K. E. Ogunsola and C. O. Ilori
Miracle berry is an evergreen tropical shrub which modifies sour food to produce a sweet taste. Its propagation is, however, hindered by seed recalcitrance and difficulty of stem to root. Thus in vitro propagation was investigated through embryo and nodal explants using different levels and combinations of auxins and cytokinins in MS medium.
All efforts to induce rooting on the buds formed from nodal explants proved abortive.
INTRODUCTION
Miracle berry (Synsepalum dulcificum Daniell) is a tropical West African shrub of the family Sapotaceae. It is reported to be indigenous to tropical West Africa and commonly found growing in the wild in fringes of virgin forest while it also grows naturally in farms and secondary bushes. The fruits are small, approximately 2 to 3 cm long ellipsoid berries that are bright red when ripe and composed of a thin layer of edible pulp surrounding a single seed. The most unusual thing about the fruit is the extra- ordinary effect the fleshy pulp of the fruit has on the taste buds of the tongue that causes every sour food eaten to taste very sweet. The taste-modifying effect lasts for 30 min to 1 h or more, causing acidic food substances such as sour lime, lemon, grape fruits and even vinegar to taste sweet.
Various studies have shown that the sweetening property is due to the presence of miraculin, which is a glycoprotein, in the pulp of the berry. The interest in natural sweeteners, which do not con- tain carbohydrates, has been reawakened because of the health hazards associated with the use of some artificial sweeteners like saccharine and the suspicion that these synthetic sweeteners, especially the cyclamates, are carcinogenic. A natural sweetener which is high in protein can be found in the miraculin from Synsepalum dulcificum.
However, despite the need for large-scale production of miracle berry to exploit its potential and for further genetic studies required to enhance its improvement, commercial production of the plant has been a constraint. Miracle berry is also usually found growing more in the wild than cultivated and its growth rate is very slow. Hence, there is a need for an alternative method of propagation that will overcome these growth constraints. Although some trees and shrubs have been successfully micro-propagated using Murashige and Skoog (MS) medium with minimum phytohormone modifications, there has been no report on established protocols for in vitro regeneration of miracle berry.
The objective of this study is to regenerate plantlets of miracle berry through mature embryo and nodal cultures by determining the appro- priate growth regulators (auxin-cytokinin) combinations required to modify Murashige and Skoog (MS) medium for in vitro propagation of the plant.
Materials and Methods
This study was carried out in the Tissue Culture Laboratory. Miracle berry seeds used in the embryo culture. Nodal explants were from a year old seedlings.
Sterilization of Materials and Explants
Glasswares and dissecting tools were sterilized for 30 min by autoclaving at 121 0C and 1.06 kgcm -2 pressure. Nodal cuttings (1.0 cm long) were surface-sterilized with 70% ethanol for 5 min, 25% solution of sodium hypochlorite (NaOCl) with 2 drops of Tween 20 for 30 min and 10% NaOCl solution for 10 min and then rinsed thrice with sterile distilled water. Miracle berry seeds, after removal of peels and fleshy pulp, were surface-sterilized with 70% ethanol for 5 min, 10% NaOCl for 20 min and 5% NaOCl for 10 min.
Media preparation
Murashige and Skoog (MS) (1962) basal medium was used in all cultures. Forty eight (48) treatments were used in all which comprise of MS basal medium without modification and MS medium supplemented with varied levels of growth regulators which include BAP, NAA, IBA, 2,4-D, kinetin and GA3 prepared in stock solutions and used at different combinations to initiate culture, form and proliferate buds and to induce root (Tables 1, 2 and 3).
Embryo Culture
Miracle berry embryos were aseptically excised from the sterilized seeds by cracking the seed coat and dissecting out the embryos with small parts of the endosperm. The embryos, which are located at the anterior end of the seeds, were immediately implanted into the culture initiation medium and 10 replicates were prepared per treatment. Also, after bud proliferation of the shoot-regenerated embryos, the regenerated shoots were excised into nodal segments and transferred into the rooting medium.
Nodal Culture
Nodal explants obtained from both mature field grown miracle berry plants and one-year old screen-house raised seedlings were surface sterilized and aseptically dissected into 1.0 cm long nodal cuttings, containing at least one node with either terminal or axillary buds. These were cultured into the entire 48 MS medium modifications prepared with and without the addition of 0.02 mg/l of GA3 and 20 replicates were prepared per treatment.
Growth Parameter Studied and Data Analysis
Growth parameters measured include: radicle length, shoot length, number of leaves, number of buds formed (proliferated buds); root number and root length all from the regenerated embryos while the number of terminal or auxiliary buds formed were studied from the nodal culture. Percentage embryo germination (radicle emergence), percentage rooted plantlets and the frequency of cultures that formed terminal or axillary buds were also studied. Data taken were subjected to Analysis of Variance (ANOVA) and means with significance differences separated by Duncan Multiple Range Test (DMRT) at 5% confidence level.
RESULTS
Culture Initiation from Mature Embryo
Five out of the twenty-six different MS medium modifications supported normal germination of embryos as shown by emergence of radicals and plumules by the third week of culturing. MS medium supplemented with a range of 0.05 – 0.1 mg/l of NAA plus 0.04 – 0.2 mg/l BAP induced embryo culture initiation but combination of 0.1 mg/l NAA plus 0.2 mg/l BAP was shown to be optimum for morphogenesis of embryo. MS medium modified with 2,4-D and kinetin completely inhibited radicle growth and some of these hormone combinations (0.5 mg/l 2,4-D + 0.4 mg/l kinetin and 0.02 mg/l 2,4-D + 0.01 mg/l kinetin) induced callus formation that could not be induced to differentiate shoot buds. MS basal medium without modification could not support full germination of embryos because it could not induce shoot regeneration.
Lateral Bud Proliferation of Shoot Regenerated Embryo
Germinated embryos transferred at six weeks to modified MS medium produced rapid shoot growth with bud multiplication. A range of BAP (0.6 – 3.0 mg/l) plus NAA (0.1 – 0.2 mg/l) induced node proliferation while the bud number appeared to be highest in MS medium with 3.0 mg/l BAP + 0.1 mg/l NAA in 80% of the culture. Adventitious shoot multiplication could not be achieved through embryo culture but lateral bud proliferation was obtained through which the plant was multiplied by transferring excised nodal segments with growing bud into rooting medium.
Root Formation of In Vitro Germinated Embryo
Half-strength MS medium with 20 gl -1 sucrose without vitamin and with NAA and BAP hormone supplements (0.8 – 3.0 mg/l NAA + 0.2 mg/l BAP) induced a minor level of root formation on the germinated embryo while full-strength MS with vitamins, supplemented with 1.0 – 2.0 mg/l IBA + 0.1 mg/l BAP induced a better rooting. MS medium supplemented with 2.0 mg/l IBA + 0.1 mg/l BAP was shown to be optimum for root induction. The plantlets from embryo cultures were acclimatized in the screen house and were successfully established on soil.
Nodal Culture
From the nodal explants cultured in all the 48 MS media modifications. A high occurrence of microbial contamination was noticed in the nodal cultures even at 30% NaOCl sterilization and there was secretion of exudates on the nodal explants. Efforts to induce rooting on the few buds formed to regenerate whole plantlets from nodal explants were futile.
DISCUSSION
Miracle berry was successfully regenerated through mature embryos in MS medium supplemented with minimum levels of NAA, BAP and IBA. MS medium without hormonal modification could not support embryo germination of miracle berry.
It was difficult to fully regenerate miracle berry through nodal explanations from mature plants and young seedlings. This might be attributed to the difficulty in rejuvenating mature tissues of 248 Afr. J. Biotechnol, woody species and production of exudates of the latex producing miracle berry.
Meanwhile, the progress made on the regeneration of whole plantlets of miracle berry from mature embryos will enhance cultivation and in vitro conservation of the plant that will remedy the inability to store the seeds in the genbank due to the seed’s recalcitrance to germination. It will also reduce the breeding cycle of this slow growing plant, promote germplasm movement of pathogen-free miracle berry plantlets and enhance germplasm diversity studies of miracle berry for the much needed characterization and classification of the plant, provide stocks for micro-grafting and promote breeding for improvement.
Further studies on the growth rate under field conditions (in vivo) as well as further modifications of the in vitro physical and chemical environments of the nodal culture are recommended to enhance propagation of the plant.
Reference:
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