Factors affecting the in-vitro establishment of Jamaican yams (Dioscorea spp) from nodal pieces

J Sci Food Ayric 1995,67,541-550

Factors Affecting the In-Vitro Establishment of Jamaican Yams (Dioscorea spp) from Nodal Pieces Sylvia A Mitchell, Helen N Asemota and Mohammad H Ahmad* Biotechnology Centre, University of the West Indies, Mona, Kingston 7, Jamaica

(Received 25 May 1994; revised version received 11 October 1994; accepted 30 November 1994)

Abstract: Factors such as inorganic ammonium, growth regulator supplements, propagule type and subculture period affecting the in-vitro establishment of Dio- scorea cayenensis and D trifda have been examined. The results indicate that for D cayenensis, transfer of whole plantlets to fresh initiation media prior to cutting into nodal segments and transfer to establishment media was beneficial. Addition of 800 mg litre-' NH,NO, to basal medium (BM) containing 0.5 mg litre-' benzylamino purine (BAP) caused a significant improvement in nodal numbers and resulted in 11.8 propagules per original explant after 8 weeks or 184.1 propagules after 16 weeks in-vitro. Rapid establishment of D trifida cv Short Neck Yampie depended on the original explant source. Explants obtained from small tuber vines grew faster on establishment than those obtained from leafy cuttings. Initiation on BM supplemented with 0.5 mg litre-' kinetin (BMO.5K) and establishment on BM supplemented with 0.5 mg litre-' BAP (BMO.5BAP) produced the optimum result for the vines obtained from small tubers (24.0 propagules after 8 weeks of culture). For explants obtained from leafy cuttings, initiation on BMO.SBAP and establishment on BMO.5K gave optimum results (15.8). Quick establishment of initiated plantlets shown by increased shoot response and high multiplication rates were achieved in this study.

Key words: yam, Dioscorea, nodal position, growth regulators, tissue culture, establishment.

INTRODUCTION

In the Caribbean, Jamaica is the leading producer of yams with over 15 local varieties of about six cultivated species (Dioscorea alata, D cayenensis, D rotundata, D trijida, D esculenta and D bulbifera). Traditional methods of growing yam are inefficient and associated with virus, nematode and other disease infections that result in decreased yields. Increases in yam production can come from improved cultural methods, improved cultivars by selection and breeding and by improved propagation methods such as in-vitro micropropagation.

An important and often neglected stage of micropropagation is the establishment of the plantlets once they have been successfully initiated in culture. The first stage (initiation) is concerned with obtaining aseptic fast

* To whom correspondence should be addressed.

growing in-vitro cultures of explants taken from in-vivo conditions. The second stage (establishment) is concerned with maintaining the optimum stable growth rate of the initiated plantlets. It is only after plantlet growth has stabilised that scaling up for production can be considered. The literature lacks detailed information about establishment of yam plants in culture. Chatur- vedi (1975) mentioned that explants of D Joribunda initiated on media containing adenine sulphate and naphthalene acetic acid grew more vigorously on transfer to the same media while Mantel1 et a1 (1978) neither reported any differences in growth rates of explants on subculture nor mentioned an establishment stage. Arnolin (1980) identified an establishment stage for in- vitro yam cultures of D cayenesis-rotundata, D alata and D triJida with secondary explants producing more root, stem and nodal growth than primary explants. However, further subculture of D cayenensis-rotundata on the same media (MS169) resulted in decreased node

54 1 J Sci Food Agric 0022-5142/95/$09.00 0 1995 SCI. Printed in Great Britain

542

numbers. No explanation was given in these papers for the difference in growth of primary and secondary explants. Preliminary experiments in the authors' labor- atory indicated that explants of D cayenensis produced shoot growth very slowly on subculture. There is thus a need to define an efficient establishment regime for yam micropropagation. This paper reports studies carried out on cultures of D cayenensis and D trijida during their establishment to in-vitro conditions.

EXPERIMENTAL

Sampling

The nodal pieces used in this study were obtained from cultures of D cayenensis and D trijida explants initiated at different times of the year on basal medium (Murashige and Skoog (1962) salts, 100 mg litre-' ino- sitol, 0.1 mg litre-' thiamine, 0.5 mg litre-' pyridox- ine, 0.5 mg litre-' nicotinic acid, 2 mg litre-' glycine, 30 g litre-' sucrose and 1.8 g litre-' gelrite), BMO.SK, BMS.OK, BMO.SBAP, BMO.SBAPO.OSNAA, 2BMO. 5BAP or 3BMO.5BAP (BM stands for basal medium; the numbers are the concentrations (mg litre-') of: K, kinetin ; BAP, benzylamino purine; NAA, naphthalene acetic acid). The explant cultures were 4 weeks old unless otherwise mentioned.

Culture conditions

All solutions and media used were autoclaved at 121°C under 1.05 kg m-* pressure for 20 min. Plantlets were grown in test tubes containing 10 ml of media covered with Magenta opaque caps. The cultures were transferred to the growth room with a 16 h photoperiod at 24-27"C, with light provided by cool white fluorescent tubes (1200 lux intensity). A 16 h photoperiod was chosen as previous studies (Mantel1 and Hugo 1989) have shown that yam has a tendency to early tuberisation under shorter photoperiods. Nodes with leaf were transferred at 4-weekly intervals unless otherwise stated.

Media used for establishment included BM, BM2 (BM and 0.3 mg litre-' thiamine, 1 g litre-' charcoal, 100 ml litre-' coconut water), and MS169 (Murashige and Skoog (1962) salts, Morel vitamins, 0.23 mg litre- ' 6-BAP, 30 g litre- ' sucrose and 1.8 g litre- ' gelrite. Details on growth regulator supplements (mg litre- I )

given in methods.

Methods

Factors affecting the estabishment of D cayenensis or D trijida have been examined in six different sets of experiments.

S A Mitchell, H N Asemota, M H Ahmad

D cayenensis cv Round Leaf Yellow Yam Explant cultures (initiated 1 March) were placed on fresh initiation media for 4 weeks and then cut into nodes. All nodes from each fresh initiation media were transferred at 4-weekly intervals to establishment media BM, BM2, BM, BM and MS169. At each subculture, all nodes were used. Twelve-week-old explant cultures (initiated 2 March) from initiation media BMO.SBAP, 2BMO.SBAP or 3BMO.SBAP were cut into nodes and transferred to establishment media BM or BMO-SBAP. Explant cultures (initiated October) were cut into nodes (whether subtended by cataphyll, small unextended leaf or an extended leaf and tips) where possible. All nodes from each initiation medium were transferred for establishment to the same media or BM supplemented with 0.5 mg litre- ' zeatin (BMO.5Z). All explant cultures (initiated late January) were placed on fresh initiation media for 4 weeks. Fifteen nodes cut from explant cultures on fresh initiation media were transferred to establishment medium BMO.5BAP. In this experiment the effect of nodal position of initiated plantlets was examined. Ten explant cultures (initiated late January) from fresh initiation medium BMO.5BAP having at least three shoots were cut into nodes and transferred to establishment medium BMO.5BAP and then to either BM or BMO.5BAP. Thirteen nodal positions were used. Twelve-week-old explant cultures (initiated early January) from BMO.5BAP or BM0.5BAP0.05NAA initiation media were cut into nodes. Ten nodes were transferred to each of the establishment media $BM, ;BMO.SBAP, BM, BMO.SBAP, BM1650NH4NO3 (2BM), 2BMO-SBAP, BM3300NH4N03 (3BM) and 3BMO.SBAP. Explant cultures (initiated late January) from fresh initiation media BMO.5BAP or BM0.5BAP0.05NAA were cut into nodes. Ten nodes with or without leaves were transferred to establishment media BM, BMO.SBAP, BM800NH4N03, BM800NH4NO30.5BAP, 2BMO-SBAP, BM2.0BAP0.01 IBA, BMO.1 BAPO. SNAA and BM3.02i-PO.4NAA. All nodes from establishment media were then transferred to either BM or BMO-SBAP.

D trifida cv Short Neck Yampie (6) Explant cultures of D trzjida cv Short Neck

Yampie (initiated from sympodial or mono- podia1 vines of leafy cuttings, or monopodial vines from small tubers) were cut into nodes. Nodes from each initiation medium were transferred to establishment media BM, BMO.SK,

Factors affecting establishment of yam from nodal pieces 543

BM5-OK and BMO.5BAP. Five nodal positions from explant cultures originating from mono- poidal vines were used.

Parameters of growth such as swelling of the nodal region, microtuber development, callus production, number of buds, nodes, leaves, tips and roots were recorded at 4-weekly intervals for all experiments. Two criteria were used to assess the effect of in-vitro treatments on culture establishment: number of responsive nodes in regard to shoot production (those producing at least one leaf) and the multiplication rate (MR) (MR = original node + new nodes + new tips). Unre- sponsive nodes either remained green, produced callus or died. Experiments were done in a random block design with a minimum of five replicates per treatment. Results were statistically analysed using GENSTAT (Lawes Agricultural Trust 1986) and Duncan’s t-test. Least significant difference (LSD) values were calculated using Student’s t-test.

RESULTS

It was observed that during establishment, growth of plantlets was slower than during initiation and bacterial contamination was found to occur even from presum- ably clean nodes.

Establishment of D cayenensis

Cultures initiated at different times of year Explants initiated in March established more slowly that those initiated at the other times. When explant cultures were transferred to fresh initiation media, those initiated 1 March on BM or BMO.5K gave more responsive cultures (84% and 57% shoot growth) than those initiated late January (33% and 36%). New growth (MR) from 1 March (0.9 and 0.6) or late January (1.0 and 0.3) was low. On BMS.OK, however, shoot response (61%) and MR (1.0) were lower from 1 March than late January (8 1 YO, 5.2).

Effect of sequential establishment on BM (Expt I ) Shoot response of the nodal pieces increased with subculturing. The number of nodal pieces producing shoots at the first subculture from initiation media BMS.OK, BMO.5K and BM was 41, 61 and 62%, respectively. Most nodal positions from explants initiated on BMO.5K produced microtubers while those initiated on BM5.OK gave more unresponsive nodes. The highest shoot response was on BM2 (100, 98, 97%) and remained at between 90 and 100% on subculturing. Only on MS169 (Arnolin 1980) was the average MR above two. After 28 weeks in-vitro culture approx- imately the same number (28-30%) of the original nodal explants survived on each medium (Table 1). After five

subcultures, nodal explants that had been initiated on BM produced more nodes in establishment culture than those initiated in other media (3.4 propagules after 16 weeks in culture). The total number of propagules per original explant after 28 weeks was 20 from BM, 13.4 from BMO.5K and 6.9 from BM5.OK.

Eflect of nodal position For all experiments the most new growth on subculture was found to develop from the basal nodes cut from plantlets.

When cultures initiated 1 March were used, the original nodal position (position of node on minisett vine) did not influence the subsequent growth of nodes beyond the first subculture on establishment media although more growth was noted from positions 7-9 after 28 weeks of culture. The two youngest nodal positions did not produce viable cultures from initiation media (Table 1).

When cultures initiated October were used, nodes cut from the older nodal explants showed enhanced growth after 4 weeks especially on BMO-SBAPO.05NAA establishment media. The nodes that responded during the first 4 weeks on establishment media continued to grow well and by 16 weeks in vitro the effect of original nodal position was not as obvious. Nodal position 3-9 produced the highest number of nodes (Fig 1).

There was a significant (P < 0.05) effect of nodal position of initiated plantlets on fresh initiation media (Expt 3). Shoot production from the basal node was 100% and significantly (P < 0.05) more roots, leaves and new nodes were produced than from all the other nodes (Table 2). On many of these nodes (21%) no axillary buds were visible. Nodes where buds were visible had well-developed basal nodal region (swelling and bud development) but fewer new nodes by 4 weeks than from the basal node. The tips produced a lot of browning. On transfer, the basal nodes produced a lot of browning and no new growth. After transfer to BM there was a significant difference (P c 0.05) between the MR of nodes taken from the basal node cultures (1.4) and those taken from the other nodal cultures (2.7). On BMO.5BAP the effect was not significant (7540% shoot response, 2.7-3.2 MR). Growth obtained after 16 weeks in culture was 11.8 (MR after fresh initiation medium BMO.5BAP) x 1.73 (nodes placed on BMO-SBAP) x 2.95 (transfer to BMO.5BAP) = 60.2 propagules per original explant.

Length of time on establishment media (Expt 2) The nodes that responded during the first 4 weeks on the higher cytokinin establishment media showed mainly monopodial growth. The nodes where the leaves were removed were less responsive and did not produce leaves or roots in the first 4 weeks. The only sign of growth was swelling and buds. On all media used, shoot response and MR increased with time (Table 3). By 12

544 S A Mitchell, H N Asemota, M H Ahmad

weeks

TABLE 1 Effect of initiation media and original nodal position on establishment of D cayenensis cv RLYY plantlets

Original' Multiplication rateb ( M R ) Percentage of Average

explant media explants per original position surviving explant after

nodal on sequential establishment original number of propagules

BM BM2 BM BM MS169 after 28 28 weeks weeks

Initiation on BM 2 3 4 5 6 7 8 9

2 3 4 5 6 7 8 9

2 3 4 5 6 7 8 9

Initiation on BMO.5K

Initiation on BM5.OK

0.0 1 .o 2.1 2.0 2.1d 2.0 3.3 2.0

0.0 1 .o 1.2 1.0 1.3d 1.2 1.7 1.5

1 .o 1.1 1 .o 1 .o 1.ld 1.7 1.1 1 .O'

0.0 1 .o 1.7 3.8 0.0 1 .o 1.6 1 .o

0.0 1.8 1 .o 1.5 0.0 3.5 1.1 1.5

1 .o 1.3 1.1 1 .o 0.0 1.9 1 .o 0.0

0.0 1 .o 1.4 1.4 0.0 1.7 1.5 2.0

0.0 1.5 2.0 1.5 0.0 1.6 1.8 2.3'

1 .O' 1.7 1.5 2.5 0.0 1.3 1.7 0.0

0.0 0.0 1 .o 0.0' 1.4 1.9 1.5 2.0 0.0 0.0 1.5 2.4 1.6 2.4 3.2 2.9

0.0 0.0 1.2 1.8 1.2 2.9 2.2 2.0 0.0 0.0 1.6 2.2 2.0 2.4 0.0 0.0

0.0 0.0 1.3 1.9 1.5 1.7 2.2 1.5 0.0 0.0 2.3 2.1 1.4 1.1 0.0 0.0

0 0

70 50 0

70 70 10

0 40 60 30 0

80 40

0

0 60 60 20 0

80 40 0

0.0 0.0

12.2 15.0 0.0 16.7 30.9 46.0

0.0 6.0 8 .O 8.3 0.0

22.6 14.3 0.0

0.0 7.3 3.0 4-5 0.0 103 6.5 0.0

Nodal position 1 all explants died during initiation. Multiplication rate = number of new propagules (original node + new nodes + new tips). For each nodal posi-

All dead or produced callus. All nodes at nodal position contaminated. All dead.

tion at least seven replicates were used, all nodes used on subculture.

on establishment media, shoot response was after 4 weeks on BM5OK (81%), BMO.5BAP (97%) and highest (91Y0) on BMO.5K but so also was micro- tuberisation (89%). Root numbers were low on establishment from BM5.OK or BMO.SBAPO.OSNAA. MR values were reduced when established on BMO.5Z (Fig 1). Among the conditions used, growth was best when explants were initiated and established on BM0.5BAP0.05NAA giving 19.2 propagules per original explant after 16 weeks in culture (Table 3).

E f e c t of propagule type on establishment Plantlets developed on initiation media and cut up into nodes for establishment gave a low shoot response (Table 3). Transfer of whole plantlets to fresh initiation media (Expt 3), however, gave a higher shoot response

BM0.5BAP0.05NAA (94%). New growth in the second month (0.3-5.4) was similar to the first (0.2-5.4), with a 30-80% shoot response when nodes were cut from the plantlets. It was observed that explant cultures on fresh initiation media followed the same pattern of growth as during initiation with cultures on BMO.5BAP producing the most growth. Nodal pieces from fresh initiation media on transfer to establishment medium BMO.5BAP gave higher MR and shoot response from BM (64%) and BMO.5K (80%) than from BM5OK (50%) or BMO.5BAP (30%) (Table 4). However, when the MR after establishment on BMO.5BAP is multipled by the number of propagules obtained after initiation, BMO.5BAP was still the better initiation medium (Table 4).

Factors affecting establishment of yam from nodal pieces

fn 10 x al :

545

. -3

!x E

0) L

0 1 I I I I I I 1 I I 0 1 2 3 4 5 6 7 8 9

Original nodal explant position

35 t

P

0 ' I 1 I I I I 1 I I 0 1 2 3 4 5 6 7 8 9

Original nodal explant position Fig 1. Effect of hormonal treatments on establishment of D cayenensis cv RLYY Number of propagules (MR) per original explant after 4 weeks on initiation media and (a) 4 weeks, or (b) 12 weeks on establishment media. Establishment media: 0, BM; 0, BMO.SK; V, BMS.OK V, BMO.SZ from BM5.OK; 0, BMO.SBAPO.OSNAA; D, BMO.5Z from BMO.SBAPO.05NAA. At least five replicates used per treat-

ment.

Egect of ammonium nitrate concentration and hormone supplements Nodes from 12-week-old explant cultures did not establish well, whether initiated 2 March or early January (0-30% shoot response). More browning of the media was observed at 12 weeks than from cultures on establishment media for 12 weeks (Expt 2). On transfer of the nodes to establishment media there was no significant difference (P < 0.01) between treatments (Duncan's t-test) and none of the establishment media tried produced more than 1.9 nodes in 4 weeks. The best com-

bination, initiation on BMO.5BAP for 12 weeks (4.1 MR) and establishment on BMO5BAP (1.8MR) gave 7.4 propagules per original explant after 16 weeks in-vitro growth.

When explant cultures initiated late January on media with BAP were used, growth during establishment was highest when explants from fresh initiation media BMO.SBAPO.OSNAA were established on BMO.5BAP (3*OMR, 60% shoot response). When explant cultures from BMO-5BAP were used, significantly (P < 0-05) higher M R s were obtained from nodal pieces without leaves established on BMO.5BAP with NH,NO, supplements (2.0-2.6MR). Addition of 800 mg litre-' NH,NO, increased shoot response from 30 to 60%. Subculture from BM0.5BAP800NH,N03 to BMO.5BAP produced very satisfactory growth (6.OMR, 100% shoot response). The other hormone com- binations were unsatisfactory, media supplemented with NAA resulted in callus production on 10-50% of nodes. Overall, the best combination of media was found to be as follows: initiation on BMO-5BAP then transfer to fresh BMO.5BAP (1 1.8MR), establishment of nodal pieces without leaves on BM0.5BAP800NH,NO3 (2.6MR) followed by transfer to BMO.5BAP (6.OMR) giving 184.1 propagules per original explant after 16 weeks in-vitro growth (Fig 2).

Establishment of D trifida

Eflect of explant source and hormone supplements (Expt 6) A 100% shoot response was obtained on establishment of D trijida from all explant cultures. The pattern of nodal growth varied with explant source. Nodes from sympodial vines of leafy cuttings of D trzjida cv Short Neck Yampie did not establish well. Initiation on BMO.5BAP and establishment on BM gave the most propagules (3.3) after 8 weeks in culture. None of the cultures from this explant source established as well as those originating from monopodial vines from leafy cuttings or small tubers.

Explants from monopodial vines from leafy cuttings developed significantly (P < 0.01) more roots on establishment medium BM than on the other media. Root numbers and percentage of nodes producing roots or sympodial shoots decreased significantly (P < 0.01) when cytokinin was added to the media (Table 5). The youngest node produced significantly (P < 0.01) less new nodes and roots than the older nodes.

Growth during initiation from monopodial vines of leafy cuttings or small tubers was highest on BMO-5BAP (Mitchell et al 1994). Lower multiplication rates were obtained on this medium (BMOeSBAP) during establishment. The effect of initiation media on subsequent growth during establishment differed with vine source. When leafy cuttings were used, explants


TABLE 2 Effect of nodal position on shoots grown on fresh initiation media BMO.5BAP during

establishment of D cayenensis cv RLYY

Growth after 4 weeks on establishment medium BM0.5BAPb Nodal position"

Root numbers Leaf numbers MR'

(1) Tip 0.oc 0 . k 1 .oc First node 0 . k 0.k 1 .oc Second node 0.6 f 1.4b 0.4 f 0.7bc 1.2 f 0.4bc Third node 0.2 f 0.7bc 1.0 f 1.8bc 1.7 f 1.3bc

(2) Tip 0.oc 0.3 f 0.5bc 1.5 f 1-2bc First node 0.oc 0 . k 1 ac Second node 0.oc 0.3 f 0 . 7 ~ 1.2 f 0.4bc Third node 0.k 1.8 f 2.7bc 2.3 f 2.3bc Fourth node 0.oc 1.0 2.4bc 1.5 f 1.2bc

(3) Tip 0 . k 1.1 f l.Obc 1.5 f 0.5bc First node 0.oc 0.9 f 1.7bc 1.3 f 0.5bc Second node 0.oc 1.7 & 2.5b 2.4 f 2.3b

Basal node 1.1 0.9a 4.6 f 2.8a 4.9 f 2.2a

(1) first shoot formed, nodes counted from tip down; (2) second shoot formed; (3)

Values (mean & standard error) followed by the same letter are not significantly dif-

MR, multiplication rate.

third shoot formed; Base, original explant and buds after shoots removed.

ferent (Duncan's I-test); 10 replicates used per treatment.

initiated on BMO.5BAP did produce the most growth on establishment. Establishment was significantly (P < 0.01) better on BMO.5K. There was increased browning of the establishment media BM5.OK or BMO.5BAP regardless of the initiation media used. The best combination of media, initiation on BMO.5BAP and establishment on BMO.5K gave 15.8 propagules per original node after 8 weeks in culture (Table 5).

When small tubers were used, explants initiated on BMO.5BAP produced the least growth on establishment media. Significantly (P < 0.01) better growth was obtained by 8 weeks from explants initiated on BM or BMO.5K and established on BM5OK or BMO.5BAP. More browning of the media occurred when BM5.OK or BMO.5BAP was used for initiation and establishment media. The best combination of media was initiation on BMO.5K and establishment on BMO.5BAP giving 24.0 propagules per original explant after 8 weeks in culture (Table 5).

DISCUSSION

It is evident from this study that a different establishment regime is required for each species of yam. This is not surprising as a wide diversity occurs among yam cultivars in their biochemical composition, storability, sprouting and growth in culture (Mitchell et a1 1989; Mitchell and Redway 1991; Mitchell and McLaughlin 1992; Asemota et a1 1992; Muzac-Tucker et a1 1993;

Wellington and Ahmad 1993). Several factors were found to influence the speed of establishment of D cayenensis in culture.

Explants initiated in March from young minisett vines ( - 6 weeks old) did not establish well especially from media with high kinetin concentration. Mantell et al (1978) found that explants of D alata from yam vines grown under long days for 4 months initiated well. Comparison with these results is difficult as no figures are given for establishment. It is possible, however, that the physiology of the minisett vines differed from the mature vines used by Mantel1 et a1 (1978).

During establishment higher multiplication rates were obtained on media supplemented with cytokinin, although growth was possible on basal medium. On initiation high multiplication rates and shoot response were obtained on media with 0.5 mg litre-' BAP but growth decreased on subculture suggesting that the BAP concentration became supraoptimal. Arnolin (1980) used a lower BAP concentration (0.23 mg litre-I) which could explain why he only found a decrease in growth after the second subculture. Addi- tion of ammonium nitrate to media with BAP increased shoot response and growth. This suggests that the ammonium nitrate concentration in the basal medium was insufficient to sustain the growth rate possible from D cayenensis during establishment.

Transfer of plantlets to fresh initiation media prior to subculturing resulted in better shoot response and growth on establishment. After four weeks on initiation

Factors affecting establishment of yam f rom nodal pieces 547

TABLE 3 Growth response and organ development of D cayanensis cv RLYY after various times on establishment media"

Initiation Establishment Length Growth after various times on E M Total number mediab mediab of time propagules

( E M ) on EM Percentage of nodes Root M T Percentage of nodes MRd from (week) with numbers (%) with initiation

roots shoots

BM

BM 0.5K

BM 5.OK

BM O.5BAP O.05NAA

BM

BM 0.5K

BM 5.OK

BM 0.5Z

BM O.5BAP O.05NAA

BM 0.5Z

4 8

12

4 8

12

4 8

12

4 8

12

4 8

12

4 8

12 Approx SE roots (0.32), MR (0.33)

81 83 89

84 93 93

41 53 -

28 38 -

22 37 -

31 37 -

2.5 3.7 5.1

2.7 4.8 6.5

1.3 2.5 -

1 .o 1.6 -

1 .o 1.6 -

0.8 1.3 -

27 45 59

28 72 89

10 20 25

5 8

14

0 0 8

0 4

12

51 73 73

54 83 91

37 59 64

49 58 61

43 65 72

43 51 71

1.2 2.1 2.5

1.6 2.3 2.9

1.9 2.5 3.0

1.9 2.3 2.8

2.4 2.9 4.3

1.6 2.0 2.5

1.7 2.9 3.5

2.2 3.2 4.1

7.2 9.5

11.4

7.2 8.7

10.6

10.8 13.3 19.2

7.2 9.0

11.3

~~~

Data analysed by regression. All available nodes used from initiation; no single LSD value could be given as replicates per media

K, kinetin; BAP, benzylamino purine; NAA, naphthalene acetic acid; Z, zeatin. MT, microtuber. MR, multiplication rate (original node + new nodes + new tips).

varied between 45 and 73; data averaged over nodal position.

media, browning of the media was noticed and transfer to fresh media was beneficial to the explants. The lower response of nodes after prolonged periods on initiation media may be due to the increase of brown phenolic

substances in the media which may adversely affect their growth. It is possible, on the other hand that nodal response increased with time on establishment media due to increased starch accumulation or develop-

TABLE 4 Growth of D cayenensis cv RLYY plantlets on establishment medium BMO.5BAP

Initiation Growth after 4 weeks on Number of propagules mediab establishment medium (BMO.5BAP) per original node

after 12 weeks Root numbers Leaf numbers MR'

BM 0.5 f 0-8a 3-1 f 3.0a 3.9 f 2.9a 11.3 BMO.5K 0.7 f 1-5a 3.4 f 2.5a 3.0 2.4ab 4.5 BM5.OK 0.4 1.3a 1-5 f 1.6b 2.1 1.5b 16.2 BMO.5BAP 0.2 f 0.3ab 1-0 f 1.2b 1.7 f 1 . 1 ~ 20.1

Values (mean f standard error) followed by the same letter are not significantly different

(4 weeks on initiation media, then roots removed and placed on fresh initiation media for

MR, multiplication rate.

(Duncan's t-test); 15 replicates used per treatment.

another 4 weeks).


1 2 3 4 5 6 7 8

Establishment media

d Z @

40

20

0 1 2 3 4 5 6 7 8

Establishment media Fig 2. Effect of ammonium nitrate and hormonal supplements on in-vitro growth of D cayenensis cv RLYY after initiation on (a) BMO.SBAP, and (b) BMO.5BAP O.OSNAA, establishment of nodes without leaves on eight different media and then transfer to either m, BM; or m, BMO.5BAP. Ten replicates per treatment on establishment media, all available propagules were then transferred. 1, BM; 2, BMO.5 rng litre-' BAP; 3, BM 800 mg litre-' NH,NO,; 4, BMO.5 mg litre-' BAP 800 mglitre-' NH,NO,; 5, BM 0.5 mg litre- ' BAP 1600 mg litre-' NH,NO,; 6, BM2.0 mg litre-' BAP 0.01 mg litre-' IBA; 7, BMO.1 mg litre-' BAP

0.5 mg litre- ' NAA; 8, BM3.0 mg litre-' 2-iP 0.4 mg litre- NAA.

mental processes (Mantell, pers comm). For commercial purposes, however, it was noted that more propagules were obtained with short subculture periods.

The older nodes of in-vivo grown minisett vines produced enhanced in-vitro growth during initiation (Mitchell et a1 1994). This enhanced growth was also obvious on the first establishment media from all nodes cut from the older explants but especially from the basal node and on media with BAP. The basal node (original explant piece) was capable of producing successive crops of plantlets but growth decreased at each subculture until no new growth could be obtained. The older nodes from plantlets grown in-vitro, regardless of nodal origin, also produced more shoots. The younger nodes produced less growth but there was more devel- opmental of the nodal region in terms of swelling and

bud development which on subculture gave increased growth. This increased growth was only obvious on basal medium as addition of BAP increased shoot production from all nodes. Shoot growth was also inhibited by NAA, suggesting that the lower growth of the younger nodes is due to higher endogenous auxin levels due to apical dominance. Gould et al (1987) found that in-vitro growth of pea axillary buds was faster from the basal nodes. He concluded that the growth potential of buds at a node is a consequence of the number of buds and number of primordia within a bud that are established while the terminal shoot is growing. He further suggested that apical dominance not only inhibits the outgrowth of lateral shoots but can also suppress the initiation and early development of axillary buds. In the present study the axillary nodal area of the basal nodes

Factors a fec t ing establishment of y a m from nodal pieces 549

TABLE 5 Growth of D trifda cv Short Neck Yampie after 4 weeks on establishment media"

Initiation Establishment Type of growth after 4 weeks on EM Total number media media of propagules ( I M ) (EM) Percentage of nodes Root Percentage of M R ~ after 8

with numbers sy mpodial weeks roots shoots

From leafy cuttings BMO.5K BM

BMO.5K BM5.OK BMO.5BAP

BMO.5BAP BM BMO.SK BM5.OK BMO.5BAP

From small tubers BMO.5K BM

BMO.5K BM5.OK BMO.5BAP

BMO.5BAP BM BMO.5K BM5.OK BMO.5BAP

Interaction between IM and EM

85 60 15 0

55 10 15 0

65 61

5 19

2.1 94 1.9 1.3 100 2.1 0.2 50 1.2 0.0 30 1.9

1.2 56 2.2 0.3 34 3.1 0.2 22 2.8 0.0 0 1.8

1.9 63 2.5 1.9 60 3.0 0.1 50 3.4 0.4 3 5.0

32 1.5 41 1.6 23 0.7 18 2.1 1 1 0.1 0 1.8 0 0.0 0 2.9

root numbers LSD ( P > 0.01) 0.7 (leafy cuttings), 1.7 (small tubers) node numbers LSD ( P > 0.01) 0.8 (leafy cuttings), 1.8 (small tubers)

7.0 7.8 4.4 7.0

11.2 15.8 14.3 9.2

12.0 14.4 16.3 24.0

11.4 14.9 12.8 20.6

At least 20 replicates used per treatment, results averaged over nodal position. MR, multiplication rate.

was larger and more developed while near the tip there was decreased axillary bud development and no buds could be identified at some of the nodes.

In general, plantlets initiated on media containing BAP established well. For D trijida, however, the combination of cytokinins found to give optimum growth after 8 weeks depended on the explant source. While all the nodes of D trijida showed continuous growth in culture (100% shoot response) there seemed to be an inhibitory effect on addition of 0.5 mg litre-' BAP to both initiation and establishment media. With D cayenensis, this inhibitory effect of BAP was not noticed when whole plantlets were transferred instead of nodal pieces. Nodes of D trijida obtained from monopodial shoots from small tubers gave the highest multiplication rate followed by monopodial shoots and then sympodial shoots of leafy cuttings. The effect of explant source seemed more important than cultivar as D trijida cultures gave similar multiplication rates once fully accli- matized to in-vitro conditions. It is possible that the sensitivity of the nodal explants to growth regulator supplements changes with the stage of growth of the plant.

This work has shown that fast establishment of yam cultivars in culture is attainable under specified conditions. This is vital for the development of a commercial propagation scheme for yam using micropropagation techniques.

ACKNOWLEDGEMENTS

The authors are grateful to European Development Fund (EDF), Jamaican Agricultural Research Project (JARP) for funding and financial support; to Dr Sinclair Mantel1 of UAPS, Wye College, UK for useful sugges- tions and detailed scrutiny of this work; and to Mrs Heather Reid of Caribbean Agricultural Research and Development Institute (CARDI) for help with statistical analysis.

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Factors affecting the in-vitro establishment of Jamaican yams (Dioscorea spp) from nodal pieces