In Vitro Cell. Dev. Biol. 29P:135-142, July 1993 © 1993 Tissue Culture Association 1054-5476/93 $01.50+0.00

I N V I T R O C L O N A L M U L T I P L I C A T I O N O F 4 - Y E A R - O L D P L A N T S O F T H E B A M B O O ,

D E N D R O C A L A M U S L O N G I S P A T H U S K U R Z

SANJAY SAXENA ANO SANT S. BHOJWANI l

Tata Energy Research Institute, 90 Jor Bagh, New Delhi-l l O 003, and Department of Botany, University of Delhi, Delhi-llO 007, India (S. S. B.)

(Received 5 July 1992; accepted 25 January 1993; editor 1. Y. E. Chu)

SUMMARY

A complete protocol for micropropagation of 4-yr-old plants of the bamboo Dendrocalamus Iongispathus is described. Culture initiation was strongly influenced by the nature of the explant and the season. In vitro multiplication was achieved through forced axillary branching. Single node segments from the young lateral branches produced multiple shoots on agar-solidified Murashige and Skoog (MS) medium supplemented with 12 gM benzylaminopurine (BAP) and 3 #M kinetin. The shoots have been multiplied for 15 passages in hquid and thereafter for over 5 passages on semisohd MS + 15 #M BAP + 1 #M indolebutyric acid (1BA) + 10% coconut water at a rate of 3.2- and 2.8-fold, every 4 wk, respectively. The nature of the propagule was a critical factor for shoot multiplication and rooting. Seventy-three percent of the shoots rooted on a modified MS medium (major salts reduced to half strength) containing 1 #M indoleacetic acid, 1 #M IBA, and 68 #M coumarin. Through a simple in vitro hardening step, more than 85% of the tissue culture-raised plants were successfully transferred to soil.

Key words: Dendrocalamus longispathus; bamboo; micropropagation; tissue culture.

llWI'RODUC']?ION

Dendrocalamus longi~pathus, a large tufted bamboo, is one of the 15 commercially important species of bamboos growing in India that have been used in the paper and pulp industries. It is ideally suited for the manufacturing of craft paper. It is also used in a variety of other ways, such as thatching, roofing, construction, bas- ket making, furniture, floats for timber, rafts, fencing, containers, and musical instruments (CSIR, 1952). Young shoots are eaten raw or pickled.

Natural regeneration of D. longispathus occurs both sexually and vegetatively. However, like most other bamboo species, both con- ventional methods of propagation suffer from certain drawbacks that restrict the large-scale multiplication of this species. The sexual method is unreliable because of long (30 to 60 yr) and often erratic flowering cycle, short viability of seeds (55 days under natural con- ditions) (Banik, 1987), and large-scale consumption of seeds by wild animals; propagation by vegetative means is difficult on ac- count of fewer and bulky propagules and season specificity.

This paper describes a reproducible and efficient protocol for in vitro clonal muhiplication of 4-yr-old plants of D. longispathus. Most of the earlier studies on tissue culture of bamboos deal with seedling material (see Saxena and Bhojwani, in press).

Msaxmst.s AND METHODS

Initiation of aseptic cultures. Single node segments were excised from young lateral branches of the main eulm of 4-yr-old plants of D. /on#-

1 To whom correspondence should be addressed.

spathus growing at Gwal Pahari in Haryana State. After carefully removing the thin and fibrous leaf sheath that envelops the axillary bud and a part of the upper internode, the stem pieces were given a quick rinse in 95% ethanol, washed with 2% solution of "Cetavlon" (IEL, Limited, India) for 15 rain followed by thorough washing under running tap water for 20 min. The nodal segments were then surface sterilized by treating them with 0.1% solution of mercuric chloride for 10 rain. After three washings in sterile distilled water, the cut ends of the segments were trimmed, leaving behind 1.0 to 1.5 cm of internodal portions on either side of the node, and planted on different nutrient media. MS (Murashige and Skoog, 1962) and B s (Gamborg et al., 1968) basal media containing 3% sucrose and 0.8% agar were tried for bud-break. The two media were variously supplemented with eytokinins [benzylaminopurine (BAP) and kinetin], either individually or in different combinations with the auxins indoleacetic acid (IAA) and indole- butyric acid (IBA). The pH of each medium was adjusted to 5.8 before autoclaving. All cultures were initiated in 150-ml glass bottles containing 30 ml of medium.

Multiplication of shoots. The entire clusters of axillary shoots produced by 2-wk-old primary cultures of nodal segments were transferred to liquid MS medium for shoot multiplication. The basal medium was supplemented with BAP alone or in various combinations with other cytokinins (kinetin and 2-ip) and auxins [IAA, IBA, and 1-naphthaleneacetic acid (NAA)]. Unless mentioned otherwise, the pH of the media was adjusted to 5.0 before autoclaving. As with initiation, multiphcation of shoots was done in 150-ml glass bottles containing 30 ml of medium.

In stationary hquid cultures, submerged shoots died, whereas in shake cultures they invariably became vitrified. To prevent the shoots from getting submerged into the liquid medium, a small piece of rubber foam (4.5 X 4.5 X 1.0 cm) was placed inside each bottle and the base of the shoot was fixed on top of it. Before using, the foam pieces were washed with 5% Teepol solution for 10 min followed by thorough washing under running tap water for 20 min. The foam pieces were then autoclaved in 2000-ml conical flasks containing distilled water Oust enough to keep the foam pieces soaked). After autoclaving, the foam pieces were manually squeezed to remove the water containing leachouts, if any. After three washings with

135

136 SAXENA AND BHOJWANI

distilled water, the foam pieces were dried in an oven at 80 ° C and stored until use.

The shoots were subcuhured at 4-wk intervals. Inasmuch as individual shoots failed to survive in subcultures, at the end of each passage the proliferated shoot clusters were cut into discrete units, each containing three to five shoots, hereafter called a propagule (Fig. 1 D), and transferred to fresh medium. The multiplication rates have been calculated on the basis of the number of propagules derived from one culture at the end of each passage. All experiments were repeated twice with at least 12 cultures per treatment. Wherever possible, the effect of different treatments was quanti- fied on the basis of percent cultures showing the response and the degree of response per culture. For the latter, standard error of means was calculated, which is presented in the tables as "+" and as bars in the graphs (rigs 4, 5).

Rooting. Rooting of the shoots was attempted both under in vivo and in vitro conditions. For in vivo rooting, the entire cluster of multiple shoots, obtained at the end of a 4-wk cycle of shoot multiplication, was used. Each cluster was given a pulse treatment with IAA (280 gM, 570 ttM) or IBA (250 #M, 490 ~tM) alone or in combination with coumarin (340 gM, 680 ttM) in liquid MS medium for 1 to 4 days. During treatment the cultures were maintained on a rotary shaker at 140 rpm. The treated shoots were directly transferred to a potting mix consisting of peat moss and soilritc in a 1:1 ratio (vol/vol) and irrigated with the inorganic nutrients of MS basal medium (pH 5.0).

For in vitro rooting, the propagules bearing three to five shoots were used. Initially, liquid and semisolid MS media were tried. Subsequently, modified MS media with the major inorganic salts reduced to half strength (MS1) or quarter strength (MS2) were also tested. The media were variously supplemented with IAA, IBA, NAA, activated charcoal (AC), coumarin, and boric acid. The liquid media (pH 5.0) were tested with and without rubber foam, and the semisolid media (pH 5.8) were gelled with 0.2% gelrite (Scott Laboratories, Carson, CA). Medium (25 to 30 ml) was poured into 150-ml glass bottles. Each hormonal combination was tested 3 times with 12 cultures per experiment. However, with modified MS media, 50 cultures were used in each treatment.

Transplantation. Rooted shoots from 4-wk-old cultures on MS 1 + IAA (10 #M) + IBA (10 #M) + coumarin (68 #M) were transferred to a potting mix comprised of equal quantities of soil, soilrite, and farmyard manure (vol/vol) in black polythene bags, either directly or after in vitro hardening. For in vitro hardening, the plantlets from the rooting medium were trans- ferred to 200-ml screw cap glass bottles one-third filled with soilrite (Fig. 1 F) and irrigated with 40 ml of inorganic salt solution of MS medium (major salts reduced to half strength) with the pH adjusted to 5.0. After 7 to 8 days, the caps of the bottles were removed (Fig. 1 G) and the plantlets allowed to grow in the glass bottles for another 2 days. During this period the plantlets were irrigated as before. The in vitro hardened plants were transferred to the polythene bags. The potted plants (Fig. 2 A) were reared for 3 too. inside a glasshouse, maintained at 28 ° _+ 2 ° C and relative humidity 75 to 85%, before transfer to field conditions. The media and soilrite were steam sterilized at a pressure of 1.06 kg/cm 2 for 15 min.

Culture conditions. All cultures were maintained at 26 ° +- 2 ° C under a 12-h photoperiod with light intensity of 4000 lux provided by cool, white fluorescent tubes of 40 W (Philips, Calcutta. India).

RESULTS

Initiation of shoot cultures. The sterilization procedure de- scribed in Materials and Methods yielded 70% aseptic cultures. Bud-break frequency was strongly influenced by the juvenility of lateral shoots, position of axillary bud on the branch, and season in which cultures were initiated. Only the young lateral branches showed bud-break. Older branches with partially sprouted axillary buds did not respond in culture. The first five to six nodal cuttings from the shoot tip and one to three cuttings from the base of the branch (leaf number was assumed as the corresponding node num- ber) did not develop shoots. Therefore, for all subsequent experi- ments, nodal cuttings were taken from the middle of the young lateral branches.

On basal media the frequency of bud-break was very low. Incor-

poration of BAP (1, 3, 6, 9, 12, or 15 #M) to the basal media improved the incidence of bud-break and promoted multiple shoot formation (Table 1). The frequency of bud-break and the number of shoots developed on an explant were much higher on MS than on B5. Consequently, in all subsequent experiments only MS basal medium was used. On the optimum MS medium, containing 12 #M BAP, 58% cultures showed bud-break within 2 wk with over four shoots per explant. Subsequently, two other cytokinins (kinetin and 2-ip) and auxins (IAA and IBA) were tested, at different concentra- tions (1, 3, and 6 #M), in conjunction with 12 #M BAP. Of all the treatments, BAP combined with 3 /zM kinetin induced maximum bud-break (75%) as well as shoot yield per explant (Table 2) and shoot growth (Fig. 1 A,B). The addition of either IAA or IBA was inhibitory. In all the combinations the incidence of bud-break was much lower than that with BAP alone (Table 2).

The results described above refer to the experiments conducted during the monsoon season (July to September). However, during other months the bud-break frequency varied considerably. To study the effect of season on bud-break, fresh cultures of nodal segments derived from plants growing under irrigated conditions were raised on MS medium containing 12 /~M BAP and 3 #M kinetin, at regular intervals throughout the year. The cultures initi- ated during the rainy season (July to September) showed best re- sponse not only in terms of the frequency of bud-break (Fig. 3) but also in the vigor of the shoots. Another advantage of initiating the cultures during July to September is the availability of large number of explants.

Shoot multiplication. During the first four cycles of shoot mul- tiplication, 85% of the cultures showed browning of the medium. Addition of 1000 mg/liter of AC or soluble polyvinyl pyrrolidone (PVP) to liquid MS + 15 #M BAP + 1 ~tM IBA + 10% coconut water (CW) checked browning to some extent, but the presence of AC also inhibited shoot growth and multiplication. Fortnightly transfer of shoots to fresh medium proved most effective to circum- vent the browning problem. By Passage 5, the incidence of brown- ing was reduced to 42% (Fig. 4), and thereafter regular four weekly subcultures could be made.

On basal medium the shoots started turning pale and necrotic from Day 4 and finally died after 10 to 15 days of culture. Incorpo- ration of BAP (1, 5, 10, 15, or 20 #M) to MS basal medium induced shoot multiplication, the frequency of which varied with the concentration of the cytokinin (Table 3). At its optimum level (15 gM), 1.8-fold multiplication occurred every 4 wk. The presence of kinetin or 2-ip in conjunction with 15/.tM BAP improved the multi- plication rates marginally. The most effective combination of BAP (15 #M) and kinetin (3 #M) yielded a prohferation rate of 2.1- fold in 4 wk. Higher concentrations of kinetin proved inhibitory

(Table 4). Of the different auxins (IAA, IBA, and NAA) tested in conjunc-

tion with 15/.tM BAP, at three concentrations (1, 3, or 6 #M), 1 #M IBA proved to be most effective, resulting in a multiplication rate of 2.4-fold. Although IAA was promotory only at the lowest concentra- tion (1 ~tM), NAA proved inhibitory at all the tested concentrations

(Table 5). Addition of 10% CW to MS + 15 #M BAP + 1 #M IBA en-

hanced the rate of shoot multiplication to 3.2-fold every 4 wk (Fig. 1 C). On this medium, even the shoot growth appeared more vigor- ous. At higher concentrations (15 and 20%), CW proved inhibitory and often caused vitrification of shoots.

Gelling of the multiplication medium (MS + 15 gM BAP + 1 #M

MICROPROPAGATION OF MATURE BAMBOO 137

Fie. 1. Micropropagation of adult Dendrocalamus longispathus. A, a nodal segment showing bud-break after 5 days ofeuhure on MS + 12/.tM BAP + 3/ tM kinetin. B, 12-day-old culture showing multiple axillary shoots on the explant. C,D, a propagule (D) derived from a cluster of shoots (C) 4 wk after culture, on polyurethane foam, in MS + 15 ,aM BAP + 1 #M IBA + 10% CW. E, a clump of shoots rooted on MSI + 10 ttM IAA + 10 #M IBA + 68 ~M coumarin. F, a micropropagated plantlet 6 days after in vitro hardening. G, decapped culture bottle 7 days after in vitro hardening; plantlet is ready for transfer to pot.

138 SAXENA AND BHOJWANI

FIG. 2. Transplantation, A, a tissue culture raised plant of D. longispathus 3 rap. after transfer to soil. B, a batch of micropropagated plants in open nursery.

IBA + 10% CW) with 0.8% agar or 0.2% gelrite caused a decline in the rates of multiplication to 2.5- and 3.0-fold, respectively. On liquid as well as semisolid media about 5 to 15% of the propagules

formed callus at their basal end, which occasionally differentiated small adventitious shoot buds. Some of the buds were albino. The shoots of callus origin, along with the callus, were discarded at each subculture.

During each shoot multiplication passage, 10 to 15% of the ap- parently healthy propagules failed to multiply. Most of such shoot clusters remained dormant even in subsequent passages and did not root on the optimum rooting medium.

TABLE 1

EFFECT OF BASAL MEDIA (MS AND Bs) AND VARIOUS BAP CONCENTRATIONS ON BUD-BREAK IN THE CULTURES OF NODAL CUTTINGS OF BAMBOO °

Percent Bud-break No. of Shoots/Explant BAP

Concentration MS B s MS B s

Control 11.11 8.33 2.25 + 0.16 1.66 + 0.60 1 t~M 22.22 16.66 2.37 + 0.30 2.00 + 0.58 3 ~tM 36.11 27.77 3.15 + 0.36 2.80 ± 0.44 6 #M 41.66 33,33 3.80 + 0.27 3.16 + 0.20 9 #M 52.77 38.88 4.31 + 0.60 3.57 + 0.52 12 #M 58.33 41.66 4.80 + 0.54 3.68 + 0.39 15 #M 55.55 44.44 4.65 + 0.33 4.13 + 0.33

For 15 passages on the liquid medium the shoots remained healthy and there was no decline in the rate of multiplication. How- ever, thereafter some of the shoots exhibited vitrification. This prob-

TABLE 2

EFFECT OF ADDING CYTOKININS (2IP AND KINETIN) OR AUXINS (IAA AND IBA) TO MS + 12 gM BAP (CONTROL)

ON BUD-BREAK FREQUENCY AND THE NUMBER OF SHOOTS PER EXPLANT IN THE CULTURES OF

SINGLE NODE SEGMENTS OF BAMBOO"

Percent Number of Shoots Treatment Bud-break per Explant

Control 58.33 4.76 + 0.20 2-ip

1 #M 55.55 4.65 + 0.44 3 #M 61.11 5.04 + 0.28 6 #M 61.11 4.86 + 0.54

Kn 1 #M 66.66 5.66 + 0.18 3 tiM 75.00 6.33 + 0.31 6 #M 72,22 6.15 -+ 0.26

IAA 1 gM 55,55 4.15 +- 0.35 3 ~tM 47,22 4.00 ± 0.28 6 #M 41.66 3.33 -+ 0.16

IBA 1 #M 50.00 3.55 ± 0.47 3 #M 41.66 3.20 ± 0.23 6 #M 36.11 3.07 ± 0.28

a Semisolid MS and B s basal media served as the controls. Culture pe- riod: 2 wk a Culture period: 2 wk.

MICROPROPAGATION OF MATURE BAMBOO 139

8O

6 0 -

i "O

..Q 4 0 -

o

2 0 - Q.

0 t ~ | i , i i • t I Jan Feb Mar A lar May Jun dul Aug SeD Oct Nov Dec

Month of culture initiation

F~G. 3. Frequency of bud-break as affected by the time of initiation of cultures of bamboo. Culture medium: MS + 15 ~M BAP + 3 ~M kine~n. Growth period: 2 wk.

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Concentration of Boric acid (rag/I)

Fit;. 5. Effect of boric acid on in vitro rooting of shoots of bamboo, Boric acid was added to MS A- 10 #M IAA + 10 #M IBA + 68 #M coumarin. Culture period: 4 wk.

lem was overcome to a great extent by transferring the shoots to semisolid medium (pH 5.8) gelled with 0.3% gel.rite. However, this also caused a slight decline in the multiplication rates, from 3.2- to 2.8-fold every 4 wk.

Rooting. In vivo rooting was not successful. In all the treat- ments, the shoots turned pale within 5 to 7 days and eventually died. In vitro, the shoots failed to root on a semisolid MS basal medium. Of the three auxins (IAA, IBA, NAA) added to the basal medium individually at 1, 6, 10, or 12 #M, only IBA proved induc- tive. With 10 or 12 #M IBA, 11% of the shoots formed three to five roots within 3 to 4 wk (Table 6).

Addition of 34 or 68 #M coumarin to the basal medium resulted in 17 and 22% rooting, respectively. The combined presence of 68 /.tM coumarin and 10/.tM IBA proved more effective, resulting in 36% rooting. Only 14 to 19% of the shoots rooted on medium containing coumarin in combination with IAA or NAA. However, 47% rooting occurred when MS basal medium was supplemented with 10/.tM 1AA, 10 gM 1BA, and 68/.tM coumarin (Table 6).

To further improve the rooting frequency, AC (200, 500, 1000, and 2000 rag/liter) and boric acid (10, 25, 50, and 100 rag/liter)

e-

o c-

o

a .

1 2 3 4 5 6 7 19

Multiplication cycle

FIG. 4. Extent of browning of medium in relation to the shoot multiph- cation cycle in bamboo. The first five cycles were of 2 wk each and thereaf- ter of 4 wk each. Culture medium: liquid MS + 15 gM BAP + 1 /.tM IBA + 10% CW.

were added to MS + 10 #M IAA + 10 #MIBA + 68/.tM coumarin. However, irrespective of its concentration, AC strongly suppressed rooting. On AC containing medium, the shoots turned yellow and displayed extremely poor growth. As AC, boric acid also inhibited rooting. The rooting frequency dechned with the increase in boric acid concentration in the rooting medium (Fig. 5).

Thus, the MS medium with the best combination of growth regula- tors (10/zM IAA, 10/.tM IBA, and 68/zM coumarin) induced 47% rooting. Modification of the above medium by reducing the concen- tration of MS salts to one half (MSI) or one quarter (MS2) consider- ably enhanced the rooting frequencies; 73 and 76% shoots rooted on MS1 and MS 2 media, respectively (Table 6). Maximum rooting occurred on MS 2 medium but on this medium the shoots exhibited very poor growth and often turned pale and necrotic. Occasionally, some of the propagules on this medium died even after forming roots. In contrast, on MS1, which induced a slightly lower rooting rate, the shoots remained green and healthy (Fig. 1 E). MS~ + 10 /.tM IAA + l0 #M IBA + 68 #M coumarin is, therefore, regarded as the best rooting medium. On this medium, the shoots generally developed two to six roots from their basal end within 2 wk. Occa- sionally, multiple roots (more than six) were formed. After 3 wk some of the roots had developed laterals. No callusing or browning was observed during root formation (Fig. 1 E).

TABLE 3

EFFECT OF BAP CONCENTRATION ON MULTIPLICATION AND GROWTH OF SHOOTS OF BAMBOO =

Percent Cultures Rate of Maximum

BAP Showing Mulfiplation Shoot

Concentration Mukiplication (× ) Length, cm

0 (control) 0 0 0 BAP

1 #M 5.55 0.83 + 0.06 3.66 + 0.23 5 #M 19.44 1.16 + 0.08 3.83 + 0.35 10/.tM 52.77 1.42 + 0.14 4.53 + 0.12 15/.tM 66.66 1.80 + 0.15 5.96 + 0.26 20/.tM 61.11 1.72 +- 0.18 6.03 + 0.31

a Control: liquid MS basal medium. Culture period: 4 wk.

140 SAXENA AND BHOJWANI

TABLE 4 TABLE 6

EFFECT OF SUPPLEMENTING LIQUID MS + 15 #M BAP (CONTROL) WITH KINETIN (KN) OR 2-IP ON SHOOT

GROWTH AND MULTIPLICATION OF BAMBOO"

Treatment

Percent Cultures Rate of Maximum Showing Multiplication Shoot

Multiplication (×) Length, cm

Control 61.11 1.72 + 0.22 5.80 ± 0.18 Kn

1 #M 69.44 1.86 ± 0.26 5.83 ± 0.23 3 ttM 72.22 2.13 ± 0.18 6.13 ± 0.27 6/.tM 63.88 1.83 ± 0.25 6.03 ± 0.40

2-ip 1 ttM 58.33 1.91 ± 0.30 5.66 ±_ 0.31 3 #M 61.11 1.86 + 0.14 6.06 ± 0.17 6 #M 63.88 2.02 + 0.23 6.16 ± 0.24

Culture period: 4 wk.

Rooting frequency in liquid medium (with or without foam) was relatively lower than that on semisolid medium. Moreover, in liquid media vitrification of leaves and leaf sheaths sometimes occurred. The use of foam for rooting proved disadvantageous because the developing roots pierced through the foam pieces and were severely damaged during transplantation. Therefore, the rooting medium was gelled with 0.2% gelrite.

Transplantation. None of the plantlets directly transferred from rooting medium to the potting mix and grown under natural conditions survived. However, 65% of such plants survived if the potted plants were reared inside the glasshouse. The survival per- centage rose to 85% if the plantlets were hardened under aseptic conditions (Fig. 1 F,G) before being transferred to the polythene bag. During in vitro hardening, shoots elongated, leaves turned greener, and their lamina expanded. Consequently, the plants seemed much healthier after in vitro hardening. Such plants also

TABLE 5

EFFECT OF ADDING IAA, IBA, OR NAA TO LIQUID MS + 15 #M BAP (CONTROL) ON SHOOT GROWTH

AND MULTIPLICATION OF BAMBOO ~

Treatment

Percent Cultures Rate of Maximum Showing Multiplication Shoot

M uldplieation IX) Length, cm

Control 63.88 1.66 + 0.19 5.60 + 0.23 IAA

1 ~M 69.44 2.05 + 0.08 5.46 + 0.37 3 ttM 63.88 1.83 + 0.16 5.23 + 0.21 6 gM 52.77 1.47 + 0.21 4.86 + 0.48

IBA 1 ttM 75.00 2.41 + 0.23 6.16 + 0.29 3 ttM 77.77 2.27 + 0.12 5.70 + 0.30 6 ttM 58.33 1.61 + 0.17 4.56 _+ 0.28

NAA 1 /.tM 52.77 1.53 + 0.14 4.23 + 0.32 3 #M 41.66 1.19 + 0.15 3.66 + 0.29 6 ttM 27.77 0.83 ± 0.18 2.13 + 0.20

a Culture period: 4 wk.

EFFECT OF SOME TREATMENTS ON IN VITRO ROOTING OF SHOOTS OF BAMBOO

Rooting Treatment Percentage

MS (control) 0 MS + IAA (1 ~M) 0 MS + IAA (6 #M) 0 MS + IAA (10 gM) 0 MS + IAA (12 #M) 0 MS + NAA (1 #M) O MS + NAA (6 gM) 0 MS + NAA (10 #M) 0 MS + NAA (12 #M) 0 MS + IBA (1 ttM) 0 MS + IBA (6 ~M) 2.77 MS + IBA (10 #M) 11.11 MS + 1BA (12 gM) 11.11 MS + coumarin (34 #M) 16.66 MS + coumarin (68 gM) 22.22 MS + IAA (10 #M) + coumarin (34 #M) 16.66 MS + IAA (10 #M) + coumarin (68 #M) 19.44 MS + NAA (10 ~M) + coumarin (34 #M) 13.88 MS + NAA (10/.tM) + coumarin (68 #M) 19.44 MS + IBA (10/.tM) + coumarin (34/.tM) 27.77 MS + IBA (10 gM) + coumarin (68 #M) 36.11 MS + IAA (10 #M) + IBA (10 #M) + coumarin (68 #M) 47.22 MS1 + IAA (10 #M) + IBA (10 #M) + coumarin (68/.tM) 73.33 MS2 + 1AA (10 #M) + IBA (10 gM) + coumarin (68/.tM) 76.00

grew more vigorously in the glasshouse. The surviving plants pro- duced new culms after 5 to 6 wk. So far, over 150 plants have been transferred to soil (Fig. 2 A,B).

DISCUSSION

Several authors have reported the micropropagation of bamboos from embryonal tissues (see Saxena and Bhojwani, in press). How- ever, in these highly cross-pollinating plants, both quantitative and qualitative gains could be expected only if the explants are derived from adult plants. Moreover, the use of vegetative tissues would relinquish dependency on seeds whose availability is always doubt- ful. Many attempts have been made to micropropagate adult mate- rials of the bamboos Bambusa beecheyana var. beecheyana (Yeh and Chang, 1986b), B. glaucescens (Banik, 1987), B. multiplex (Huang et al., 1989), B. oldhami (Huang et al., 1989; Yeh and Chang, 1986a), Dendrocalamus latiflorus (Zamora et al., 1989), D. strictus (Nadgir et al., 1984), Phyllostachys aurea (Hdang et al., 1989), and Sinocalamus latiflora (Tsay et al., 1990). In a recent paper, Prutpongse and Gavinlertvatana (1992) reported the micro- propagation of 54 species of bamboos. However, the paper lacks details regarding the rate of proliferation in recurrent cycles of shoot muhiphcation, frequency of rooting, etc., and in that study transplantation was not attempted. A complete protocol for in vitro multiplication of 4-yr-old bamboo is reported in this paper for the first time. Moreover, in all the published reports, except for D. strictus (Nadgir et al., 1984), a callus phase was involved which may endanger genetic fidelity of the progeny. In the present study, shoot multiplication was achieved stricdy through axillary bud prolifera- tion. The only other case where this approach was followed to multi- ply an adult bamboo is D. strictus (Nadgir et al., 1984). In that

MICROPROPAGATION OF MATURE BAMBOO 141

report maximum rooting frequency obtained was 20% and trans- plantation was not attempted. In contrast, rooting and transplanta- tion survivals achieved in the present study are 73% and over 85%, respectively.

The nature of the propagule was a crucial factor in the muhiplica- tion of shoots of D. longispathus. On shoot mulfiphcation medium the new shoots emerged as discrete units each comprising of three to five shoots. At the end of each cycle these units were excised and used as propagules, because their further dissection into individual shoots was always detrimental. This is in contrast to the observation made by Nadgir et al. (1984) in D. strictus where individual shoots were used as the propagules.

In the present study, liquid medium supported shoot muhiphca- tion better than semisolid medium. However, more than 15 pas- sages in the liquid medium caused vitrification of the shoots. High relative humidity is known to promote vitrification even when the shoots are not submerged (Ziv, 1986; Ziv et al., 1987a; Ziv et at., 1987b). As in the present case, vitrification in Dianthus caryophyl- lus (Davis et at., 1977; Sutter and Langhans, 1979) and Vitis vinif- era (Monette, 1983) could be effectively controlled by transferring the shoots from liquid to semisolid medium. However, this caused a slight decline in the rate of multiphcation. It may be possible to circumvent this problem to some extent by introducing one or two cycles of shoot multiphcation on semisolid medium after every 10 to 12 cycles on liquid medium. The use of Paclobutrazol to reduce the problem of vitrification in hquid medium, as suggested by Ziv (1989), is also being tested.

The rooting frequency of D. longispathus shoots was much higher in the combined presence of IAA and IBA than individually. Ryugo and Breen (1974) have suggested that IBA favors conjugation be- tween endogenous IAA and amino acids which leads to the synthesis of specific proteins necessary for the formation of root initials.

Coumarin, a phenol, is regarded as a naturally occurring inhibi- tor of IAA oxidase. It has been reported to promote rooting of various bamboo species both in vivo (B. tulda, Kumar et al., 1988; B. balcooa, Seethalakshmi et al., 1983; and B. arundinacea, Su- rendran et al., 1983) and in vitro (B. tulda, Saxena, 1990; B. balcooa, Roohi et al., 1991; D. hamiltonii, Sood et al., 1991). In combination with the auxins, IAA and IBA, coumarin considerably improved the rooting frequency in D. longispathus (present study).

Promotory effect of reducing salt concentrations of MS, to one half, one third, or one quarter on in vitro rooting of shoot has been described in several reports (Constantine, 1978; Hammerschlag, 1982a,b; Lane, 1979; Quoirin and Lepoivre, 1977; Skirvin et al., 1980). According to Hyndman et al. (1982), the favorable effect of reduced macroelement concenu'ation on rooting is probably less due to reduction in total ionic concentration than to the need for only a small amount of total nitrogen. In vitro rooting of the shoots of D. longispathus was more than doubled by dropping MS salt concentration to one half or one fourth.

ACKNOWLEDGEMENTS

This study was supported by a grant from the International Development Research Centre, Canada.

REFERENCES

Banik, R. L, Seed germination of some bamboo species. Indian For. 113:578-586; 1987.

Banik, R. L. Techniques of bamboo propagation with special reference to

pre-rooted and pre-rhizomed branch cuttings and tissue culture. In: Rao, A. N.; Dhanarajan, G.; Sastry, C. B., eds. Recent research on bamboos. Chinese Academy of Forestry, Beijing and IDRC, Singa- pore: 1987:160-169.

Constantine, D. In vitro multiplication of woody species. In: Round Table Conf., Gembloux, Belgium; 1978:134.

CS1R. The Wealth of India: A dictionary of Indian raw materials and indus- trial products: Raw Material-3, New Delhi: CSIR; 1952:32-33.

Davis, M. J.; Baker, R.; Hanan, J. J. Clonal multiphcation of carnation by micropropagation. J. Am. Soc. Hortic. Sci. 102:48-53; 1977.

Gamborg, O. L.; Miller, R. A,; Ojima, K. Nutrient requirements of suspen- sion cultures of soybean root cells. Exp. Cell Res. 50:151-158; 1968.

Hammerschlag, F. A. Factors influencing in vitro multiplication and rooting of the plum rootstock Myrobalan (Prunus cerasifera Ehrh.). J. Am. Soc. Hortic. Sci. 107(1):44-47; 1982a.

Hammerschlag, F. A. Factors affecting establishment and growth of peach shoot tips in vitro. Hortscience 17:85-86; 1982b.

Huang, L. C.; Huang, B. L.; Chen, W. L. Tissue culture investigations of bamboo--IV. Organogenesis leading to adventitious shoots and plants in excised shoot apices. Environ. Exp. Bot. 19:307-315; 1989.

Hyndman, S. E.; Hasegawa, P. M.; Bressan, R. A. Stimulation of root initiation from cultured rose shoots through the use of reduced con- centration of mineral salts. Hortscience 17:82-83; 1982.

Kumar, A.; Dhawan, M.; Gupta, B. B. Vegetative propagation of bamboo using growth promoting substances. Ind. For. 114:569-575; 1988.

Lane, W. D. Regeneration of pear plants from shout meristems. Plant Sci. Lett. 16:337-342; 1979.

Monette, P. L. Influence of size of culture vessel on in vitro prohferation of grape in a liquid medium. Plant Cell Tissue Organ Cult. 2:327- 332; 1983.

Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant. 15:473-497; 1962.

Nadgir, A. L.; Phadke, C. H.; Gupta, P. K., et al. Rapid multiplication of bamboo by the tissue culture. Silvae Genet. 33:219-223; 1984.

Prutpongse, P.; Gavinlertvatana, P. In vitro micropropagation of 54 species from 15 genera of bamboo. Hortscience 27:453-454; 1992.

Quoirin, M.; Lepoivre, PH. Improved medium for in vitro culture of Prunus sp. Acta Hortic. 78:437-442; 1977.

Roobi, F. N.; Shamsi, F. Q.; Rao, 1. V. R., et al. Clonal propagation of mature Bambusa balcooa in vitro. In: Abstracts IV International Bamboo Workshop, Chiangmai, Thailand. Kasetsart University, Bangkok, Royal Forest Department Bangkok and IDRC, Singapore; 1991:71,

Ryugo, K.; Breen, P. J. Indoleacetic acid metabolism in cuttings of plum (Prunus cerasifera X P. munsoniana cv. Mariana 2624). Proc. Am. Soc. Hortic. Sci. 99:247; 1974.

Saxena, S. In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation. Plant Cell Rep. 9:431-434; 1990,

Saxena, S.; Bhojwani, S. S. Regeneration and mass propagation of bamboo species through tissue culture. Proc. of IV International Bamboo Workshop, Chiangmai, Thailand, Nov. 24-29, 1991. Kasetsart Uni- versity, Bangkok, Royal Forest Department Bangkok and IDRC, Singapore; in press.

Seethalakshmi, K. K.; Venkatesh, C. S.; Surendran, T. Vegetative propaga- tion of bamboo using growth promoting substances-1. Bambusa balcooa Roxb, Ind, J. For. 2:98-103; 1983.

Skirvin, R. M.; Chu, M. C.; Rukan, H. Tissue culture of peach, sweet and sour cherry, and apricot shoot tips. In: Proc. lII State Hortic. Soc. 113:30-38; 1980.

Sood, A.; Palni, L. M. S.; Sharma, M., et al. Micropropagation of Dendroca- lamus hamiltonii Munro using single node cuttings taken from elite seedling plants. In: Abstracts IV International Bamboo Workshop, Chiangmai, Thailand. Kasetsarl University, Bangkok, Royal Forest Department, Bangkok and IDRC, Singapore; 1991:65.

Surendran, T.; Venkatesh, C. S.; Seethalakshmi, K. K. Vegetative propaga- tion of the thorny bamboo Bambusa arundinacea (Retz.) Willd. us- ing some growth regulators. J. Tree Sci. 2(1&2):10-15; 1983.

Sutter, E.; Langhans, R. W. Epicuticular wax formation on carnation plant-

142 SAXENA AND BHOJWANI

lets regenerated from shoot tip culture. J. Am. Soc. Hortic. Sci. 2:327-332; 1979.

Tsay, H. S.; Yeh, C. C,; Hsu, J. Y. Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora [Munro] MeClure). Plant Cell Rep. 9:349-351; 1990.

Yeh, M. L.; Chang, W. C. Plant regeneration through somatic embryogene- sis in callus cultures of green bamboo (Bambusa oldhami Munro). Theor. Appl. Genet. 173:161-163; 1986a.

Yeh, M. L.; Chang, W. C. Somatic embryogenesis and subsequent plant regeneration from inflorescence cells of Bambusa beecheyana Munro var. beecheyana. Plant Cell Rep. 5:409-411; 1986b.

Zamora, A. B.; Grezo, S. S.; Damasco, O. P. Callus induction and plant regeneration from internode tissues of Dendrocalamus latiflorus cv. Machiku. In: Rao, A. N.; Yusoff, A. M., eds. Proceedings of the

Seminar Tissue Culture of Forest Species. Forest Research Institute of Malaysia, Kuala Lumpur and IDRC, Singapore; 1989:76-82.

Ziv, M. In vitro hardening and acclimatization of tissue culture plants. In: Withers, L. A.; Anderson, P. G., eds. Plant tissue culture and its agricultural apphcations. London: Butterworths; 1986:187-196.

Ziv, M. Enhanced shoot and cormlet prohferation in liquid cultured gladio- lus buds by growth retardants. Plant Cell Tissue Organ Cult. 17:101-110; 1989.

Ziv, M.; Schwarts, A.; Fleminger, D. Malfunctioning stomata in vitreous leaves of carnation (Dianthus caryophyUus) plants propagated in vitro; implications for hardening. Plant Sci. 52:127-134; 1987a.

Ziv, M.; Schwarts, A.; Fleminger, D. Hardening aspects of micropropagated carnation plants having malfunctioning stomata. In: Ducate, G.; Ja- cob, M.; Simeon, A., eds. Plant micropropagation in horticultural industries. Liege, Belgium: Presses Universitaires; 1987b:47-54.