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Research Article
 

Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)



Hemalatha Kudikala, Raghu Ellendula, Shama Nazrin, Abhiteja Sirikonda, Kasim Mood and Venkateswar Rao Allini
 
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ABSTRACT

Background and Objective: Annona reticulata Linn. (Bullock’s Heart) is traditionally an important ethnomedicinal plant of Annonaceae. It having potential role in ayurvedic for the treatments of several diseases. Different parts of this plant have various pharmacological activities. Seed germination is a major hurdle for this plant. In this sense, this study was aimed to optimize the in vitro germination procedures. Materials and Methods: In vitro seed germination of A. reticulata was done by evaluating different pretreatments such as mechanical scarification, hot water treatment with different timings and GA3 treatment with different concentrations. Results: About 8.67 μM GA3 for 24 h pre-treatment was shown better results pertaining to the maximum percentage (60%) of germination, minimum germination period (46.67 days) and maximum length (10.5 cm) of the seedling. Conclusion: The present investigation described an enhanced in vitro seed germination protocol for the first time in this woody species. It will strengthen large scale plantation and useful in the field of pharmacology.

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Hemalatha Kudikala, Raghu Ellendula, Shama Nazrin, Abhiteja Sirikonda, Kasim Mood and Venkateswar Rao Allini, 2018. Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.). Asian Journal of Plant Sciences, 17: 142-149.

DOI: 10.3923/ajps.2018.142.149

URL: https://scialert.net/abstract/?doi=ajps.2018.142.149
 
Received: August 27, 2018; Accepted: September 05, 2018; Published: October 25, 2018


Copyright: © 2018. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The species A. reticulata belongs to the family, Annonaceae. It is native to tropical regions of America, particularly in West Indies and South America1. It is commonly called as custard apple and has many common names like Bullock’s heart (English), Ramphal (India), Buah nona (Indonesia), Ramaseeta (Tamil), Rramasitapalam (Telugu), Manilanilam (Malayalam)2. It can grow up to a height of near about 6-7.5 m with many lateral branches and fruits are heart shaped and edible3.

The tree has many ethnomedicinal properties such as roots are used for mental depression, spinal disorders and blood dysentery. Leaves are used for antispasmodic, anthelmintic, insecticidal and destroying lice. Fruits are used to enrich the blood, increasing muscular strength. Seeds are used in making detergents and insecticides. The bark is used as an astringent and tonic1. Scientifically various phytochemical and pharmacological medicinal properties have been investigated by several researchers2. Phytopharmacological properties such as Antipyretic4, anthelmintic5, antihyperglycemic6, antiulcer7, in vitro cytotoxic and recombinant caspase inhibitory8, antinociceptive9, analgesic and CNS depressant10, analgesic and anti-inflammatory11, antiproliferative12,13, wound healing and anti-marking14, antioxidant and antimicrobial15, larvicidal16 activities indicated in A. reticulata.

Annona reticulata plant contains a wide range of useful secondary metabolites and minerals. An efficient regeneration method from juvenile material would allow the possibility to enhance the secondary metabolites could be responsible for different therapeutic activities. Rapid propagation may be useful in the field of pharmacology to develop new drugs for human welfare. Prerequisite for this research is to establish an efficient, rapid, reliable and simple method for germination of Bullock’s heart seeds in vitro. Exogenous dormancy of the seeds can be removed by treating the seeds with physical, mechanical and chemical methods17. The growth substance most commonly used for better germination for various plant species are Auxin (IAA, IBA, NAA), Gibberellic acid (GA3), etc. Among these, GA3 has proved to be the best for seed germination and proper seedling growth. In various species, Gibberellins have been used for dormancy breakage along with accelerating germination of non-dormant seeds18. The present study was carried out to improve the seed germination and to assess the efficiency of gibberellic acid (GA3 ) on seed germination in A. reticulata.

MATERIALS AND METHODS

Obtaining the seeds: The seeds were collected from trees located in Suraram village, Karimnagar district of Telangana state in the month of February and they were separated from the pulp and washed with running tap water and cleaned with paper towels. Cleaned seeds were stored at room temperature (28°C) for further use.

Ex vitro germination: A sample of 60 seeds was sown in trays containing cocopeat and sand. They were incubated at 25°C with 16/8 h photoperiod in greenhouse .

In vitro germination: Paper bridges19 were used as a scaffold for in vitro germination of seed. Medium pH was adjusted to 5.6-5.8 and 15 mL of liquid medium was dispensed into 50 mL test tubes before autoclaving at 121°C for 15 min. Culture conditions were maintained at 25±2°C under 16 h photoperiod. The cultured seeds were daily observed to check for germination.

Intact seeds were sterilized with 0.1% HgCl2 for 5 min. followed by rinsing with sterile distilled water for 2-3 times. Later, they were cultured on paper bridges in DW, MS20 liquid medium and ½ strength MS liquid medium. Observed results were used as the control.

Seed pre-treatment methods
Mechanical scarification (removal of seed coat): The seed coat was carefully removed by using the surgical blade without damaging the embryo. Later, the de-coated seeds were sterilized with 0.1% HgCl2 for 5 min followed by rinsing with sterile distilled water for 2-3 times. These sterilized seeds were soaked for 24 h in sterile distilled water and cultured on paper bridges having DW, MS liquid medium and ½ strength MS liquid medium.

Hot water pre-treatment: The intact seeds were exposed to hot water treatment (100°C) for 3, 5 and 7 min. Treated seeds were immediately transferred to the normal water and sterilized with 0.1% HgCl2 for 5 min. followed by rinsing with sterile distilled water for 2-3 times. Later, they were soaked in sterile distilled water for 24 h before inoculating on to paper bridges having DW, MS liquid medium and ½ strength MS liquid medium.

GA3 hormone pre-treatment: Intact seeds were sterilized with 0.1% HgCl2 for 5 min followed by rinsing with sterile distilled water for 2-3 times and subsequently soaked in 3 different concentrations of GA3 solutions i.e., 2.89, 5.78, 8.67 and 11.56 μM for 24 h.

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 1(a-d):
In vitro seed germination of A. reticulata, (a) Seeds, (b) Germinating seed on paper bridge, (c) In vitro raised seedling on MS basal media and (d) In vitro raised seedlings

Later they were cultured on paper bridges having DW, MS liquid medium and ½ strength MS liquid medium supplemented with the same concentration of hormone..

All above treatments were repeated thrice with 20 seeds in each and the cultured seeds were daily observed for 9 weeks for the germination. Seeds were considered as germinated when a radicle is visible (Fig. 1).

Data analysis: Data on the effect of pretreatments on seed germination in vitro was subjected to IBM SPSS software statistical version 20. All the results were stated as the Mean±Standard Error (SE). Statistical analysis was done by the Analysis of Variance (one way ANOVA between the groups) and means of comparative analysis was made by (DMRT) duncan’s multiple range tests (p<0.05).

Table 1:
Ex vitro seed germination of A. reticulata under greenhouse in different months (60 seeds were tested per month)
Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)

RESULTS AND DISCUSSION

The month of March seems the most favorable time for seed germination in the greenhouse. Ex vitro seed germination of A. reticulata was observed to be very poor (13.3%) (Table 1). The same was also reported by Padilla and Encina21 in A. cherimoya (52.5%) and Campbell and Popenoe22 in A. diversifolia (30-80%). In contrast, George and Nissen23 opined a high germination rate (90-95%) in Annona squamosa.

Experiments were carried out with untreated and decoated seeds for in vitro germination and the results were presented in Table 2.

Table 2: Effect of different pre-treatment methods on measured characteristics in A. reticulata seed germination
Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Mean±SD. Error, DW: Distilled water, MS: Murashige and Skoog’s medium

Results shown that the seeds were grown on three different medium i.e., distilled water, MS medium and ½ strength MS medium shown similar germination (%). It was also recorded that the variation was observed in the number of days to germination (Fig. 4, 5) and an average length of seedlings. Decoated seeds were germinated 3-4 days earlier than untreated seeds and their average length of the seedling is also considerably increased. Decoated seeds of A. muricata were germinated on half strength MS medium24. The seed coat removal followed by 24 h pre-soaking in DW enhanced the early germination than intact seed germination. This may be due to seed coat involvement in prevention of water uptake exerting a mechanical restraint on the growth of the embryo. Similarly, removal of seed coat is necessary to A. squamosa seeds to germinate them in vitro25. In A. diversifolia26, A. squamosa and Atemoya27 the germination process was not regulated by the permeability of seed coat.

Later, A. reticulata seeds were treated with hot water at different time periods i.e., 3, 5 and 7 min and allowed them to germinate on different media. Results indicated that low germination (%) was recorded in all the hot water treatments (DW 8.3, 10 and 5%, in MS 8.3, 15 and 5% and in ½ MS 6.6,11.6 and 5%, respectively) (Fig. 2). Immersing in hot water for 5 min significantly resulted in earlier germination (Fig. 4, 5) compared to control and mechanical scarification. Whereas as in 7 min treatment, it was recorded that germination (%) was decreased and days of germination was considerably increased. The reason may be a longer period contact of seed with hot water effects the damage of embryo. Similarly, Amusa28 reported in Afzelia africana a 12 h treatment with (100°C) hot water. Hence hot water pre-treatment was not appropriate pre-treatment for the seeds of A. reticulata. It perhaps leads to the seed embryo being killed because of prolonged contact with boiled water.

In GA3 treatment, 8.67 μM GA3 concentration produced a high percentage (in DW 53%, in MS 60% and in ½ strength 50%, respectively, Fig. 2) of germination in all three media tested and substantially improved plantlet development (highest length of seedling i.e., 10.5 cm. Fig. 6). GA3 effects in stimulating cell division, cell elongation, auxin metabolism, cell wall plasticity and permeability of cell membrane leading to enhanced growth29. About 8.67 μM GA3 treatment also has considerably decreased the number of days to germination (Fig. 3-5) when compared with other treatments earlier tested. For these reasons, it was selected as standard.

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 2: Effect of pre-treatment methods of seed germination (%) in A. reticulata
  DW: Distilled water, MS: Murashige and Skoog’s medium

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 3: Effect of pre-treatment methods on No. of days for germination in A. reticulata using distilled water

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 4: Effect of pre-treatment methods on No. of days for germination in A. reticulata using MS medium

An improved germination in cherimoya seeds also has been shown by GA3 treatment30. The increasing seed germination parameters might be due to the involvement of GA3 in the activation of cytological enzymes along with increase in cell wall plasticity and better water absorption. GA3 acts as a directly on embryo relieving them from dormancy through promoting protein synthesis and elongation of coleoptiles and leaves and also helps in the production of ethylene.

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 5: Effect of pre-treatment methods on No. of days for germination in A. reticulata using ½ MS medium

Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)
Fig. 6: Effect of pre-treatment methods on length of A. reticulata seedlings
  DW: Distilled water, MS: Murashige and Skoog’s medium

Table 3: Ex vitro and in vitro seed germination of A. reticulata
Image for - Effect of Pre-treatment Methods on in vitro Seed Germination of Bullock’s Heart (Annona reticulata L.)

This ethylene invokes the synthesis of hydrolases, especially amylase, which favours the seed germination31. GA3 also stimulates seed germination by formation of a-amylase enzymes which converts insoluble starch into soluble sugars and it also initiates the radical growth by removing some metabolic blocks32. In the case of A. reticulata GA3 could increase the germination rate considerably.

Pre-soaking period of 24 h improved in vitro germination of A. reticulata seeds. Ferreira et al.27 reported pre-soaking period for 4-5 h was beneficial to A. squamosa seeds and for Atemoya (A. squamosa×A. cherimola) seeds more than 12 h allowed the best results. Seeds treated with GA3 for 24 h pre-soaking and standard method of sterilization with 0.1% HgCl2 solution has given no contamination. Elongated pre-soaking (more than 24 h) produced more contamination. Lemos et al.33 stated that a longer pre-soaking period required more than 24 h for Annonaceae seeds.

Finally, the results revealed that the ex vitro seed germination percentage was very low when compared with GA3 treatment of in vitro seeds germination (Table 3). Treatment of the seeds with GA3 resulted in earlier germination, well-developed seedling and high germination (%) compared with seeds without GA3 treatment. Therefore the GA3 treatment of A. reticulata seeds was only effective for increasing the speed and high percentage of seed germination.

CONCLUSION

An efficient protocol has been optimized for in vitro seed germination of A. reticulata using different seed pre-treatment methods. According to our observations, pre-soaking of seeds in 8.67 μM GA3 for 24 h has shown maximum germination percentage and highest length of seedlings in short time duration. Therefore in this study, it concluded that GA3 treatment can only be used to accelerate the in vitro seed germination of A. reticulata.

SIGNIFICANCE STATEMENT

For the First time, procedures were established by applying several pre-treatments. GA3 observed to have an important role in improving germination percentage. This will be useful for exploration and development of the plant for pharmaceutical use.

ACKNOWLEDGMENT

The authors are grateful to the Head, Department of Biotechnology, KU for providing necessary facilities, working environment and Prof. A. Sadanandam, Department of Biotechnology, KU for his valuable suggestions and encouragement.

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