In the present study an efficient in vitro adventitious shoot formation and organogenesis protocol was developed for Myrica rubra. Embryonic axes were excised and cultured on MS medium fortified with 0.6 mg L-1 TDZ for different time intervals. Explants not exposed to TDZ concentration did not germinate. Exposure of explants for 2-3 weeks was found the optimal time interval for induction of adventitious shoots. Shoot regeneration was suppressed by longer exposures. Highest shoot induction frequency (99.80%) was observed when explants were exposed for 21 days followed by 14 days (97.00%) exposure. Maximum number of shoots (16.66) were recorded when explants were exposed to TDZ containing media for 21 days. Shoots were tremendously elongated with proper regulation of BA concentrations alone or in combination with NAA. Longer shoots (4.60 cm) were harvested with 0.5 mg L-1 BA in combination with 0.9 mg L-1 NAA. Roots were induced with a high frequency of 100% in MS medium supplemented with 0.6 mg L-1 IBA. Plants were successfully acclimatized to the green house conditions.
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Myrica rubra, a member of Myricaceae is an endemic species of Far East Asia, grown under warm and humid climates. It is grown in China, Japan, India, Thailand, Burma and Vietnam as fruit tree; in America and Europe as an ornamental tree[1,2]. It is the first Actinorhizal family to have emerged during the late cretaceous period and M. rubra is one of 28 Actinorhizal species of Myrica genera. It is a stone fruit with a berry like edible part, developed from the exocarp, consisting of capsule like cellules termed flesh segments. This species is highly valued in Japan and China not only for fresh fruits but also for its medicinal properties. Fruits and leaves are used for treating congestion, coughs, digestive problems and diarrhea while bark is used for treatment of arsenic poisoning, skin diseases, wounds and ulcers[2,7,8]. Tannin, flavonoids, polyphenols and perfumery compounds have been extracted from different parts of this tree[9-11]. Seed is used for extraction of edible oils. Each year, during the fruit season of Red bayberry, there is high competition among the local alcohol making industries. Due to long lifespan, great economic value, low production costs and nitrogen-fixing activity, Red bayberry is regarded as an important economic source for the growers[7,8].
Conventional propagation techniques for Red bayberry such as seed, grafting and air layering are not rapid to meet the need of elite varieties in time. In vitro organogenesis and somatic embryogenesis offer prospects of faster mass multiplication. For improvement of qualitative and quantitative traits, a highly efficient regeneration method is one of the pre-requisite in determining the success of genetic transformation. Shoot regeneration from mature and immature cotyledon explants have been reported in tree plants such as Anacardium occidentale[14,15], Aegle marmelos, Cuphea tolucana, Olea europae, Juglans regia and Carica papaya; however, there are very few reports on plant regeneration from mature embryonic axes. Tonon et al. reported that embryonic axes explants showed a greater regenerative potential than cotyledons in Fraxinus angustifolia. There is no report on in vitro adventitious bud induction and shoot regeneration from embryonic axes explants of M. ruba. The objective of this study was to establish an efficient shoot regeneration system of red bayberry in the shortest possible time. To date, recalcitrant nature, difficulties in adventitious bud induction and their subsequent conversion into shoots have restricted work on genetic transformation of Myrica rubra. This highly regenerative protocol from embryonic explants would be a useful tool in Agrobacterium mediated genetic transformation of Myrica species.
MATERIALS AND METHODS
Plant material and culture conditions: Mature seeds of M. rubra var. Biji were procured from Cixi County, Zhejiang, China during June, 2004. Seeds were chilled under -2°C for 144 h. Endocarps were removed and disinfected with 60% alcohol for 0.5-1 min and rinsed 3-5 times with double distilled water and sterilized with 0.05% HgCl2 for 6-7 min followed by 5-7 rinses under aseptic conditions. Seed testa was removed and the pointed ends of the seeds consisting embryos were cut apart form the rest of cotyledons. Embryonic axes were excised using a sharp knife and incubated with the cut end towards the medium. Murashige and Skoog medium was used throughout the experiments and the explants were incubated under 50 μ E m-2 s-1 of illumination provided by cool white fluorescent tubes, 16/8 h light/dark and 26±2°C. All the media were fortified with 30 g L-1 sucrose, gelled with 0.8% agar and the pH was adjusted to 5.8 prior to autoclaving at 1.5 kg cm-2 and 121°C for 20 min. Growth regulators were filter sterilized and added in the medium in aseptic conditions.
Induction of adventitious shoots: Embryonic axes explants were exposed to MS media containing optimal concentration of TDZ (0.6 mg L-1) for different time intervals (0, 7, 14, 21 and 28 days). Explants were immediately transferred to hormone free medium and cultured for 7 weeks. There were 50 explants in each treatment replicated five times. Entire experiment was repeated three times. Data on number of responding explants and number of adventitious shoots were recorded.
Elongation of adventitious shoots: Embryonic explants with adventitious multiple shoots were transferred to MS medium fortified with different concentration of BA (0, 0.1, 0.5, 1.0 mg L-1) alone or in combination with NAA (0.3, 0.6 and 0.9 mg L-1). There were 45 explants per treatment with 5 replications and the entire experiment was repeated at least three times. Shoots were counted and harvested after 5 weeks in culture.
Rooting and acclimatization: Before transferring the micro-shoots to root induction medium, single shoots were transplanted into growth regulators free medium and cultured for 2 weeks with the aim to eliminate the effect of growth regulators. Healthy shoots (>2 cm) were transferred to full strength MS medium containing various concentrations of IBA (0.3, 0.6 and 0.9 mg L-1). Data on root induction was recorded after 25 days in culture. Once roots were initiated, plantlets were transferred to hormone free MS medium.
After 4 weeks of culture, plantlets recovered were washed with running tap water and agar sticking to the roots removed and individually planted in pots containing 1:2 of per liter and sand. Plants were maintained under tissue culture conditions for 3 weeks covered with thin plastic sheet and then transferred to the green house. After 15 days, slits were made in the plastic to lower the humidity and sheets were completely removed on shifting them to the green house. Plants were maintained in the greenhouse for 3 months and showed normal growth and development without showing any morphological abnormalities.
Statistical analysis: Data were analysed using SAS statistical package (SAS Inst., Cary, N.C.) and differences between treatment means were compared using LSD at p≤0.05.
Induction of adventitious shoots: Results in Table 1 show that exposure of embryonic axes explants to 0.6 mg L-1 TDZ containing media for different time interval had a significant effect on the in vitro multiple shoot induction of Myrica rubra. Embryonic axes explants exposed to the optimal concentration of TDZ were germinated (Fig. 1A), while those not exposed did not germinate. All explants became swollen twice their original size during the second week. After 5 weeks of culture, masses of adventitious buds emerged from the cotyledons bud point, which gradually extended to the whole explants (Fig. 1B). Exposure of explants for longer intervals had a toxic effect on shoot regeneration and the cotyledon buds did not emerge that led to the browning of maximum explants. Highest shoot regeneration frequency (99.80%) was observed when explants were exposed for 21 days to 0.6 mg L-1 TDZ followed by 14 days (97.00%).
|Table 1:||Effects of 0.6 mg L-1 TDZ exposure for 0, 7, 14, 21 and 28 days on adventitious shoot induction from embryonic axes of Myrica rubra after 7 weeks in culture|
|Means within each column followed by the same letter are not significantly different by LSD at p≤0.05, Data was recorded after 7 weeks in culture|
|Fig. 1A-E:||Shoot regeneration and organogenesis from embryonic axes explants of Myrica rubra|
|A: Germinating embryonic axes after exposure to 0.6 mg L¯1 TDZ for 21 days, incubated on MS medium, B:Shoot induction on explants after exposure to 0.6 mg L¯1 TDZ and subsequent culture on hormone free medium for 5 weeks, C: Shoot development on MS medium supplemented with 0.5 mg L¯1 BA plus 0.9 mg L¯1 NAA after 5 weeks in culture, D: Well-developed root system on MS medium fortified with 0.6 mg L¯1 IBA, E: Plants acclimatized to the green conditions|
|Table 2:||Effect of BA alone and/or in combination with NAA on shoot elongation of Myrica rubra after 5 weeks in culture|
|Means within each column followed by the same letter are not significantly different by LSD at p≤0.05|
|Table 3:||Effect of IBA on rooting of microshoots of Myrica rubra after 3 weeks in culture|
|Means within each column followed by the same letter(s) are not significantly different by LSD at p≤0.05|
Maximum number of shoots (16.60) were recorded when explants were exposed to TDZ containing media for 21 days.
Shoot clusters obtained from embryonic axes explants in hormone free media were growing very slow and did not attained the standard size to be transferred direct to the rooting media. Therefore it was necessary to culture these micro shoots in shoot elongation media.
Elongation of adventitious shoots: For shoot elongation, explants with clusters of shoots were transferred to MS medium supplemented with different concentration of BA alone in combination with NAA (Table 2). BA in combination with NAA significantly increased shoot length. In hormone free media shoot length was only 0.40 cm, which was improved to 4.60 cm with the addition of 0.9 mg L-1 NAA (Fig. 1C). For shoot elongation, BA was found to be a necessary growth hormone.
Root induction: Multiple shoots (>2 cm) were separated and cultured in MS medium without growth regulators for 2 weeks to eliminate the effects of cytokinins. Transfer of shoots from elongation medium directly to the rooting medium drastically reduced rooting efficiency. After two weeks, shoots were transferred to full strength MS medium containing various concentrations of IBA for root initiation. Root induction started within 2 weeks in the media containing IBA. IBA had a significant effect on root formations (Table 3) (Fig. 1D). Roots formation did not take place in the media without IBA. Roots were induced with a high frequency of 100% in the medium supplemented with 0.6 mg L-1 IBA. Comparing different levels of IBA, 0.6 mg L-1 was found to be the optimum level with maximum (6.60) roots as compared to the minimum (3.53) with 0.3 mg L-1 IBA. Root length was found maximum (4.34 cm) in the media containing 0.9 mg L-1 IBA as compared to shortest roots (3.00 cm) with 0.3 mg L-1 IBA concentration.
Acclimatization: Rooted plants were removed from the jars; residues of the media were washed out and individually planted in pots containing 1:2 of per liter and sand. Plants were maintained under tissue culture conditions for 3 weeks covered with thin plastic sheet and then transferred to the green house. After 15 days, slits were made in the plastic to lower the humidity and sheets were completely removed on shifting them to the green house. Plants were maintained in the greenhouse for 3 months and showed normal growth and development without showing any morphological abnormalities (Fig. 1E).
Results of this study indicated that adventitious shoots in M. rubra could be induced from embryonic axes explants exposed to MS media containing optimal TDZ level (0.6 mg L-1) for different time intervals. The embryonic axes and cotyledons excised from mature seeds of F. angustifolia underwent adventitious shoot regeneration after exposure to growth regulators, the former showing higher organogenetic capacities than the latter. TDZ is among the most active cytokinin-like growth substance for tissue culture of woody plant species and its low concentrations promote formation of adventitious shoots. Shoots were tremendously elongated with proper regulation of BA levels in combination with NAA in this study. Peddaboina et al. also reported that for shoot elongation, micro-shoots were cultured in media containing different BA and NAA concentrations. In this experiment it was necessary to transfer microshoots to growth regulators free medium before root initiation to eliminate the effects of cytokinins. Transfer of shoots from elongation medium directly to the rooting medium drastically reduced rooting efficiency. Naser et al. suggested that microshoots before transferring to rooting media should be grown on a growth regulator-free medium to eliminate any carry over effect of cytokinins, which inhibit root formation.
Root induction on microshoots was initiated in the medium containing IBA. Sahoo et al. reported that of the three auxins tested (IBA, IAA and NAA) with O. basilicum microshoots, IBA was most effective in inducing roots. This efficient regeneration system from embryonic axes explants could be used as a tool for possible genetic transformation of M. rubra.
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