J. Appl. Hort., 2(2):84-87, July-December, 2000
2,4,5-T induced somatic embryogenesis in papaya (Carica papaya L.)
J. Bhattacharya and S.S. Khuspe
Plant Tissue Culture Division, National Chemical Laboratory, Pune - 411 008, INDIA, Email: sskhuspe@dalton.ncl.res.in
Abstract
A protocol for high frequency somatic embryogenesis in Carica papaya L. was developed using immature zygotic embryo explant of
cultivar Honey Dew and CO 2. Somatic embryos were induced in immature embryos, cultured on MS basal medium supplemented
with 3 mg/l of 2,4,5-T, and incubated in dark for a period of 3-6 weeks. Loosely attached globular somatic embryos appeared from
apical domes within 3-6 weeks of incubation. Development of somatic embryos was asynchronous which passed through globular,
heart and torpedo shaped stages. Embryos continued to proliferate with regular subculture and remained morphologically competent
for up to one year. Maturation of the embryos was achieved in medium supplemented with ABA (0.1 mg/l). The cotyledonary stage
embryos germinated (71.33% in Honey Dew and 59.33% in CO 2) on phytohormone free MS basal medium. The regenerated
plantlets were established in the greenhouse and hardened plants were transferred to soil.
Key words: Somatic embryogenesis, Papaya, Carica papaya L., 2,4,5-Trichlorophenoxyacetic
Abbreviations: ABA: Abscisic acid. BAP: 6- Benzylaminopurine. 2,4-D: 2,4-Dichlorophenoxy acetic acid. 2,4,5-T: 2,4,5Trichlorophenoxyacetic acid. Kinetin: 6-(Furfurylamino)-purine. MS: Murashige and Skoog’s basal medium. Zeatin: ( 6-[4-Hydroxy3-methyl-2-butenylamino] purine).
Introduction
Materials and methods
Papaya (Carica papaya L.), a popular fruit crop, is valued highly
for vitamin A and for the industrially important digestive enzyme,
papain. Papaya is highly susceptible to the aphid transmitted Papaya
Ring Spot Virus (PRSV) disease that severely limits yield and for
which adequate level of native resistance is not available. A solution
to the problem is genetic engineering of the crop. One of the major
prerequisites for this is an efficient plant regeneration system.
Among all the pathways of morphogenesis, somatic embryogenesis
is the obvious choice of regeneration because of their single cell
origin or are produced from a single cell within the proembryonic
mass (Litz and Gray, 1995).
Plant material: Immature fruits of Carica papaya L. cv. Honey
Dew and CO 2 were collected from field grown plants of our
campus. The fruits were washed in running tap water for one
hour and then with a liquid detergent (Extran, E. Merck, India)
for 10 minutes. These were then placed in a 1% solution (v/v) of
Savlon (Johnson and Johnson, USA) for 5 minutes. The fruits
were then washed repeatedly till free of Savlon, rinsed with 70%
ethyl alcohol for 30 seconds and finally with 0.1% mercuric
chloride for 5 minutes. The fruits were thoroughly washed at
least three times with sterile double distilled water, cut
transversely and the immature seeds were collected in a sterile
petridish. These seeds were used to isolate the immature embryos,
which were used as explant.
Somatic embryogenesis in papaya although has been reported
from immature zygotic embryo (Fitch and Mansherdt, 1990),
seedling pieces (Yamamoto and Tabata, 1989) and petioles (De
bruijne et al., 1974), complete regeneration into plantlet with high
frequency germination is still limited. However, in other related
species of papaya, i.e. C. papaya x C. cauliflora, C. papaya x C.
pubescens, C. stipulata, regeneration of somatic embryo derived
plantlets were found to be a quite common phenomenon (Litz and
Conover, 1980; Chen, 1988; Chen et al., 1991).
2,4,5-Trichlorophenoxyacetic acid, more commonly known as
2,4,5-T has been used to initiate somatic embryogenesis in other
crops earlier (Suhasini et al. 1994, Mckersie and Brown 1996). To
our knowledge, the potentiality of this growth regulator has not
been utilized in papaya somatic embryogenesis.
The present study was therefore undertaken i) to develop rapid
and high frequency somatic embryo induction in papaya (Carica
papaya L.) and their germination in to normal plantlet and ii) to
evaluate the effect of 2,4,5-Trichlorophenoetic acid in papaya
somatic embryogenesis.
Induction of embryogenesis: Ten immature embryo axes were
cultured in 85 mm plastic petridishes (Laxbro, India) on MS
basal medium (Murashige and Skoog, 1962), supplemented with
30g/l sucrose and various concentrations of 2,4,5trichlorophenoxy acetic acid (2,4,5-T). The pH of the medium
was adjusted to 5.8 before autoclaving at 121oC for 20 minutes.
The medium was solidified with 0.7% agar (HiMedia, India).
Thirty ml medium was poured in each pre-sterilized petridishes
and allowed to solidify. Fifty explants were cultured in each
treatment. For the induction of somatic embryos the cultures
were incubated at 25±2 oC for 3-6 weeks in both light (16 hr
photoperiod under cool white fluorescent light, 25 mol m-2s -1)
and dark condition. The experiments were repeated thrice.
Development of somatic embryos: Globular embryos induced
on MS medium with 3.0mg/l 2,4,5-T after 3-6 weeks were
separated from the explant and subcultured on the same medium
in 85 mm petridishes for further development. The cultures were
2,4,5-T induced somatic embryogenesis in papaya (Carica papaya L.)
incubated in the same conditions as mentioned above for 2-3
weeks for formation of cotyledonary stage somatic embryos.
Maturation of somatic embryos: Cotyledonary structures were
separated and transferred to MS medium with 3% sucrose, 0.75%
agar and 0.1 mg/l ABA (maturation medium) in petridishes and
incubated for 5 days under conditions mentioned above.
Germination of embryos: For germination, matured
cotyledonary stage embryos were transferred to MS medium
supplemented with sucrose (30g/l) and zeatin (1mg/l), BAP (1.0
mg/l), IBA (1.0 mg/l), BAP (1.0 mg/l) + NAA (0.1 mg/l) and
GA3 (1 mg/l). The embryos were cultured at 25±2oC under cool
white fluorescent light at 140 mol. m-2 s-1 with 16 hr photoperiod
for 2-3 weeks when they formed well developed root and shoot
systems.
Regeneration of plantlet: Fully germinated embryos were
transferred to growth regulator free MS basal medium for further
elongation of shoots and roots. The cultures were incubated for
2-3 weeks in 250 ml Erlenmeyer’s flask with 100 ml or in 350
ml jam bottle with 50 ml medium under conditions described
for embryo germination. Plants with well developed roots were
transferred to 8 cm pot containing a mixture of peat, vermiculite
and soil (1:1:1) and hardened for 2 weeks at 25±2oC under above
mentioned conditions. After establishment plants were
transferred to greenhouse.
Histology: For histological studies the samples were fixed in
formalin: acetic acid: alcohol (5:5:90) (v/v) for 48 hours and
then dehydrated by passing through ethanol-tertiary butyl alcohol
series. Paraffin embedding of tissue samples was done as
described by Sharma and Sharma (1980). Longitudinal sections
of 10 mm were stained with hematoxylene-eosin and mounted
with DPX-4 189-(2-chloro-N- (methoxy-6-methyl-1, 3-5-triazin2-yl amino carbonyl benzene sulfonamide) (Qualigens, India)
and observed microscopically.Observations recorded were
statistically analyzed using student’s t- test.
Results and discussion
Various concentration of 2,4,5-T (1.0, 2.0, 3.0, 4.0, 5.0, 8.0 and
10.0 mg/l) was evaluated for their induction potential. After 5
85
days of incubation the explant became swollen and cotyledons
expanded. Among them, best response was obtained on MS basal
medium supplemented with 3.0 mg/l 2,4,5-T for both Honey
Dew and CO 2 cultivar, when cultured both in light and dark.
Illumination time played an important role in papaya somatic
embryo induction. Cultures incubated in dark responded more
in percent embryo production and also produced more number
of embryos per explant in both the cultivars, Honey Dew and
CO 2 (Fig. 3) as compared to the cultures kept in 16 hr
photoperiod condition. Similar positive effect of darkness in
somatic embryogenesis was also observed in citrus (Perez et al.
1998). Explants that did not initiate somatic embryos developed
unorganized friable callus and sometimes with adventitious roots.
Within 3-6 weeks of incubation 79.33% and 71.00% embryos
of both the cultivar showed swelling and formation of loosely
arranged globular embryos. During that period, 29.33 and 30.33
embryos per explant were counted for both Honey Dew and
CO 2, respectively. The globular embryos either continued to
develop into torpedo stages or underwent budding at their surface
to give rise to additional globular embryos. Somatic embryos
originated both directly and via an intermediary callus phase
from the explants.
The type of regeneration response was found to be dependent
on the concentration of the auxin used. General observation was
that lower concentration up to 3 mg/l 2,4,5-T favored direct
somatic embryogenesis whereas higher concentration tested lead
to the formation of somatic embryos accompanied by callus
proliferation. Though embryo formation was also observed in
higher concentrations of 2,4,5-T (5.0,8.0 mg/l) number of
embryo formation per explant was less (13.66, 15.0) and (4.33,
4.66) for both concentrations in Honey Dew and CO 2,
respectively. However no embryo formation was observed with
lower concentration (0.1 mg/l) and higher concentration (10.0
and 20.0 mg/l) of 2,4,5-T (Table1). While shoot tip region of
explant formed somatic embryo the root pole region turned into
brown callus.
Pale greenish loosely arranged globular embryos were separated
and transferred to same induction medium where the cotyledons
expanded and thickened to give mature somatic embryos. An
established culture usually consisted of greenish somatic embryos
Table 1. Effect of 2,4,5-T on somatic embryogenesis in papaya cv. Honey Dew and CO 2
2,4,5-T
% explant forming
No. of globular embryos
Conc.
globular embryo
per explant
HD
CO 2
HD
CO 2
0.0
G
G
0.1
0.0
0.0
0.0
0.0
0.5
8.33±1.52 ef
3.66±1.53 f
10.66±2.30 b
11.00±1.0 b
1.0
51.00±3.60 c
39.66±3.055 d
16.66±2.51 b
22.66±3.0 a
2.0
66.66±5.68 b
53.33±4.72 b
25.33±4.50 a
23.66±3.51 a
3.0
79.33±5.03 a
71.00±3.60 a
29.33±3.21 a
27.66±4.04 a
5.0
40.66±2.51 d
41.00±2.64 cd
13.66±3.05 b
15.00±1.73 b
8.0
3.66±2.08 f
8.66±1.53 ef
4.33±4.04 c
4.66±2.08 b
10.0
0.0
0.0
0.0
0.0
20.0
0.0
0.0
0.0
0.0
% embryos that formed globular
to cotyledonary stage
HD
CO 2
0.0
0.0
23.33±1.53 c
12.00±2.0c
42.33±4.50 b
17.66±1.15c
52.33±1.53 a
31.66±4.16b
53.33±1.53 a
45.66±3.21a
38.33±3.51 b
33.00±4.59b
7.66±4.04 d
15.66±5.03c
0.0
0.0
0.0
0.0
All data were recorded after 4 weeks in culture. Data represent mean for three sets of experiment, each set composed of ninety explant. ±
Standard deviation. MS medium without 2,4,5-T served as control. Values with same letter (a-f) in a column are not statistically different at
P = 0.05. G Germination
86
Journal of Applied Horticulture
at various developmental stages (Fig. 1A). The developmental
pattern of the somatic embryos was continuos and essentially
asynchronous passing through various developmental stages such
as heart, torpedo and cotyledonary structures (Fig. 1B). Basal
medium supplemented with 3mg/l 2,4,5-T supported formation
of highest number of explant forming globular embryos and
number of globular embryos per explant in both the cultivar
tested. In terms of both explants forming globular embryos and
number of embryos per explant 3 mg/l 2,4,5-T was found to be
the best. Number of somatic embryos increased with the auxin
concentration upto 3 mg/l 2,4,5-T, which started declining with
the increasing concentration and finally stopped at 10.0 mg/l
and 20.0 mg/l.
Table 2. Effect of illumination period on percent embryo forming
globular embryos and induction of papaya somatic embryos in
Honey Dew and CO 2
Culture
% Embryos forming
No. of globular
condition
globular embryos
embryos explant-1
Honey Dew
CO 2
Honey Dew
CO 2
16 hr light
24 hr dark
15.00±2.00
72.33±3.05
22.00±4.00 13.0±3.0
65.66±2.08 33.0±3.6
3.33±1.52
3.00±3.60
Table 3. Effect of different media on germination of papaya
somatic embryos of cv Honey Dew and CO 2
Germination
% Germination
media
Honey Dew
CO 2
A
0.0
0.0
B
16.33±2.51
0.0
C
71.33±2.08
59.33±1.50
D
61.33±2.08
54.66±1.15
average of 300- 500 embryos per explant could be counted.
Evidence of morphological competence of these tissues was
judged by the germination of somatic embryos isolated from
them.
Use of 2,4,5-T in papaya somatic embryogenesis is found to be
more effective than earlier reports (Fitch and Mansherdt, 1990;
Chen et al., 1987) in terms of time taken and number of somatic
embryos produced per explant. Litz and Conover (1982) obtained
mature somatic embryos of C. papaya x C. cauliflora after 7
months of culture. Also in the earlier reports the germination
percentage and origin of the somatic embryos were not very
clear. With this protocol, a complete somatic embryo derived
plantlet is ready in about 13 weeks, which is comparable to the
previous report of papaya somatic embryogenesis in liquid
medium (Castillo et al. 1998). Moreover our system results in
both origin of direct somatic embryos and high germination
percentage. Additionally secondary embryogenesis was a
common phenomenon. Thus, these facts can advantageously be
used for any clonal propagation or transformation studies in
papaya.
Acknowledgements
The research fellowship awarded by C.S.I.R., Government of
India, New Delhi to J. Bhattacharya is acknowledged. Financial
support in the form of research grant by Department of
Biotechnology (DBT), Govt. of India is also gratefully
acknowledged. We are thankful to Dr K .V. Krishnamurthy for
providing the central facility of tissue culture laboratory.
A
B
A-MS medium with BAP (1 mg/l), B- MS medium with Zeatin (1 mg/l),
C- MS medium without phytohormone, D-MS medium with GA 3 (1 mg/l)
In general, it took about 8-10 weeks to obtain cotyledonary
structures starting from the culture of explants in the induction
medium. A large number of cotyledonary structures formed were
morphologically abnormal. Typical abnormalities included
missing, extra or fused cotyledons, fusion of several embryo at
their hypocotyl or overall grossly misshapen embryo
morphology. Many a times such structures failed to germinate
with proper shoot apex. Shoot and root development in papaya
somatic embryo rarely occurred in parental zygotic embryo
cultures. To encourage their germination the mature somatic
embryos were shifted to various media formulations.
Germination of cotyledonary stage somatic embryos was
maximum when MS basal medium without any growth hormone
was used. GA3 at concentration of 1mg/l was found to be the
next best treatment (Fig. 2). Positive effect of GA3 in embryo
germination was also reported earlier (Chang and Hsing,1980).
Freshly transferred somatic embryos started expanding within
5-10 days and germinated (Fig. 1C) within 3 weeks of incubation,
with adventitious root initiation and shoot extension. Plants
obtained on growth regulator free medium showed vigorous
growth of both root and shoot. Regenerated plants were
morphologically similar to seed derived plants (Fig.1D) and were
transferred to soil in pots and hardened in green house.
Embryogenic cultures were maintained in a state of proliferation
by regular culture to a fresh medium. By the end of 6 months an
C
D
Fig. 1. Stages of regeneration of Carica papaya L. through somatic embryogenesis from immature zygotic embryo cultured on
MS basal medium supplemented with 3% sucrose and 3 mg/L
2,4,5-T. A–Asynchronous developmental stage somatic embryo
derived from immature zygotic embryo after 4-5 weeks of culture
(g-globular stage; c-cotyledonary stage). B– Prolific development
of somatic embryos from a single explant. C–Oerminating single
somatic embryo. D–Somatic embryo regenerated plant.
2,4,5-T induced somatic embryogenesis in papaya (Carica papaya L.)
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