Plant Cell, 1"issue and Organ Culture 44: 189-194, 1996. 189 © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Improvement of somatic embryogenesis in highland-papaya cell suspensions

M. Jo rdan & J. Velozo Departamento de Ecolog£a, Facultad de Ciencias Bioldgicas, Pontificia Un&ersidad Cattlica de Chile, Casilla 114-D, Santiago, Chile

Received 18 October 1994; accepted in revised form 9 November 1995

Key words: adventive embryogenesis, Carica pubescens

Abstract

Axillary buds (2 ram) from 3-year-old Caricapubescens Lenn6 et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in N-N-medium supplemented with different growth regulators (naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2-3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.

Abbreviations: 2,4-D-dichlorophenoxyacetic acid; CH-casein enzymatic hydrolysate; BA-benzyladenine; FAA- formalin:acetic acid:alcohol; Glu-L-glutamine; IAA- indoleacetic acid; N A A - naphthaleneacetic acid; N N - Nitsch and Nitsch-medium (1969); TDZ- thidiazuron; SD- standard deviation

Introduction

Carica pubescens Lenn6 et Koch, one of the "high- land papayas", growing in the Andes Mountains is an important fruit species which gives higher yields under cooler climate conditions than C. papaya up to 2500 meters elevation (Cardenas, 1989; National Research Council, 1989). A characteristic feature of this species is the existence of different sex forms that affect fruit yield, especially when seeds are chosen for propaga- tion. Recently it has been reported that sex forms can be identified in C. papaya at the seedling stage (Mondal,

1992), making it possible to maintain these characters earlier by vegetative propagation. However, adult and mature field productive papaya plants with desirable traits are still of major interest for asexual propaga- tion of selected or proved material, although in vitro regeneration appears then more difficult as compared to juvenile explants (Litz & Conover, 1978; Drew & Smith, 1986; Rajeevan & Pandey, 1986; Drew, 1988) and establishment of explants is affected by sex type, seasonal responses and microbial infection (Litz & Conover, 1981). For C. pubescens the morphogenic potential of cells and of other explant types~ of juve-

190

nile condition has previously been shown (Jordan et al., 1983; 1986; Jordan, 1989; 1990; 1992). Mor- phogenic responses leading to a high yield of regener- ated plantlets by means of somatic embryogenesis and adventitious embryo formation from adult axillary-bud explants are presented in this report.

developing embryos was recorded using acetocarmin and Evan's blue according to Durzan (1988).

Results and discussion

Axillary buds

Material and Methods

Axillary buds (2 mm) from 3-year-old fruit-bearing plants grown in the greenhouse and collected during March (Fall) were cultured on NN medium containing NAA (ranging from 0.05 to 5.37/aM) or IAA (0.29- 5.71/aM) in combination with BA (4.44/aM), kinetin (4.65 #M), zeatin (4.56 /aM) or TDZ (4.54 /aM)). Explants were maintained during 3 months in culture at 24-t-1 °C under an 18-h photoperiod at 48 /amol m -2 s - l provided by daylight fluorescent lamps (Gen- eral Electric F40T10-SS). After this period, growth responses of axiflary buds, callus initiation and somat- ic embryo development were evaluated.

Cell suspensions were obtained from one-month- old calluses originated from axillary buds, grown on NN-medium (NAA 2.69/aM; BA 4.44 #M) not show- ing embryo initiation. For induction, calluses were transferred to a WPM-medium (Lloyd & McCown 1981) containing sucrose and mannitol, both at 3%, and 2,4-D (0.0 or 4.97/aM) in combination with BA, TDZ or zeatin (1.78, 1.82 and 1.82/aM, respective- ly) under constant agitation (Lab-Line Orbit Environ- Shaker, 100 rpm at22 °C, at 10/amol m -2 s-l). After a 6-day induction phase, free cells were diluted (1:2 v/v) in the same WPM-medium, but devoid of mannitol and growth regulators and finally 2 ml of this suspen- sion was resuspended in this medium containing 3% sucrose, supplemented with 450 mg 1 - l Glu, 500 mg 1-1 CH or 0.44/aM BA, and cultured at constant agi- tation (50 rpm) for 3 months. Alternatively, free cells were also plated on 0.6% agarose solidified (Sigma Type I) WPM-medium containing the same addenda as indicated before.

After 3 months of culture, the clusters of globular embryos formed in the more effective treatments (2,4- D plus BA), were isolated and subcultured for 45 days more, to evaluate further increases in embryo yield. This material was later plated in Magenta flasks. His- tological studies were carried out by fixing explants in FAA, embedding in Paraplast, cutting 10/am thin sec- tions and staining with Safranin/Fast-Green. Early evi- dence of adventitious embryogenic-occurring sites of

Results from axillary bud tests are summarized in Table 1. Several types of responses were found after 3 months

• of culture; namely, sprouting of preformed buds, bud branching into multiple shoots, callus formation at the

• distal end of the explants, and indirect embryogene- sis, derived frorn this tissue. AxiUary bud sprouting occurred in most treatments, except in combinations with BA. After 3 months, calluses formed in all treat- ments, becoming 0.7-1.6 cm. in diameter. Somatic embryogenesis was observed on these calluses only after two and one-half months; initially showing green embryos distinguishable by their green color from the pale ground callus tissue. NAA or IAA in combination with BA, kinetin or zeatin triggered embryogenesis in a range of concentrations tested with the exception of TDZ, which was always ineffective (not in Table). Further embryo development was not observed over a period of 4 months in culture, unless calluses were transferred to fresh media, when were formed.

Cell suspensions

After 3 months of continuous agitation, suspended cells developed embryos directly (Fig. la), in addi- tion to friable calluses. Mixed globular, heart-shaped and torpedo stages (Fig. lc) were found in samples in addition to multiple globular embryos attached to old- er embryos developing toward more advanced forms. Embryos at the cotyledonary stage had green or yellow cotyledons and development of radicles; older embryos showed root elongation and germinated on solid media. (Fig. ld).

The effect of different growth regulators on embryogenesis induction is shown in Table 2. Results show that the presence of 2,4-D (4.97/aM) only, but not cytokinin-like growth regulators, is required to trigger embryogenesis. Preliminary results (not shown) sug- gested that the selected culture period of 6 days in the presence of 2,4-D, or in combination with cytokinins, seemed satisfactory for induction. However, the inclu- sion of BA (1.78/aM) or zeatin (1.82 #M) to 2,4-D, enhanced embryo formation; i.e, supplementing with BA yielded 13.8 embryos per ml of suspension, as

Table 1. Axillary bud growth, callus and somatic embryos originated from axillary bud explants in culture after 3 months. (Only treatments showing embryogenesis are shown).

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Growth regulators Callus initiation Axillary growth Somatic

(tzM) embryos NAA BA % Formed Diam(cm) % Sprout Aver.Nr Length(cm) % Formed

0.05 4 . ~ 53.0 1.6 0.0 35.3

0.27 4 . ~ 1~.0 1.3 0.0 72.2 0.54 4 . ~ 53.3 1.5 0.0 ~ . 6

2.69 4 . ~ 62.5 1.3 0.0 43.7

5.37 4 . ~ 33.3 1.5 0.0 33.3

NAA Zeatin

0 . ~ 4.56 1 ~ 1.0 15.4 1.0 1.0 23.0

0.27 4.56 1 ~ 1.0 8.3 1.0 0.5 41.6

0.54 4.56 1 ~ 1.6 14.3 2.0 0.8 28.6

2.69 4.56 1 ~ 1.3 6.3 1.0 0.6 18.8

NAA ~ n e ~

0 . ~ 4.65 1 ~ 1.2 ~ . 4 1.0 0.8 33.3 0 . ~ 4.65 1 ~ 1.1 ~ .9 1.0 0.5 42.9

IAA BA

0.57 4 . ~ 1~.0 0.7 ~ . 7 1.0 0.5 23.5

5.71 4 . ~ 55.5 1.2 55.5

IAA Zeatin

0.29 4.56 1 ~ 1.1 61.5 1.0 0.5 ~ . 2

0.57 4.56 1 ~ 1.3 27.3 1.0 0.5 36.4 2.85 4.56 1 ~ 1.4 50.0 1.0 0.6 ~ . 0

5.71 4.56 1 ~ 0.9 0.0 ~ . 0

Aver. of 20 replicates/Ireatment.

TaMe 2. Callus and embryo formation in cell suspensions after a three- month-subculture in a growth regulator-free medium, after pre-treatment for 6 days with 2,*D and combinations of cytokinins. I

Growth regulators (#M)

2,4-D BA Zeatin TDZ

Morphogenic responses

Callus Callus Embry formation diam. formation

% (cm) %

Aver. No. embryos ml- 1

+S.D.

0.0 0.0 0.0 0.0 0.0 - 0.0

4.97 0.0 0.0 0.0 1 ~ 0.7 ~ . 0 4.97 0.0 0.0 1.82 1 ~ 1.0 60.0 4.97 0.0 1.82 0.0 1~ 0.6 83.3 4.97 1.78 0.0 0.0 1 ~ 0.5 87.5

0.0 4.34-1.2 3.6-1-0.5

6.54-2.3 13.8±3.5

IResults o f 15 Erlenmeyer/treatment.

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Fig. 1. Embryogenesis from cell suspensions. Several developing stages of embryo-formation after a 3-month-culture in liquid medium. (a) Globular embryos with suspensor (s) formed from single cells in suspension (bar=0.1 mm). (b) Further stage with several globular embryo clusters developing from suspensor (bar=0.5 ram). (c) Globular, heart and torpedo stages, and adventitious embryos (a) derived from a more developed embryo (bar=-l.0 mm). (d) Close-up showing root development in single germinating embryo (bar=l.0 mm). (e) Three-month-old suspension showing different stages of embryo development after globular embryo cluster subculture.

compared with an average of 4.3 embryos per ml when 2,4-D was used alone. Enhancement by means of zeatin was less effective, while TDZ had no effect.

Except for a few proembryos detected under the microscope (Table 3), cells grown in a medium with- out 2,4-D were unable to initiate embryo, although microcolonies and callus were formed. Also, supple-

mentation of the medium with 1.78 # M BA (best treat- ment Table 2) or organic addenda (Glu and CH) did not lead to differentiation into embryos, but enhanced cell proliferation and microcolony formation. Previous results with juveni le explants (hypocotylderived cal- lus) showed the ability of C. pubescens single cells to initiate embryogenesis in the presence of N A A or BA

Table 3. Effect of glutamine, casein hydrolysate and BA on plated cells cultured for 3 months in a WPM medium I .

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Treatment (/~M) % Micro- Aver. No. Micro % Callus Aver. No. % Somatic

colonies -colonies formation callus per embryos

per plate plate

Control (no growth.reg.) 30 3.8

BA (1.78 ~M) 22 10.4 Glu (400 mg 1- I)-I-CH (500mg 1 - I ) 55 13.1 Gin (400)+CH(500)+BA(1.78~M) 20 9.5

70 3.3 0.0 89 3.6 0.0

90 3.3 0.0 84 3.8 0.0

16-day pre-treatment without 2-4-D; results obtained from at least 18 Petri dishes per treatment, except control which utilized only 10 dishes.

Table 4. Characterization of different embryo developmental stages formed from globular embryo clusters in subculture after 45 days I

Normal development Abnormal development

Development Aver. No. Size (mm) Aver. No. Size (mm)

stages 4- S.D. 4- S.D. 4- S.D. 4- S.D.

Globular cluster 53.54-11.3 0.34-0.1 0.0 0.0

Globular, single 33.44-8.5 0.94-0.3 1.24-2.6 1.94-0.4

Heart 1.24-2.5 1.64-0.1 0.0 0.0 Torpedo 19.64-8.4 1 . 5 4 - 0 . 5 10 .54-8 .0 3.24-0.5

Cotyledonary 13.44-3 .4 2.24-0.5 9.44-4.3 3.54-1.0

embryos Germinated 2.24-2.7 4.84-1.1 1.44-2.2 4.84-1.1

1Cells were initially grown in 4.97/zM 2,4-D and 1.78 pM BA for6 days, later subcultured onto media without growth regulators; after 3 months, clusters with adventitious embryos were transferred to the same, fresh media for another 45 days and examined. (Data based on 30, {lml cell suspension} samples). S.D. - Standard Deviation

alone or together (Jordan, 1986). Also; for C. stipula- ta somatic embryogenesis, NAA has been considered essential and BA acted in promoting embryogenesis in this species (Litz & Conover, 1980). These growth regulators were not effective to initiate embryogen- esis in adult C. pubescens tissue. Despite the initial NAA supplement to start calluses, results show a fur- ther requirement of an auxin-like regulator for embryo induction (in this case 2,4-9) during the single cell sus- pension phase. As 2,4-D treatment resulted in a much faster tissue softening and cell loosening, as compared to NAA, leading to single cells after 6 days in papaya callus, the higher cell density achieved by means of this treatment may have favoured induction during the subculture phase.

A remarkable embryogenic effect, which appeared in previously formed embryos after subculturing in suspension for 45 days, was the formation of numer- ous adventive embryos on embryo clusters at the glob- ular stage (0.3 mm diam.). These structures originated

@ H T ~ E I h T ~ L d m l q m I m la lem

Fig. 2. Distribution of normal and abnormal embryos formed from subcultured embryo clusters after 45 days. (G: globular stage; H: heart; T: torpedo; CE: cotyledonary embryos and GE: germinated embryos).

from the remains of the suspensor or from proembry- onic tissue of the developing embryos (Fig.1 b) and represented a significant increase in embryo yield. In the best medium for induction (2,4-D 4.97/zM and BA, 1.78/zM), over 100 embryos per ml were found (sum- ming up all developmental stages) (Table 4). Globular clusters attached to older embryos and single globular embryos were the most abundant stages in suspension, but heart stages were less frequent.

194

M o r p h o g e n i c reg ions on embryos were clearly observable . Us ing the ace toca rmin /Evan ' s blue stain,

the n e w e m b r y o g e n i c zones showed up in red, contrast-

ing with the n o n - e m b r y o g e n i c por t ions that turned vio-

let, r e sembl ing co lor patterns repor ted dur ing somatic

po lyembryogenes i s in some conifers (Durzan, 1988).

At this t ime, e m b r y o product ion in subcultures can be increased 10-fold v ia advent i t ious embryony by a short

cul ture per iod (45 days) in an embryogen i c m e d i u m

(F ig . l e ) . Howeve r , a s ignif icant number o f embryos

with abnormal d e v e l o p m e n t (approx. 20%) were also

found; this ef fec t is greater in the m o r e deve loped

torpedo and co ty ledonary stages (Fig. 2). Abnorma l

embryos are p resumably a result o f a gradual imbalance

in g rowth regulators , which could eventua l ly be pro-

duced by the same embryos in cul ture in more devel-

oped stages or, due to the lack o f a regula tory factor in

e m b r y o n i c d e v e l o p m e n t (Kreuge & van Hoist , 1993).

Acknowledgement

The authors thank P S T C / U S A I D Grant N ° 8007, the

A.W. M e l l o n Founda t ion and Fondecy t Grant. N °

069/89 for fund ing and are also grateful for the valu-

able he lp g iven by M m e . A. Goreux.

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