Tissue culture of some woody species

T I S S U E C U L T U R E O F S O M E W O O D Y S P E C I E S *

BY R. NARASIMHAN, BHARATI DHRUVA, S.V. PARANJPE, D . D . KULKARNI,

A. F. MASCARENHAS (National Chemical Laboratory, Poona)

AND

S. B. DAVID (Department of Botany, University of Poona, Poona)

Received August 22, 1969

(Communicated by Professor T. S. Mahabale, F.A.SC.)

ABSTRACT

The investigations embodied in the paper describe different methods o f culturing tissues of Tectona grandis (teak), Artocarpus heterophyllus (jack), Morus alba (mulberry) and Populus nigra (poplar). Of these, teak, jack and mulberry tissues have been established in culture for the first time. Teak, mulberry and poplar tissues grow well on Murashige and Skoog's medium containing 1.0 ppm glycine, while jack tissue grows well on Blaydes' medium. The tissues of teak, grown on Marashige and Skoog's medium containing 3"2 rag. glycine and gibberellic acid, have good pigmentatioa. Jack tissue requires a specific higher temperature 130 ° C.) for its growth.

INTRODUCTION

THE heart-woods of many woody species have been investigated for their phenolic contents (Goodwin, 1965; Harborne, 1964). A scheme for the biogenesis of some of these compounds has also been suggested on the basis of structural relationships and other evidences (Grisebach, 1965; Radha- krishnan et al., 1965). The present investigations were undertaken mainly to locate the site of synthesis of some of these compounds or their main precursors within the plant. Since plant tissue and organ culture techniques have been used with advantage in such studies (Street, 1965), attempts were made to establish in culture the tissues of Tectona grandis (teak), Artocarpus heterophyllus (jack), Morus alba (mulberry) and Populus nigra (poplar). Of these, the culture of some species of poplar have been reported earlier (Morrel, 1948; Heller, 1953; Jacquiot, 1964; Gautheret, 1959).

*NCL Communication No. 1377. 204

Tissue Culture of Some Woody Species 205

MATERIALS AND METHODS

Basal media suggested by White (1963) (WB), Murashige and Skoog (1962) (MSB), Reinert and White (1956) (RWB), simplified Reinert and White (Wolter and Skoog, 1966) (RWBS), Blaydes (1966) (BL) and Knop (1865) (Kn) were used. Modifications were made with regard to growth supplements. In MSB medium glycine was included at 1.0 ppm (instead of 2.0 ppm in the original medium) as lower concentration was found to be better for the growth of many ether tissues in this laboratory. Blaydes medium was made up with 2 per cent sucrose, instead of 3 per cent in the original medium. Agar at 0.75 per cent was included to solidify the medium and the PH adjusted to 5.8 -4- 0.1 prior to autoclaving at 15 lbs. pressure for 20 minutes. 20 ml. of the medium were used per 6" × 1" tube.

Explants were made according to the suggestions of White (1963). Small segments were taken from the cambia region in all cases. Alternatively, longitudinally cut, wedge-shaped pieces of young twigs or of the epicotyl region (in the case of jack) were taken for inoculation. The pieces were directly placed on the medium.

RESULTS

T. grandis.--Initial explants were cultured on WB and RWB media. The different supplements included in the medium were coconut milk (CM), kinetin (Ki), yeast extract (YE), malt extract (ME), casein hydrolysate (CH), biotin (Bio), calcium pantothenate (Ca pan-), ascorbic acid (Vit. C), indole- acetic acid (IAA), naphthaleneaeetic acid (NAA), 2-benzothiozole oxy- acetic acid-(2-BTOA), 2, 4-dichlorophenoxy-acetic'acid (2,4-D) and gibberellic acid (GA). These supplements were used either singly or in various combinations. In all, nineteen different combinations were used for culturing explants. Of these, the following three combinations proved satisfactory.

(a) WB q- CM (15 percent) + NAA (1.0 ppm) q- YE (1000 ppm) (b) WB + CM (15 per cent) + Ca. pan- (1.0 ppm) + Bio (0.1 ppm) (c) RWB -t- Ki (1 '0 ppm) -t- 2, 4-D (0.06 ppm) q- CH (200 ppm)

In all the media callus formation commenced after about 10 days. The incubation period for all cultures was 40--50 days.

On subculturing the growth rate diminished in subsequent transfers. Other media were, therefore, tried which included MSB, Kn and RWBS with different combinations of growth substances. Of these only five ~ f t

206 R. NARASIMHAN AND OTHERS

(Table I) proved useful for the third subculture. The tissue grown on MSB were highly pigmented and since our aim was to use this technique ultimately for the eludication of the biosynthetic pathways of different phenolics, different combinations of growth supplements were tried with this medium. Addition of CM, CH, CH and GA, CH and Vit. C gave good growth, Tissues have been continuously grown in these media for ever 10 subcultures without any fall in the growth rate.

TABLE I

Growth of teak tissue in MSB and RSWB medium with supplements Incubation period: 30 days. Initial inoeulum: 50 nag.

Sub- BASAL Supplements Wet culture medium weight in g.

3rd

MSB MSB

MSB (-IAA)

RWBS

RWBS ~-Ki,-2, 4-D)

CH (200 ppm) 1-258 CH (200 ppm.) + GA (10 ppm)+ 1.071

Vit. C (0.02 ppm) 2, 4-D (0.06 ppm) + CH (200 ppm) 0.245

+ GA (10 ppm) + Vit. C (.01 ppm) CH (200 ppm) + GA (10 ppm) + 0.419

Vit. C (0.01 ppm) CM (15 per een 0 + IAA (5 ppm.) 0.609

+ CH (200 ppm) + GA (I0 ppm) + Vit. C (0.01 ppm)

It has als0 been possible to eliminate thein fluence of unknown factors in growth supplements like CH by a study of amino-acids as they occur in 200 ppm: CH (Street and Henshaw, 1966). The results are presented in Table II. It may be seen that glycine alone can be a good growth supplement and can fully substitute for the activity of CH. The other growth supplement included is GA.

The growth pattern of teak cultures grown on different media varies. On RWB medium the growth was compact and had a brownish appearance, while the growth in MSB medium though compact varies in colour from pale yellow to light oranse, particularly when GA or Vit. C is included.

Tissue Culture of Some Woody Species

TABLE II Study of the effect of amino-acids present in 200 ppm casein

in MSB medium in the growth of teak tissue In(mbation period: 40 days.

207

hydrolysate*

Weight expressed No. Media composition as percentage

over control

1 MSB + CH (Control) .. 100 2 MSB q- all amino-acids present in CH .. 120 3 MSB q- Arg HCI q- Lys HCI q- Met q- Gly ... 69 4 MSB q- Glu HC1 + Tyr q- Thr .. 72 5 MSB q- Asp q- His q- Val .. 70 6 MSB q- Leu q- Ph ala q- Iso leu .. 36 7 MSB q- Arg HC1 q- Gly HCI + D1 Asp q- D1 Leu .. 60 8 MSB -}- Lys HCI q- Tyr + His q- Ph ala .. 54 9 MSB -t- Met q- Thr -t- D1 Val q- DI Isoleu .. 110

10 MSB q- Gly .. 130

* See Wilmer, 1966.

The characterisation of the pigments in the tissues has been undertaken and while the details of characterisation are being completed, it may be men- tioned that acetone extracts of tissues grown in different media gave two yellow bands, when monitored on preparative silica plates (solvent system- benzene containing 5 per cent acetone). The major band was a single compound which contained a little impurity of the nearest band. The compound is needle-shaped yellow crystals. Presently, the tissues are grown on MSB containing 3.2 ppm g|ycine and GA 10 ppm. The growth of the tissues are quite satisfactory and the pigmentation is quite good.

A. heterophyllus.--Initial explants were cultured on 41 different media of which some growth was obtained in the following media:

(a) WB q- CM 10 per cent + YE (1000 ppm) q- ME (1000 ppm) -F inositol (10 p p m ) q - N A A (1.0 ppm):

(b) WB -}- CM (10 per cent) + YE (1000 ppm) + ME (1000 ppm) + Ino (t0 ppm) + 2-BTOA (1.0 ppm);

(c) MSB + CM (15 per cent) + YE (1000 ppm) + ME (1000 ppm) + 2-BTOA (1.0 ppm).

208 R. NARAS1MHAN AND OTHERS

Although growth took place in these combinations, it was not quite satisfactory. It was thought that incorporation of some known Artocarpus pigments would enhance the growth in the earlier stages and once habi- tuated the tissues could be grown on pigment-free media, as was done by Jacquiot (1950) with caffein. The compounds included were artocarpin and cycloartocarpin. Inclusion of 1 ppm in medium (c) (see above) gave reasonable growth in some tubes. Since the reponse of tissues was variable it was thought necessary to reinvestigate the conditions for growth of tissues de novo. In the course of these investigations it was found that jack tissues have a higher temperature requirement compared to other tissues so far investigated. Tissues were grown at temperature ranging from 20 ° to 35 ° C. The most ideal temperature for the growth of the tissues was 30 ° C. Also at this temperature the apparent beneficial effect of artocarpin dis- appears. All jack fruit cultures are now being maintained at 30 ° C. irrespective of the media in which they are grown.

Since the media contained some unknown growth factors, an attempt was made to devise a medium the composition of which was chemically defined. It has been possible to grow jack tissues on BL medium supple- mented with 2, 4-D or tyrosine, or phenylalanine or phenylalanine and tyrosine. The tissues now in their 14th passage are being maintained in all the five media (Table III) with a view to compare the effect of different supplements on the production of phenolics in the tissues. The callus pro-

M. alba. Incubation

TABLE I I I

Growth of jack tissue in BL medium with supplements The explants grew well on:

period; 45-50 days. Initial inoculum: about 80 rag.

Subculture NO.

Wet weight in grams

Bladyes' Bladyes' Bladyes' Bladyeo' Bladyes' medium + 2, 4-D + phenyl Tyrosine +Phe + Tyr

( lp pm) (10 ppm) (10 ppm) (10 ppm each)

8 1"10 1 "16 0"85 0.93 0.85 9 0"99 1 '01 1 "02 1.02 0.92

10 1 '03 0.98 1-10 0.90 0.99 11 1 "01 1'1t 1.02 0.89 0.97 12 0"98 0'99 1.30 1.10 0-98 13 0"97 0.91 0"938 1.00 1.20


duced in all cases is light to dark brown and the pigment sometimes diffuses into the medium. The pigments are now being examined.

(a) MSB + Ca-pan (1.0 ppm) + Bio 0-1 ppm + 2, 4-D (0 "06 ppm) ;

(b) WB + C M (15 per cent) + C a - p a n 1.0 p p m + B i o (0.1 ppm) + 2, 4-D (0.06 ppm) ;

(c) RWB + CH (15 per cent) + Ca-pan (1.0 ppm) + Bio (0"1 ppm) + 2, 4-D (0.06 ppm).

Further subculture was possible in all these media. Growth in a defined medium was possible when Ca-pan and Bio are included in MSB medium (Tables IV and V).

TABLE IV

Growth of mulberry tissues in MSB medium with supplements: Ca-pan (1.O ppm) and biotin (O. l ppm)

ncubation period: 40-50 days. Initial inoculum :'about 80 rag.

Subculture No. 11 12 13 14 15

Wet. wt. in g. 1.183 1.129 1.331 1.328 1.512

TABLE V

Growth of poplar tissue in MSB medium containing O" 1 ppm Ca-pan, biotin 0"01 ppm and NAA 1.0 ppm

Incubation period: 40-50 days Inttial inoeulum : about 80 mg.

Subculture Ne. 17 18 19 20

Wet. wt. in g. 4-627 6-399 6-140 6.681

Populus nigra.--Tissue cultures have been previously reported by Heller (1953), Morel (1948) and Jacquiot (1964). In this laboratory the tissues have been successfully grown on MSB with the addition of Ca-pan 0. l ppm q- Bio 0.01 ppm and NAA 1.0 ppm. Good growth of callus was obtained within 20 days. Viability of cultures is not affected even if subcultures are made after 40 days. Subculture of the tissue has been possible for twenty passages (Table V). On this medium the growth is rapid and the tissue is

B4

210 R. NARASIMHAN AND O1HERS

friable and white in colour. In later passages the tissues become pignlented, particularly if they are exposed to light.

DISCUSSION

Although it has been possible to establish in culture tissues of woods being investigated for their phenolics in this laboratory, the growth requirements raise certain problems. For instance, in the case of teak, the inclusion of glycine apparently replaces the stimulatory effect of CH. MSB medium which was developed for tobacco tissues contain glycine at 2 mg. per litre. But its inclusion was on an arbitrary basis. Later Linsmeir and Skoog (1965) found that glycine at 2 mg. per litre or at lower concentrations had no visible effect on the tissues while at higher concentrations, i.e., up to 20rag. it was inhibitory. On this basis Linsmeir and Skoog (1965) excluded glycine from their revised medium. For the culture of excised roots of groundsel (Skinner and Street, 1954; Charles and Street, 1959), and of lucerne (David, 1957) glycine was found to be inhibitory. Many tissues have been ~rown in this laboratory on media containing only 1 mg. per litre of glycine. On the other hand, White (1967) had always made it a point to include glycine in all his media and had shown glycine to be essential for the growth of excised roots and tissues. The absolute requirement of glycjne for leak tissue will, therefore, have to be examined further.

The anthraquinone isolated from leak tissues has not so far been reported to occur naturally in teak. Its chemistry will be discussed elsewhere. However, it raises some interesting problems regarding its origin in tissue cultures and its non-occurrence in normal tissues ; whether the occurrence is a character of the family or the product occurs as an artefact. If natural, whether it would be possible to trace its biosynthetic pathway by feeding precursors. These problems are now under investigation in our laboratory.

Higher temperature requirement for jack tissue (30 ° C.) suggests that the culture of some of the tropical species may have proved refractive because they need higher temperatures for their incubation.

ACKNOWLEDGEMENT

This work has been financed in part by a grant made by the United States Department of Agriculture under PL-480.

Grateful acknowledgement is made to Professor K. Venkataraman and Dr. V. Jagannathan for their continued interest. Thanks are also due to Mr. S. G. Barawarkar for his technical help.


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Tissue culture of some woody species