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 %)

References AmmiratoPV, (1987).In: PlantTissueAndCellCulture(GreenCD, Somer DA,HackettWP, BiesboerDD eds) AR Liss Inc. 57-81.

differentiated to a plantlet. There are several reports of

Baker CM, Wetzstein HY (1992). Plant Cell Rep., 11:71-75.

apparently well-formed embryos that failed to produce

Hazra S, SathayeSS, MascarenhasAF (1989). Biotechnology,7:949-951

plantlets (Ammirato 1987) which have been attributed to malformation of epicotyl pole (Kerns et al. 1986) or

Kerns HR, BarwaleUB, Meyer MM Jr, WildholmJM (1986). Plant Cell Rep.,5:140-143.

abnormal shoot apex development (Trigiano et aI. 1988).

Murashige, T, SkoogF. (1962). Physiol. Plant., 15:473-497

Incorporation of ABA, GAs, Z, BAP and Kn in the embryo

Ozias-AkinsP (1989). Plant Cell Rep., 8:217-218.

induction medium was ineffective in improving the frequency of somatic embryo formation (data not shown).

Parrott WA, MerkleSA, 'WilliamsEG (1991). AdvancedMethodsin Plant Breeding and Biotechnology(MurrayDR ed) CAB Int. 158-200.

Though 100% of the embryos formed well-differentiated

Ramdev ReddyL, Reddy GM (1993). Ind. J. Expt. Biol., 31:57-60.

roots (fig f), the frequency of conversion remained poor.

SenaratnaT (1992) Biotech. Adv., 10:379-392

The conversion frequency was increased to 24.7% by

Trigiano RN, BeatyRM~ GrahamET (1988). Plant Cell Rep., 7:148-150.

with or without charcoal, the differentiation of the plumule

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.).

Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos d...
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