In vitro somatic embryogenesis and plant regeneration in Acacia arabica

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PiantCell Reports

Plant Cell Reports (1987) 6:248-251

© Springer-Verlag1987

In vitro somatic embryogenesis and plant regeneration of cassava Lfiszl6 Szabados, Rodrigo Hoyos, and William Roca Biotechnology Research Unit, CIAT, A.A. 6713, Call, Colombia, South America Received October 21, 1986 / Revised version received March 13, 1987 - Communicated by G. C. Phillips

Abstract An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4-16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.i mg/l BAP, img/l GAq, and 0.01 mg/l 2,4-D. Regenerated plants were tranffferred to the field, and were grown to maturity. Introduction Cassava (Manihot esculenta Crantz) is most important staple food crops of tropics. It constitutes a major source to nearly 500 million people in over of Africa, Asia, and Latin-America (FAO,

one of the the lowland of calories 60 countries 1979).

Tissue culture methods have been developed at CIAT, for virus elimination, vegetative propagation, conservation and exchange of germplasm collections (Roca 1984). Implementation of molecular and cellular genetic methods in breeding programs requires an efficient regeneration system from somatic tissues. Leaves and shoots have occasionally been regenerated from callus grown from stem sections (Tilquin 1979), or from calli developed from leaf mesophyll protoplasts (Shahin and Shepard 1980). Plant regeneration through somatic embryogenesis is well documented and usually requires a two-step procedure. In many species in the induction period proembryos and embryos are formed on the explants in the presence of auxin. Embryo maturation and germination takes place when auxin is removed, or its concentration is lowered drastically (Ammirato 1983). In cassava, somatic embryos have been induced on cotyledons, embryonic axes of seeds and on young leaf lobes of in vitro cloned plants (Stamp and

Offprint requests to: W.M. Roca, CIAT, Miami, c/o Fernando Posada, 1380 N.Y. 78 Avenue, Miami, FL 33126, USA

Henshaw 1982, Stamp 1984, and Henshaw 1986). Cassava is a vegetatively propagated plant, therefore plant regeneration from vegetative tissues of well characterized, clonally propagated plants, is important. The aims of our study were: - to establish a plant regeneration system through somatic embryogenesis from vegetative tissues of in vitro propagated cassava plants; - to test a range of cassava genotypes and wild Manihot species for their capacity to produce somatic embryos; - to investigate the possibility of inducing secondary embryogenesis and to establish continuously proliferating, long-term embryogenic cultures either on solid or in liquid media. Materials and methods Plant material: Fifteen varieties of cassava (.Manihot esculenta) and one genotype of each M.cecropiaefolia and M.aesculifolia were selected from the in vitro germplasm collection of CIAT (see Table i). Aseptic shoot cultures were maintained on standard propagation medium, which is a modification of the medium devised by Kartha et al. (1974): Murashige and Skoog (MS) basal medium (1962) with 1 mg/l Thiamin-HCl, I00 mg/l m-inositol, 2% sucrose, 0.02 mg/l NAA (B-Naphthaleneacetic-acid), 0.05 mg/l BAP (6 Benzylaminopurine), 0.05 mg/l GA 3 (Gibberellic-acid) solidified with 0.6% Difco agar, routinely used for cassava meristem culture in our laboratory (Roca 1984). Shoot cultures were grown under a 12 hr photoperiod (300 lux, Sylvania fluorescent tubes), at 28°C during the light period and 25°C during the dark period and were micropropagated by nodal cuttings every 12 months. Induction of somatic embryogenesis and plant regeneration was based on the procedure described by Stamp and Henshaw (1982, 1986). Induction of somatic embryogenesis: Immature leaves of 3-6 mm, and shoot tips of 2-3 mm length were excised and placed on solid induction medium in i0 cm diameter petri dishes. Induction medium was the basal culture medium of Murashige and Skoog (1962), usually supplemented with 8 mg/l 2,4-D and solidified by 0.8% agar (Difco). The effect of different concentrations of 2,4-D (1,2,4,8,16 mg/1) was tested in several experiments. Inoculated petri dishes were sealed with Parafilm and incubated

249 at 27°C in continuous darkness.

Results and Discussion

Secondary embryogenesis: To induce secondary embryogenesis, somatic embryos were subcultured on solid or liquid induction medium under the same conditions as described for the induction period. Secondary cultures on solid medium were maintained in the dark and transferred to fresh induction medium every 4-6 weeks. When liquid cultures were established, groups of fast proliferating somatic embryos were transferred into 125 ml Erlenmeyer flasks, containing 20 ml of liquid induction medium. Liquid cultures were maintained on a rotary shaker at 120 rpm, at 25-27°C and subcultured every 7-10 days.

Since seeds of cassava show high genetic diversity and sexual propagation results in high genetic segregation, immature tissues of in vitro vegetatively propagated plants were chosen as explant.

Plant regeneration: Explants with developed somatic embryos and proliferating secondary embryos were transferred to solid regeneration medium, in order to encourage plant development. Regeneration medium was MS basal medium, supplemented by 0.I mg/l BAP, 0.01 mg/l 2,4-D and 1 mg/l GA 3 unless otherwise stated. The effects of different combinations and concentrations of BAP, NAA, 2,4-D and GA. on plant regeneration was tested J . in several exper!ments. Regeneratmng cultures were incubated in continuous 3000 lux light, at 25-27°C for 4-6 weeks. Developing plantlets (1-3 cm in size) were transferred to standard propagation medium for further growth and for vegetative propagation. Plantlets with 4-6 leaves were potted and transferred to a greenhouse as described before (Roca 1984). Histology: Embryos were fixed in FAA (formalin/glacial acetic acid/ethanol, 5:5:90, v/v), dehydrated through ethanolisopropyl alcohol-xylol series and embedded in paraffin. The tissues were sectioned at a thickness of 5 um and stained with Harris hematoxilin and eosin (Luna 1968).

Fig. i. Somatic embryos developing on immature leaf explant (20X). Arrow shows somatic embryo initials. Fig. 2. Cassava somatic embryo (80X). Fig. 3. Longitudinal section of a somatic embryo with initials of secondary embryos (IOOX).

Induction of somatic embryogenesis: After one week on induction medium, cultured immature leaflets became swollen and nodular embryogenic tissue formed near the main vein (Fig. I). Somatic embryos developed from these embryogenic tissues after two more weeks of culture. (Fig. 2). Most of the explants produced friable callus, which proliferated at the cut surface of the explant (Fig. i)o Somatic embryos developed only from the nodular, embryogenic tissues and never from the friable, disorganized callus. Longitudinal sections of the developed somatic eNbryos showed typical bipolar meristems and provascular strands (Fig. ~). The process of somatic embryo formation and the morphology of the developed embryos were similar to that, described by Stamp (1984) and Stamp and Henshaw (1982, ]986). The frequency of somatic embryo formation on shoot tip explants was generally lower than that on immature leaf explants. In four experiments, performed with varieties M Col 22, M Col 1505, M Col 1940 and M Ven 270, the average frequency of somatic embryo formation on shoot-tip explants was 29.5%, while on immature leaf explants, it was 47%. Older, fully expanded leaves, 20 mm in size, have never formed somatic embryos. This observation emphasizes the importance of the juvenility of the explant tissue in the induction of somatic embryogenesis in cassava. Young leaves have prove~ to be embryogenic in other plant species, such

as in millet (Haydu and Vasil 1981). Somatic embryogenic potential has been reported to vary from one species to another and often differs between varieties of the same species (Vasil 1982, Brown and Atanasov (1985). Stamp (1984) noted differences in embryogenic responses of several cassava cultivars using sexual embryonic axis and cotyledon

250 explants. In order to compare the embryogenic capacity of different Manihot genotypes, we tested the induction of somatic embryogenesis in immature leaf explants of 15 cassava (M. esculenta) cultivars and in one accession of each of M.cecropiaefolia and M.aesculifolia (Table i). Wide variation was found among the genotypes tested. The best respOnse was observed in cultivars M Ven 270 and M Col ]505. Usually 6070% and sometimes as high as 80% of the explants formed somatic embryos. Other cassava vayieties showed moderate to low embryogenic responses. Somatic embryogenesis was not observed in M.cecropiaefolia or M__~. aesculifolia (immature leaves or shoot tipsy when they were subjected to the same induction treatment.

8O

"E 6O X ¢,,..

40

E o 20 E uJ

0 1

Table i: Embryogenic capacity of immature leaf explants of cassava (Manihot esculenta) and wild Manihot genotypes.

High M M M M

Ven Col Col Col

270 1505 22 2215

Moderate

Low

No

M Mal l M Bra 12 MGI 1940 M Col 1684 M Per 302 CM 955-2

M Col 1468 M Ven ~5 M Col 1438 CM 976-15 CM 430-37

M.cecropiaefolia M.aesculifolia

In table 1 high embryogenic capacity indicates that repeatedly more than 50% of the explants formed somatic embryos when cultured on the induction medium. In the moderate category, embryo formation was usually between 25-50%, while with low embryogenic capacity, less than 25% of the explants produced somatic embryos. Among the components of the culture medium 2,4-D appears to be important for the induction of somatic embryogenesis in different species(Stamp and Henshaw 1982, Ammirato 1983, Brown and Atanassov 1985). We compared the effects of different 2,4-D concentrations (1-16 mg/l) in the induction medium. The proportion of leaf explants showing embryo formation significantly increased with higher auxin levels up to 8 mg/l 2,4-D (Fig. 4). The rate of somatic embryo formation on media with 8 mg/l and 16 mg/l 2,4-D was similar. No significant differences have been found at 5% level between the three cassava varieties tested (M Col 1505, M Ven 25, M Col 1468) with respect to their 2,4-D requirements for somatic embryo formation (data not shown). Secondary embryogenesis: When somatic embryos were separated from primary explants and transferred onto fresh induction medium (usually with 4 or 8 mg/l 2,4-D), secondary embryos developed, as described before (Stamp 1984). First, small swellings appeared on the apical region of the somatic embryo, then new embryos developed from these initials (Fig. 3 and 5). Continuous proliferation of secondary embryos could be achieved by subculturing these embryos monthly on induction medium (Fig. 5). Secondary embryogenic cultures have been maintained for a cultu[e period of up to 20 months without the loss of regenerative capacity. Secondary embryos could proliferate in liquid induction medium with continuous shaking (Fig. 6). Embryogenic cultures usually grew as big clumps which were easily separated into individual embryos using forceps. Packed volume measurements indicated, that the doubling time of the secondary embryo proliferation was approximately I0 days in liquid medium (data not shown).

2 4 8 16 2,4-D concentration (rag/I)

Fig. 4. The effect formation of somatic leaf explants of the bars at each point (data from 3 repeated

of 2,4-D concentration on the embryos on cultured immature cultivar M Col 1505. Vertical represent the standard error experiments).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Secondary embryogenesis and liquid cultures have been initiated with four cultivars (M Col 1505, M Col 22, M Col 1940, M Ven 270) but none of the cultivars allowed the establishment of a fine, embryogenic cell suspension. Although cell suspensions were formed easily in liquid medium; ~hey were composed of loose aggregates of nonembryogenic, callus derived cells. No embryo formation has been observed in such cell cultures. Plant regeneration: To promote the further differentiation of the somatic embryos and the development of plants, explants were transferred to regeneration medium and were incubated under continuous light. Green, folious structures were formed first and plantlets with cotyledon-like leaves emerged by the outgrowth of the shoot and root axis, after 20-30 days of incubation (Fig. 7). Normal leaves and shoots were formed after the plantlets were transferred onto standard propagation medium, or medium supplemented with 1 mg/l GA 3. Among the hormone combinations tested, a medium with 0.i mg/l BAP, 0.01 mg/l 2,4-D, 1 mg/l GA~ was found to be significantly superior (at 5% l~vel) in supporting plant regeneration from somatic embryos (Table 2). Callus formation was more intense on media with higher BAP concentrations, and more adventitious roots formed in the presence of GA 3. Table 2: Regeneration of cassava plantlets somatic embryos, attached to parental explants.

from

Medium

1

2

3

4

5

6

Regenerating explants (%)

35.3 +8.2

50.0 +7.9

30.8 +7.4

31.6 +7.5

22.2 +6.9

12.5 +8.3

No. of plants /explants

0.50 1.15 0.90 0.89 0.13 0.19 +__0.09 +__0.07 +--0.05 +__0.05 +__0.08 +-0.1

Leaf explants with somatic embryos were placed on MS regeneration medium with different hormone combinations: Medium l:O.img/l BAP; 0.01 mg/l 2,4D (Stamp and Henshaw 1982). Medium 2: 0.i mg/l BAP; 0.01 mg/l 2,4D; 1 mg/l GAq. Medium 3: 0.5 mg/l BAP; 0.I mg/l NAA (Shahin and~ Shepard 1980). Medium 4: 0.5

251

Fig. 5. Proliferating secondary embryos of a 15months-old culture (20X). Insert shows a secondary embryo with new embryo initials (50X). Fig. 6. Clumps of secondary somatic embryos grown in liquid medium (50X). Fig. 7. Germinating somatic embryos. Fig. 8. Regenerated cassava plants in the field. .

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Acknowledgements We thank Dr. J.Cock and Dr. J.Reeves for critical reading of the manuscript and to Mrs. L.A. Cartagena for careful typing.

References

.

mg/l BAP; 0.I mg/l NAA; 1 mg/l GA~. Medium 5: 1 mg/l GAn. Medium 6: 0.04 mg/l BAP; J0.02 mg/l NAA; 0.05 mg/~l GA 3 (Roca 1984). Data represent the average of 6 independent experiments made with cultivars M Col 22 and M Col 1505. Under our conditions 40-60% of the embryo containing explants formed at least one plantlet (Table 2). Usually 2 or 3 and sometimes as many as 15-20 plantlets emerged on one explant. The number of somatic embryos on one explant usually was higher than that which actually germinated. This means, that only a portion of the somatic embryos germinated, and developed into plants. The rest produced either cotyledon -like structures, adventitious roots, or callus. Rooted plantlets have been transferred to greenhouse and to field for further growth and evaluation (Fig. 8). Mature plants have been succesfully regenerated in all of the genotypes tested so far: M Col 1505, M Col 22, M Col 2215, M Ven 270, M Col 1940. Preliminary examination of the regenerated plants revealed no obvious phenotypic variation. Plants were able to flower and set seeds normally. Our results indicate, that plant regeneration of a number of cassava cultivars is possible, using the previously described procedure of somatic embryogenesis (Stamp and Henshaw 1982). Secondary embryogenesis have also been established, permitting the the plant regeneration from long term embryogenic cultures. This achievement may help the future adaptation of cellular and molecular genetical methods in cassava breeding programs.

An~virato PV (1983). In: DA Evans, WL Sharp, PV ~Ammirato, Y Yamada (edls.), Handbook of Plant: Cell:Culture Vol I, MacMillan Publishing Company, New York, pp. 82-123. Brown DCW, Atanassov A (1985) Plant Cell Tissue Organ Culture 4: ]11-122. FAO (1979) Agriculture Commodity Projections 19751985. Food and Agricultural Organization o£ the United Nations. Rome. Haydn Z, Vasil IK (1981) Theor.Appl.Genet. 59: 269273. Kartha KK, Gamborg OL, Constabel F, Shyluk JP (1974) Plant Sci. Lett. 2: 107-113. Luna LG (1968) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw Hill Book Company, New York, p.32. Murashige T, Skoog F (1962) Physiol. Plant. 15: 473-497. Roca WM. (1984) In: WR Sharp, DA Evans, PV Ammirato, Y Yamada (eds.), Handbook of Plant Cell Culture vol. 2, MacMillan Publishing Company, New York, pp. 269-301. Shahin EA and Shepard JF (1980) Plant Sci. Lett. 17: 459-465. Stamp JA and Henshaw GG (1982) Z.Pflanzenphysiol. 105: 183-187. Stamp JA and Henshaw GG (1986) In: LA Withers, PG Anderson (eds.) Plant Tissue Culture and its Agricultural Applications, pp. 149-157. Stamp JA (1984) Ph.D. thesis, University of Birmingham U.K., 1984. Tilquin JP (1979) Can.J.Bot. 57: 1761-1763. Vasil IK (1982) In: IK Vasil, WR Scowcroft, KJ Frey (eds.) Plant Improvement and Somatic Cell Genetics, Academic Press, New York, p.179-204.

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