ELSEVIER Scientia Horticulturae 61 (1995) 227-239 SCIENTIA HORTlCULTUR& Efficient plant retrieval from alginate-encapsulated vegetative buds of mature mulberry trees S.K.Pattnaik, Y. Sahoo, P.K. Chand* Plant Tissue and Cell Culture Facility, Post-Graduate Department ofBotany, Utkal University, Bhubaneswar-751004, Orissa,India Accepted 15 September 1994 Abstract Axillary vegetative buds of 3-year-old mature mulberry trees of three indigenous and two Japanese varieties from the open field were successfully encapsulated in calcium algin- ate beads. The best gel complexation was achieved using 4% sodium alginate with 75 mM CaC12.2Hz0. Use of semi-solid MS medium resulted in 100% conversion of encapsulated shoot buds into plantlets, especially when buds were pretreated with 1.O mg l- ’ BA for 36 h. Although sprouting was faster on medium fortified with 1.0 mg I-’ BA and 0.3 mg 1-l GA,, one-step germination, i.e. both shoot and root induction, was better on a MS basal medium without added phytohormones. Among several plating substrates assessed for in vivo germination, the maximum response was elicited on artificial soil moistened with half-strength MS medium devoid of sucrose. A varietal difference was evident with respect to germination potential between encapsulated meristems under in vitro and in vivo situ- ations. The encapsulated buds could be stored for 60 days at 4°C without loss of viability only when the gel matrix contained MS nutrients, vitamins and sucrose. There was a neg- ligible ( < 10%) mortality of complete plants while they were being transferred from arti- ficial soil to natural soil and finally to the open field. Keywords: Encapsulation; Genotypic variation; In vitro germination; In vivo germination; Mulberry 1. Introduction Recently, the encapsulation technique for producing synthetic seeds or artifi- cial seeds has become an important asset in micropropagation. Encapsulation of * Corresponding author. Abbreviations: BA, benzyladenine; GA,, gibberellic acid; IBA, indole butyric acid; MS, Murashige and Skoog ( 1962) medium. 0304-4238/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0304-4238 (94)00742-X
Transcript
ELSEVIER Scientia Horticulturae 61 (1995) 227-239
SCIENTIA HORTlCULTUR&
Efficient plant retrieval from alginate-encapsulated vegetative buds of mature mulberry trees
S.K.Pattnaik, Y. Sahoo, P.K. Chand* Plant Tissue and Cell Culture Facility, Post-Graduate Department ofBotany, Utkal University,
Bhubaneswar-751004, Orissa, India
Accepted 15 September 1994
Abstract
Axillary vegetative buds of 3-year-old mature mulberry trees of three indigenous and two Japanese varieties from the open field were successfully encapsulated in calcium algin- ate beads. The best gel complexation was achieved using 4% sodium alginate with 75 mM CaC12.2Hz0. Use of semi-solid MS medium resulted in 100% conversion of encapsulated shoot buds into plantlets, especially when buds were pretreated with 1 .O mg l- ’ BA for 36 h. Although sprouting was faster on medium fortified with 1.0 mg I-’ BA and 0.3 mg 1-l GA,, one-step germination, i.e. both shoot and root induction, was better on a MS basal medium without added phytohormones. Among several plating substrates assessed for in vivo germination, the maximum response was elicited on artificial soil moistened with half-strength MS medium devoid of sucrose. A varietal difference was evident with respect to germination potential between encapsulated meristems under in vitro and in vivo situ- ations. The encapsulated buds could be stored for 60 days at 4°C without loss of viability only when the gel matrix contained MS nutrients, vitamins and sucrose. There was a neg- ligible ( < 10%) mortality of complete plants while they were being transferred from arti- ficial soil to natural soil and finally to the open field.
Keywords: Encapsulation; Genotypic variation; In vitro germination; In vivo germination; Mulberry
1. Introduction
Recently, the encapsulation technique for producing synthetic seeds or artifi- cial seeds has become an important asset in micropropagation. Encapsulation of
somatic embryos or apical and axillary shoot buds, and regeneration of whole plants from them, has been reported for a number of species (Redenbaugh et al., 1986; Mathur et al., 1989; Ganapathi et al., 1992; Lulsdorf et al., 1993). In view of the significance of mulberry (Morus spp. ) in the sericulture industry, there has been considerable interest in mulberry micropropagation (Ivanicka, 1987; Jain et al., 1990; Hossain et al., 1992). However, to date, information regarding the feasibility of the practical application of this encapsulation technique to this eco- nomically important plant species is limited (Bapat et al., 1987; Bapat and Rao, 1990). Whilst studies in recent reports are restricted to nodal segments from al- ready established axenic shoot cultures, the present paper reports on a much sim- pler method of utilising the resting vegetative buds from 3-year-old mature mul- berry trees from the open field for the encapsulation process. A thorough study was also carried out on the regeneration of complete plantlets from such encap- sulated buds in axenic conditions as well as directly on soil. In addition, we have examined one indigenous (M. indica) and two Japanese (M. multicaulis and M. bombycis) species with respect to their germination potential both in vitro and in vivo, and a genotypic difference was found. Some work was also carried out on the long-term storage of these encased shoot meristems to ensure satisfactory re- tention of viability. The in vivo strategy described in this paper would enable marginal farmers from developing countries to carry out large-scale propagation of elite mulberry varieties.
2. Materials and methods
2.1. Explant source
Healthy axillary buds were collected from the Mulberry Germplasm Unit es- tablished and maintained in the Post-Graduate Department of Botany, Utkal University. Three indigenous varieties, namely ‘Kanva-2’, ‘S- 1635’ and ‘S-36’, belonging to M. indica and two Japanese varieties, namely ‘Goshoerami’ (M. multicaulis) and ‘Kenmochi’ (M. bombycis) served as the source material. The vegetative buds were washed thoroughly under running tap water for 20-30 min, followed by treatment with 10% bleaching powder (CaOCl,) solution in distilled water for 4-5 min, and then rinsed with tap water (3-4 changes). The buds were then transferred to a laminar airflow cabinet and treated with 0.1% HgC& (BDH, India) for 12 min, followed by lo-12 min in a solution of 7% NaOCl and 5% Teepol in distilled water. Finally, shoot buds were rinsed in autoclaved water (5- 6 changes). A set of buds were soaked in a range of concentrations of BA (0.5- 2.5 mg 1-l ) for different time periods ( 12, 24,36 or 48 h). During soaking the shoot buds were incubated at 26’ C in the dark.
2.2. Encapsulation
Sodium alginate (CDH, Bombay) was added in the range of 2-8O/6 (w/v) either to distilled water or to MS liquid medium with or without 3% sucrose and 100
mg l- ’ myo-inositol. The solutions were supplemented with BA ( 1 .O mg 1-l). GAS (0.3-0.5 mg l- ’ ) was added when winter buds served as the source material. A bactericide, rose bengal (4,5,6,7-tetrachloro-2’,-4’,-5’,-7’-tetraiodofluorescin, BDH, England) and a fungicide, bavistin, were added to the gel matrix at a con- centration of 250 mg 1-l and 1 O-20 mg I-‘, respectively, for in vivo germination. For complexation, different concentrations of CaC12.2H20 (50-l 25 mM) were prepared in either distilled water or liquid MS medium containing the same growth adjuvents as the sodium alginate matrix but excluding rose bengal and bavistin. Both the gel matrix and the complexing agent were autoclaved after adjusting the pH to 5.8. Encapsulation was accomplished by mixing the shoot buds into the sodium alginate solution and dropping these into the calcium chloride solution. Each drop contained one bud. The drops set as small, white beads when left in CaCl, solution for 30 min. The beads containing the entrapped shoot buds were retrieved using a sterilized tea-strainer.
In special cases when germination response was to be recorded following pro- longed storage, vegetative buds encased in a calcium alginate capsule were main- tained in a laboratory refrigerator at 4°C and data was collected up to 3 months following encapsulation.
2.3. Medium and culture conditions for in vitro germination
In order to induce sprouting in vitro, the encapsulated shoot buds were plated on one of the following culture media: ( 1) MS basal medium + 3% sucrose t- 0.8% agar (MSO); (2) MS+BA (0.5-2.0 mg l-‘)-t3% sucrose+0.8% agar; (3) MS+BA (0.5-2.0mgl-‘)+GA3 (0.2-0.5mg1-‘)+3%sucrose?0.8%agar; (4) Tap water + 0.8% agar.
Each treatment consisted of five buds and was repeated three times. Beads were inoculated either in Petri dishes ( 12 + 2 beads per 9 cm diameter Petri dish) or in jars (4 +- 1 beads per 6 oz. wide-mouthed screw-capped glass jar) and main- tained under continuous light (cool white fluorescent tubes, 2.0 W me2) and at a constant temperature regime (25 2 1 “C). After sprouting the encapsulated shoot buds were transferred to 150 ml flasks (two sprouted buds per flask) containing the same medium as that on which the encapsulated buds were sprouted. Three- week-old shoots with fully expanded leaves emerging through the ruptured cap- sule were placed in half-strength MS medium fortified with a range of IBA con- centrations (0.25-2.0 mg 1-l) to trigger root development. A set of non-encap- sulated shoot buds were plated on the media described above and maintained under similar conditions in order to serve as a control.
2.4. Medium and culture conditions for in vivo germination
For germination under in vivo conditions, four artificial and natural plating substrates were tested: ( 1) Soihite mix (Kelperlite, Kamataka Explosives Ltd., Bangalore, India) moistened with half-strength MS medium t- 3% sucrose; (2)
natural soil + half-strength MS medium 5 3Oh sucrose; ( 3 ) Soilrite mix + tap water. (4) natural soil + tap water.
Each treatment consisted of live replicates and was repeated three times. The substrates were autoclaved prior to use. Vegetative buds entrapped in calcium alginate capsules were plated onto the media, which were contained in Petri dishes or glass jars. Substrates ( 1) and (2) were moistened with autoclaved half-strength MS solution at 2-day intervals in the first week, and subsequently with quarter- strength MS solution. Cultures were maintained in the glasshouse under incan- descent bulbs supplemented by daylight, and adjusted to the natural photoper- iod. A set of non-encapsulated shoot buds were plated onto the same substrates and maintained under similar conditions in order to serve as a control.
2.5. Transfer of plantlets
The 6-week-old plants, grown to a height of ca. 8 cm and with well-developed roots, were transferred to plastic pots (7.5 cm diameter) containing Soilrite mix that had previously been autoclaved and irrigated with quarter-strength MS liq- uid medium. Plantlets were maintained inside a plant growth chamber (SICO, India) kept at 26 + 1 “C, 85-90% relative humidity and 16 h daylength for 2 weeks, and in natural soil for a further 1 week, before transfer to the field.
In the case of in vivo germination, 5-6-week-old plantlets with well-developed shoot and root systems from alginate-encapsulated shoot buds were maintained in the glasshouse in earthenware pots (9 cm diameter), each covered with a po- lythene bag to ensure maximum humidity. The available illumination, from in- candescent bulbs supplemented by daylight, was 5.0 W me2 at the pot level. After acclimatization in natural soil for 2 weeks while keeping the plants covered by polythene bags, the complete plants were eventually transplanted into the open field.
3. Results
3.1. Characteristics of encapsulated buds
About 200-220 characteristic beads (hydrogels) were formed using 50 ml of sodium alginate and 120 ml CaC12.2H20 solution. The beads differed morphol- ogically with respect to texture, shape and transparency, with different combina- tions of sodium alginate and calcium chloride, and with diameters ranging from 4.5 to 6.2 mm. An encapsulation matrix of 4% sodium alginate with 75 mm CaC12.2H20 was most suitable for the formation of ideal beads (Fig. 1). Na- alginate concentrations of l-3% were not suitable for encapsulation because the resulting beads were without a defined shape and were too soft to handle, whilst at higher concentrations the beads were isodiametric but were hard enough to cause considerable delay in sprouting. Lower concentrations of calcium chloride
Fig. 2. (a-d). Progressive stages of sprouting of encapsulated buds ( X 1.55 ) .
caused delays in the preparation of the gel matrix in addition to adversely affect- ing the bead quality.
3.2. In vitro germination
Liquid media were unsuitable because both encapsulated and unencapsulated (control) buds failed to sprout, irrespective of the nutrient supplement. On agar- solidified MS medium containing BA at an optimal concentration of 1 .O mg l- I, the encapsulated buds sprouted within 8- 10 days (Fig. 2 (a-d ) ) . Sprouting took 12-15 days on MS0 and 20-25 days on agar-solidified tap water. This period was lengthened to 28-30 days for sprouting of buds collected in the winter season. However, the sprouting period was reduced to 12- 14 days by supplementing GA3 (0.3 mg I- ’ ) in the encapsulation matrix with BA ( 1 .O mg I-’ ) . No significant stimulatory effect was recorded with GA3 supplement alone, regardless of con- centration. The unencapsulated shoot buds sprouted within 8- 10 days on MS + BA ( 1 .O mg I- ’ ) . The sprouting period was lengthened to 18-20 days on MS0 in
232 S.K. Pattnaik et al. /Scientia Horticulturae 61 (1995) 227-239
indigenous varieties. The Japanese varieties did not sprout on MSO. On agar- solidified tap water, both indigenous and Japanese varieties failed to sprout. In indigenous varieties the percentage of germination of encapsulated shoot buds was 85-909/o, 70-80% and 55-6596 in MS+BA, MS0 and tap water+agar, re- spectively. In Japanese varieties the germination percentage was 70-75% in MS+BA, 40-45% in MS0 and 20-25% in tap water+ agar. In indigenous vari- eties the percentage of sprouting of unencapsulated shoot buds was 80-90% in MS+BA and 35-40% in MSO. In Japanese varieties 65-70% of the unencapsu- lated buds sprouted in MS +BA. Roots developed from the sprouted buds in MS0 within 2 1 days of culture. However, the shoot buds cultured in MS + BA ? GA3 did not show any sign of rhizogenesis even after 5-6 weeks. Rooting was induced in the latter case in half-strength MS0 fortified with 1 .O mg l- ’ IBA.
3.3. In vivo germination
Among the different plating substrates assessed for germinating alginate-en- trapped shoot buds in vivo, the best result was recorded with Soihite mix mois- tened with half-strength MSO. The percentage germination was 55-65% in indig- enous varieties and 45-50% in Japanese ones. The encased shoot buds inoculated onto this substrate germinated within lo- 12 days, producing leafy shoots imme- diately followed by rooting. However, exclusion of sucrose from the gel matrix delayed the germination up to 20-25 days. About 70-7596 shoot buds encapsu- lated in sucrose-free gel turned necrotic within 14 days of inoculation onto Soil- rite mix+ half-strength MS0 and failed to germinate. In indigenous varieties the percentage of germination was 45-50%, 30-40% and 15-20% in natural soil+ half- strength MSO, Soilrite mix + tap water and natural soil + tap water, respectively. However, for Japanese varieties the percentage of germination was 30-40% in natural soil + half-strength MSO, 15-2096 in Soihite mix + tap water and lo- 15% in natural soil + tap water (Fig. 3 ) . The unencapsulated buds (control) inocu- lated onto the above plating substrates turned necrotic within 6-7 days and failed to germinate. Maintenance of a high humidity was essential before and immedi- ately after germination. Failure to do so meant that the alginate beads suffered from intense desiccation.
The inclusion of rose bengal was essential, as this chemical satisfactorily pre- vented bacterial contamination. A concentration of 250 mg 1-i was most effec- tive without adversely affecting the germination of encapsulated buds. The bac- tericidal role was most pronounced in the winter season when pathogenic attack was greatest. Addition of bavistin to the encapsulating matrix at an optimum range of 1 O-20 mg l- ’ successfully prevented fungal contamination. A combina- tion of rose bengal (250 mg 1-l ) and bavistin ( 10 mg 1-l ) was essential in situ- ations in which alginate-encased shoot buds were allowed to germinate directly on artificial/natural soil.
Fig. 3. In vivo germination of different genotypes on various plating substrates. K-2, ‘Kanva-2’; KN, ‘Kenmochi’; GS, ‘Goshoerami’; NS, natural soil; SM, Soilrite mix; TW, tap water.
3.4. Eflect of BA pretreatment of buds on germination
Vegetative buds soaked in BA prior to encapsulation exhibited early sprouting (6-8 days) under both in vitro and in vivo situations. Also, there was a remark- able enhancement in the germination frequency. An optimal concentration of 1 mg l- ’ BA boosted the germination up to 100% in indigenous cultivars and up to 85-90% in Japanese ones. Although sprouting was promoted as a result of soak- ing in higher concentrations of BA (2.0 or 2.5 mg 1-l ), root development was considerably delayed and often inhibited, thereby affecting gross germination fre- quency. A pretreatment for 36-48 h was ideal, as shorter durations were not sig- nificantly effective. In addition, BA presoaking of buds stimulated prolific mul- tiple shooting (Fig. 4)) especially in the case of the indigenous cultivars tested, i.e. ‘S-1635, ‘S-36’ and ‘Kanva-2’. This promotion of germination of encapsu- lated buds after a BA pretreatment was comparable between in vitro and in vivo situations. A kA presoaking was a necessary prelude to encapsulation, especially when the shoot buds were to be germinated on a basal medium without added phytohormones (MSO). The percentage of germination of unsoaked buds on MS0 was 7080% in indigenous varieties and 40-45% in Japanese ones. How- ever, the germination percentage was increased to 95-10096 in indigenous vari- eties and 85-90% in Japanese ones when the shoot buds were soaked with 1 .O mg l- ’ BA prior to encapsulation.
3.5. Long-term storage
High humidity and low temperature were essential conditions for adequate re- tention of viability and hence for storage of encapsulated buds. It was noticed
234 S.K. Pattnaik et al. /Scientia Horticulturae 61(1995) 227-239
Fig. 4. Multiple shoots emerging from a BA-presoaked encapsulated bud ( x 2.2).
100
S-1635 S-38 K-2 m GS 4 d
Mulberry variety
Fig. 5. Variation in genotypic responses for in vitro germination at 30, 60 and 90 days following encapsulation. K-2, ‘Kanva-2’; KN, ‘Kenmochi’; GS, ‘Goshoerami’.
that there was no striking difference in germination rate when the beads were kept in a refrigerator (4” C ) for 60 days, but that longer periods resulted in a marked decline. This trend held good for both in vitro and in vivo germinations (Figs. 5 and 6). A comparative response of the buds entrapped in an alginate matrix that had been prepared either in MS medium or in distilled water was studied. One set of beads prepared in MS/distilled water was immediately placed on MS + BA ( 1 mg l- ’ ) for germination. Another set was kept at 4 o C in a refrig- erator for 60 days and subsequently allowed to sprout. In the former case, the germination of encapsulated buds prepared in distilled water was lo- 15% lower
S.K. Pattnaik et al. /Scientia Horticulturae 61 (1995) 227-239 235
S-1635 S-36 K-2 KN GS
Mulberry cv
Fig. 6. Variation in genotypic responses for in vivo germination at 30, 60 and 90 days following en- capsulation. K-2, ‘Kanva-2’; KN, ‘Kenmochi’; GS, ‘Goshoerami’.
Table 1 Comparative response of encapsulated buds (‘S-l 635’) prepared in MS medium or distilled water for in vitro germination
(Data Rk SD) recorded from three independent experiments, each with three replicatess.
compared with those in MS medium, but in the latter case the difference was even more marked (Table 1) .
3.6. Eflect of genotypic variation
Three indigenous varieties (‘Kanva-2’, ‘S-1635’ and ‘S-36’) of Torus indica and two Japanese varieties, ‘Goshoerami’ (M. multicaulis) and ‘Kenmochi’ (M. bombycis), were assessed for variation in their genotypic responses during in vi- tro and in vivo germination at 30,60 and 90 days following encapsulation (Figs. 5 and 6). In general, the three Indian varieties performed better than the Japa- nese ones, regardless of the duration of storage. Nevertheless, there was a retro- gressive trend in sprouting responses as the inoculation of encapsulated buds was
236 S.K. Pattnaik et al. /Scientia Horticulturae 61 (1995) 227-239
Fig. 7. A well-rooted plant 2 months after potting ( x 0.12 )
delayed for 30,60 or 90 days. This trend held good for both Indian and Japanese varieties. Out of the three Indian varieties tested, the sprouting percentage was highest in ‘S-1635’, followed by ‘S-36’ and ‘Kanva-2’. Among Japanese varieties, ‘Kenmochi’ responded better than ‘Goshoerami’.
3.7. Acclimatization and transfer of rooted plants to the field
Rooted plantlets with 4-5 fully expanded leaves from in vitro germinated en- capsulated shoot buds were successfully hardened off in the plant growth cham- ber for 2 weeks in artificial soil, followed by 1 week in natural soil. For vegetative buds germinated directly (in vivo ), complete plants with well-developed shoots and roots were acclimatized in natural soil (Fin. 7 ) and successfully transplanted into the field. The mortality was < 5% in ilaits ‘germinated < 10% in the case of plants retrieved via an in vitro set-up.
in vivo as against
4. Discussion
Fundamental to a successful propagation programme routed through encap- sulation is the selection of the appropriate plant part as the starting experimental material, the critical evaluation of the factors influencing the formation of the gel matrix, and above all optimisation of the process of germination for plant re- trieval. Axillary shoot buds were suitable for encapsulation studies as they were excellent material for the preparation of artificial seeds or synthetic seeds, besides possessing great potential for plant development from pre-existing meristematic tissues. This strategy was of particular significance for a plant like mulberry, in which the process of somatic embryogenesis has not been documented, and there- fore somatic embryos are not available for production of synthetic seeds. In ad- dition, the use of axillary shoot buds would ensure a genetic uniformity and sta- bility in the regenerants. In the present studies, shoot buds for encapsulation were
SK. Pattnaik et al. /Scientia Horticulturae 61(1995) 227-239 237
obtained directly from 3-year-old mature field-grown plants, which differs from previous reports in which axenic shoot cultures provided the explants, e.g. MOWS indica L. (Bapat et al., 1987; Bapat and Rao, 1990) and Vakriana wallichii D.C. (Mathur et al., 1989). In order to simplify the micropropagation strategy for en- capsulation, it would be desirable to have direct access to the propagules from garden plants growing in vivo. Such a study could ensure the maintenance of the propagules as near to the natural plant level as possible, in order to facilitate sub- sequent in vivo germination of the encapsulated meristems directly on soil. It could also bypass the in vitro step of plant maintenance in the form of axenic shoot cultures, which might be necessary and desirable from an economic stand point.
As assessment of the effects of various concentrations of sodium alginate ( l- 8%) and calcium chloride (50-125 mM) on the texture, shape and size of the beads was a prerequisite in order to standardise the preparation of characteristic beads. An optimal ion exchange between Na+ and Ca*+ producing firm, clear, isodiametric beads was achieved using a 4% solution of sodium alginate upon complexation with 75 mM CaC12.2H20, forming an insoluble gel matrix of cal- cium alginate. Sodium alginate preparations at lower concentrations became un- suitable for encapsulation, probably because of a reduction in its gelling ability after exposure to high temperatures during autoclaving (Larkin et al., 1988 ) . The use of agar as a gel matrix was deliberately avoided because it has been described as inferior to alginate with respect to long-term storage (Bapat et al., 1987). Al- ginate was chosen for ease of capsule formation as well as for its proven low tox- icity for somatic embryos (Fujii et al., 1987). Also, the rigidity of the alginate beads provided better protection to the encased meristems against mechanical injury than did agar.
Entrapped vegetative buds sprouted within 8- 10 days on MS medium contain- ing a cytokinin ( 1 mg l- ’ BAP ), whereas sprouting took 12- 15 days on MS basal medium. However, the latter medium was preferable because it supported shoot and root formation in one step and eventually whole plant recovery. Buds failed to sprout in the unagitated liquid media; submergence may have impeded respi- ration. An additional supplement of gibberellic acid in the gel matrix as well as in the culture medium had a clear role in stimulating the germination of vegeta- tive buds collected in the winter season, possibly by breaking their dormancy. This promoting effect of GA3 corroborates germination studies using unencap- sulated winter buds as explants (Pattnaik et al., 1993).
From a comparative study on the sprouting behaviour of encased vegetative buds of three Indian (‘S-1635’, ‘S-36’ and ‘Kanva-2’) and two Japanese (‘Ken- mochi’ and ‘Goshoerami’) varieties, a clear genotypic response was evident with respect to bud-to-plant conversion frequency.
The ultimate realisation of the synthetic seed system is when these are trans- lated into whole plants in soil. The major limiting factor for continued develop- ment of artificial seeds to grow into whole plants is probably the availability of nutrients. It is therefore imperative adequately to build up the nutrient reservoir for the encased key tissue (axillary shoot buds) either endogenously or exoge-
nous]y. A nutrient-cum-cytokinin loading could be accomplished by a pretreat- ment, which is a very useful technique in plant conversion (Redenbaugh et al,, 1986). In mulberry, treating the vegetative buds in a solution of BA ( 1 .O mg l- I ) for 36-48 h prior to encapsulation accelerated the germination process and con- sequently enhanced plant recovery. A more pronounced effect of a BA pretreat- ment was the prolific development of multiple shoots from germinating buds-a very desirable feature in micropropagation. This BA stimulation corroborates findings from studies on explants pretreated in a similar way with (Bapat et al., 1987) or without (Mhatre et al., 1985) encapsulation. However, an adequate exogenous supply of nutrients must be ensured to create an ideal synthetic endo- sperm. An interesting feature of the encapsulated buds was their ability to retain viability in terms of germination potential even after a considerable period of storage. A low temperature (4” C) made it possible to store the encapsulated buds for as long as 60 days without significant loss of viability. Retrieval of plants after such a storage period was feasible only when the encapsulating matrix was pre- pared using MS media with the necessary phytohormone additives. Such a cap- sule gel must therefore potentially serve as a reservoir of nutrients, behaving as an artificial endosperm and supplying the necessary nutrients to the entrapped explants as the natural endosperm does to the growing zygotic embryo. Con- versely, the use of distilled water in making sodium alginate failed to support sprouting because of the very poor nutrition provision.
Incorporation of a suitable combination of bactericide and fungicide, as given by rose bengal and bavistin in the encapsulation matrix, markedly improved the plant retrieval ability of the alginated shoot buds, especially under in vivo con- ditions. Similar use of such antimicrobial agents was beneficial for in vivo ger- mination of encapsulated buds of Vuleriana wullichii (Mathur et al., 1989).
In the case of mulberry, only 30-4OW of cuttings survive the time period be- tween pruning, transportation and final transplantation, whereas the encapsu- lated buds could easily be packed in bottles or polythene bags, thus limiting the space needed and ensuring a high viability and survival rate. Thus, this synthetic seed technology could be a revolutionary propagation strategy for many of the elite and difficult-to-root mulberry cultivars.
As compared with previously published methods, our procedure is much sim- pler because it does remove the need to establish a tissue-culture facility in order to obtain propagules from axenic cultures. In the protocol given in this paper, resting vegetative buds can be picked from 2-3-year-old mature trees growing in the open field, surface-cleaned, encapsulated in a simple nutrient gel with cheap antimicrobial agents, and finally sown directly onto soil. Such an approach could considerably reduce the cost of production of artificial seeds in the case of fruit trees in general, thereby bypassing the involvement of a sophisticated laboratory, presently unaffordable by most marginal farmers in developing countries.
S.K. Pattnaik et al. /Scientia Horticulturae 61(1995) 227-239 239
Acknowledgement
The financial assistance provided by Central Silk Board, Ministry of Textiles, Government of India, through a National Sericulture Project is gratefully acknowledged.
References
Bapat, V.A. and Rao, P.S., 1990. In vivo growth of encapsulated axillary buds of mulberry (Torus indica L.). Plant Cell Tissue Organ Cult., 20: 69-70.
Bapat, V.A., Mhatre, M. and Rao, P.S., 1987. Propagation of Mom indica L. (mulberry) by encap- sulated shoot buds. Plant Cell Rep., 6: 393-395.
Fujii, J.A., Slade, D.T., Redenbaugh, K. and Walker, K.A., 1987. Artificial seeds for plant propaga- tion. TIBTECH, 5: 335-339.
Ganapathi, T.R., Suprasanna, P., Bapat, V.A. and Rao, P.S., 1992. Propagation of banana through encapsulated shoot tips. Plant Cell Rep., 11: 571-575.
Hossain, M., Rahman, SM., Zaman, A., Joarder, 0.1. and Islam, R., 1992. Micropropagation ofMo- rus laevigata Wall. from mature trees. Plant Cell Rep., 11: 522-524.
Ivanicka, J., 1987. In vitro micropropagation of mulberry (Morus nigra L. ) . Scientia Hortic., 32: 33- 39.
Jain, AK., Dandin, S.B. and Sengupta, K., 1990. In vitro propagation through axillary bud multipli- cation in different mulberry genotypes. Plant Cell Rep., 8: 737-740.
Larkin, P.J., Davies, P.A. and Tanner, G.J., 1988. Nurse culture of low number of Medicago and Nicotiana protoplasts using calcium alginate beads. Plant. Sci., 58: 203-210.
Lulsdorf, M.M., Tautorus, T.E., Kikcio, S.I., Bethune, T.D. and Dunstan, D.I., 1993. Germination of encapsulated embryos of interior spruce (Picea glauca engelmanii complex) and black spruce (Pi- tea mariana Mill.). Plant Cell Rep., 12: 385-389.
Mathur, J., Ahuja, P.S., Ial, N. and Mathur, A.K., 1989. Propagation of Valeriana wallichii DC. using encapsulated apical and axillary shoot buds. Plant Sci., 60: 11 l-l 16.
Mhatre, M., Bapat, V.A. and Rao, P.S., 1985. Regeneration of plants from cultured leaves and axillary buds in mulberry (Morus indica L.). Plant Cell. Rep., 4: 78-80.
Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant., 15: 473-497.
Pattnaik, S.K., Sahoo, Y., Panda, M., Pattnaik, S.N. and Chand, P.K., 1993. Gibberellic acid-stimu- lated winter bud cultures of mulberry in vitro. XVIIth Annu. Conf. Orissa Botanical Society, ab- stract. 17 pp. (unpublished).
Redenbaugh, K., Paasch, B.D., Nichol, J.W., Kossler, M.E., Viss, P.R. and Walker, K.A., 1986. So- matic seed: Encapsulation of asexual plant embryos. Biotechnology, 4: 797-80 1.
