Influence of Desiccation Time on Survival and Regeneration of Embryonic Axes of Groundnut (<i>Arachis hypogaea</i> L.) Immersed in Liquid Nitrogen

doi:10.4236/ajps.2013.49211

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American Journal of Plant Sciences, 2013, 4, 1725-1730

http://dx.doi.org/10.4236/ajps.2013.49211 Published Online September 2013 (http://www.scirp.org/journal/ajps)

Influence of Desiccation Time on Survival and

Regeneration of Embryonic Axes of Groundnut (Arachis

hypogaea L.) Immersed in Liquid Nitrogen

M. M. Abdulmalik, I. S. Usman, J. D. Olarewaju, D. A. Aba

Department of Plant Science, Ahmadu Bello University, Zaria, Nigeria.

Email: [email protected]

Received June 17th, 2013; revised July 17th, 2013; accepted August 5th, 2013

cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Cryopreservation, the storage of biological materials in liquid nitrogen (LN), is a useful method for long term conserva-

tion of plant germplasm. This study was carried out with the objective of establishing an efficient desiccation technique

for successful cryopreservation and recovery of embryonic axes of groundnut. Embryonic axes of four groundnut (Ara-

chis hypogaea L.) genotypes were evaluated. The excised embryonic axes were dehydrated by air current of a laminar

air flow cabinet for different duration (0, 1, 2, 3, 4 & 5 hrs) before being plunged in LN (−196˚C) and held for 1 hr.

Samples were thawed in water bath at 40˚C for 2 min, thereafter cultured on MS medium supplemented with 15 mg/L

BAP for recovery. Highest survival (96.67% - 100%) and shoot formation (91.67% - 96.67%) were obtained at an av-

erage moisture content of 17% after 4 - 5 hr desiccation. Among the genotypes evaluated, Samnut 22 and Samnut 23

recorded the highest survival and shoot formation. This technique therefore appears promising for cryopreservation of

groundnut germplasm.

Keywords: Cryopreservation; Groundnut; Embryonic Axes; Desiccation

1. Introduction

Groundnut (Arachis hypogaea L.) is an important source

of protein and edible oil in the world. Nigeria ranks third

after India and China in terms of production [1]. Conser-

vation of groundnut ensures availability of germplasm

for future breeding needs, and seeds are the most pre-

ferred propagule used by seed bank curators for its stor-

age. However, even under seed bank condition, long term

storage of groundnut is not feasible as viability losses

frequently occur [2]. This is because of its high oil con-

tent (45% - 50%), which makes it more perishable and

prone to rapid loss of both quality and viability in storage

[3]. For this reason, groundnut germplasm is maintained

by planting every season in the Institute for Agricultural

Research (IAR). This is not only laborious, time con-

suming and expensive, but also plants are exposed to the

possible risk of pest, disease and environmental stresses.

Newly improved genotypes of crops are fast replacing

traditional genotypes or landraces which are often the

source of diversity that breeders use for crop improve-

ment. The need to conserve groundnut and other crop

biodiversity therefore becomes imperative. Thus, cryo-

preservation should be considered as important compli-

mentary strategy for ex situ conservation. Cryopreserva-

tion is used for long-term storage at ultralow temperature

of −196˚C [4]. Cell division and metabolic activities are

stopped when plants are exposed to ultra-low tempera-

tures, allowing storage without alteration for an indefi-

nite period of time [5]. Desiccation of excised embryos

and embryonic axes is one of the most practicable tech-

niques for cryopreservation [6]. Desiccation technique

has been applied to a wide range of plant taxa which in-

clude embryonic axes of citrus [7,8] almond [9] and em-

bryos of maize [10]. The present work is aimed at estab-

lishing an efficient desiccation technique for cryopreser-

vation of embryonic axes of groundnut.

2. Materials and Methods

Seeds of four groundnut (Arachis hypogaea L.) geno-

types were obtained from the groundnut breeding unit of

IAR and used for this experiment. The seeds were sur-

face sterilized by sequential treatment for 5 min in 70%

Influence of Desiccation Time on Survival and Regeneration of Embryonic Axes of Groundnut

(Arachis hypogaea L.) Immersed in Liquid Nitrogen

1726

alcohol, 20 min in 10% NaOCl (commercial bleach) plus

2 - 3 drops of tween 20, rinsed thrice with sterile distilled

water and immersed in 5% NaOCl plus 2 - 3 drops of

tween 20 for 10 min with occasional stirring and washed

three times with sterile distilled water. Thereafter seeds

were soaked in sterile distilled water for 3 hr. Embryonic

axes were excised and subjected to desiccation under the

air current of a laminar flow cabinet for 0, 1, 2, 3, 4 and 5

hr exposure period. Moisture content (MC) was deter-

mined on fresh weight basis after drying in a 100˚C oven

for 24 hr with 3 replicates of 10 embryonic axes per du-

ration. Desiccated and nondesiccated (0) embryonic axes

were placed in 2 ml sterile cryovial and directly immersed

into liquid nitrogen (−196˚C) and held for 1 hr. Thawing

took place in a water bath at 40˚C for 2 min. Embryonic

axes were cultured individually in test tubes containing

10 ml of MS medium [11] supplemented with 15 mg/L

6-benzylaminopurine (BAP) and solidified with 8 g/L

agar. Cultures were maintained in a growth chamber at

26˚C ± 2˚C under 16 hr light/8 hr dark photo period pro-

vided by white inflorescence. Three replicates of 10 em-

bryonic axes were used per treatment. Survival was de-

termined by the appearance of green color, increase in

size, callusing and development of the root or shoot pole

and expressed as a percentage of the total number of em-

bryonic axes that survived within two weeks of culturing.

While shoot formation was expressed as a percentage of

the total number of embryonic axes forming shoots with-

in one month of culturing. Data collected were subjected

to analysis of variance (ANOVA) and means compared

using Duncan’s Multiple Range Test [12].

3. Results and Discussion

ANOVA for moisture content, survival and shoot forma-

tion of cryopreserved embryonic axes of groundnut is

presented in Table 1. The main effects of desiccation and

genotype were highly significant (p ≤ 0.01) for all the

characters. Two-way interaction between the desiccation

time and genotype was also significant for all the char-

acters.

Desiccation rates significantly influenced the extent of

water loss of embryonic axes of groundnut, their survival

and subsequent shoot formation after storage in liquid

nitrogen (Table 2). Nondesiccated embryonic axes of

groundnut failed to survive liquid nitrogen storage. This

result corroborates that of Gagliardi, who observed non-

survival of nondehydrated and cryopreserved embryonic

axes of Arachis species [13]. Lack of germination of

nondesiccated plant material has also been reported in

other crops [14,15]. Similarly, embryonic axes desiccated

for 1 hr also failed to survive the cryogenic treatment.

Table 1. Analysis of variance for moisture content, survival and shoot formation of cryopreserve d embryonic axes of ground-

nut.

Mean Square

Source of variation Degree of freedom

Moisture content (%) Survival (%) Shoot formation (%)

Desiccation(D) 5 1060.57** 131597.01** 116536.55**

Genotype (G) 3 99.60** 2539.94** 2410.05**

D x G 15 150.15** 3603.66** 2163.56**

Error 48 156.00** 1575.00** 489.99**

**P ≤ 0.01.

Table 2. Effect of desiccation rate on moisture content, survival and shoot formation of cryopreserved embryonic axes of

groundnut.

Treatment Moisture content (%) Survival (%) Shoot formation (%)

Time of desiccation (hr) (D)

0 27.33a 0.00d 0.00e

1 22.50b 0.00d 0.00e

2 18.25c 76.12c 59.35d

3 17.42c 83.75b 78.75c

4 17.25c 96.67a 91.67b

5 16.67c 100a 96.67a

SE± 0.74 2.34 1.30

Mens followed by the same letter(s) within a column are not significantly different at P < 0.05 level of significance using DMRT. a

Influence of Desiccation Time on Survival and Regeneration of Embryonic Axes of Groundnut

(Arachis hypogaea L.) Immersed in Liquid Nitrogen

1727

Their nonsurvival could be attributed to the insufficient

loss of moisture at this period which caused formation of

lethal ice crystals that damage the cells during liquid ni-

trogen storage or thawing. Insufficient dehydration of the

explants prior freezing may cause the formation of ice

crystals during freezing or warming leading to the de-

struction of cellular structures and death of the explant

[16]. Desiccation of embryonic axes from 2 hr to 5 hr

rapidly caused significant loss of moisture content. This

greatly improved the survival and shoot formation of cryo-

preserved embryonic axes. The highest survival (96.67%

- 100%) and subsequent shoot formation (91.67% -

96.67%) was obtained at an average moisture content of

17% after 4 - 5 hr desiccation rates. [17] reported 100%

germinability of embryonic axes when moisture content

was reduced from 25% to 8.5% which was achieved at

2.5 hr desiccation time. While [13], reported 80% shoot

development at 18% moisture content after 1 hr desicca-

tion rate in Arachis species. The improved survival and

shoot formation with decreasing moisture content could

be due to increased accumulation of sugars during drying.

As it is possible that accumulation of sugars may serve to

maintain cellular integrity by osmotically decreasing cell

volume, or act directly to protect by stabilization of

membranes [18]. Another possibility is the probable ac-

cumulation of abscisic acid (ABA) in the desiccated em-

bryos. ABA is reported to promote desiccation tolerance

in mature embryo through the synthesis of late embryo-

genesis abundant (LEA) proteins encoded by mRNA [19],

ABA has also been implicated in cold acclimation in

plants [20].

The moisture content of excised embryonic axes is the

most critical factor influencing the success of cryopre-

servation using desiccation protocol. This to some extent

is influenced by the desiccation rate. Results from corre-

lation studies (Table 3) indicate that moisture content

was negatively correlated with survival and shoot forma-

tion of embryonic axes after cryopreservation (r = −0.41

and −0.35, respectively). This implies that any significant

loss of moisture will greatly improve survival and sub-

sequent shoot formation of cryopreserved embryonic axes

of groundnut. Therefore samples to be cryopreserved

must be sufficiently dehydrated to avoid lethal intracel-

lular freezing. [21] reported that, survival and emergence

of post-thaw embryos were closely related to their mois-

ture contents prior to freezing.

Results obtained from this study showed a significant

difference among the genotypes evaluated (Figure 1).

Samnut 22 embryonic axes recorded the highest moisture

content, which was statistically the same as that of Sam-

nut 10 and Samnut 21. This was followed by Samnut 23.

Similarly, significant survival and shoot formation of

Table 3. Correlation coefficie nts between moisture content, surv ival and shoot formation of groundnut as influenced by des-

iccation.

Moisture content (%) Survival (%) Shoot formation (%)

Moisture content (%) 1.00

Survival (%) −0.41 1.00

Shoot formation (%) −0.35 0.98 1.00

Samnut 10

Samnut 21Samnut 22Samnut 23

Genotype

Shoot formation (%)

Moisture content (%)

Survival (%)

Moisture content, Survival, Shoot formation (%)

Figure 1. Influence of genotype on moisture content, survival and shoot formation of cryopreserved embryonic axes of

groundnut.

Influence of Desiccation Time on Survival and Regeneration of Embryonic Axes of Groundnut

(Arachis hypogaea L.) Immersed in Liquid Nitrogen

1728

cryopreserved embryonic axes was observed among the

genotypes. Samnut 22 and Samnut 23 which are at par

with each other had the highest survival and shoot for-

mation followed by Samnut 21. While Samnut 10 had a

relatively lower survival and shoot formation. The ob-

served differences among the genotypes could be due to

genotypic influence, as tissue culture response in ground-

nut is strongly influenced by the plant genotype [22,23].

Genotypic influence has also been reported in cryopre-

served embryonic axes of maize [24]. There was a sig-

nificant interaction between the genotypes and time of

desiccation for moisture content of cryopreserved em-

bryonic axes of groundnut. Samnut 10 had the highest

moisture content prior to desiccation which was compa-

rable to that recorded by Samnut 22, followed by Samnut

21 and Samnut 23. At 1 hr desiccation time Samnut 10

significantly had higher moisture content compare to the

other genotypes, from 2 hr to 5 hr desiccation time all the

genotypes recorded comparable low moisture content

(Table 4). The genotype x desiccation interaction on sur-

vival clearly indicated that embryonic axes of all the

genotypes did not survive cryopreservation when none

desiccated and when desiccated for 1 hr (Table 5 ). How-

ever, at 2 hr and 3 hr desiccation time Samnut 22 and

Samnut 23 comparably recorded the highest survival

rates compare to the other genotypes. While from 4 hr to

5 hr desiccation time, all the genotypes comparably re-

corded very high survival rates. Effect of genotype x

desiccation interaction on shoot formation of cryopre-

served embryonic axes was significant (Table 6). Sam-

nut 21, Samnut 22 and Samnut 23 comparably produce

more shoots than Samnut 10 when desiccated for 2 hr. At

3 hr desiccation time Samnut 22 and Samnut 23 had

higher shoot formation than Samnut 21 followed by

Samnut 10. All the groundnut genotypes comparably re-

corded high shoot formation at 4 hr desiccation time,

with the exception of Samnut 10 which significantly re-

Table 4. Effect of genotype x desiccation interaction on the

moisture content of cryopreserved embryonic axes of

groundnut.

Treatment level

Time of desiccation

Genotype

0 1 2 3 4 5

Samnut 10 31.33a 24.67b 18.67c 17.67c 16.00c 15.67c

Samnut 21 26.00b 22.00bc 18.33c 17.00c 17.67c 18.33c

Samnut 22 27.67ab 26.00b 19.00c 18.67c 18.00c 17.00c

Samnut 23 24.33b 17.33c 17.00c 16.33c 17.33c 15.67c

Means followed by the same letter(s) are not significantly different at P <

0.05 level of significance using DMRT.

Table 5. Effect of genotype x desiccation interaction on the

survival of cryopreserved embryonic axes of groundnut.

Treatment level

Time of desiccation

Genotype

0 12 3 4 5

Samnut 100e0e53.83d 60cd 86.67ab 100a

Samnut 210e 0e71.43c 75bc 100a 100a

Samnut 220e 0e88.89ab 100a 100a 100a

Samnut 230e 0e88.33ab 100a 100a 100a

Means followed by the same letter(s) are not significantly different at P <

0.05 level of significance using DMRT.

Table 6. Effect of genotype x desiccation interaction on the

shoot formation of cryopreserved embryonic axes of

groundnut.

Treatment level

Time of desiccation

Genotype

0 12 3 4 5

Samnut 100f 0f47.50e 60.00cd 70.00bc 90a

Samnut 210f 0f62.50cd 75b 100a 100a

Samnut 220f0f66.67bcd 90a 100a 100a

Samnut 230f 0f60.71cd 90a 96.67a 96.67a

Means followed by the same letter(s) are not significantly different at P <

0.05 level of significance using DMRT.

corded the least shoot formation. However, at 5 hr desic-

cation time, all the groundnut genotypes recorded com-

parably high shoot formation after cryopreservation. This

suggests that the effect of time of desiccation was not

uniform over the different genotypes.

The survived and regenerated shoots after 4 weeks

were rooted on MS medium supplemented with 1mg/L

1-naphthaleineacetic acid (NAA) [25]. These were then

transferred to potty soil (riverside sand) for hardening.

No morphological abnormalities were observed in the

plants developed from cryopreserved embryonic axes by

desiccation (Figures 2-5).

4. Conclusions

Results obtained in this study indicate significant geno-

typic differences in response to the desiccation rates.

However, in view of the very high survival (96.67% -

100%) and shoot regeneration (91.67% - 96.67%) ob-

tained in the groundnut genotypes, it can be concluded

that cryopreservation using desiccation protocol can be

successfully applied to groundnut, and the provided

moisture content is reduced to about 17%. Hence, this

Influence of Desiccation Time on Survival and Regeneration of Embryonic Axes of Groundnut

(Arachis hypogaea L.) Immersed in Liquid Nitrogen

1729

Figure 2. Microshoot regeneration from cryopreserved em-

bryonic axes on MS + 15 mg/L BAP after 2 wk culture.

Figure 3. Microshoot on MS + 15 mg/L BAP after 4 wk

culture.

Figure 4. Regenerated Plantlet rooted on MS + 1 mg/L

NAA.

Figure 5. A potted plant in green house.

technique could be routinely employed for conservation of

groundnut germplasm.

5. Acknowledgements

The authors acknowledge the Institute for Agricultural

Research, Ahmadu Bello University Zaria, Nigeria for

funding this research.

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