Plant Cell Reports

Plant Cell Reports (1988) 7:385-388

© Springer-Verlag 1988

In vitro plant regeneration of leguminous trees (Albizia spp) Uttar K. Tomar and Shrish C. Gupta Department of Botany, University of Delhi, Delhi 110007, India Received April 29, 1988/Revised version received May 2, 1988 Communicated by J. M. Widholm

ABSTRACT

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B 5 basal medium. M a x i m u m number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 ~aM) in A. amara (2) and BA (I0 jaM) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B + 1 ~ M IAA developed roots (up to 30-40%). T~ese plants have been successfully transferred to the field. 2,4-D, 2,4-dichlorophenoxyacetic acid ; BA, 6-benzylaminopurine ; BM, Gamborg 's B~ medium with 0.9% agar + 3% sucrose; IAA, Indole3-~acetic acid ; IBA, Indole-3-butyric acid ; Kn, Kinetin, and NAA, ~-naphthaleneacetic acid. Abbreviations:

Key Words: Albizia amara, A. lucida, A. richardiana, in vitro plant regeneration. INTRODUCTION

Leguminous forest trees are of special interest because of their economic and ecological importance. Therefore, the application of tissue culture techniques for their clonal propagation and improvement is urgently warranted. However, they are recalcitrant to regeneration under in vitro conditions and, therefore, we initiated these investigations in tree taxa. A few successful reports on their regeneration have appeared during the last decade. Among Albizia spp., encouraging results have been achieved by Crizaldo (1980), Gharyal and Maheshwari (1981, 1983), Roy and Datta (1985), as well as Tomar and Gupta (1986, 1987, 1988). The in vitro expression of organogenesis is a reflection of the intrinsic genetic constitution of a taxon. With this background, a comparative account of the morphogenic responses of hypocotyl explants of three m°derate-sized'timber-yielding' leguminous forest tree species of Albizia namely, A. amara Boivin, A. lucida Benth. and A. richardiana King is presented here, with a view to ascertain the

Offprint requests to. S. C. Gupta

genotypic variations, if any, within the individual species.

between

as

well

as

MATERIALS AND METHODS

Seeds of the three species were procured from Pratap Nursery and Seed Stores, Dehra Dun. They were surface sterilized with freshly prepared chlorine water (3.5 +0.5 gm/l) for 30 minutes and then washed thoroughly with sterilized distilled water. The concentration of chlorine was estimated by iodometric method (cf. Vogel, 1973). Seeds were germinated aseptically on Knop ts medium (Knop, 1865), modified by adding 25 mg/l MnSO 4. 4H20, I0 mg/l H~BO~, 0.5 mg/l ZnSO..7H20 , 5.56 mg/l FeSO4.?H 0, 3 7.J44 mg/l Na~EDTAa, I00 mg/l • . 2 z myo-lnosltol and 1 mg/l thiamlne-HCl, l~o agar (Centron, Bombay) and 1% sucrose (BDH, London) were added to the medium and pH was adjusted to 5.8 before autoclaving. The seedlings were reared in continuous diffuse light of 20 ; W / c m 2 at 25 i2°C and 55 +10% RH. One centimeter long segments of hypocotyl (without axillary buds) were excised from 10-12 days old seedlings and cultured on BM (Gamborg et al., 1968). The pH was adjusted to 5.8 before autoclaving. Cultures were maintained at 25 +2°C under continuous light of 450 ~W/cm 2 produced by Crompton 40W day light fluorescent tubes. A minimum of 40 cultures were raised for each treatment. The morphogenic responses have been represented in terms of (i) percentage of explants producing shoots or roots, (it) number of shoots or roots per explant, and (iii) relative amount of callus,i.e, nil (-), little (+), moderate (++), good (+++) and profuse (++++). The data were recorded up to 40 days after inoculation. The significance of differences in responses of the three species for various treatments was checked by employing the Chi-square test (P = 0.05). RESULTS AND DISCUSSION

An influence of genotype on differentiation under in vitro conditions has been reported earlier at species and cultivar levels (Tremblay and lalonde, 1984; Rubluo and Kartha, 1985; Brown and Atanassov, 1985 ). Presently t o o , variation in morphogenic potential of three species of Albizia has been experimentally established. However, they reveal almost similar trends for root and shoot differentiation under the influence of varying levels of

386 Table I: Morphogenic responses* of hypocotyl explants of A. amara (A), A. lucida (B) and A. richardiana (G) at different levels of IAA added to BM.

Table 3: Morphogenic responses of hypocotyl explants of A. amara (A), A. lucida (B) and A. richardiana (C) at different levels of 2,4-D added to BM.

BM + IAA (pM)

BM + 2,4-D

Number Explants Shoots Explants Roots of producing per preduc- per explants shoots explant ing roots explant

(%)

0

0.I

45(A)- ** 22 2 bcd 41(B)35"3 abc 40(C)2510 abed

I0

100

0

0.I

48(A)++ 48(B)++ 48(C)-

1

48(A)+++ 43 8 bcd

0.0 f ,

Of c 0.4b

54(B)+ 40(C)-

44[0de 15

1.3 a. 0.3 e1

20.4c~ 15.0 cae

0:3bc d 0

208c

02{

40(B)++ 44(C)+

20.0c~ 4.6 el

0.3 ~ 0.I g

30.0°~ 54.6 a°

0 Ia

57(A)++ 49(B)++ 50(C)+

15.4c d 2010cd e 16

0"4 ef 0.4e f .

26.5 ca

0. 4~ Ia I.

0:1°6

60.0 a

0.0~ ~ ll.6d~ 0 5°ca 15 0 eae 0:3e f 14:3cd e

42(C)+

I.

2 cd 0.2c d 013bc d

0

Table 2: Morphogenic responses of hypocotyl explants of A. amara (A), A. lucida (B) and A. riehardiana (C) at different levels of IBA ad-ded to BM. BM + IBA (pM)

40(B)45(C)-

0 Of "2d 0~0 f 0

0.8 b

0.0 f, . 25 0 a°ca 40(B)+++ 2 5 :0abcd 56(C)++

60.0a~

0 5 cde 0 0 f ^'-bc .~'^cde u.~ ~ .ii.~. 0.5 caeI 0.0 z

7 ab 46.4a

52(A)+++

40(A)-

I0

40.0bc d 5 5 .a°c 0

0.2 b

83.3 a, 65.0a~ 25.0 ca

2.0~ c 1.4 e.2 ° 0

0.0 b 20.0 a 12.5 a

0.0 c 0.3~ 0.i b

0 7 de

0.0 b

0

0

0.0 b

0 0 0.of f 0:0 f

0

14 0.0~ 0.0~

0.0 ~

0.07 0.0 c

0.0 c, 0:I b 0.0 c 0.0 c

0.0 c

Table 4: Morphogenic responses of hypocotyl explants of A. amara (A) and A. lucida (B) at different levels of NAA added to BM. BM + NAA

(pM)

Number Explants Shoots Explants Roots of producing per produc- per explants shoots explant ing roots explant

(%)

(%)

40(A)48(B)-

60 0abc 2510 d

1.1 ab 0.4 e

0.0 e 0.0 c

0.0 d 0.0 d

48(A)48(B)-

50.0~ c 54.2 aDc

0

0.0 c

0.0 d

48(A)++ 48(B)++

75.0 ab 87.4 a

1.5 a 1.5 a

6.3.c 29.2 b

0.I d 0.6 b

10

54(A)++ 50 B)++

33.3c~ 40.0 ca

5de 33.3 ab 0 [8bc d 0 36.0 aD

0.4 c 1.2 a

100

51 A)++ 50 B)++

5.9 e 4.0 e

0:11

52.9 a

0.8 b

0

56.0 a

1.3 a

(%) 0.1

50(A)46(B)40(C)-

32.0 b c d 47.8~ b

0.5 c

0.0 e

0.0 ~

0.0 t

0.0 e

0.I

42(A)+ 48(B)+ 48(C)-

21.4 ede 75.0 a 12.5 e

0.4 cd 1.4% 0.2 a

0.0% I~.O~ ° 6.3 ae

0.0 d o.2 e 0.I e

1

48(A)++ 40(B)++ 45(C)+

18.8 de 75.0~

0.3 cd 1.7~ 0.01

31.3b,c 40.0a~ 13.3 ca

0.4~ 0.9 D 0.2 c

0 3 cd 1.3a b 0:0 f

13.3 cd 65.0 a, 45.5 a °

0.3 c 1 "9a 2.0 a

0.01

(%) 1.2 b, 0.0 b 10:6cde 6 a ° I0.0~

.0e e 48(A)++++ 0.0 48(B)++++ 0.0 e

48(C)++

Number Explants Shoots Explants Roots of producing per produc- per explants shoots explant ing roots explant

(%) 0

(%)

(%)

45(A)+

1

(pM)

Number Explants Shoots Explants Roots of producing per produc- per explants shoots explant ing roots explant

00c

0 0d

ooo 0

Shoot buds were induced directly (without callusing) on hypocotyl explants of all the three species on B M within 10-15 days of culturing.

However, in six experiments, covering the three species, the percentage of explants producing shoots was some what variable on B M (control). The average percentages of explants producing shoots, after 40 days, were 40.9 +4.8, 40.2 +7.6 and 20.2 +11.6 and the average numbers of shoot buds per responding explant were 2.1, 2.0 and 1.3 for A. lucida, A. amara and A. richardiana, respectively. Roots did not develop on B M in A. amara and A. richardiana (Fig. IA) but organised only in two experiments with A. lucida (up to 12%).

* For Tables I to 6, data were scored after 40 days. Values in a column followed by the same letter(s) are not significantly different at 5% level. ** For the amount of callus produced, please see Materials and Methods.

Auxins: Among experiments involving b6th natural (IAA, Table I) and synthetic auxins (IBA, NAA and 2,4-D, Tables 2-4), 0.I p M IAA significantly enhanced the number of shoots per explant and the percentage of explants producing shoots in A. amara, but only the number of shoots per

10

26 7 bcde 45(A)+++ 40(B)+++ 45.0abc 44(C)++ 0~0 f

phytohormones (Tables 1-6).

387

Fig. 1 (A-F). Morphogenic responses of Albizia richardiana hypocotyl explants after 40 days of culture (A-D). A: Shoots developed on BM, B: Shoots and roots arising from opposite ends of an explant on B M + 0.I ~M IAA, C: only roots developed on B M + 1 ~ M IBA, D: Shoots and callus originated on B M + 1 ~ M BA E: Complete plantlet developed after one of the in vitro differentiated shoots was excised and cultured on B M + 1 ;IM IAA, F: In vitro regenerated plant of A. richardiana growing in soil for over a year. Bars in A-E represent 4 m m and in F, 5 cm.

Table 5: Morphogenic responses of hypocotyl explants of A. amara (A), A. lucida (B) and A. richardiana (C) at different levels of BA added to BM. BM + BA (~M)

Number of explants

Explants producing shoots (%)

Shoots per explant

Explants producing roots (% )

0

52(A)42(B)40(C)-

38 5 bcd 2318di 35.0 ca

0.6 e 0.4 e 0.5 e

0.0 0.0 0.0

54(A)+ 44(B)+ 48(C)-

66.7~bI 36 4 Dc~ 37~5 bcd

I bce 016d

49(A)++ 44(B)++ 42(C)+

61.2~c~ DC~ 43.1, ~ 47.6 aca

l0

60(A)+++ 44(B)+++ 46(C)++

I00

40(A)+++ 40(B)+++ 48(C)+++



0 1

1

1

Table 6: Morphogenic responses of hypocotyl explants of A. amara (A) and A. lucida (B) at different levels of Kn added to BM. BM + Kn (~M)

Number of explants

Explants producing shoots(%)

Shoots per explant

Explants producing roots(%)

0

40(A)44(B)-

60.0a~ 50.0 amc

1.3 a, 1.0 aD

0.0 0.0

40(A)-

4019ab c

0

.6bc 0.6 c

0.0

0.1

44(B)-

50 0 abc

0.0

0.0 0.0 0.0

1

44(A)+ 44(B)+

63.6~ 31.8 °c

1.3 a 0.5 c

0.0 0.0

1 0 cd l'2bc li0C d

0.0

I0

48(A)++ 52(B)++

41 7 abc 5010 abc

0.6c 1.4 ~

0.0 0.0

I00 86.7~ 56.8~ c 52.2 Dc

1.3 bc 2.0 a, 1.6 aD

0.0 0.0 0.0

44(A)+++ 48(B)+++

27.3 c, 40.2 aoc

0.4 c 1 .2 a

0.0 0.0

35.0c~ 35.0 ca 0.0 e

0.6~ 1 3 mc 010 f

0.0 0.0 0.0

0.5 e

0.0 0.0

explant improved in A. lucida over the control. Roots were induced with 0.I ;IM IAA in A. riehardiana (Fig. IB) and with 1 pM in A. amara. The optimal concentrations of IAA for rhizogenesis were 1 ~ M for A. lucida and I0 pM for both A. amara as well as A. richardiana. Significant differences were observed among all the three species at 0.I ;~M IAA for number of shoots per explant and 1 ;~M for number of roots per explant. However, shoots were completely suppressed by I00 ~M IAA

in A. amara

(Table

I).

IBA did not significantly enhance the percentage of explants producing shoots of A. amara at any level tried (Table 2). However, it increased the number of shoots per explant in A. richardiana at 0.1~M and A. lucida at both 0.I and 1 pM. Shoot bud differentiation was completely suppressed by 1 and I0 ;~M IBA in A. richardiana (Fig. IC). The optimum concentration for rooting was 1 ~M for A. amara and I0 ~ M for both A. lucida and A. richardiana. Considering the overall growth of roots (length and thickness), IBA was most effective. All species differ significantly in number of shoots per explant both at 1 and I0 ;IM. H o w ever, significant variation in the percentage of

388 explants producing ; M (Table 2).

shoots

was

noted

only

at

1

Only in A. amara, the number of shoots per explant enhanced significantly at 0.I ~M 2,4-D without rhizogenesis (Table 3). However, 0.I and 1 ~M 2,4-D favoured root development in A. lucida and A. richardiana. All the three species differ significantly in the number of shoots as well as roots per explant at 0.I ,uM 2,4-D. A higher concentration of 2,4-D (I0 ;M) inhibited organogenesis and yielded massive white friable calli in all the three species. The effect of NAA was studied only on A. amara and A. lucida hypocotyl explants. Significant differences were observed in percentages of explants producing roots at 1 ~ M and in number of roots per explant at I-I00 ~ M NAA (Table 4). NAA increased the percentage of explants producing shoots at 0.I and 1 ~ M as well as the number of shoots per explant at 0.I-I ~ M only in A. lucida. Auxins in general, increase the rooting and production of white friable calli in the three species. However, with increasing levels, their shoot forming potential is decreased. Cytokinins: Roots did not develop at any concentration of BA and Kn tried but green compact calli were formed in higher concentrations of cytokinins (Tables 5,6). Addition of BA 0.I-I0 ~ M enhanced the percentage of explants producing shoots as well as number of shoots per explant in all the three species. Significant increase in the percentage of explants producing shoots was observed in A. amara and A. lucida only at I0 ~M BA. The number of shoots per explant was enhanced significantly at I-I00 ~ M in A. lucida, at 0.I-I0 ~ M in A. amara and at 1 and I0 ; M in A. richardiana (Fig. ID). Most of the shoots developing on BA containing media were stunted in all the species. Further, they were suppressed completely at I00 ~M in A. richardiana (Table 5). Kn did not enhance the percentage of explants producing shoots or the number of shoots per hypocotyl explant of A. amara and A. lucida (Table 6).

Rooting of Excised Shoots: The excised shoots of the presently investigated species did not easily develop roots unlike the earlier report on Albizia lebbeck (Gharyal and Maheshwari, 1983). However, a two-step treatment proved helpful in the three species. The in vitro induced shoots, along with the mother explants, were first cultured on B M + 1 ~ M IAA for 30 days. Then shoots were excised and placed on the same but a fresh medium,

where 30-40% of them rooted (Fig. IE). The in vitro grown plants of A. amara and A. richardiana have been transferred to vermiculite and finally to soil where they are progressing well (Fig. IE). Present studies have revealed that the most efficient media for obtaining shoots are B M + 0.I ~ M 2,4-D for A. amara and B M + I0 ~ M BA for both A. lucida as well as A. richardiana. Rooting, though not profuse, has now been possible at relatively high frequency by a two-step treatment on B M + 1 )~M IAA medium. Further work to formulate a medium for profuse and high frequency of rooting is in progress. The most significant contribution of the present report is the successful regeneration of three additional forest tree legumes through tissue culture techniques. ACKNOWLEDGEMENTS

This work was supported by the PL-480 research scheme entitled "Improvement of Angiospermous Forest Trees Through Tissue Culture" sanctioned to SCG. UKT is grateful to the Council of Scientific & Industrial Research, N e w Delhi, for the award of a Senior Research Fellowship. REFERENCES

Brown DCW, Atanassov A (1985) Plant Cell Tissue Organ Culture 4: 111-122 Grizaldo EN (19807 For. Res. J. 5: 123-138 Gamborg OL, Miller RA, Ojima K--(1968) Exptl Cell Res. 50: 151-158 Gharyal PK, Maheshwari SC (1981) Naturwissenschaften 6 8 : 3 7 9 Gharyal PK, Maheshwari SC (1983) Plant Cell Tissue Organ Culture 2:49-53 Knop W (1865) Landwirtsch. Vers.-Sta. 7:93-107 Roy SK, Datta SK (1985) Bangladesh --J. Bot. I__44: 127-131 Rubluo A, Kartha KK (1985) J. PI. Physiol. II___~9: 425-433 Tomar UK, Gupta SC (1986) VI Int. Gongr. PI. Tissue & Cell Culture, Univ. Minnesota, Minneapolis, U.S.A., pp 41 Tomar UK, Gupta SC (1987) II Ann. Conf. Int. PI. Biotech. Network, Bangkok, Thailand, Abs. No. 55 Tomar UK, Gupta SC (1988) Plant Cell Reports 7: 70-73 Tremblay FM, Lalonde M (1984) Plant Cell Tissue Organ Culture 3: 189-199 Vogel AI (1973) A textbook of quantitative inorganic analysis, English Language Book Society and Longman Group Limited, London, pp 364-365

In vitro plant regeneration of leguminous trees (Albizia spp).

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B5 basal me...
649KB Sizes 0 Downloads 0 Views