Centre of Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta - 700019, India. Received May 9, 1990/Revised ...
PlantCell Reports
Plant Cell Reports (1991) 9:667-670
9 Springer-Verlag 1991
Plant regeneration through somatic embryogenesis from spear callus culture of Asparagus cooperi Baker Biswajit Ghosh, and Sumitra Sen Centre of Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta - 700019, India Received May 9, 1990/Revised version received January 23, 1991 - Communicated by G. C. Phillips
ABSTRACT Somatic embryogenesis and plantlet formation were obtained from callus derived from the subapical region of spears of Aspara$us cooperi Baker. Callus was obtained in Murashige and Skoog's medium supplemented with l-naphthaleneacetic acid and kinetin. Increase in the concentration of potassium nitrate in subsequent subcultures resulted in the formation of embryos. Rapid multiplication of embryos was secured on transfer to a medium containing a different source of nitrogen and a low level (0.01 rag/l) of gibberellic acid. Media containing zeatin or gibberellic acid led to the formation of complete plantlets from embryos. Regenerated plants were cytologically and phenotypically stable.
ABBREVIATIONS IBA, Indole-3-butyric acid; NAA, l-Naphthaleneacetic acid; 2ip, (2-1sopentenyl) adenine; BAP, 6-Benzylaminopurine, GA 3 , Gibberellin ; ABA, Abscisic acid; MS, Murashige and Skoog (1962) medium. INTRODUCTION Plant regeneration through somatic embryogenesis has been reported in a few species of the Liliaceae, including Asparagus officinalis(Reuther, 1977), Allium sativum (Abo E1 N i l , 1977), Haemerocallis sp. (Krikorian and Kann, 1981), Urginea indica (Jha and Sen, 1986) and Scilla indica(Chakravarty and Sen, 1989). Somatic embryos may develop directly from the explant, from callus tissue or from cell suspension cultures (Ammirato, 1983). This process is dependent on culture conditions, manipulation of growth regulators, and principally on the source and type of nitrogen in the medium (Ammirato, 1987). The formation of somatic embryos in A. cooperi has been reported recently by the present authors (Gho~h and Sen, 1989). This paper reports a detailed study of embryo development and plant regeneration through somatic embryogenesis in A.cooperi and the cytological status of regenerated plants. MATERIALS AND M E T H O D S The plant Asparagus cooperi Baker, collected from Ghandra Nursery, Sikk--~m, india, was grown at the experimental garden of the University of Calcutta. Rapidly elongating, 15-20 cm tall, young spears were used as sources of explant. The apical region(2-2.5 cm from the apex) was discarded and the subjacent 6 cm section of each spear, denuded of lateral leaves, was cut into small discs(4-6 mm)
Offprint requests to. S. Sen
and used as explants. The explants were sterilized with 0.1% mercuric chloride for 6 rain and placed on basal media with macro- and micro-elements of MS medium supplemented with 100 mg/l myo-inositol, o.5 mg/l nicotinic acid, 0.5 mg/l pyridoxJne HCI, 1 mg/l thiamine HCI, 2.0 mg/l glycine, 30,000 mg/l sucrose, 0.7% agar and various growth regulators. The p was adjusted to 5.8 prior to autoclaving at 121~ 1.8 Kg/cm- for 15 rain. The growth regulators (CA^, zeatJn ABA) and amino acids used as addltives were filter sterilized. Cultures were kept under 16/8 h light/dark cycle at 25~ _+l~ under artificial fluorescent 40W tubes providing 3000 lux intensity. Callus was induced a f t e r four weeks of explantation on MS basal medium containing 1 mg/l NAA and 1 mg/l kinetin. Small pieces of calli(10-15 mg fresh weight) were transferred onto basal medium with the same concentration of growth regulators and subcultured at 40 d interval. After the third subculture, somatic embryos were induced in four month old non-morphogenic calli by increasing the concentration of KNO 3 from 1900 mg/l to 2900 mg/l in the basal media with no alteration of growth regulators. For study of mitotic chromosomes, the early and developmental stages of embryos and root tips of regenerants were pretreated with 0. 002 M 8hydroxyquinoline for 4 h, fixed in 1:3 acetic acid : dehydrated ethanol and stained with 2% aceto-orcein. RESULTS AND DISCUSSION j
"
.
,
Formation and Development of Somatic Embryos Somatic embryos were induced in non-morphogenic four month old calli when transferred from M S media containing 1900 mg/l to a higher concentration of KNO 3. The m a x i m u m efficiency of induction of embryo was noted in the medium with 2900 mg/l KNO~, in which embryos could be induced in 65% of~ the cultures. On increasing the concentration of K N O _ the callus became smooth, glossy, nodular and yello~wish green in colour. The globular structures appeared on the callus(Figs. I-2), and gradually became enlarged and elongated(Fig. 3). After 2-3 weeks of induction of globular embryos, the concentration of KNO~ was reduced from 2900 mg/l to 1900 mg/l and typical monocotyledonous embryo structures with a lateral shoot tip and a terminal cotyledon was formed(Figs. 4-5 ) . Further development of these structures eventually gave rise to germinating embryos(Figs. 67). A few embryos were torpedo shaped, simulating two cotyledons which did not form plantlets(Fig. 8). Similar types of embryo development have also been
668 Table I. Effect of GA 3 and different nitrogen sources at various concentrations on A. cooper• somatic embryo multiplication(after 30 d incubation). Initial inoculum = l0 mg morphogenic portion of the call• Basal medium(MS) + Supplements
Concentration (mg / ! )
Embryogenic cal I i. F r e s h wt. (mg) • S.D.
Type of embryo
Control
0
10.8
(•
Normal
L-glu a)
200
15.8
(•
Normal
L-glu
400
30.4
(•
Normal
L-glu
600
18.2
(•
Aberrant
L-leu b)
120
17.0
(•
Normal
L-leu
140
21.4
(•
Normal
L-leu
150
15.4
(•
Normal
L-pro c)
15
20.4
(•
Normal
L-pro
!8
32.6
(il.3)
Normal
L-pro
20
18.6
(•
Normal
200
18.0
(•
Normal
Fig 1 - Globular somatic embryos of A~cooperi. Bar = 400 p m . Fig 2 SEM of a cluster of globular somatic embryos of A.cooperi. Bar = I00 ~ m .
L-ser d) L-ser
250
30.0
(•
Normal
L-set
300
26.2
(•
Normal
reoprted in bamboo(Hassan and Debrag, 1987). In the present casa, the requirements of somatic embryogenesis were more complex than in the typical model where callus is transferred from an auxin-enriched medium to a hormone-free medium (Ammirato, 1983; Raghavan, 1986) There was induction of callus in a medium supplemented with a cytokinin and ~uxin, followed by transfer of ca]lus to a similar mediL1m with a high level of KNO_ for induction of somatic embryos. The role o# enhanced levels of KNO 3 in somatic embryogenesis has been discussed in carrot (Tazawa and Reinert, 1969).
C~ e)
1800
20.8
(•
Normal
CH
2000
25.2
(•
Normal
CH
2400
20.6
•
Normal
0.002
12.0
•
Normal
GA 3
0.01
21.8
•
Normal
GA 3
0.03
18.0
•
Normal
63.2
•
Normal
Normal
GA 3
L-glu CH
of Embryos To increase the frequency of embryos, the effect of casein hydrolysate, NH.NO-, GA and the amino 9 . 4 3 aclds L-prollne, L-serlne and L-glu#amine at various concentrations in MS basal medium was studied. In this experiment, I0 mg of embryogenic call• grown on embryo induction medium with 2900 mg/l KNO 3 was used as inoculum and data were taken after 30 d incubation. With L-alanine and Larginine, there was no noticeable stimulation in embryo multiplication. The combination of Lglutamine (300 rag/l), casein hydrolysate(1000 rag/l) and NHaNO2(1200 rag/l) showed remarkable enhancement in the rate of multiplication. It was thus possible to increase the frequency of embryos by using the appropriate nitrogen sources and levels. The multiplication of embryos was also accelerated at a low level ( 0.01 mg/l ) of GA 3 ( Table 1 ). Multiplication
+
+
300 I000
+ +
NH4NO 3
1200
NH4NO 3
1400
26.6
•
NH4NO 3
1600
35.4
•
Normal
2000
30.8
•
Aberrant
NH4NO 3
a) glu; g l u t a m i n e proline d) ser; hydrolysate Establishment
b) leu; serine
leucine e) CH;
c) pro; casein
of P l a n t l e t s
Complete plantlets could not be obtained by transferring the mature embryoids with single cotyledon to MS medium depleted of all growth regulators. In this medium, the embryoids developed up to 2-3 m m in length and the basal region formed callus. Finally, a large portion of the callus produced globular secondary embryos. Of the cytokinins tested, BAP at lower levels
669
Fig 3 - Elongated somatic embryo of A. cooperi. Bar = 700 7~m. Fig 4 - Elongated single cotyledon with small shoot initial in A.cooperi somatic embryo. Bar = 700 ~m. Fig 5 - S E M of the single cotyledon with shoot initial in A.cooperi somatic e m b r y o . Bar = 200 ~ m . Fig 6 - A germinating somatic e m b r y o of A.cooperi. (S = Shoot region, R = Root region). Bar = 700 ~ m .
Fig
? - A germinating somatic e m b r y o of A.cooperi with clearly developed shoot and root regions. Bar = 1 cm. Fig 8An abnormal somatic e m b r y o of A.cooperi with two cotyledons. Bar = 800 ~ m . Fig 9 - Somatic e m b r y o - d e r i v e d complete plant of A. cooperi with well formed root and shoot. Bar = I cm. Fig 1O - Metaphase plate of root tip cell of somatic embryo-derived plant of A. cooperi (2n = 40). Bar = 1 ~m.
(0.5 to 1 mg/l) induced shoot elongation in 10% of e m b r y o s but no roots were formed. Most e m b r y o s b e c a m e abnormal w h e n 0.05 to 0.5 mg/l kinetin was used. Only 2-3% o f the e m b r y o s developed shoots but the embryos became swollen with abnormal development. At a low concentration (0.75 mg/i) of 2ip, complete plantlets were obtained from only 2% of the embryos. In 1 mg/l of 2ip, 10% of the e m b r y o s developed shoots but b e c a m e abortive. On the other hand, zeatin led to the development of complete plantlets (table If). The m a x i m u m number of plantlets (38%) was obtained at the level of 1 mg/I zeatin. Moreover, GA^ also stimulated the development of complete plan#lets with a root and a shoot (Fig. 9). The shoots or roots were obtained from e m b r y o s cultured in MS basal m e d i u m with ABA, containing either zeatin or GA~. The shoots transferred to basal m e d i u m suppleme~nted with IBA also developed roots. A m a x i m u m of 300 complete plantlets were secured from a single explant over a
period of six months. Prior to transfer to the field, plantlets were kept in half-strength liquid M S basal m e d i u m with 1% sucrose. After two weeks sucrose was omitted, and after another two w e e k s plants were transferred to the pots containing sandy soil in the greenhouse. In most species, embryos germinate in the original basal m e d i u m (Ammirato, 1983) whereas in A.cooperi, plantlets could not be obtained in M S basal medium only. The optimum results were secured w h e n e m b r y o s were cultured in the basal medium(MS) supplemented with zeatin or GA_ alone, similar to reports in Glycine max (G~azi et ai.,1986) and sugarcane (Ho and Vasil, 1983). Gytological Study was the
The c h r o m o s o m e n u m b e r in the original explant noted to be diploid with 2n=40 chromosomes. In embryogenic calli, there was no variation of
670 Table II.
The effect of different concentrations from A__. cooperi somatic embryos.
Growth regulators
Concentration of Growth Regulator (mg/l)
of
Zeatin and
Complete plantlets / treatment (%)
GA 3
for
complete
Time taken for regeneration
plantlet
formation
Phenotype
(d)
Control Zeatin Zeatin Zeatin Zeatin Zeatin
0 0.2 0.5 1.0 2.0 3.0
0 3 16 38 12 2
20-22 18-22 18-20 21-24 28-30
Normal Normal Normal Normal Normal
GGA3 GA ~ GA~ GAf
0.2 0.5 1.0 2.0 2.5
3 6 17 9 2
20-24 17-23 18-24 22-26 28-32
Normal Normal Normal Normal Normal
chromosome number after 30 d of subculture. All nuclei were diploid. Embryos at different stages of development showed diploid cells only. In all the 80 regenerated plants derived from embryogenic calli that were analyzed, 2n=40 chromosomes (Fig. I0) with no irregularities in chromosome behaviour was noted. Plants regenerated by somatic embryogenesis were thus euploid, and were free of any noticeable phenotypic variability. In general, plants regenerated from pre-existing or axillary meristems tend to be similar to the parent plant(Ammirato, 1987). The appearance and extent of variability observed may depend on the explant used, the methodology of culture, and the mode of regeneration. In A. cooperi regenerated plants from somatic embryo are cytogenetically stable and normal, similar to that of control plants noted among species of the Gramineae(Haydu and Vasil, 1981; Lu and Vasil, 1981, 1982). The genetic uniformity of plants regenerated from the embryogenic cultures of A. cooperi may be due to the homogeneity of the explant, or to preferential selection and regeneration from normal diploid cells.
ACKNOWLEDGEMENT
The authors are grateful to Prof. A. K. Sharma, Programme Co-ordinator, Centre of Advanced Study, for facilities provided. B.G. is indebted to University Grants Commission, New Delhi, for financial assistance.
REFERENCES
Abo E1 Nil, N M (1977) Plant Sci Lett 9:259-264 Ammirato, PV (1983) Embryogenesis. In:Handbook of Plant Cell Culture Vol 1 : 82-123 Ammirato, P V (1987) In : Plant Tissue and Cell Culture, Alan R. Liss Inc 57-81 Chakravarty B, Sen S (1989) Plant Cell Tissue Organ Culture 19 : 71-75 Ghazi TD, Cheema HV, Nabors M W (1986) Plant Cell Reports 5 : 452-456 Ghosh B, Sen S (1989) Curr Sci 58(5):256-257 Hassan E1 A, Debergh P (1987) Plant Cell Tissue Organ Culture I0 : 73-77 Haydu Z, Vasil Ik (1981) Theor Appl Genet 59:269272
Ho W, Vasil Ik (1983) Ann Bot 51 : 719-726 Jha S, Sen S (1986) J Plant Physiol 124:431-439 Krikorian AD, Kann RP (1981) Ann Bot 47:679-686 Lu C, Vasil IK (1981) Theor Appl Genet 59:275-280 Lu C, Vasil IK (1982) Amer J Bot 69:77-81 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Raghavan V (1986) In : "Embryogenesis in Angiosperms. A Developmental and Experimental Study" Cambridge Univ Press pp 115-151 Reuther G (1977) Acta Hortic 78:217-224 Tazawa M, Reinert J (1969) Protoplasma 68:157-173
