Amelioration of mangosteen micro propagation through leaf and seed segments ( Garcinia mangostana l . )

A micro-propagation system was developed for mangosteen (Garcinia Mangostana L.) using leaf sections and mature seeds. This study investigated the effect of various modified Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP) alone or in combination with -naphthalene acetic acid (NAA) on multiple shoot induction from leaf and half seed explants. Among the treatments, leaf explants on medium supplemented with 2.0 mgL 1 (w/v) of BAP gave the highest percentage of shoot formation (70.07%), highest number of shoots formed per explant (46.5%) and showed high mean shoot height (0.48 cm). It was observed that medium with 0.5 mgL 1 NAA, did not induce shoot organogenesis, but induced callus in 90% of the leaf explants. Seed explants gave the highest percentage of shoot formation (60.2%) and mean number of shoots per explant (9.5) on medium supplemented with 20 mgL 1 (w/v) BAP and 10 mgL 1 (w/v) NAA. However, highest mean shoot height (0.69 cm) was observed on medium supplemented with 10 mgL 1 (w/v) BAP and 10 mgL 1 (w/v) NAA. Callus induction from seeds was also observed in most of the treatments. Medium-supplemented 20 mgL 1 (w/v) NAA gave the highest percentage of callus formation (83.36%). Subsequently, roots were induced from the shoots by pretreating these shoots in various indol-3-butyric acid (IBA) and NAA concentrations in various MS salt strengths. Root formation was obtained on one-quarter strength MS salt medium supplemented with 0.1 mg/L NAA (90.4%). Plantlets were acclimatized on media composed of soil + sand+ organic matter+ vermiculite (2: 2: 1: 1) with 80% survival.


INTRODUCTION
Mangosteen is usually propagated by apomictic seeds; but propagation by seeds is insufficient due to its seasonal fruiting and low yield of seeds.Usually, it produces a maximum of two seeds per fruit but some fruits are seedless.Furthermore, the recalcitrant nature of the seeds causes difficulties in producing planting materials throughout the year.Conventional vegetative propagation methods have been commercially unsuccessful because of the occurrence of abnormal growth.Alternatively, in vitro culture of mangosteen has been established using seeds (Goh et al., 1988;Te-chato and Aengyong, 1988), *Corresponding author.E-mail: hossein955@yahoo.com,mbjavadi2002@yahoo.com.
Tel: 0389464148.Fax: 0389464115.young leaves from in vitro (Te-chato et al., 1992b) and field grown seedlings and mature trees (Goh et al., 1990).However, the efficiency of plantlet multiplication by methods described in those studies was not sufficient for the constant and mass supply of planting materials year round.Recently, compact nodular callus formation from young leaf culture was reported in mangosteen (Te-chato et al., 1995a).The nodular callus was efficiently induced by adding thidiazuron (TDZ) to the culture medium (Techato et al., 1995b).Histological studies revealed that the nodule was composed of a meristematic dome and a pair of leaf primordial and the nodules originated from two main sources of cells, epidermis and vascular bundle (Te-chato et al., 1992b).Due to the limited availability of seeds, evergreen mangosteen leaves serve as favorable explants for in vitro mass propagation of planting materials via shoot organogenesis.The increased induction of nodular calli per leaf explant would therefore facilitate mass production of shoots for every cycle of production.However, there are many factors affecting shoot bud morphogenesis from leaf tissues such as ethylene (Goh et al., 1977), auxin and time of benzyladenine (BA) exposure (Lakshmanan et al., 1977).Therefore, this study investigated the effects of different 6-benzylaminopurin (BAP) concentrations alone or in combination with -naphthalene acetic acid (NAA) on shoot organogenesis from mangosteen leaf and seed.Subsequently, the effect of various auxins and their concentration, and Murashige and Skoog (MS) salt strengths on root initiation of the shoots was investigated.Finally, an acclimatization protocol was established for the plantlets.

Plant material and sterilization protocol
Mangosteen leaves were obtained from mature trees and seeds from mature fruits.Leaves were excised to 1 x 1 cm square segments with midribs and seeds were removed from fruits and cut in two halves.The explants were disinfected by immersion under running tap water with 5% (v/v) Teepol solution for 15 min.Under sterile environment, explants were sterilized with 0.5% (v/v) Benlate solution for 15 min; 70% ethanol for 3 min; washed 3 times in sterile distilled water; sterilized in 20% Clorox solution, 2 drops of Tween 20 for 15 min; washed with sterile distilled water 5 times and dried on filter paper.The solid MS (Murashige and Skoog, 1962) medium used was supplemented with 30 gL -1 sucrose and 3.79 gL ¹ Gelrite and various plant growth regulators [BAP, and NAA] as required.The medium was adjusted to pH 5.8 before autoclaving.
Cultures were kept at 25 ± 2 o C under 16 h of light at intensity of 15.8 molm -2 s -1 .

Explant preparation and treatments
Leaf explants were cultured on MS media supplemented with various concentrations of BAP (0, 1.0, 2.0, 4.0 and 8.0 mg L -1 ) alone or in combination with NAA (0 and 0.5 mg L -1 ), and on hormone free MS media as control.Seed explants were cultured on MS media supplemented with various concentrations of BAP [0, 10, 20, and 30 mg L ¹ (w/v)] alone or in combination with NAA [0, 10, 15 and 20 mg L -1 (w/v)] and on hormone free MS media.In the root initiation study, shoots were cultured on MS medium supplemented with different concentrations of IBA (0. 0, 0.1, 1.0 and 2.0 mg L -1 ) or  NAA [0, 0.1, 0.5 and 1 mg L ¹ (w/v)].

Variables observed
Leaf and seed cultures were observed for the percentage of explants that responded with shoot organogenesis, number of shoots produced per explant, shoot height (cm) and the percentage of explants that responded with callus formation.Data were collected every two weeks until the twelfth week of culture.In the root initiation study, shoots were observed for the percentage of explants producing root, the number of roots produced per explants and root length (cm).Data were collected until the 6 months of culture.In the acclimatization study, the percentage of plant survival and plant height (cm) were recorded for 6 months.

Experimental design and statistical analysis
All treatments had ten replications and the experimental units were arranged in a Completely Randomized Design (CRD).Data were analyzed using the analysis of variance (ANOVA) and Duncan New Multiple Range Test (DNMRT) at =5% for comparison between treatment means.

The effect of various media on shoot induction and callus formation from leaf explants
The production of shoots from leaf explants on all media tested was slow until the fourth week.By the sixth week, the number of shoots began to increase for treatments either under BAP or NAA alone, but the number of shoots on media with NAA alone began to decrease by the end of the twelfth week of culture.Relatively, media supplemented with both BAP and NAA produced more shoots.There was no treatment interaction between BAP and NAA until the fifth week of culture, because NAA showed no significant difference in mean number shoots with the various concentrations of BAP.This result indicated that BAP was the plant growth regulator influencing shoot formation from the leaf explants.Although, BAP supplemented at 2 mgL ¹ (w/v) produced relatively the highest percentage of responding explants, but the treatment significantly produced the highest number of shoots per explant (46.5%) (Figures 1 and 5B).Among treatments supplemented with BAP alone, the number of shoots produced per explant was significantly high at 2 mgL ¹ BAP, but as BAP concentrations increased at 4 and 8 mgL ¹, the number of shoots per explants decreased to 39.25 and 35.5 number of shoots, respectively.Similarly, among the combined BAP and NAA treatments, NAA and 2 mgL ¹ BAP treatment gave significantly higher number  of shoots per explant as compared to treatments with higher BAP concentrations (4 and 8 mgL ¹).However, treatment 2 mgL ¹ BAP alone was sufficient to produce significantly higher number of shoots per explant compared to its combination with NAA.Furthermore, shoot height was significantly high in treatment supplemented with BAP 2 mgL ¹ (0.48 cm; Figure 5C) which was also not significantly different when the treatment was combined with NAA (0.44 cm).Nonetheless both treatments had relatively higher shoot height compared to the rest of the treatments, whether BAP alone or in combination with NAA.In general, the callus induction on all medium was slow up to the fourth week.The induction was apparent from the sixth week onwards on media supplemented with 0.5 mgL ¹ NAA.Treatments supple-Sirchi et al. 2027 mented with NAA alone and in combination with BAP, showed callus induction.This indicated that NAA was important for the induction of callus from leaf explants.However, 0.5 mgL ¹ NAA treatment alone was sufficient to produce significantly high percentage of induction compared to the combined treatments (Figure 5D).

Shoot proliferation per seed explant
After two weeks of culture, the seed explants began to swell and shoots emerged (Figure 6A).Shoots were separated from proliferating clumps by the twelfth week and transferred to MSO medium for rooting.The highest percentage of shoot formation (60.2%, Figure 6A) occurred on medium containing 20 mgL ¹ (w/v) BAP and 10 mgL ¹ (w/v) NAA (Figure 1).The presence of BAP in the medium either alone or in combination with NAA stimulated the half seeds to produce shoots.On medium without BAP, shoot induction was not observed.The highest number of shoots produced per half seeds (9.5) also occurred on medium containing 20 mgL ¹ (w/v) BAP and 10 mgL ¹ (w/v) NAA (Figure 2).However, medium incorporated with 10 mgL ¹ (w/v) BAP and 10 mgL ¹ (w/v) NAA resulted in the highest shoot height (0.69 cm; Figure 6B) followed by 0.66 cm shoot height on medium containing 20 mgL ¹ (w/v) BAP and 10 mgL ¹ (w/v) NAA (Figure 3).Both treatments showed were significantly highest shoot height compared to other treatments.In most treatments the explants also produced callus and the highest percentage of callus formation (83.36%) occurred on medium containing 20 mgL ¹ (w/v) NAA (Figures 4 and 6C

Conclusion Leaf
Leaf explants on medium supplemented with 2 mgL ¹ of BAP resulted in significantly the highest shoot induction response (70.07%).The same treatment also produced the highest number of shoots per explant (47) and relatively high shoot height.The highest percentage of callus formation (90%) occurred on medium containing 0.5 mgL ¹ (w/v) of NAA, while calli were not formed in media without NAA.

Seed explant
Hartmann and Kester (1983) stated that cytokinin at relatively high concentrations (about 13 mg/l) promoted bud formation.Specifically, high levels of endogenous cytokinin have an important role in the initiation of proliferation centers in the explants and in turn can promote the subsequent bud primordial formation (Valdes et al., 2001).Average shoot height produced on media supplemented with BAP and NAA was relatively higher than on media supplemented with BAP alone.This indicates that NAA tends to stimulate the height of shoots.Therefore, BAP induced shoot formation from seed explants, while NAA improved the shoot height.
According to Bhojwani and Razdan (1983), BAP is the most useful and reliable and the cheapest cytokinin.Increasing level of BAP produced more number of shoots.The use of NAA did not promote the increase of mangosteen shoot numbers.Apparently, the effect of NAA was on the height of shoot rather than the shoot numbers.

Figure 1 .
Figure 1.Effect of BAP in combination with NAA on percentage of shoot per half seeds after twelve weeks of culture.

Figure 2 .
Figure 2. Effect of BAP in combination with NAA on mean number of shoots produced per half seed explant after twelve weeks of culture.

Figure 3 .
Figure 3.Effect of BAP in combination with NAA on mean shoot height (cm) attained after twelve weeks.Words represented ±SD; followed by the same letter(s) are not significantly different using DNMRT at p=0.05

Figure 4 .
Figure 4. Effect of BAP in combination with NAA on percentage of half seeds that responded to callus formation.Words represented ±SD; followed by the same letter(s) are not significantly different using DNMRT at p=0.05.

Figure 6 .
Figure 6.Effect of BAP and NAA either alone or in combination (high concentration) on mangosteen half seed explant.(A) Effect of 20 mgL ¹ (w/v) of BAP in combination with 10 mgL ¹ (w/v) NAA on shoot induction after two week of culture (Bar = 0.57 cm).(B) Effect of 10 mgL ¹ (w/v) BAP and 10 mgL ¹ (w/v) NAA on shoot elongation after 12 weeks of culture (Bar = 0.66 cm).(C) Effect of 20 mgL ¹ (w/v) NAA on callus formation after 12 weeks of culture (Bar = 0.9 cm ).

Table 1 .
Effect of BAP or NAA concentrations as well as their interactions on mangosteen leaf explant after 12 weeks of culture.Mean followed by the same letter (S) in the same column are not significantly Different using DNMRT (p=0.05).