic callus, globular, heart, torpedo shapes to cotyledonary stage was the best in the medium containing 0.5 mg/l 2,4-D compared to the medium containing 1.0 and 2.0 mg/l 2,4-D. At concentration of 0.5 mg/l, 2,4-D significantly gave high somatic embryos production compared to the addition of 2,4- D at 1.0 and 2.0 mg/l (Fig. 2). This treatment could be applied to other cultivars to find out the optimum level of 2,4-D which give the best development of somatic embryos in each cultivar. The addition of 2,4-D was also the influenced the development of somatic embryos on Arka Anmol cultivar after 1 month in culture. In this cultivar the best embryogenic response was found in the medium containing 3.0 mg/l 2,4-D (Thomas, 1999). Level of 2,4-D was also affected in some other cultivars (Litz et al., 1998). No. of embryos ,5 mg/l 1,0 mg/l 2,0 mg/l week Figure 2. Effects of 2,4-D at mg/l on the production of somatic embryos on Kensington Pride cultivar. Somatic embryos at torpedo and cotyledonary stages was recorded after 7 weeks in culture. Different cultivars had different responses to the Phytagel concentration added into the medium. Phytagel at concentration of 3 g/l was the best for embryo maturation of Lalijiwo, Gadung 21, and Bapang compared to those grown in medium with addition of 2.5, 3.5 and 4 g/l Phytagel, however, Phytagel at 4 g/l was the best for Gedong 115, and that at 2.5 g/l was the best for Irwin cultivar (Table 3). Phytagel at 2.5 g/l was the best for development of somatic embryos of Kensington Pride, but this medium became liquid after 3 weeks in culture. Therefore, subculturing was needed to be carried out every 2-3 weeks before the medium was melting. In medium containing 3.5 g/l Phytagel also gave high response for embryo maturation on Kensington Pride. At 4.0 mg/l Phytagel reduced embryo development in this cultivar (Tabel 3; Figs. 3 and 4). The level of gelling agent was also affected somatic embryo maturation after treatment with liquid medium on Hindi cultivar (Monsalud et al., 1995). Addition of GA 3 (5 mg/l) alone or in combination with TDZ (0.01 and 0.05 mg/l) enhanced embryo germination of Kensington Pride compared to the use of medium containing 2,4-D (1.0 mg/l) in combination with kinetin (1.0 mg/l). More than 20 embryos formed single shoot and root in medium containing GA 3 and TDZ, but only 12 plantlets were obtained from medium containing 2,4-D in combination with kinetin. In Arka Anmol cultivar, low level of TDZ in combination with GA 3 stimulated shoot and root development compared to addition of BA or TDZ in combination with GA 3 in higher concentration (Thomas, 1999). Combination Establishment of somatic embryogenesis of some mango cultivars (Mangifera indica L.) grown in Indonesia 3

4 of 2,4-D and kinetin was the best for germination and plantlet development of Amprapali cultivar regenerated from protoplast culture (Ara et al., 2000). Table 3. Effects of Phytagel concentrations ( g/l) on the production of mango somatic embryos. Somatic embryos at torpedo and cotyledonary stages was recorded after 7 weeks in culture. Number of somatic embryos on Cultivar medium with different level of Phytagel (g/l) Bapang Gadung Gedong Irwin Kensington Pride Lalijiwo Figure 2. Effects of Phytagel concentrations ( g/l) on the production of somatic embryos on Kensington Pride cultivar. Somatic embryos at torpedo and cotyledonary stages was recorded after 7 weeks in culture. Fig. 4 represents the development of embryogenic callus to plantlets but using different mango cultivars as an example. The efficiency of regeneration from nucellar tissue of mango has been genotype dependent in both polyembryonic (Litz et al., 1982; Litz, 1986) and monoembryonic cultivars (Litz, 1984; Jana et al., 1994; Litz et. al., 1998). This could be the case of mango cultivars tested in this experiment. Figure 4. Development of somatic embryogenesis on mango. Embryogenic callus induced from Gedong 115 cultivar after 8 weeks cultured in 3M medium (left); cotyledonary stage of Piit cultivar after 10 weeks grown in 3M medium (centre); shoots regeneration of Kensington Pride cultivar after 6 weeks cultures in medium containing 5 mg/l GA 3 and 0.01 mg/l TDZ (right). Growth and development of somatic embryos tested in this experiment is still slow. Shoot regeneration and plantlet development need to be stimulated. Ethylene biosynthesis should be investigated in the mango cultivars tested as in some other cultivars ethylene biosynthesis proven to have great significant effect on the development of somatic embryos (Litz and Yurgalevitch, 1997). Photoautotrophic culture including modification of in vitro environment, such as light intensity and CO 2 enrichment could be attempted in this experiment to increase the embryo germination as well as to stimulate the plantlet development. This technique had great beneficial to promote growth in many species including woody plant species (Ermayanti et al., 1999). 2.5 g/l 3.0 g/l 3.5 g/l 4.0 g/l Figure 3. Development of somatic embryos on Kensington Pride cultivar after 7 weeks cultured in the maturation medium containing different levels of Phytagel ( g/l). CONCLUSIONS 1. Somatic embryogenesis on mango was affected by size (length) of immature fruits used as explants. 2. Medium compositions, level of 2,4-D and Phytagel affected the maturation of somatic embryos and different cultivars gave different responses to development of the embryos. 3. Addition of GA3 alone or in combination of TDZ enhanced plantlet development on Kensington Pride cultivar. 4. This report contributes to the embryogenesis research on mango done in some other cultivars grown in India, USA, Australia and others Ermayanti and Rantau 4

5 initiated from nucellar tissues which was very strongly genotype-dependent. Acknowledgements The authors would like to thank Dr. R. Brettell from CSIRO Division of Plant Industry, Australia for the technical assistance on induction of mango embryogenesis and head of Germplasm Collection Garden at Puspiptek, Serpong and Cukurgondang, Pasuruan for providing mango fruitlets used as explants. We also would like to thank D.R. Wulandari for explants collection and culture preparation, E. Al Hafiizh for poster preparation and Mr. Jitno Rijadi for taking photographs. References Ara H, Jaiswal U, Jaiswal VS (1999). Germination and plantlet regeneration from encapsulated somatic embryos of mango (Mangifera indica L.). Plant Cell Reports. 19: Ara H, Jaiswal U, Jaiswal VS (2000). Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis. Plant Cell Reports. 19: Ermayanti TM, Imelda M, Tajuddin T, Kubota C, Kozai T (1999). Growth promotion by controlling the in vitro environment in the micropropagation of tropical plant species. Proceeding the International Forum on Conservation and Sustainable Use of Tropical Bioresources. NEDO-JBA. Tokyo Ermayanti TM, Rantau DE, Wulandari DR (2003). Effects of size of explants on the embryogenesis frequency on mango cultivars. National Biotechnology Seminar. Purwokerto, Indonesia. Gamborg OL, Miller RA, Ojima K (1968). Plant cell culture. I. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research. 50: Husen S, Wardiyati T, Sitompul SM (2001). Induction of somatic embryogenesis of mango (Mangifera indica L.) through in vitro. The 2 nd Indonesian Biotechnology Conference. Yogyakarta, Indonesia. IBPGR (International board for plant genetic resources) (1986). Genetic resources of tropical and subtropical fruits and nuts. (Excluding Musa). Rome Jana MM, Nadgauda RS, Rajmohan, K, Mascarenhas, AF (1994). Rapid somatic embryogenesis from the nucelli of monoembryonic mango varieties. In Vitro Cellular Development Biology-Plant. 30P: Litz RE, Yurgalevitch C (1997). Effects of 1-aminocyclopropane-1-carboxylic acid, aminoethoxyvinylglycine, methylglyoxal bis-(guanylhydrazone) and dicoclyhexyl-ammonium sulfate on induction of embryogenic competence of mango nucellar explants. Plant Cell, Tissue and Organ Culture. 51: Litz RE (1984). In vitro somatic embryogenesis from nucellar callus of monoembryonic mango. Hort Science. 19: Litz RE (1986). Mango. In: Handbook of plant cell culture. 4. Techniques and applications. (Evans DA, Sharp WR, Ammirato PV. Eds). Macmillan Publishing Co. New York. USA Litz RE, Hendrix RC, Moon PA, Chavez VM (1998). Induction of embryogenic mango cultures as affected by genotype, explanting, 2,4-D and embryogenic nurse culture. Plant Cell, Tissue and Organ Culture. 53: Litz RE, Knight RL, Gazit S (1982). Somatic embryos from cultured ovules of polyembryonic Mangifera indica L. Plant Cell Reports. 1: Litz RE, Mathews VH, Moon PA, Pliego-Alfaro F, Yurgalevitch C, DeWald SG (1993). Somatic embryogenesis of mango (Mangifera indica L.). In: Redenbaugh K (Ed.). SYNSEEDS: applications of synthetic seeds to crop improvement. CRC Press, Boca Raton, USA Monsalud MJ, Mathews H, Litz RE, Gray DJ (1995). Control of hyperhydricity of mango somatic embryos. Plant Cell, Tissue and Organ Culture. 42: Pliego-Alfaro F, Litz RE, Moon PA, Gray DJ (1996). Effect of absisic acid, osmolarity and temperature on in vitro development of recalcitrant mango nucellar embryos. Plant Cell, Tissue and Organ Culture. 44: PROSEA (Plant resources of south-east asia) (1992). Edible fruits and nuts. 2 (Verheij EWM, Coronel RE. Eds.): Thomas, P (1999). Somatic embryogenesis and plantlet regeneration from nucellar tissue of monoembryonic mango. J Hort Sci Biotechnol. 74: Establishment of somatic embryogenesis of some mango cultivars (Mangifera indica L.) grown in Indonesia 5