Stable regeneration in Asparagus cooperi Baker as controlled by different factors

Plant Science, 82 (1992) 119-124

119

Elsevier Scientific Publishers Ireland Ltd.

Stable regeneration in Asparagus cooperi Baker as controlled by different factors Biswajit Ghosh and Sumitra Sen Centre of Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta 700019 (India) (Received July 23rd, 1991; revision received November 13th, 1991; accepted November 13th, 1991 )

For in vitro regeneration spear, shoot tip, internode, node and root of mature plants of Asparagus cooperi were used as explants. Induction of callus from different explants depended on the photoperiod in addition to a specific combination of c~-napthalene acetic acid (NAA) and kinetin (Kn) in the basal medium. The development of shoots from callus required 6-benzylaminopurine (BA), Larginine, adenine and a low level of NAA. The individual shoots produced roots in the presence of indole-3-butyric acid (IBA) or indole-3-butyric acid containing potassium salt (KIBA). Regenerated plants were cytologically and phenotypically stable.

Key words: Asparagus cooperi; tissue culture; explant type; callus; organogenesis; plant regeneration; photoperiod; cytology

Introduction

Micropropagation is an effective tool in plant biotechnology. The genus Asparagus is well known for its economic value [1]. There is no protocol for in vitro regeneration of this genus except for two species, A. officinalis [1] and A. racemosus [2]. The species A. cooperi Baker is important for its high horticultural and as well as medicinal value due to the presence of sapogenin [3]. There is no report of in vitro studies on this species. In the course of our studies on in vitro propagation in A. cooperi, we have recently reported plant regeneration through somatic embryogenesis in this species [4,5]. In this paper, we report plant regeneration through organogenesis in A. cooperi and cytological studies along with organogenesis. Materials and Methods

Explant Juvenile spear segments (2-4 mm), phyllode Correspondence to: Biswajit Ghosh, Centre of Advanced Study, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Calcutta 700019, India.

(2-4 mm), node (2-5 mm, 3-6 number from apex), internodal segments (4-6 mm) and root segments were used as explant. The explants were disinfested by 0.1% mercuric chloride solution for 9 min and rinsed five times in sterile double distilled water.

Culture media Three different basal media along with different growth regulators, amino acids and additives were used for regeneration of complete plants through callus culture. These basal media were supplemented with various auxins (NAA; 2,4-dichlorophenoxyacetic acid (2,4-D); indoleacetic acid (IAA); IBA and KIBA), cytokinins (BA, N6-(2-isopentenyl)-adenine (2iP), Kn, adenine) and amino acids (L-arginine, L-proline, L-leucine) singly or in different combinations for callus, shoot, bud and root induction. The compositions of the basal media were as follows: (1) MSC-1 media containing Murashige and Skoog (MS) macro-salt, 100 mg 1-1 Ca(NO3)2.4H20, MS micro-salt, 125 mg 1-I myo-inositol, 3 mg 1-l glycine, 0.4 mg 1-t thiamine HC1, 3% sucrose and 0.8% agar. (2) MS-S media containing MS macro-

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salts, 180 mg l-~ Na2SO 4 MS micro-elements, 0.1 mg 1-1 thiamine HCI, 1 mg l-I nicotinic acid, 4 mg 1-1 L-glutamine, 150 mg 1-I myo-inositol and 3% sucrose. (3) MS-R media containing halfstrength of MS macro, full strength of MS micro, MS vitamin, 3 mg 1-1 riboflavin, 100 mg 1-l myoinositol, 1 mg 1-1 glycine, 2% sucrose and 0.4% agar.

Cultural condition The pH of the media was adjusted to 5.7 before autoclaving. The media were sterilized by autoclaving for 15 min at 121°C and amino acids were filter sterilized and added to the media. The cultures were maintained at 25 ± 2°C and with 60% relative humidity. Some sets of cultures were exposed 16 h per day to an illumination of 3000 Ix provided by fluorescent lamps. A few sets of cultures were kept in full darkness.

Chromosome stud), For mitotic chromosome study, the early and subsequent passages of callus culture and root tips of regenerants were pretreated with 0.002 M 8-hydroxyquinoline for 4 h at 10-12°C and fixed in 1:3 acetic acid:dehydrated ethanol. Acetoorcein staining technique was adopted.

Results

Induction of callus from different explant For callusing MSC-1 basal medium with variable concentrations and combinations of NAA and Kn were found to be effective in all the explants. In spear and internodal segments healthy callusing was obtained within 12 days in medium containing 1 mg 1-1 NAA and 1 mg 1-1 Kn. Significant response was noted in shoot tip explant in 15-18 days of culture, in medium containing 1.5 mg 1-1 NAA and 1.5 mg 1-1 Kn. Optimal induction from the nodal tissue was noted in 2.0 mg 1NAA and 1.0 mg 1-1 Kn in 14-17 days of culture. Phyllode and root tissue responded well in the presence of 2.5 mg 1-l NAA and 2.0 mg 1-1 Kn within 18-21 days. The optimum callus induction was obtained from spear explant. The calli derived from different explants were morphologically different too. The calli derived from spear and node were soft and white, from shoot tip they were soft and yellowish green, and from root segments they were compact white nodular. The rate of callus induction from different explants was significantly high in full darkness (Table I) rather than 16/8 h light/dark period.

Table I. Analysis of variance (ANOVA) for frequency of callus induction for six explant sources of A. cooperi grown in different combinations of NAA and Kn growth regulators in different photoperiod. Source

Replication Explant (A) Error (a) Photoperiod (B) A x B Error (b) Growth regulators combination ratio (C) B × C A × C A x B × C Error (c) *Significant at P = 0.05% **Significant at P = 0.01% *** Significant at P = 0.001% n.s., not significant.

Degrees of freedom

Sum of square

Mean square

F

2 5 10 1 5 12 13

16.86 25 228.5 17.44 2716.07 63.37 73.01 105 211.19

8.43 5045.7 1.74 2716.07 12.67 6.08 8093.16

4.84* 2899.82**

13 65 65 312

1104.54 238 770.11 658.84 758.72

84.96 3673.38 10.13 2.43

34.96*** 1511.67"** 4.16"**

446.72*** 2.08 n.s. 3330.5***

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Induction of shoot from callus derived from different explants Cytokinin in combination with L-arginine was effective in shoot differentiation from calli in MSS basal medium. Of the different cytokinins (viz. BA, Kn, 2iP, adenine) applied, the combination of BA, adenine, L-arginine accelerated shoot initiation. The callus produced from nodal explant gave large numbers of shoots (8-11) from 1 g of callus in the medium containing 2.5 mg 1-1 BA, 100 mg 1-1 adenine, 100 mg 1-l L-arginine and 0.02 mg 1-1 N A A after 18-22 days of culture. Maximum shoots were obtained in calli derived from spear explants (20-24 shoots/g calli) and internodal explants (16-18 shoots/g calli) within 10-14 days in MSS medium containing 2.0 mg 1-1 BA, 80 mg 1-I adenine, 100 mg 1=1 L-arginine and 0.02 mg l-l N A A (Fig. 1). In the callus derived from shoot tips and phyllodes nearly 9-11 and 12-15 shoots per g calli respectively, could be obtained within 13-16 days of inoculation in 1 mg 1-1 BA, 80 mg l-1 adenine, 50 mg l-l L-arginine and 0.02 mg i -1 N A A containing media. After several trials,

only 1-2 shoots per g calli could be obtained in root callus in the presence of 2 mg 1-1 BA, 100 mg 1-1 adenine, 100 mg 1-1 L-arginine and 0.02 mg 1-l N A A in 35-42 days. The number of shoots was significantly higher when calli for organogenesis were incubated in a 16/8-h light/dark cycle than in complete darkness (Table II). When calli induced under a 16/8 h photoperiod were used for shoot induction under a similar photoperiod, the rate of shoot induction was much lower. The shoot induction rate was nearly 20% (±2.5) higher when calli induced under complete darkness were used for shoot induction under a 16/8 h light/dark period. This was the response irrespective of the explant type from which calli were derived.

Shoot proliferation For further growth and proliferation, 1-2 cm long shoots were detached from the callus mass and recultured on MS-S basal medium containing 280 mg 1-l Na2SO 4, 1 mg 1-1 BA, 40 mg 1-I adenine, 40 mg 1-~ L-arginine and 0.02 mg ! -~

II

• II qil'i

t ~ ~ i:¸7!!~ ¸¸

Fig. I. Fig. 2. Fig. 3.

Multiple shoots on spear-derived callus• Complete regenerated plant with well formed roots. Metaphase plate (2n = 40) of root tip cell of regenerated plant.

3

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Table II. Analysis of variance ( A N O V A ) for the number of shoot(s) formed from l-g calli pieces of different explant sources of A. cooperi g r o w n in several combinations of adenine, BA, L-arginine and NAA in different photoperiods Source

Replication Explant (A) Error (a)

Photoperiod (B) A x B E r r o r (b)

Medium(C) B x C A x C A x B x C E r r o r (c)

Degrees of

Sum of

freedom

square

Mean square

2 5 10 1 5 12 10 10 50 50 240

22.37 2928.06 10.29 1015.04 293.59 3.28 2707.08 155.21 2654.19 330.37 33.52

11.18 585.6 1.02 1015.04 58.71 0.27 270.7 15.52 53.08 6.6 0.13

F

10.96" 574.11"* 3759.4*** 217.44"* 2082.3*** 119.38"** 408.3*** 50.76***

*Significant at P = 0.05% **Significant at P = 0.01% ***Significant at P = 0.001%

N A A . Shoots proliferated from the basal part of the detached shoots and axillary branching resulted into the formation of small clumps of rootless shoots.

Induction of root in excised shoots F o r initiation and growth of the root, the individual shoots were cultured on MS-R medium supplemented with different auxins. Of the different auxins tested 2 mg 1- l IBA produced 86.4% roots within 9 - 1 0 days. But 2 mg 1-1 KIBA produced 94.4% roots within 6 - 7 days of treatment (Fig. 2). Dark condition was found to be effective for rooting (Table III).

Cytological study The callus cells of different passages revealed 2n = 40 chromosomes and no irregularities in number and structure could be noted. R o o t tips of ten regenerated plants derived from each line of calli were analysed. All plants revealed 2n = 40 chromosomes (Fig. 3). Plants regenerated by organogenesis from all types of callus line were thus euploid and were free of any noticeable phenotypic variability. Discussion The in vitro organogenesis depends to a great

Table III. Effect of IBA and K1BA on the r o o t i n g of A. cooperi excised shoots in a 16/8 h light/dark cycle and in complete d a r k culture (50 shoots per treatment, average of 3 replications). Growth

L i g h t / d a r k (16/8 h cycle)

D a r k (24 h)

regulator (rag l -I)

IBA (0.5) IBA (1.0) IBA (2.0) IBA (3.0) K I B A (0.5) K I B A (1.0) K I B A (2.0) K I B A (3.0)

Percentage

Number of

Days taken

Percentage

Number of

Days taken

of rooting (4- S.D.)

roots (4- S.D.)

for r o o t i n g

of r o o t i n g ( 4- S.D.)

roots ( 4. S.D.)

for rooting

58.0 67.2 81.4 72.4 62.4 73.6 87.2 79.4

2.8 2.8 3.6 3.0 3.2 3.4 4.2 3.8

14-16 13-15 12-13 12-13 12-13 10-11 8-10 10-11

62.0 71.4 86.4 77.2 67.6 78.2 94.4 83.6

3.0 3.4 4.2 4.0 3.8 5.2 6.4 5.8

12-13 10-12 9-10 9-11 9-11 8-9 6-7 8-10

444+ 44. + 4-

2.73 3.50 3.36 3.40 2.07 2.00 1.92 3.84

4. 44. 4. 4444-

0.80 0.83 0.89 1.00 1.30 0.50 0.80 0.80

+ 44. 4444. 4-

4.60 2.50 2.00 3.00 2.20 2.68 2.60 3.80

4. + 4. 44444-

1.00 0.54 0.84 1.20 0.83 1.30 1.14 1.20

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extent on the choice of explant, composition of medium and control of the physical environment [6]. In the present study, cultural conditions have been standardized which could consistently induce plant regeneration from all explants excepting the roots of A. cooperi. For callusing, different ratio of NAA and Kn was required in different explants. This may be due to the variable levels of endogenous hormones. Complete dark culture was noted to be essential for healthy callusing. It is evident from Table I that explant (A), photoperiod (B) and growth regulator (C) have characteristic interaction patterns. The interaction of explant and photoperiod (A x B) was insignificant irrespective of the type of explant. All types of explant show similar response to the same photoperiod. As far as callusing is concerned the interaction between explant and the medium (A x C) and the medium with the photoperiod (B x C) were highly significant. All these factors namely explant, photoperiod and medium taken together (A x B x C) show significant interaction as far as induction of callus is concerned. It is likely that, for the production of callus, NAA and Kn act as vigorous triggers, in the absence of light. In A. officinalis, NAA was found to be more effective in dark conditions for callusing rather than in light conditions [7]. The organogenic response of callus also varied depending on the supplements in the basal medium. In several species, BA has been widely used for organogenesis [8]. In this study BA in combination with adenine, also triggers the shooting process. With the addition of L-arginine, there was rapid production of large numbers of shoots. A similar response with L-arginine has been reported in Agavefourcroydes [9]. The interaction between explant and photoperiod (A x B), photoperiod and medium (B x C) as well as explant and medium (A z C) were highly significant as far as organogenesis is concerned. Lastly, interaction between explant, photoperiod and medium (A x B x C) too showed high significance for shoot formation (Table 2). Thus efficiency of shoot production depends on the culture medium, the type ofexplant from which calli originated and also the photoperiod. This type of response has also been reported in Abelmoschus esculentus [10],

Citrus mitis [11], Dioscorea alata and D. bulbiJera [121. For the induction of root KIBA was found to be very effective. The differential effect of IBA and KIBA may be due to the conjugate form of K ÷ to IBA, leading to a faster triggering action. The genetic stability following regeneration from any tissue is a prerequisite for all plant breeding programmes. Plants regenerated here via organogenesis were stable in their cytology and showed no apparent variation in morphology. The protocols developed during the present study can be effectively employed for rapid regeneration of stable individuals of A. cooperi.

Acknowledgement The authors are grateful to Prof. A.K. Sharma for useful advice during this work. BG is thankful to the University Grants Commission, New Delhi for financial assistance.

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