Cloning of adult trrees of Jamun (Syzygium cuminii)
Vinod Rathore, N S Shekhawat*, R P Singh, J S Rathore and H R Dagla Biotechnology Unit, Department of Botany, Jai Narain Vyas University, Jodhpur 342 001, India
For cloning of adult tree(s) of Jamun (Syzygium cuminii), explants were harvested from rejuvenated shoot sprouts produced by lopped tree(s). Multiple shoots regenerated by activation of axillary meristems of the explants on MS + 9.0 µM BAP + growth additives. Shoots were proliferated in culture by (i) repeated transfer of original explants, and (ii) subculture of shoots on amended medium with combination of BAP (4.50 µM) + kinetin (2.25 µM). The cloned shoots were rooted (i) in vitro on half-strength MS + 0.1% activated charcoal supplemented with IBA (10 µM) or NAA (15.0 µM), and (b) ex vitro by pulse treatment with 2.50 mM of IBA. The shoots root 100% under ex vitro conditions. The cloned treelets were hardened and pot transferred. The ex vitro rooting of cloned shoots was highly effective; it saved time and resources, and can be used for cloning of Jamun.
Keywords: Cloning, Jamun, mature tree, nodal shoots, Syzygium cuminii IPC Code: Int.Cl.7 A 01 H 4/00, 5/00
Introduction
Jamun [Syzygium cuminii (L.)], highly adapted to diverse environmental conditions, is widely distributed in India up to an altitude of 1800 m. It is cultivated commercially for fruit and timber, and also for medicinal uses1-4. A patent, US Pat 5900240, has been granted on an edible herbal composition comprising mixtures of herbs, one of which is S. cumini5. Polyembryonic jamun, propagated in summer from seeds or from semi-ripen cuttings, requires well- drained soil and plenty of Sun.
Tissue culture of Syzygium species is attracting considerable attention for its ability to produce clonal population under in vitro conditions. Micro- propagation of Syzygium cuminii using 10-15-day-old seedlings is reported6. However, this protocol was reported to be non-reproducible7. Micropropagation of S. alternifolium is also reported8. The cultures were initiated from shoot tips/nodal bud explants harvested from seedlings. In vitro process using 1-2-year-old plants of S. travancoricum was also developed9. No protocol is available for cloning of mature/adult tree(s) of any of the Syzygium spp including S. cuminii. This paper presents cloning of adult trees of S. cuminii.
Materials and Methods
Selection and Surface Sterilization
Trees of Syzygium cuminii (< 10-year-old) were selected in and around Jodhpur. Some trees were
regularly watered and lopped during May-June and November-December. The fresh shoots/sprouts were collected to establish the cultures.
Healthy nodal stem segments each with 2-3 nodes were pretreated with 0.4% Bavistin (a systemic fungicide manufactured by Aktiengesellshaft, Ger- many) for 30 min, and then with 0.1% tetracyclin (Hi- Media, Mumbai) solution prepared in autoclaved distilled water for 10 min. This was followed by 0.1%
HgCl2 for 3-5 min depending upon the types of explants. The surface sterilized explants, thoroughly washed several times with autoclaved water, were then kept in chilled, sterile aqueous solution of antioxidants (0.1% ascorbic acid + 0.05% citric acid + 0.1% PVP) for 10-15 min.
Bud Breaking and Multiple Shoot Induction
Surface sterilized explants were inoculated horizontally/vertically on MS medium10 + growth additives (100 mgl-1 ascorbic acid + 50 mg l-1 citric acid) with various concentrations of cytokinins [Kinetin (Kn) or BAP] for shoot initiation. The cultures were kept under controlled conditions of temperature (26-28±2°C) and light (10-40 µmol m-2s-1SFP). For each experiment, minimum of 10 replicates were incubated and each experiment was repeated thrice. Observations were recorded after every 2-3 weeks of inoculation.
Multiplications and Subculturing of in vitro Regenerated Shoots
Mother explants were repeatedly transferred every 18-20 days on either the fresh medium or on a suitably
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amended medium. On first transfer, the mother explants were cultured on fresh medium along with all the in vitro proliferated shoots and shoot buds.
Subsequently, the basal portions remained after harvesting the in vitro generated shoots (2-3 cm), were sub-cultured.
After optimizing the conditions for proliferation of axillary shoots from the mature tree, explants were amplified and multiplied as follows: (i) Repeated transfer of the mother explants; and (ii) In vitro produced shoots were cut into nodal shoot segments each with 2-3 nodes and cultured on various culture media. The shoot multiplication media were supplemented with growth additives. Various concentrations and combinations of cytokinins (BAP, Kn) and auxins (IAA or NAA) were evaluated. To check tip burning, premature leaf fall and leaf curling, several modifications, made in shoot multiplication media, included reduction in levels of nitrates (NH4NO3 and KNO3) to 50% concentration of MS medium and incorporation of (NH4)2SO4 (250 mg l-1), KCl (70 mg l-1) and K2SO4 (100 mg l-1). The modified MS medium was used for shoot multiplication.
Rooting and Hardening of in vitro Produced Shoots
In vitro produced (cloned) shoots were rooted and hardened both in vitro and ex vitro. In vitro rooting and hardening method included in vitro produced shoots (4- 5 cm long), which were excised and transferred individually on various strengths (full, half, one-third and one-fourth) of MS medium containing 0.1% of activated charcoal and different concentrations of IBA and NAA (1.23 to 16.1 µM). Cultures were incubated at 28±2°C under diffused light conditions. In vitro rooted treelets were removed from the culture vessels and washed with sterile water to remove adhered agar.
These were transferred to bottles containing autoclaved soilrite (moistened with one-fourth strength of MS basal salts solution). The bottles were properly capped and kept near pad section of greenhouse [relative humidity (RH) 80-85% and temp 28-30±30°C].
In ex vitro rooting and direct hardening method, strong shoots were harvested and pulse treated with different concentrations of auxins (IBA and NAA) for different time durations. After rooting under green- house conditions, the plantlets were gradually exposed to ex vitro conditions (by loosening and subsequently removing caps of bottles).
Acclimatization and Pot Transfer of Micropropagated Plantlets
Hardening and acclimatization of micropropagated plantlets was done by gradually exposing the plantlets
from high humidity and low temperature to high temperature and low humidity by gradually shifting of plantlets in different zones of greenhouse. The acclimatized plantlets were transferred to poly-bags containing mixture of organic manure, garden soil and sandy soil (1:1:1; V/V). These plantlets were kept in greenhouse for 5-10 days and then transferred to nursery.
Results and Discussion
Establishment of Cultures from Nodal Explants
Shoot cultures were established from nodal stem segments obtained from pruned trees. Sustainable growth and multiplication of shoots was achieved by:
(i) Selection of responsive (reinvigorated) shoots from rejuvenated source plants; and (ii) Modification of nutrient medium. The source plant was lopped during winter or severe summer. The explants were prepared from shoot sprouts (flushes) that regenerated from lopped tree during active growth phase. Such explants exhibited bud breaking on modified MS medium supplemented with 8.87 µM BAP. The explants derived from non-lopped tree were difficult to sterilize as these carried recalcitrant contaminants. The explants, which leached phenolic substances that caused browning of culture medium, did not respond in culture and deteriorated and died. Perennially growing shoots exposed to the seasonal/environmental stresses, accumulate metabolites/inhibitors/bud arresting factors that probably help the plant survive the stress. Unless these factors are diluted/removed, the meristems remain arrested/inactive and therefore, poor response of explants derived from such shoots. Lopping of tree during winter removed the inhibitory substances and allowed rejuvenation and regeneration of invigorated shoot sprouts. These shoots, when used as explants, responded in culture as these contained no/low anti- inhibitors. Effects of season/stresses on the morphogenic competences of mature trees have been investigated11. A variety of rejuvenation pretreatments to activate the meristems/buds for in vitro/in vivo cloning have been suggested, including treatments with cytokinins (BAP in most of the cases), pruning, severe lopping and hedging12,13. Of the rejuvenated nodal stem explants of S. cuminii, 90% responded by exhibiting bud breaking and produced multiple shoots. On modified MS + 9.00 µM of BAP, maximum number of shoots were produced within 7-8 days at 28±2°C (Table 1, Fig. 1). BAP is reported the most active, the cheapest and the only one that can be autoclaved14.
Proliferation of Shoots In vitro
Shoots of S. cuminii could be multiplied in vitro by repeated transfers/serial culture of the mother explants on the fresh medium but with half of the concentration of BAP. Thus once the meristem was activated, for further growth and multiplication of shoots, low concentration of BAP was required. An improved capacity of the cultures to multiply and to form plantlets can be obtained by in vitro procedures.
Rejuvenation by serial culture is the most common of these. Improved performance after several transfers was observed in cultures of numerous tree species14. Subculturing of in vitro generated shoots on modified MS medium+4.50 µM of BAP resulted in
differentiation of 2-3 shoots from each node.
However, the shoots multiplied on full strength MS medium showed symptoms of hyperhydaration and defoliation. The levels of nitrates were reduced to half of the concentrations of MS. (NH4)2SO4 (100 mg/l), KCl (70 mg/l), and K2SO4 (250 mg/l) were added to the culture medium. A combination of BAP (4.50 µM) and Kn (2.25 µM) was incorporated to the culture medium to promote healthy growth of the shoots (Fig. 2). Subculture was regularly done every 20-25 days. Delay in subculture adversely affected both the shoot growth and multiplication of the shoots. Maximum growth and multiplication occurred at 28+2°C under 50-60 µmol m-2 s-1 SFP.
Rooting of Shoots and Hardening of Cloned Plants
In vitro generated shoots of S. cuminii rooted on half-strength MS + 0.1% activated charcoal supplemented with IBA and NAA. The shoots rooted (95%) in 10.0 µM of IBA, whereas on 15.00 µM of NAA rooting percentage was 82%. The effect of IBA at 1.25 µM, 2.50 µM and 15.00 µM concentrations on root number per shoot and length of individual root was not significantly different. However, the number of roots produced per shoots and the length of root were significantly higher on 5.0 and 10.0 µM IBA (Table 2). The shoots rooted 100% under ex vitro conditions if pulsed with 2.50 mM of IBA (Fig. 3).
However, by treatment with 2.50 mM of NAA, only 65% of the shoots rooted (Table 3). Both the types of plantlets rooted in vitro and ex vitro were hardened in the green house by gradually shifting from high RH/low temperature regimes to declining RH to increasing temperatures. The ex vitro rooted plantlets were hardened with greater ease as compared to in vitro rooted and transferred to soilrite (Fig. 4).
Adventitious rooting has been considered and manipulated as a single phase processes, in which auxin was considered to play a major role15. IBA is very effective in promoting rooting of a wide variety of plants, and it is used commercially to root many plant species worldwide16.
While studying the effects of in vitro root initiation on subsequent root quality of in vitro generated shoots of three woody plants, greater resistance of ex vitro rooted plants to stress was suggested17. The main advantage of ex vitro rooting is that the root damage during transplantation to soil is less, rooting rates are often higher and root quality is better15. In the case of S. cuminii, ex vitro rooted plantlets grew faster than the in vitro rooted plantlets. There are reports18,19 on
Figs 1-4⎯ Cloning of trees of S. cuminii: 1, Multiple shoot production by bud breaking from nodal shoots explants on MS + 9.0 µM BAP + additives; 2, Amplification of shoots on amended medium + BAP (4.5 µM) + kinetin (2.25 µM); 3, Ex vitro root regeneration from the cloned shoots treated with 2.50 mM of IBA;
& 4, Micropropagated/cloned treelets ready for field transfer Table1 ⎯Effects of concentrations of BAP on multiple shoot
induction from nodal sten explants of S. cuminii Concentrations
µM
Response
%
Shoot number (mean± SD)
Shoot length (cm; mean± SD) BAP
1.125 - - -
2.250 25 1.20±0.42 0.68±0.24 4.500 47 1.40±0.48 1.12±0.11 9.000 95 2.20±0.40 1.84±0.48 22.500 82 1.60±0.39 0.96±0.33 Comp: F
BAP
Replication: 2.086ns 0.2253ns Treatment : 27.304** 18.02**
CD : 0.645 0.671
ex vitro shooting of woody plants. Authors suggest that ex vitro is very effective in establishment of cloned plants of Azadirachta indica (Neem), Simmondsia chinensis (Jojoba), Citrus limon, Rauvolfia serpentina and even difficult to root species like Acacia nilotica and Tecomella undulata. Ex vitro root saves both tissue and resources.
The protocol described in this paper is being used for production of Jamun plants. This can be used by foresters for cloning of desirable trees of Jamun.
Acknowledgement
Authors gratefully acknowledge the Department of Biotechnology, Govt. of India, New Delhi for providing support for establishment regional facilities of micropropagation and greenhouse for arid areas.
Thanks are also due to the University Grants Commission, Govt. of India, New Delhi for providing support and facilities for research under Special Assistant Programme (SAP-DSA) to the Department of Botany, JNV University, Jodhpur (Rajasthan).
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Table 2⎯ Root induction in shoots of S. cuminii on different auxins incorporated in half-strength MS + 0.1% activated charcoal Auxins con-
centration µM
Response %
Root number (mean± SD)
Root length (cm, mean±SD) IBA
1.25 40 1.20±0.40 0.86±0.19 2.50 80 1.60±0.48 0.80±0.25 5.00 90 2.80±0.45 1.80±0.36 10.00 95 2.20±0.44 1.24±0.11 15.00 85 1.60±0.31 0.86±0.24 NAA
2.50 50 1.60±0.40 1.04±0.30 5.00 65 1.60±0.00 1.36±0.32 10.00 72 1.70±0.48 1.20±0.79 15.00 82 1.80±0.41 1.10±0.41 Comp F:
IBA Replication: 0.528ns 3.30 Treatment : 8.07** 16.96**
CD 0.979 0.436
NAA
Replication: 3.40 4.32**
Treatment : 2.66ns 3.26*
CD 0.780 0.898
Table 3⎯Effects of concentrations of auxins (pulse treatment) on ex vitro root induction from isolated shoots of S. cuminii Auxin con
centration (μM)
Response % Root number (mean±SD)
Root length (cm, mean±SD) IBA
0.125 32 1.20±0.40 1.86±0.43 0.250 45 1.60±0.48 1.52±0.39 0.500 60 1.80±0.32 2.38±0.36 1.000 92 2.80±0.97 3.06±0.49 2.500 100 3.00±0.63 3.04±0.69 NAA
0.025 35 1.20±0.40 1.12±0.04 0.500 42 1.40±0.48 1.36±0.37 1.000 54 1.60±0.48 2.22±0.09 2.000 65 2.20±0.39 2.14±0.67 Comp F:
IBA
Replication 3.89* 1.22ns Treatment : 12.31** 9.36**
CD: 0.829 1.03
NAA
Replication: 2.59ns 0.274ns Treatment : 5.33* 6.61**
CD: 0.78 1.05
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