PlantCell Reports
Plant Cell Reports (1994) 13:578-581
9 Springer-Verlag1994
Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.) K. Chengalrayan, Sangeeta S. Sathaye, and Sulekha Hazra Division of Plant Tissue Culture, National Chemical Laboratory, Pune - 411 008, India Received 24 June 1993/Revised version received 22 March 1994 - Communicated by J. J. Finer
Summary. Embryogenic masses were obtained from
To be of practical value, somatic embryogenesis should
immature leaves of peanut (Arachis hypogaea L.) cultured
culminate in the formation of plantlets. Recently, there has
on a medium containing 20 mg/l 2,4-D. Somatic embryos
been a great deal of interest in in vitro regeneration of peanut
developed from these masses following transfer to a
(Hazra et al. 1989, Ozias-Akins 1989, Durham and Parrott
medium containing 3 mg/l 2,4-D. The embryo morphology
1992, Baker and Wetzstein 1992, Ramadev Reddy and
was quite variable. Following transfer to hormone-free
Reddy 1993) via somatic embryogenesis. Direct somatic
medium, these embryos germinated. Shoot elongation was
embryogenesis from immature zygotic embryos and 50%
obtained in 25% of the embryos following transfer to a
conversion was reported from this laboratory (Hazra et al.
medium supplemented with 0.5 mg/l each of BAP and Kn.
1989). A similar report (Ozias-Akins 1989) also indicated
The plants grown in vitro by this method survived in
problems in conversion of somatic embryos to plants. Since
sand:soil mixture and were grown to maturity.
immature zygotic embryos are not always readily available, attempts have been made to regenerate plants from other
Abbreviations : ABA: abscisic acid; BAP: 6-benzyl amino
explants including leaflets (Baker et al. 1992). In spite of
purine; 2,4-D: 2,4 dichlorophenoxyacetic acid; GA3:
these studies, there are no efficient protocols for
gibberellic acid; K_n: kinetin; NAA: 1-naphthaleneacetic
regeneration
acid; 2,4,5-T: 2,4,5-trichlorophenoxyacetic acid; Z:zeatin.
embryogenesis in vitro.
of
plantlets
of
peanut
via
somatic
There are certain limitations in using immature zygotic
Key words : Mature embryo derived - Conversion -Arachis
embryos of peanut as initial explants. These include the need
hypogaea
to maintain excessive greenhouse plantings and the difficulty in obtaining the
Introduction In vitro regeneration of plants via somatic embryogenesis
has much potential for use in plant propagation and gene transfer (Ammirato 1987, Parrott et aL 1991, Senaratua 1992). However, the efficient conversion of somatic embryos into plants remains a problem (Ammirato 1987).
Correspondence to: S. Hazra
embryo at the proper
developmental stage. Isolation of sterile cultures from immature embryos of peanut also poses a problem due to a high contamination rate because of its underground fruiting habit. In this report, we describe a protocol for somatic embryogenesis from mature embryo-derived leaflets of
579 peanut without the associated callusing. In addition,
C). H e r e a f t e r , t h e s e b u l g e s are r e f e r r e d as
embryogenic
experiments were conducted to improve the rate of
m a s s e s , s i n c e t h e d e v e l o p m e n t o f e m b r y o s w a s restricted to
conversion.
these protuberances. The frequency of embryogenic mass f o r m a t i o n v a r i e d w i t h the t y p e a n d c o n c e n t r a t i o n o f a u x i n
Materials and Methods A high yielding cultivar of Peanut (Arachishypogaea.J.L.24) was used in this investigation.The pods were obtained from Agriculture College Pune, India. Seeds were removed from the pods, and embryo axes were excised and surface sterilized with 0.1% mercuric chloride (HgClz) for 3-5 minutes. Excess HgCI2 was removed by repeated washing (5 times) with sterile double distilled water under aseptic conditions. Embryo axes were soaked in sterile double distilled water for 12-16 hours prior to dissection of immature leaflets. Thirty leaflets were excised and placed in each Petri dish containing Murashige and Skoog's (MS) basal medium (Murashige and Skoog 1962). Three auxins (NAA, 2,4-D, 2,4,5-T) at varying concentrations was tested. Concentrations of auxins tested were 1, 10, 20, 30, 40 rag/1 for NAA, 1, 5, 10, 15, 20, 25, 30, and 40 rag/1 for 2,4-]3 and 1, 5, and 10 mg/l for 2,4,5-T. Total number of explants in each of these 16 treatments is given in Table-1. All media were supplemented with 6% sucrose (Qualigens fine chemicals, Bombay), solidified with 0.45% agar (Qualigens fine chemicals, Bombay) and the medium pH was adjusted to 5.8 before autoclaving. The cultures were incubated at 25_.+2~ under diffuse cool white fluorescent lights (32 pE/mZ/sec.) with a 16 hour photoperiod. After 4 weeks the number of explants that responded in each Petri dish was recorded. Mean and standard deviations were calculated. To obtain further development some leaf explants with rounded structures were transfered to the same medium while most explants were transfered to MS basal medium with 3 mg]l 2,4-D, 6% sucrose and 0.45% agar. Cultures were incubated for 4 additional weeks. Developed embryos were then transferred to half or full strength MS medium with or without activated charcoal (0.3%) either singly or in clusters for germination. Eighty to ninety embryos were inoculated in each of these four combinations in test tubes. After 4 weeks of incubation in light, 170 rooted embryos without shoots were transferred to MS medium supplemented with 0.5 rag/1 each of BAP and Kn, 2% sucrose and 0.45% agar for conversion to plantlets. Cultures were incubated for 4-8 weeks depending on the growth of shoots. These plantlets were transferred to sand soil mixture (1:2) after attaining the height of 3-4 centimeters.
used.
Table-1 Effect of various concentrations of NAA, 2,4-D and 2,4,5-T on induction of embryogenic mass: Hormone
Cone. mg/l
Total No. of Explants Inoculated
No. of Explants Responded
Mean + S.D/ Petridish~
NAA
1 10 20 30 40
250 183 285 273 275
0 0 62 85 82
0 _.+0.000 (8) 0 + 0.000 (6) 7 + 3.674 (10) 9 _+3.640 (9) 9 _+3.104 (9)
1 5 10 15 20 25 30 40
183 174 223 229 623 167 227 220
0 2 34 50 492 35 42 30
0+0.000(6) 5 + 2.480 ( 7 ) 7 + 3.720 23 __.5.167 7 + 3.774 (6) 6 + 3.311 4 _.+2.227
1 5 10
209 189 207
28 67 43
4 + 1.769 /7/ 11 -+ 6.312 6 +_3.331 (7)
2,4-D
2,4,5-T
1 _ 1.000
a = Thirty explants per Petri dish. Figures in parenthesis indicates number of replicates.
The analysis of data shows that the embryogenic mass induction was highest in 2,4-D at 20 mg/1 (Table 1). At lower concentrations of 2,4-D (5-15 mg/l) the initial hump formation was noted at low frequency but it was associated with callusing. Similar observations were noted in NAA and 2,4,5-T. At higher concentrations of 2,4-D (25-40 mg/l), initial embryogenic mass induction at low frequency was observed but, by the end of the third week, the growth was minimal and the tissues turned brown. Thus, 20 mg/l 2,4-D was selected as the optimum. If the embryogenic masses were transferred to fresh medium containing 20 mg/l 2,4-D, the bulges did not grow further and in one week, the explants
Results and discussion
Upon soaking in sterile double distilled water, the embryo axis swelled and the embryonal leaves protruded from the plumular end (fig a) of the axes. Embryonal leaves were isolated (fig b) and cultured on various auxin-containing media. Two weeks after culture initiation a pair of rounded structures developed from either side of midrib of the explants cultured on medium containing 20 mg/l 2,4-D (fig
turned brown. Somatic embryos developed within 20 days from 90% of the embryogenic masses transfered to medium containing 3 mg/1 2,4-D. Embryos of various shapes and sizes were visible in the same clusters indicating that the process of embryogenesis was asynchronous. The presence of abnormal
shapes
and
fused structures suggests
developmental anomalies in the embryos (fig d). In earlier studies, direct somatic embryogenesis was obtained
580
Figa
Mature'embryo axis isolated from dry seed after a 12-16 hour soaking period: p = Plumule end, r = Radical end
Fig b
Immature leaves dissected from a soaked embryo axis - initial explant X 11
Fig c
Pair of bulges (embryogenic mass) developed on either side of the midrib at the basal end of immature leaves after 20 days in 20 mg/l 2,4-D X 13
Fig d
Somatic embryos of various shapes and sizes developed from embryogenic masses after 30 days in 3 mg/l 2,4-D. Germination of a somatic embryo 15 days after transfer to germination medium, plumule end elongated and root emerged.
Fig e Fig f
A germinated somatic embryo with fully developed root and stunted shoot after 30 days in germination medium.
Fig g
Plantlets raised by conversion of germinated somatic embryos in medium supplimented with 0.5 mg/1 each of BAP and KN.
Fig h
Planflets raised via somatic embryogenesis growing in soil:sand mixture.
581 following culture of immature zygotic embryos on a
transfering the rooted somatic embryos to MS medium
medium containing 3 mg/l
containing cytokinins (0.5 mg/1 each of BAP and Kn). The
2,4-D (Hazra et al. 1989).
Approximately 50% of these
zygotic embryo-derived
faciated embryos obtained in this study formed multiple
could be converted to plantlets in a
shoots and roots. These embryos required an exposure to
germination medium composed of half-strength MS
cytokinins for development of normal plantlet (fig g). Forty
medium with 2% sucrose and 0.25% activated charcoal.
plants that grew to 3-4 cm survived transfer to a soil-sand
However, in contrast, somatic embryos that developed from
mixture (fig h) and grew to maturity.
immature leaves did not convert to
Acknowledgement. We acknowledgeC.S.I.R.India for grant of
somatic embryos
plantlets in this
germination medium. Although all the embryos germinated
fellowshipsto K. Chengalrayanand S.S. Sathaye.
and gave rise to roots in half or full-strength MS medium end was restricted (fig e). Only 1 out of 170 embryos (0.59 %)
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roots (fig f), the frequency of conversion remained poor.
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with or without charcoal, the differentiation of the plumule