Key words: Psidium guajava L., micropropagation, nodal explant. Abstract. In vitro clonal propagation of guava 'Banaras local' was achieved by culturing nodal.
Plant Cell, Tissue and Organ Culture 9:235-243 (1987) © Martinus Nijhoff Publishers, Dordrecht Printed in the Netherlands
Short communication
Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees M U H A M M A D N. AMIN* & VIJAI S. JAISWAL** Department of Botany, Banaras Hindu University, Varanasi IND 221 005, India (**addressee for correspondence) Received 3 November 1986; accepted in revised form 23 February 1987
Key words: Psidium guajava L., micropropagation, nodal explant Abstract. In vitro clonal propagation of guava 'Banaras local' was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 #M 6-benzyladanine (BA) alone or in combination with either 0.6 #M indole-3-acetic acid (IAA), 0.5/tM indole-3-butyric acid (IBA) or 0.3 #M gibberellic acid (GA3). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5/~M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3-6 shoots developed on explants collected from field-grown plants and 5 10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5/~M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1/~M each of IBA and ~-naphthaleneacetic acid (NAA), and 1 g 1 ~activated charcoal. Regenerated plantlets were successfully established on soil.
Introduction Guava (Psidium guajava L.) is one of the very important fruit crops of the Indian subcontinent and serves as staple food in many countries [7]. Vegetative propagation of guava is preferred since it produces true-to type plants which bear early and produce about 3-4 times more fruit each year. A fairly common method of vegetative propagation of guava plants in India is air layering, which is expensive and the number of plants obtained is small [10]. Therefore, an elite plant which shows resistance to pests and diseases cannot be economically propagated quickly by this technique. The application of tissue culture propagation for large scale production of many temperate fruit trees has been well documented [13,14], but very little is known about in vitro clonal propagation of mature tropical fruit * On leave from: Department of Botany, University of Rajshahi, Bangladesh.
236 trees. We have, for the first time, reported plantlet formation from mature tissue of guava [5]. As the survival and response of shoot tip explants were not satisfactory, efforts have been made to find a suitable alternative explant source. The present paper describes the success of rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of adult trees.
Materials and methods
Apical portions (ca. 5-7 cm) of shoots of a 15-year-old guava plant were collected from new growth on mature branches and from the basal sprouts. Shoot apices were brought to the laboratory in water, expanded leaves were removed, washed thoroughly under running tap water and then treated with 1% (v/v) Cetavlon (20% w/v cetrimide, an antiseptic and detergent) for 5 min. For removal of phenolics, the material was agitated for 3 0 ~ 0 min in 0.5% (w/v) solution of polyvinylpolypyrrolidone (Sigma Chem. Co., USA) containing 2% sucrose. Surface-sterilization was carried out with 0.05% HgC12 for 2 min after a brief rinse in 70% ethanol. Softwood nodal explants (1.0-1.5 cm) were then prepared aseptically from these shoot pieces and were implanted on semi solid shoot proliferation medium after a dip in an antioxidant solution (75 mg citric acid and 50 mg ascorbic acid. 1-1 water). Explants were placed on the media surface vertically for the first time and after 2-3 initial changes of medium, when phenolics exudation was checked, were oriented horizontally facilitating better proliferation response. Murashige and Skoog [9] medium with full strength salts and sucrose was used for shoot proliferation and MS with 1/2 strength salts and sucrose was used for adventitious root formation. For initiation of axillary bud proliferation explants were cultured in 25 × 150mm culture tubes each containing 15 ml of medium. After 5-7 wk explants with proliferating shoot clumps were transferred to either 150 ml or 250 ml flasks each containing 25 ml or 50 ml of medium. Subsequently, cultures were transferred to the fresh medium after every 3-4 wk. An appropriate amount of all media components were mixed, except growth regulators and additives, and pH was adjusted to 5.8 + 0.1 before addition of agar (Difco Bacto, 0.7-0.8%) and sucrose (1.5-3%). After melting the agar and sucrose, growth regulators were added and media were dispensed into culture vessels. All media were autoclaved for 10min at 120 °C and 1.1 kg/cm 2. Cultures were maintained at 25 + 2 °C with 16-hr photoperiod at a photon flux density of 50-70/~E'm -2"s -j from warm white fluorescent tubes.
237 Results
Shoot proliferation and culture multiplication Explants from field-grown plants were cultured on MS medium supplemented with 4.5/~M BA alone, since this concentration gave the maximum shoot multiplication rate in previous experiments [5], or in combination with IAA, IBA or G A 3. In the present investigation also 4.5/tM BA alone produced the maximum number of shoots per culture (Table 1). In this medium explants responded by axillary bud enlargment and bursting within 4-6 wk. After proliferation started, the explants with proliferating shoot masses were subcultured in wider culture vessels with more medium to meet the increasing requirement of nutrients and space. However, at about 12 wk 3-6 shoots per culture were observed. When the nodal segments taken from in vitro proliferated shoots were cultured on the same media, a two-fold increase in number of shoots was observed in 10 wk of subculture. It was further observed that shoot multiplication rate increased with the increasing number of subcultures and then declined after 6-7 subcultures. During the proliferation stage, axillary buds grew out from the original explants (Fig. 1A) and lateral shoots developed in the axils of lower leaves of these newly produced shoots. Some laterals again showed axillary branching. As a result a dense mass of shoots was produced in each culture vessel (Fig. 1B, C). Such shoots were either cut back to stimulate further shoot formation for subsequent experimentation or these were excised and used as cuttings for rooting. For maximum production of shoots, the stock cultures were transferred to fresh medium after each batch of cuttings was harvested. At every time
Table 1. Effect o f growth regulators on shoot proliferation and n u m b e r o f shoots per nodal explant of mature guava tree. D a t a were recorded 12 wk after culture initiation. Each treatment contained 12 replicates and the experiment was repeated twice (Mean _+ Standard Error). Growth regulators (#M)
% o f proliferating cultures
No. of shoots per culture
Degree of shoot growth a
BA4.5 BA4.5 + I A A 0.6 BA 4.5 4- I A A 0.6 + G A 3 0.3 BA 4.5 + IBA 0.5 + GA3 0.3
77 ___ 6 70 ___ 5 45 ___ 7
4.6 ÷ 1.3 3.5 _+ 0.8 2.2 ___ 0.3
+++ + + + + + + +
50 + 6
2.5 + 0.3
+
a + poor; + + + good; + + + + very good
238
Fig. 1. Stages of plant regeneration from nodal explants of guava. A, B: Cultures of initial explants after 5 and 7 wk respectively, showing axillary shoot development. C: A proliferating shoot mass after 12 wk of culture. D: Same as C but inverted to show the precocious axillary branching. E, F, G: Adventitious root development on excised shoots after 1, 2 and 4 wk of culture. H: Regenerants on soil after 2-12wk of transfer.
239 during subculturing, care was taken to remove any dead or discoloured tissue. In this way stock cultures were kept in continuous production for several months. Continuous presence of 4.5#M BA in shoot proliferating medium sometimes suppressed the shoot growth. Therefore, a prior transfer of shoot clumps onto low (0.5/~M) BA medium before harvesting of cuttings for rooting was done, which allowed rapid extension growth. This practice greatly increased the number of usable shoots (Table 2) and enabled easy handling of microcuttings at the rooting stage.
Effects of explanting season on culture establishment Explants were collected from field-grown plants throughout the year to determine the most suitable time for culture establishment. This was found to be April to June. Explants collected at this time showed the minimum amount of phenolic exudation and the lowest frequency of culture contamination (5-7%). These explants also took the minimum time for sprouting and proliferation of shoot buds. Frequency of explants showing shoot proliferation was also greater among those collected at this time (Fig. 2).
Rooting of microcuttings Cuttings taken from the in vitro proliferated shoots rooted in MS medium containing 1/2 strength salts and sucrose, 1/~M each of IBA and NAA, and 1 g. 1-l activated charcoal. Eighty percent rooting with 2-4 roots (Fig. 1E, F, G) was observed in this medium. However, in the auxin medium sometimes root growth was arrested after a few mm extension and the root Table 2. Effect o f high a n d low c o n c e n t r a t i o n o f BA o n s h o o t p r o d u c t i o n o f g u a v a e x p l a n t s t a k e n f r o m in vitro proliferated shoots. T h e r e were 10 replicates a n d d a t a were recorded after 4 s u b c u l t u r e s o f 3 wk each ( M e a n _ S t a n d a r d Error). C u l t u r e period a n d B A conc. (/~M)
Total No. ofb s h o o t s per culture
No. o f usable c s h o o t s per culture
Average length of shoots (cm)
C o n t i n u o u s culture in B A 4.5 m e d i u m 3-Subcultures in B A 4.5 a n d 1-subculture in B A 0.5 m e d i a
8.0 ___ 1.1
2.5 + 0.7
1.3 + 0.5
6.7 + 0.6
4.7 _ 0.8
2.0 + 0.3
b s h o o t s with less t h a n 5 m m length were n o t t a k e n into account. c s h o o t s with 1.Ocm a n d m o r e length were considered only.
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241 tips became blunt and blackened. When these cuttings with the rudimentary roots were transferred to a lower salt medium (1/4 strength MS inorganic, 0.5% agar, no vitamins and auxin) rapid growth of roots was observed. Transfer of plantlets and their establishment on the soil The in vitro regenerated plantlets were removed from the culture tubes, washed thoroughly to remove the nutrient medium and transplanted to small pots with 1:1 non-sterile garden soil and compost (Fig. 1H). Ninety percent of transferred plantlets adapted in the soil within 2-3 wk. For the initial period of transfer, potted plantlets were kept in culture room conditions and high humidity was maintained by covering the plantlets with transparent beakers. Plantlets were subsequently transferred to larger pots and gradually acclimatized to outdoor conditions.
Discussion
The results presented in the foregoing sections reveal that rapid clonal propagation of guava is possible through in vitro culture of nodal explants. The main advantage of using nodal explants is that shoot tips can be rooted for plantlet formation and at the same time culture can be maintained by basal nodal segments. The greater responsiveness of nodal explants over the shoot tip explants can be attributed to the absence of apical dominance and presence of axillary buds at a more advanced stage of development. For apple [3] and thornless blackberry [15] also nodal cuttings were proved to be good explants for microporpagation. The maximum response with minimum contamination and browning of explant and medium was obtained from explants collected during April to June. Similar results of seasonal effects on culture establishment have also been noted for apple [4], papaya [6], red raspberry [1] and sweetgum [11]. The early response and increased shoot production capacity of nodal segments taken from in vitro proliferated shoots in comparison to that of field-grown trees might be due to the absence of lag period between explanting and adaptation of explants to in vitro conditions. Proliferating cultures maintained for extended periods in medium containing 4.5 #M BA showed suppressed shoot growth and when these cultures were transferred to low BA (0.5 #M), rapid elongation of shoots was observed. Hu and Wang [2] reported that sometimes the elongation of shoots in stage II is inhibited by high cytokinin levels thus, an intermediate shoot elongation stage becomes necessary. Blueberry shoots produced in a
242 medium containing 15 mg.1-1 6-V,v-dimethylallylamino purine showed sufficient elongation when transferred to a medium containing relatively low levels (1-5mg.1-1) of this cytokinin [16]. Our studies clearly show the shoot multiplication by precocious axillary branching (Fig. 1D) in guava. The significance of such a method in avoiding genetically aberrant plant formation has been suggsted by Hu and Wang [2], Murashige [8], and Vasil and Vasil [12].
Acknowledgements We are thankful to the Head, Department of Botany, for providing laboratory facilities and to Mr. Madan Lal for technical assistance. One of us (M.N.A.) is grateful to the Government of India for financial support.
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