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Indian Journal of BiotechnologyVol 2, April 2004, pp 203-208

Production of cloned trees ofPopulus deltoides through in vitro regenerationofshoots from leaf, stem and root explants and their field cultivation

H C Chaturvedi*, A K Sharma, B Q Agha, M Jain and M Sharma

Tissue Culture Laboratory, National Botanical Research Institute, Lucknow 226 001, India

Amongst a large number ofPopulus species, micropropagation ofP. deltoides has not been possible. The authors now

report a reproducible protocol for mass production of cloned plants of 2 important clones ofP. deltoides, viz., G3 and G48through induced shoot bud differentiation in the in vitro cultured segments of leaf, stem and root of adult tree origin.Establishment of experimental plants in aseptic culture was possible only when nodal stem segments from fresh flush ofgrowth were initially cultured in a liquid nutrient medium, a modification of Murashige and Skoog (1962) medium (MS)

supplemented with 0.25 mg l1

each of BAP and IAA and 15 mg l1

AdS. Axillary shoot growth from nodal stem segmentswas sustained in an agarified medium of the same composition following at least 2 subcultures in the liquid medium. Rate ofmultiplication got enhanced many-fold when the segments of leaf, stem and root of regenerated shoots were induced to

differentiate multiple shoot buds in the same medium instead of obtaining a single plantlet per regenerated shoot. Themaximum number of shoot buds, i.e., 28.8 was differentiated in leaf segments followed by 22.7 and 7.8 in stem and rootsegments, respectively. However, the regenerated shoots appeared similar both morphologically and in vigour irrespectiveof their origin from any of the explants. Explants with differentiated shoot buds on being subcultured in the same

morphogenic medium repeatedly produced crops of proliferated shoots. Developed isolated shoots, measuring about 3 cm in

length, rooted 100% in 20-25 days, in a liquid rooting medium having 0.25 mg l 1 IAA. The corresponding cultures onagarified medium of the same composition not only rooted poorly, but also necrosed and died. The rooted shoots required

precision during their hardening in a purely inorganic salt solution following the procedure developed earlier. It took

about.12 months to obtain hardened plants of about 30 cm in height in potted soil from the time of establishment of asepticcultures using nodal stem segments of an adult tree. The properly hardened plants survived more than 80% on theirtransplantation to soil and grew luxuriantly under field conditions. The in vitro-raised trees attained an average height of 5 mwith straight boles of an average diameter of 12 cm in 2 yrs.

Keywords: Populus deltoides, in vitro cloning, leaf segments, nodal stem segments, root segments, shoot buddifferentiation, stem segments

IPC Code: Int. Cl.7 A 01 H 4/00, 5/00

Introduction

Populus deltoides (Eastern cottonwood) is

commercially in great demand, particularly for its

ability to grow very fast even on semi-alkaline soil

and has rightly been designated as an energy plant1.

However, by the conventional method of propagation

through stem cuttings, the huge demand of its plus

trees cannot be met being limited by seasonal factors.

On the other hand, a number of other species ofPopulus have been micropropagated at very fast

rates2-4 following P. tremuloides5, the first report on

micropropagation of any tree. For meeting the ever-

increasing global demand of biomass production and

wood industry, development of in vitro methods for

rapid cloning and large-scale production of planting

material of P. deltoides throughout the year is

warranted. The earliest report, albeit fragmentary, on

shoot regeneration from cambial callus is

inconclusive, as fate of the differentiated shoots

remains unknown6

. Some reports have been publishedon in vitro regeneration of shoots from stem explants

ofP. deltoides, but formation of complete plants, their

transplantation to soil and ex vitro growth of such

plants are not reported7-10. The authors now report, for

the first time, an efficient process for rapid production

of cloned plants of P. deltoides through direct shootbud differentiation in the in vitro cultured segments of

leaf, stem and root of adult tree origin and their

luxuriant ex vitro growth under field conditions.

Whilst the clones G3, G48, D121, S7C15 and S7C20,

__________*Author for correspondence:Tel: 91-522-2205840; Fax: 91-522-2205836, 2205839

E-mail: [email protected] , [email protected]

Abbreviations:

BAP: 6-benzylaminopurine; IAA: indole-3-acetic acid; AdS:adenine sulphate; Kn: kinetin; 2iP: N6-(2-isopentenyl)

aminopurine; Z: zeatin; Na-Fe-EDTA: disodium-ferric-ethylene-diaminetetra-acetate


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INDIAN J BIOTECHNOL, APRIL 2004204

were established in aseptic culture and experimented

upon, this paper is restricted to the results of

investigations pursued on clones G3 and G48.

Materials and Methods

Establishment of Aseptic Cultures

Shoot apices and stem segments consisting of 1-2

nodes, excised during the months of March and April

from fresh flush of growth of adult trees of Populus

deltoides Marsh clones G3 and G48, grown at the

Banthra Research Station of the National Botanical

Research Institute, Lucknow, comprised the sources

of explants for establishing plants in aseptic culture.

Explants were first washed thoroughly under running

filtered (bacteria-free) tap water for 30 min, pretreated

with 5% (v/v) Laboline solution (a neutral liquiddetergent by GlaxoSmithKline Pharmaceuticals Ltd.,

Mumbai, India), to which 3 drops of tween-20 per 100

ml were added, for 5 min and given a quick dip in

rectified spirit followed by their surface-sterilization

with 0.1% HgCl2 solution for 15 min. The surface-

sterilized explants were thoroughly washed with

sterile water and left in it till their inoculation.

Surface-sterilized explants were cultured in a liquid

modified MS (1962) medium11 supplemented with

0.25 mg l1 each of BAP and IAA and 15 mg l1 AdS

on filter paper bridges for bud-break. The modified

MS differed with the original MS in respect of(concentrations in mg l

1): (NH4)2SO4 250; MgSO4

450; thiamine-HCl 0.2; pyridoxine-HCl 0.1; ascorbic

acid 5.0 and sucrose 20000.

Shoot bud Induction and Plant Regeneration

For induction of shoot bud differentiation in

segments of leaf, stem and root, excised from in vitro

proliferating shoots and rooted shoots, effects ofdifferent concentrations, i.e., 0.1, 0.25, 0.5, 0.75 and

1.0 mg l1 of BAP as well as of other kinins, viz., Kn,

2iP and Z, used individually, with 0.25 mg l1 IAA

and 15 mg l1

AdS were seen. In case of leaf, its tip,median and basal portions were excised and cultured

to assess their relative regenerative potentiality.

The isolated shoots were cultured in the same

modified MS medium, but devoid of meso-inositol

and supplemented with 0.1, 0.25 and 0.5 mg l1 IAA

for rooting. The in vitro-rooted shoots-plantlets were

acclimatized in half-strength modified Knop

solution12 having trace elements of MS and 3 ml l1

Na-Fe-EDTA solution prepared after the manner of

MS.

The pH of all the media was adjusted to 5.8 and

their sterilization was done by autoclaving at 1.08

kg/cm2 for 15 min. Cultures were incubated under 25

mol2s1 photon flux density of light for 15 hrs a day

at 271C with 705% RH maintained in the cultureroom.

Hardening and Transplantation to Soil

About 5-week-old in vitro-regenerated plantlets

were washed in running tap water and grown ex vitro

in half-strength modified Knop solution13 for

hardening following the procedure developed

earlier13. Initially, they were exposed to a humidity

regime from high to gradually low, i.e., from about

90% RH to about 70% RH, during a period of 15

days. Hardened plants were finally grown in potted

soil comprising a mixture of garden soil and compost

(3:1).

Results and Discussion

Initial establishment of in vitro cultures of P.

deltoides was a difficult proposition. Between shoot

apices and nodal stem segments employed for

establishing aseptic cultures, the former were found

unsuitable being too tender to tolerate the surface-

sterilization treatments, while the latter showed bud-

break in about 80% of explants within 20 days and

subsequently regenerated offshoots (Fig. 1). Nodal

stem segments have also been found to be the

explants of choice in other Populus spp.3,14, while insome other Populus spp. the excised axillary buds

have been regenerated to shoots15,16. In view of the

persistent problem of infection with explants of P.

deltoides, faced also by other workers7-9, for which

several antibiotics have to be incorporated in the

medium, the 80% infection-free explants obtained in

the present study by surface disinfection alone, i.e.,

without using any antibiotics in the medium was

remarkable. More so, as the presence of antibiotics in

the medium adversely affect the growth of explants

and offshoots, resulting in cultures with restricted

growth.Explants of nodal stem segments cultured on the

modified MS agarified medium supplemented with

0.25 mg l1 each of BAP and IAA and 15 mg ll AdS

did not survive despite bud-break and regeneration of

shoots. On the contrary, similar explants when

cultured on filter paper bridges with liquid medium of

the same composition not only survived, but also the

regenerated shoots proliferated (Fig. 1). Such a

requirement of liquid medium appears to be specific

to P. deltoides, since other Populus spp. and its


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CHATURVEDI et al: RAPIDIN VITRO CLONING OF POPULUS DELTOIDES 205

Figs 1-6 Cultures ofPopulus deltoides: 1, Bud-break resulting into shoot formation, in nodal stem segments taken from field-growntrees of different clones on filter paper bridges with nutrient liquid medium. Bar = 2 cm; 2, Multiple shoot bud differentiation in segments(from Lt. to Rt.) of leaf, stem and root of clone G3.Bar = 1 cm; 3, Proliferating shoot regenerated from segments (from Lt. to Rt.) of leaf,

stem and root of clone G3.Bar = 2 cm; 4, A group of in vitro-rooted shootsplantletsacclimatized in an inorganic salt solution Bar = 4cm; 5,In vitro-raised 2-yr-old trees of clone G48 grown under field conditions. Bar = 50 cm & 6, A view of the same 2-yr-old trees afterdefoliation in winter showing uniformly straight boles. Bar = 50 cm.


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hybrids show vitrification of their shoots in liquid

medium3-16.

For proper establishment, at least 2 subcultures of

explants with regenerated shoots on filter paper

bridges in liquid medium were essential for theirsubsequent survival on the same but agarified

medium. Proliferation of shoots was by a factor of 3

in 30 days, which moderately increased during

subsequent subcultures.

Differentiation of shoot buds in segments of leaf,

stem and root, taken from the in vitro-rooted shoots

greatly augmented the rate of production of cloned

plants ofP. deltoides, since each plantlet provided 3-4

leaves and each in turn provided 3 segments and 2-3

internodes and a root, each of which could be divided

into 4-5 segments. Regeneration potential of these 3

kinds of explants in BAP containing treatments isgiven in Table 1. The maximum number of shoot

buds was differentiated in segments of leaf followed

by stem and root in the decreasing order. The

variation in regeneration potentiality in leaf, stem and

root is comprehensible as controlled by intrinsic

metabolic status of these organs. Amongst the

different concentrations of BAP, used along with 0.25

mg l1 IAA and 15 mg l1 AdS, the maximum number

of shoot buds, i.e., 28.8 was formed in leaf, 22.7 instem and 7.8 in root explants, without any intervening

callusing, in its 0.25 mg l1 concentration (Fig. 2). A

further increase in the concentration of BAP from0.25 mg l1 to 0.5, 0.75 and 1 mg l

1 resulted in a

gradual decrease in the number of shoot buds

differentiated and a progressive increase in the

associated callusing. In case of leaf, shoot buds were

formed from both, the cut ends as well as the intact

surface, whereas in internodal stem segments shoot

buds were mostly confined to the cut ends and in root

segments they regenerated from all over the explant.

Other kinins, viz., Kn, 2iP and Z used at the same

optimum concentration of 0.25 mg l

1

as of BAP wereless effective than BAP with leaf and stem explants,

while they were ineffective in case of root explants.

Superiority of BAP in inducing shoot bud

differentiation is established, particularly in case of

trees17, albeit there are some exceptions too. However,

Coleman & Ernst7 selected 2iP instead of BAP for

inducing shoot bud differentiation and shoot

proliferation in P. deltoides, which may likely be due

to different clones used by them. Furthermore, though

a good number of shoot buds were differentiated from

all the three portions of leaf lamina, i.e., tip, median

and basal, a gradual decline in number of shoot budswas noticed from tip to the basal portion (Fig. 2).

However, 0.25 mg l1 concentration of BAP remained

the most effective concentration for differentiation of

optimum number of shoot buds, i.e., 39.2 in the tip,

33.7 in the median and 23 in the basal portions of the

leaf lamina (Table 2). Differences in regeneration

potentiality of tip, median and basal portions of a leaf

have also been reported in some other plant species,viz., Echeveria elegans18 and Rosmarinus

officinalis19. The situation is analogous to the one

demonstrated in a flowering plant of tobacco, where

the stem explants closest to inflorescence do notproduce shoot buds but a few flower buds instead,while the number of shoot buds produced

progressively increases in the stem explants closer to

the vegetative portion of the stem farther from the

inflorescence, which has been explained due to the

Table 1Effects of different concentrations of BAP used along with 0.25 mg ll IAA and 15 mg ll AdS on shoot bud differentiation in

segments of leaf, stem (internode) and root ofPopulus deltoides clone G3

(Incubation period: 40 days)

Response*

Leaf Stem Root

BAPconc.

mg l1 No. of shoot

buds

Callusing No. of shoot

buds

Callusing No. of shoot

buds

Callusing

0.10 24.0 0.471 - 5.0 0.471 - 6.7 0.396 -

0.25 28.8 0.712 - 22.7 0.367 - 7.8 0.249 -

0.50 23.7 0.597 + 12.5 0.401 + 7.1 0.314 +()

0.75 12.9 0.504 + 12.0 0.516 ++ 6.3 0.300+

1.00 8.1 0.536 ++ 11.4 0.581 ++ 6.8 0.327 ++*Average of 10 replicates SE

Number of + mark denotes magnitude of callusing, whilemark denotes no callusing


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CHATURVEDI et al: RAPIDIN VITRO CLONING OF POPULUS DELTOIDES 207

existence of a gradient of some morphogenic

substance florigenic factor in the plant body, in an

increasing order from base to apex, favouring flower

bud differentiation20. It is also true for the vegetative

plant axis ofSolanum khasianum, where the gradientof some morphogenic factor favouring shoot bud

differentiation has been contemplated to exist in the

increasing order from the shoot tip to the root

system21.

Shoot buds were visible within 8-10 days of

incubation and shoots of an average height of 2 cm

were formed in about 35 days in all the 3 kinds of

explants, without any difference in their rate of

proliferation (Fig. 3). Barring a single report of

embryogenesis in somatic callus ofP. ciliata22, in all

other instances of in vitro regeneration in different

species of Populus, caulogenesis has been reported.However, in early stages of shoot bud differentiation

in the present study also, the structures resembled

with embryoids, particularly in root explants, which

on further development unfolded into typical shoots

differentiated from monopolar organized shoot

meristems. In the absence of histological evidence, it

is quite possible that the organized structures formed

in cell suspension culture of leaf callus of P. ciliata,

reported to be embryoids might have actually been the

developmental stages of aberrant shoot buds.

The isolated shoots, excised from cultures of

proliferating shoots irrespective of being raised from

explants of leaf, stem or root, easily rooted 100%,

without intervening callusing, when cultured on filter

paper bridges, within about 15 days, in the rooting

liquid medium containing 0.25 mg l1 IAA. On the

other hand, the corresponding cultures of isolated

shoots on agarified nutrient medium necrosed

severely during the process of rooting, while the

rooting percentage also greatly declined to about

50%.

The rooted shoots raised from leaf, stem and rootexplants appeared similar in morphology and vigour.

While still growing on filter paper bridges in the

liquid medium, well-developed plantlets were formed

in a total period of 30 days from the time of induction

of rooting. The plantlets when hardened, following

the procedure developed earlier for Dioscorea

floribunda13 gave about 80% transplant success with

normal ex vitro growth under glasshouse conditions

followed by their transfer to field, where they grew

luxuriantly (Figs 4 & 5). It took about. 12 months to

obtain hardened plants having a height of about 30 cm

in potted soil ready for transplantation to fieldconditions from the time of bud-break in aseptically

established nodal stem segments taken from adult

trees. The in vitro cloned plants attained an average

height of 5 m with lush green foliage in 2 yrs. The

perfectly straight boles of an average diameter of 12

cm of such trees were evident in winter after the total

leaf fall (Fig. 6). There was no appreciable difference

in the responses of explants of clones G3 and G48.

The main reason of success in getting the in vitro-

raised plants grown under field conditions lay in

inducing the desirable rooting of the regenerated

shoots, for want of which the other workers dealing

with P. deltoides consistently did not succeed. The

situation can be compared with the consistent failure

in getting the in vitro-raised plants of Simmondsia

chinensis to grow ex vitro23 till it was successfully

done by controlling the intervening callusing in the

process of tailoring the induced roots to a tap root-like

system24. Control of intervening callusing in order to

obtain desirable rooting in the in vitro-regenerated

shoots of Citrus aurantifolia, C. sinensis and C.

grandis was also crucial for obtaining the in vitro-

raised plants of all these Citrus species growingsuccessfully to maturity for the first time2527. Control

of intervening callusing in the rooting process ensures

proper continuity of conducting tissues of stem with

the induced root system, which has been found

decisive, particularly in case of trees for establishment

of rooted shoots in field and their subsequent better

anchorage to soil27,28. Such examples bear out the

main reason of our success in establishing field

cultivation ofP. deltoides on a mass scale, which has

been eluding success so far.

Table 2Effects of different concentrations of BAP used along

with 0.25 mg ll IAA and 15 mg ll AdS on shoot buddifferentiation in explants of three different portions of leaflamina ofPopulus deltoides clone G3

(Incubation period: 40 days)Number of shoot buds formed in three

portions of leaf lamina*

BAP

conc.mg l1 Tip Median Basal

0.10 15.20 0.696 30.20 0.879 15.00 0.632

0.25 39.20 0.987 33.70 0.967 23.00 0.775

0.50 26.70 0.943 30.00 0.632 11.20 0.727

0.75 22.00 0.816 6.50 0.543 10.50 0.859

1.00 11.20 0.663 6.00 0.577 7.20 0.416

*Average of 10 replicates SE


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Acknowledgement

The authors thank the Director, NBRI, Lucknow,

for the facilities provided, while the corresponding

author is also thankful to the Department of

Biotechnology, New Delhi, for granting him theproject on micropropagation ofPopulus deltoides.

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