chapter - 2 review of literature -...

REVIEW OF LITERATURE

7 In-vitro propagation of C. jaunsarensis and exploring options for economizing the technology

CHAPTER - 2


2.1. GENERAL INTRODUCTION OF BAMBOOS Bamboos are an important group of plants that play a vital role in the economy

and life style of many Asian, African and American countries (Farrelly, 1984;

McClure, 1956 and 1966). Their strength, straightness, smoothness, lightness and

extraordinary hardness make bamboos most suited for many purposes. Bamboo is a

cultural feature of South-east Asia and no country in the region is without an

indigenous bamboo flora. Their versatility, rapid growth and many end-uses have

made bamboos the backbone of rural economy in many Asian countries. There seems

to be a certain degree of inseparableness between man and bamboo and the degree of

its acceptance by the people in many developing countries can scarcely be gauged.

The major representing genera of bamboo are: Arundinaria, Bambusa,

Chimonobambusa, Dendrocalamus, Gigantochloa, Indocalamus, Ochlandra,

Phyllostachys, Schizostachyum and Sinobambusa (Varmah and Bahadur, 1980;

Tewari, 1992). Unlike most timber, bamboo is a self-regenerating natural resource;

new shoots that appear annually from the rhizome ensure production after individual

culms are harvested. Based on the nature of their underground rhizomes, most

bamboos can be classified as either clump forming (Bambusa) or running bamboo

(Phyllostachys).

The bamboo plant consists of three morphological parts, the aerial part and

two underground parts (the rhizome and root). Bamboo varieties range in height from

the smallest at 15 cm to the giant variety at 40 m. The giant bamboo often reaches a

height of 20-30 m, while most bamboos are shrub like, medium or dwarf species with

a few exceptions as climbers (Wang and Shen, 1987). Some species of bamboo are

deciduous (shedding), while some are evergreen.

2.1.1. ECONOMIC IMPORTANCE Bamboos perhaps have the singular distinction of being the only natural

resource put to so many and so varied uses. Kurtz (1876) stated “no plant is known in

the tropical zone which would supply to man so many technical advantages as the



bamboo”. It is a renewable resource and its impact on environment and ecology is

immense. Below mentioned are a few examples of its usefulness:

Most suited for rehabilitation of degraded forest lands and for other soil

conservation purposes including control of landslides, flash floods and

siltation.

With carbon sequestration properties, it can lessen the pace of climate change.

Acts as a substitute of wood.

Bamboo has about 1500 documented applications (Tewari, 1992); its most

important applications are in the area of:

Building and reconstruction material.

Cottage and tiny industries like agarbatti sticks, chopsticks etc.

Food items, mainly edible shoots

Medicinal products

Handicrafts

New generation products like wood substitutes, bamboo boards, furniture,

activated carbon, charcoal

Energy production

Paper industries based on bamboo pulp

Bamboos play an important role in local economies and are growing in

national and international commercial importance in the Asia-Pacific region. Modern

manufacturing techniques allow the use of bamboo in timber–based industries to

provide bamboo flooring, board products, laminates and furniture. Bamboo is

becoming a substitute for wood in pulp and paper manufacturing, about 25% of the

fiber used in the Indian paper industry each year comes from bamboo (Anon., 1998).

Bamboo shoots are an important food crop in the international market as well as

locally and nationally. China is, by far, the leading exporter of bamboo shoot products

(Feng Lu, 2001). Bamboo furniture is an expanding business in many countries.



Considering the vast scope of bamboos as an economic asset, Government of

India has launched a “National Mission on Bamboo Technology and Trade

Development” (NMBTTD) and its action plan was released in April 2003. The

objective was to use bamboo as a means of reclaiming degraded land, conserve soil,

improve environment, aiming at raising of the forest cover to 25 % by 2007 and 33%

by 2012, organizing harvesting of bamboo from gregarious flowering areas,

expanding area under bamboo plantation in the Tenth and the Eleventh five-year plan,

improving yield, stabilizing the existing bamboo plantation and promoting plantations

of quality species needed by the industry, diversification, modernization and

expansion of the bamboo based industries and handicrafts (Anon., 2003).

National Mission on Bamboo Application (NMBA) is developing,

demonstrating and encouraging intensive and scientific cultivation practices for

higher productivity. The mission is developing improved propagation practices and

identifying and establishing a network of suppliers of plant material, including tissue

cultured material of important bamboo species.

2.1.2. DISTRIBUTION

Bamboo is an extremely diverse plant, which easily adapts to different

climatic and soil conditions. Bamboos are uneven in their geographical distribution,

and are found in tropical, sub-tropical and mild temperate regions. They occur from

sea level to high altitudes (3000-4000 m) above sea level. The distribution of

bamboos extends from 51o N latitude in Japan to 47 o S latitude in south Argentina.

About 18 million ha of bamboos are distributed in world forest ecosystems, in Asia,

Africa and America. South America and south-east Asia are considered to be the

centers of diversity of bamboo species. Their greatest abundance is in Indo-Malayan

region, China, Korea and Japan. Western Asia and Europe do not have native

bamboos. Madagascar has many bamboo species endemic to the region. South Africa

also has few native bamboo species. It is reported that over 77 genera and 1250

species occur in the world and most of which are confined to South East Asia with

largest number of species in China (300) followed by Japan (237) and India (125)

(Sharma, 1987).



Human intervention has played a significant role in the present day

distribution of bamboos. Most places where bamboos now grow have both endemic

and introduced species. India has one of the largest reserves of bamboo in the world

having 125 indigenous and exotic species falling under 23 genera in an area of 10.03

million hectares (Varmah and Bahadur, 1980; Tewari, 1992). Nine genera are native

or naturalized and around 10 species are commercially exploited. North eastern region

of India has over 50% of bamboo species. On a conservative estimate, forest area

under bamboos in India is 9.5% million hectares, which is around 12.8% or 1/7th of

the total forest area of the country.

In temperate and sub-tropical regions of Uttarakhand state four species of hill

bamboo (ringal); Drepanostachyum falcatum, Himalayacalamus falconeri,

Thamnocalamus spathiflorus, and Thamnocalamus jaunsarensis syn

Chimonobambusa jaunsarensis are reported. Ringal species grow at mid and high hills

between 1500-3500 masl in Uttarakhand. Ringal is a major raw material for rural

handicraft industries such as baskets, decorative furnitures, mats, toys, umbrella,

agricultural tools and fish rod. Leaves of ringal bamboo are considered good fodder as

well (Sundriyal and Sundriyal 2009). Chimonobambusa jaunsarensis is distributed in

North-West and Central Himalaya from Jaunsar reagion through Chamoli in Garhwal

to the source of Pindar River in Kumaon between 1800 and 3300 masl (Bahadur and

Naithani, 1976).

2.2. TISSUE CULTURE OF BAMBOOS

Bamboo, the world’s fastest growing and environment friendly giant grass, has

now gained international acceptance and priority, leading to its recognition as an

important non-timber woody resource. World-wide interest in bamboo as a source of

biomass in sustainable agriculture and agroforestry system has increased rapidly in

recent years. The multifarious uses of bamboos, especially their use as industrial raw

material, have increased their demand much beyond the availability. This trend of

increasing demand and decreasing supplies is expected to continue in future. Because

of severe deforestation and limitations of bamboo propagation and improvement,

fulfilling the ever-increasing demand is very difficult. Estimates regarding future use

of bamboo indicate that there will be a huge shortage of bamboo planting material.



While several classical propagation techniques are available, shortage of planting

material and depletion of natural resources are both increasing rapidly.

The conventional methods of propagation of bamboo, sexual as well as

vegetative are beset with many problems that restrict their multiplication on a large

scale. Large scale propagation through seed is unreliable on account of long

flowering cycle, short viability of the seeds, extremely poor seed set during

sporadic/off season flowering and large scale consumption of seeds by wild animals.

Due to the scarcity of seeds, bamboo is generally propagated by vegetative methods

that include clump division, offsets, rhizome, whole culm cuttings, layering, culm

segment cutting, branch cutting and macro proliferation. There is limited availability

of conventionally used propagules and the rate of production is beset with problems

such as the bulkiness of the propagules, their transport over long distances and low

survival. Moreover propagules obtained via. vegetative methods have been found

successful only if they root. Root promoting substances (auxins) have little effect on

bamboo rooting (mature cuttings) and planted material (vegetatively propagated

plants) has been found to develop very slowly (Hassan, 1980). Further year round,

vegetative propagation is difficult due to seasonal specificity of material (Saxena and

Bhojwani, 1993). Replanting efforts using offsets or culm cuttings are slow and

expensive. Propagation with seeds is cheaper and easier with the seedlings being

raised in nurseries and transplanted to the forest. This, however, offers only a limited

answer to the problem since most of the economically important bamboos flower only

once every 30-100 years. The sporadic flowering that takes place annually in isolated

clumps yields few viable seeds from large mass of empty florets. Viable seeds

obtained from gregarious flowering also suffer much damage due to rodents, insect

attacks and rapid loss of viability due to poor storage.

A number of successful reports documenting propagation of bamboos through

in vitro techniques have been published during the last three decades. An attempt has

been made to summarize the available information regarding micropropagation of

bamboos through tissue culture. Many groups have also attempted to induce flowering

in vitro in bamboos to study the floral details. In addition, an overview of the

information about tissue culture work done in bamboos is presented (table 1).



2.2.1. MICROPROPAGATION THROUGH AXILLARY BUD

Micropropagation is a valuable technique for rapid multiplication of difficult-

to-propagate plants, both for commercial production and germplasm conservation.

Micropropagation using tissue culture techniques offers substantial advantage over

largely insufficient and inefficient classical techniques used for mass scale

propagation of bamboos.

McClure (1966) has emphasized that with a few exceptions every node of each

segmented axis of a bamboo plant bears a bud or a branch that in turn, has a bud at

every node. Theoretically, each one of these buds has a potential to produce a

complete plant. He also observed that in nature dormancy and breaking dormancy in

buds of bamboo varied with their position in the plant, the season of the year and the

species. Enhanced axillary bud proliferation technique thus offers the potential ability

to raise thousands of plantlets in a short span of time and in a limited space.

Alaxander and Rao (1968) were the first to report in vitro culture of bamboo

embryos. They cultured the embryos excised from the mature seeds of hybrid bamboo

(Bambusa X Saccharum) on a sucrose enriched medium.

Nadgir et al., (1984) obtained multiple shoots of Dendrocalamus strictus from

seedlings on liquid MS medium supplemented with BAP and coconut milk. 80% of

these shoots rooted when treated with half strength liquid MS medium supplemented

with 0.1mg/l IBA for 48 hours in dark. They also obtained multiple shoots from nodal

segments of mature bamboo species of Dendrocalamus strictus, Bambusa

arundinacaea and B. vulgaris on MS medium supplemented with 10% coconut milk,

0.2 mg/l Kn and 0.5 mg/l BAP. Only 20% of the shoots from mature Dendrocalamus

strictus rooted within 4-5 weeks if treated with 1.0 mg/l IBA for 96 hrs. in dark. They

concluded that the method used for propagation of seedlings was not applicable to

tissues from mature plants.

Banik (1987) achieved micropropagation of Bambusa glaucescens using culm

buds, derived from mature culms, which were inoculated on MS basal medium

containing 1.0 mg/l IBA and 3.0 mg/l activated charcoal. A single shoot was



developed on this medium and multiple shoots were obtained when concentration of

BA was increased to 5.0 mg/l BA, 1.0 mg/l NAA and 3.0 mg/l activated charcoal.

Zamora et al., (1988) reported micropropagation of Dendrocalamus latiflorus

through node and callus culture obtained young lateral shoots and ground corms.

Dekkers and Rao (1989) obtained sprouting of axillary shoot bud with

multiple shoots in Bambusa ventricosa, when nodal explants cultured on MS medium

containing 3.0-4.0 mg/l BA, 0.1-10 mg/l NAA and 0.3% activated charcoal.

Saxena (1990) developed a protocol for the in vitro propagation of Bambusa

tulda through shoot proliferation of aseptically grown seedlings. Seeds were

germinated on liquid or semi solid MS medium with 70-80% germination on liquid

medium. The shoot growth and multiplication rates were better on MS medium than

on B5 medium. On MS medium containing 8 µM BA and 4 µM Kn, a continuous

shoot proliferation rate of 4-5 fold was achieved in every 3 weeks.

Chamber et al., (1991) obtained multiple vegetative shoots and flower from

the nodes of explants derived from young seedlings of Dendrocalamus hamiltonii

when cultured on MS medium supplemented with BAP. The largest number of shoots

ware obtained on medium containing 4 µM BAP. Highest frequency of flowering

(47% of nodal explants) was obtained when cultured explants were transferred from

22.2 µM BAP to a growth-free medium.

Das and Rout (1991) obtained in vitro multiple shoots from axillary buds of

nodal segments of Dendrocalamus strictus and D. giganteus. The nodal segments

were derived from seedlings, which were germinated on MS medium. An average

proliferation rate of 23 folds was obtained on MS medium supplemented with 3.0

mg/l BA and 3.0 mg/l adenine sulphate. The in vitro grown shoots were also rooted in

half strength liquid MS medium supplemented with 2.0 mg/l IBA.

Prutpongse and Gavinlertvatana (1992) tested 67 species of bamboo, out of

which 54 were successfully propagated in vitro. Multiple shoots were produced from

nodal segments when cultured on MS medium containing BAP. Rooting occurred in

media containing NAA (2.7-5.5 µM).



Sood et al., (1992 and 1994) reported micropropagation of Dendrocalamus

hamiltonii using single node cutting from elite seedling plants. Direct shoot bud

regeneration was obtained on MS medium supplemented with 1.0 mg/l BAP + 1.0

mg/l 2, 4-D. Rooting was achieved on half strength MS medium containing 1.0 mg/l

IBA or 0.5 mg/l NAA. Occasionally, callus was observed in multiplication medium,

repeated subculture on the same medium with additional 1.0 mg/l GA3 induced shoot

bud differentiation. In additional to normal green shoots, variegated shoots and albino

shoots were also differentiated from the callus.

Chaturvedi et al., (1993) regenerated Dendrocalamus strictus through nodal

segments taken from branches of 10 years old field grown culms. The nodal segments

were cultured on modified MS medium supplemented with 0.5 mg/l IAA and 15.0

mg/l adenine sulphate where 2-4 axillary shoots were produced per explant. The in

vitro rooting medium consisted of MS medium along with 1.0 mg/l each of IBA and

NAA, 0.5 mg/l 2, 4-D and 1.0 mg/l pholoroglucinol at pH 5.2. Their findings showed

that multiple shoots were more easily induced in seedlings than mature plant. Shoots

from seedlings were more easily rooted than shoots from older plants. They also put

forward the idea that differences in the metabolic status of donor plants have a

carryover effect in the excised explant cultured in vitro. The most favorable months

for experimentations coincided with vigorous growth in situ i.e. period of bud break

in culms of the donor.

Saxena and Bojwani (1993) described a complete protocol of 4 year old plant

of Dendrocalamus longispathus. They found that culture initiation was strongly

influenced by the nature of the explant and the season. Single nodal segments from

young lateral branches produced multiple shoots on semisolid MS medium

supplemented with 12 µM BAP + 3 µM Kn. The shoots have been multiplied for 15

passages in liquid and thereafter for over 5 passages on semisolid MS medium +15

µM BAP + 1 µM IBA +10% coconut water at a rate of 3.2 and 2.8 fold, respectively.

70% of the shoots rooted on a half strength MS medium + 1 µM IAA + 1 µM IBA +

68 µM coumarin.

Saxena and Bojwani (1994) successfully regenerated complete plantlets of

Dendrocalamus longispathus and Bambusa vulgaris, through axillary branching,



using single node segments from mature clumps as the explant. The shoots of

Dendrocalamus longispathus and Bambusa vulgaris have been multiplied at a rate of

3 fold for four weeks and 2.3 fold every six weeks respectively. On MS medium

containing IAA, IBA and coumarin, 68% shoots of Dendrocalamus longispathus

rooted within four weeks. The rooting frequency of the Bambusa vulgaris shoots has

been very low (10%). They also described the initiation and limited multiplication of

adult shoots of Bambusa tulda.

Huang and Huang (1995) displayed the loss of species characteristic of

bulbous internodes in Bambusa ventricosa in plant propagation from excised shoot

tips. For initiation of cultures and subsequent shoot multiplication MS medium

containing 4.44 µM BA was used. These in vitro shoots were successfully rooted on

MS medium supplemented with 5.4 µM NAA and 0.44 µM BA. The rooted shoots

were transferred to pots and none of them displayed bulbous internodes.

Hirimburegama and Gamage (1995) studied the propagation of Bambusa

vulgaris (yellow bamboo) through nodal bud culture. Single nodal segments were

inoculated on MS medium supplemented with different combinations and

concentration of growth regulators. Cytokinins were found to be essential for bud

break and gibberlic acid for multiple shoot production.

Shirgukar et al., (1996) developed simple micropropagation technique for

mass scale production of Dendrocalamus strictus using stationary liquid cultures.

Seedling cultures were initiated on half strength MS medium supplemented with 0.5

mg/l BAP and 2% sucrose. Further proliferation and production of plantlets was

obtained on simple basal medium without any growth regulators. In vitro rhizome

formation was observed in 80% of the cultures.

Maity and Ghosh (1997) standardized methods for efficient germination of old

seeds and regeneration of shoot buds from nodal segments of in vitro plantlets of

Dendrocalamus strictus. GA3 at concentrations 0.5-2.0 mg/l was most effective in

increasing in vitro seed germination, but BAP at 2.0 mg/l showed higher percentage

of shootlet formation from the nodal segments.

Ramanayake and Yakandawala (1997) reported micropropagation of

Dendrocalamus giganteus from nodal explants of field grown culms. Axillary shoots



were initiated during peak bud break periods (April and September) in semisolid

medium with 2.0 mg/l BAP + 0.1 mg/l Kn + 1.0 gm/l Benlate (benomyl). Shoot

proliferarion of 1.8 fold in 13 days was reported on liquid MS medium with 6.0 mg/l

BAP + 0.1 mg Kn. 77.5% rooting was observed on half strength MS medium with 3.0

mg/l IBA + 10.0 mg/l coumarin. Appearance of albino shoots was also observed with

over a 4 fold multiplication rate.

Yasodha et al., (1997) obtained multiple shoots of Bambusa nutans and

Dendrocalamus membranaceous from nodal segments of aseptically grown seedling

cultured on MS medium containing BAP. MS medium supplemented with 0.5 mg/l

BAP showed better multiplication for both the bamboos. In vitro rooting of 68% in

Bambusa nutans and 73% in Dendrocalamus membranaceous was obtained on MS

medium supplemented with 0.5 mg/l IBA.

Arya and Arya (1997); Arya et al., (1999 and 2002) reported rapid

micropropagation protocol for mass multiplication of edible bamboo Dendrocalamus

asper. Axillary bud break was obtained on MS medium supplemented with 1-15.0

mg/l BAP. Multiple shoots were obtained when seeds were cultured on MS medium

with BAP (1-10.0 mg/l). Multiplication rate of 15-20 folds were obtained in four

weeks, when in vitro raised shoots were subcultered on MS medium containing 3.0

mg/l BAP. More than 95% shoots were rooted, when propagule of three shoots were

transferred on to MS medium supplemented with 3.0 mg/l NAA or 10.0 mg/l IBA.

Arya and Sharma (1998) developed micropropagation technique for Bambusa

bamboos through axillary shoot proliferation. A consistent 5 fold multiplication rate

was obtained on MS medium supplemented with 3.0 mg/l BAP by regular subculture

at 4 weeks interval. Within 20 days of subculture on MS medium containing 3.0 mg/l

NAA, 80-85% rooting was achieved.

Lin and Chang (1998) developed a simple and efficient protocol for in vitro

propagation of adult plants of Bambusa edulis through shoot proliferation from nodal

explants. 3-4 folds multiple shoots were obtained on MS medium supplemented with

0.1 mg/l TDZ in every 3 weeks. Regenerated shoots rooted well on a medium

supplemented with 0.01 mg/l TDZ + 0.5 mg/l 2, 4-D. Albinism occurred at the rate of



about 30% among the regenerated shoots and isolated albino shoot also proliferated

on the medium containing TDZ.

Ravikumar et al., (1998) successfully induced multiple shoots from seedling

and axillary buds of mature plants of Dendrocalamus strictus on MS medium

supplemented with BAP and Kn. About 35-45 shoots were obtained within 20-25

days from nodal explants of seedling and 3-8 shoots were obtained from nodal

explants of mature plants in the primary culture. Rooting of shoots was achieved both

in in vitro and ex vitro methods using IBA.

Bag et al., (2000) developed an efficient protocol for in vitro propagation of

Thamnocalamus spathiflorus, through multiple shoot formation from zygotic embryos

excised from germinating seeds, as well as from nodal explants taken from a 2 year

old plant. They obtained multiple shoots in both the cases on MS medium

supplemented with 5 µM BAP + 1 µM IBA. 100% rooting was obtained on two step

rooting procedure, first the root was induced on 150-300 µM IBA containing medium

followed by root elongation on PGR-free medium.

Mishra et al., (2001) tested vipul (triacontanol) for in vitro shoot

multiplication and rice bran extract for in vitro adventitious rhizogenesis in single

node cultures derived from shoots of Dendrocalamus strictus. MS liquid medium

containing vipul (0.5 mg/l) with BAP (3.0 mg/l) induced 4.59 fold multiplication rate

whereas, application of BAP and vipul alone had corresponding value of 3.29 and

0.53 fold respectively. Maximum in vitro rooting percentage (55.66%) was obtained

on half MS medium enriched with alcoholic rice bran extract (2.5 ml/l) and NAA (3.0

mg/l).

Ramanayake et al., (2001) achieved continuous axillary shoot proliferation

and in vitro flowering using single node explants from 70 year old field clump of

Dendrocalamus giganteus. The shoots proliferated in MS medium with 6.0 mg/l BAP

and 2% sucrose. The rate of shoot proliferation increased from an initial 1.6 fold to

3.25 fold just before in vitro flowering, after which it dropped to 1.73 fold. They

concluded that the development of axillary meristems into vegetative or generative

shoots depend on the level of BAP.



Sood et al., (2002) developed in vitro protocol via. micropropagation and

somatic embryogenesis for Dendrocalamus hamiltonii. Axillary bud sprouted on half

strength MS medium with 2.5 mg/l BAP. Propagule of three to four shoots gave better

proliferation rate on liquid MS medium. Rooting response was not consistent and only

25-30% of shoots developed into plantlets. Prolonged culturing of nodular callus on

medium containing BAP; 2, 4-D (1.0 mg/l each) and GA3 (0.5 mg/l) resulted in the

formation of distinctly white embryoids.

Das and Pal (2005) first time reported in vitro regeneration of Bambusa

balcooa from mature field-grown axillary buds. Multiple shoots were obtained from

nodal segments on liquid MS medium with 11.25 µM BAP + 4.5 µM Kn. In vitro

rooting was obtained on half strength MS medium supplemented with 1µM IBA.

They observed morphogenetic competence of axillary buds in different months of the

year and obtained highest response in October. They also concluded that a

moderately high phenolic content of the nodal explant was detrimental for in vitro

morphogenesis.

Sanjaya et al., (2005) described an efficient and reproducible procedure for the

large scale propagation of Pseudoxytenanthera stocksii. High frequency multiple

shoot induction was achieved from nodal segments on liquid MS medium

supplemented with 2.68 µM NAA and 4.40 µM BAP. In vitro differentiated shoots

were multiplied on liquid MS medium with 2.68 µM NAA, 2.21 µM BAP and

additives (283.93 µM ascorbic acid, 118.10 µM citric acid, 104.04 µM cysteine and

342.24 µM glutamine). Rooting was successfully obtained on liquid MS medium

supplemented 4.90 µM IBA, 0.44 µM BAP and additives. These tissue culture raised

plants were acclimatized and established in the field and exhibited normal growth.

Arya et al., (2006) described micropropagation of Dendrocalamus giganteus

through axillary bud culture. Shoot multiplication rate of 5.44 fold was obtained on

MS medium supplemented with 20 µM BAP, while increased multiplication rate of

6.35 fold was obtained on MS medium supplemented with combination of BAP and

Kn (10 µM each). 90% rooting was obtained on MS medium supplemented with 25

µM IBA + 0.05 µM BAP.



Fengfa et al., (2006) studied the technical system of the mass propagation of

Dendrocalamus latiflorus. The optimum medium for culture initiation was MS

medium with 2.0 mg/1 BAP and 0.5 mg/1 IBA. Multiplication rate on 7.31 fold was

obtained, when propagules of four shoots were cultured on MS medium supplemented

with 2mg/1 BAP and 0.3 mg/1IBA. 95% rooting was obtained on ½ MS with 4.0

mg/1 IBA.

Guangping et al., (2006) reported tissue culture through axillary bud

proliferation of 11 ornamental bamboos. They found a great difference in rooting rate

of different species. Rooting rate for monopodial bamboo was 65-90%, while for the

sympodial was more than 95%.

Kapoor and Rao (2006) reported precocious rhizome formation in multiple

shoots of woody bamboo Bambusa bamboos var. Gigantean. Multiple shoots were

initiated from embryonic axes of caryopsis on MS basal medium supplemented with

5.0 µM BAP and 2% sucrose. Transfer of shoots to MS basal medium supplemented

with BAP 2.5 µM, 5.0 µM), GA3 (0.1 µM) and NAA (5.0 µM) and 5% sucrose led to

58% to 100% rhizome induction within four weeks of culture and these rhizomes

developed roots on auxin medium and formed shoots. The plantlets with rhizomes

were transferred to the soil.

Ndiaye et al., (2006) reported that micro shoots derived from explants of adult

trees are difficult to root in Bambusa vulgaris, addition of 5.0 mg/1 of auxins did not

induce rooting which was achieved after using high concentration (20.0 mg/1) of IBA.

They used nodal segments from adult plants for shoot differentiation on modified MS

medium supplemented with 2.0 mg/1 BAP.

Ramanayake et al., (2006) raised rapidly proliferating axillary shoots from

axillary buds of Bambusa vuigaris ‘striata’. In vitro shoots raised from adult plant and

from one year old tissue culture raised plants were continuously multiplied on MS

medium supplemented with 4.0 mg/1 BAP, 40% and 92% rooting was induced on

medium with 3.0 mg/1 IBA, respectively. They obtained enhanced rooting of 100%,

when in vitro raised shoots from adult field culms were pre-treated with 0.5 mg/1

TDZ for two to three subcultures before placing them in the root induction medium.



Zailiu and Chaomao (2006) studied tissue culture of young and mature

bamboo of Dendrocalamus sinicus. They explain that the dosage of BAP and the

shoot multiplication were proportioned positively, but if the dosage of BAP enlarged

or a long time culture made on high concentration, flower buds would be seen. They

showed that successes of explants in vitro and induced multiplication of shoots were

related to the collected time and collected section of the explants.

Jimenez et al., (2006) reported in vitro propagation of Guadua angustifolia,

through axillary shoot proliferation. They showed that to reduce contamination for

culture initiation, nodal segments were pre-treated prior to disinfection, which

included immersion in an alkaline solution (0.05% w/v) of Extran for 10 min., and a

combination of the bactericide Agri-mycin and the fungicide Benomyl, at a

concentration of 2.0 gm/1 each, for 10 min. Followed by disinfection with 1.5%

NaOCl for 10 min. They obtained 37.5% bud break response on MS medium

supplemented with 3.0 mg/1 BAP and a multiplication rate of 2.5 in every six weeks

at 5.0 mg/1 BAP concentration. Rooting occurred spontaneously in 100% of the

explants that developed lateral shoots.

Liu et al., (2007) identified two albino mutants from 8 years of subculturing of

Bambusa edulis. These mutants were spontaneously derived from multiple shoots

incubated in MS medium supplemented with 0.5 mg/1 TDZ and were maintained

their albino mutant characteristic consistently during proliferation and subculturing on

the same medium. By applying PCR methodology, they demonstrated that the albino

mutants have major deletions in their chloroplast genome.

Mishra et al., (2007) standardized an efficient in vitro propagation and

hardening procedure for obtaining plantlets from field grown culms of Bambusa

tulda. They reported MS liquid medium enriched with 100 µM glutamine, 0.1 µM

IAA and 12 µM BAP gave maximum in vitro shoot multiplication rate of two-fold.

The proliferated shoots were successfully rooted on MS liquid medium supplemented

with 40 µM coumarin resulted in a maximum of 98% rooting.

Arya et al., (2008a) reported micropropagation protocol for two economically

important bamboos Drepanostachyum falcatum and Bambusa balcooa. They

obtained axillary bud break on liquid MS medium supplemented with 5.0 mg/1 BAP.



Best shoot multiplication was observed on MS medium supplemented with 3.0 mg/1

BAP for Drepanostachyum falcatum and 3.0 mg/1 BAP with 0.5 mg/1 Kn for

Bambusa balcooa. 100% in vitro rooting was obtained on MS medium supplemented

with 7.0 mg/1 IBA in Drepanostachyum falcatum.

Arya et al., (2008b) obtained plant regeneration from cultured immature

inflorescence of Dendrocalamus asper by direct shoot regeneration on MS medium

supplemented with 7.0 mg/1 BAP. Rooting was successfully achieved on MS

medium supplemented with 1.0 mg/1 IBA.

Ramanayake et al., (2008) investigated rooting of in vitro shoots from adult

field culms of Bambusa atra, Dendrocalamus giganteus and D. hookeri and of

juvenile seedling shoots of D. giganteus. They found Bambusa atra rooted

spontaneously without exogenous auxin during axillary shoot proliferation, while both

Dendrocalamus species rooted only on transfer to rooting media with IBA. Rooting

in adult shoots of D. Giganteus was lower (45.6%) then that in the juvenile shoots

(96.7%) but adult shoots of D. hookeri rooted well (88.9%). A pre-treatment with

TDZ induced development of axillary buds, which increases rooting to 95% in adult

D. giganteus shoots.

Yasodha et al., (2008) reported a high frequency in vitro rooting method for

Dendrocalamus nutans. Two step sequential method was adopted for rooting, MS

medium with 49 µM IBA and 88 µM glucose was used in the induction phase for 3

days followed by MS salts with 88 µM sucrose. Addition of glucose along with 49

µM IBA during the root induction phase gave 85% rooting success.

Agnihotri et al., (2009) developed an efficient in vitro regeneration protocol of

a multipurpose bamboo species Dendrocalamus hamiltonii Nees et. Arn. Ex Munro

using single node cuttings taken from the lateral branches of a 20-year-old bush.

High-frequency proliferation was induced on the propagules (small clusters with 3–5

multiple shoots and rhizomatous portions). Subsequent removal of the shoots (about

1.5 cm) from the rhizomatous portion of propagules (shoot cut) influenced the plantlet

formation capacity. A multiplication of about 20-folds was achieved on MS medium

supplemented with 8.0 µM BAP and .01 µM NAA. Rooting efficiency was also

markedly enhanced (90%) when the propagules, following shoot cut, were placed on



to MS medium supplemented with 100 µM IBA for 10 days and then transferred to

IBA-free medium.

Devi and Sharma (2009) developed an in vitro propagation method of

Arundinaria callosa Munro using single axillary bud on Murashige and Skoog (1962)

medium supplemented with different concentrations of 6- benzylaminopurine (BAP).

The position of the node on the culm of lateral branches affected bud-break

percentage and multiplication, mid-culm nodes are the most suitable. The optimal

concentration of 13.3 µM BAP is found significant for shoot multiplication. Addition

of 1.0 µM 3- indolebutyric acid (IBA) enhances the shoot multiplication rate. In vitro

rooting was induced when 15 µM IBA was incorporated for three subcultures in the

shoot proliferation medium, was transferred to half strength MS containing 25 µM

IBA and 0.05 µM BAP, and finally withdrawn from the rooting medium. The

regenerants were successfully transplanted into a soil mixture for acclimatization

before field planting.

Negi and Saxena (2010) developed a protocol for large-scale multiplication of

Bambusa nutans by using liquid MS medium supplemented with 13.2 µM BA, 2.32

µM Kin, and 0.98 µM indole-3-butyric acid (IBA). In vitro raised shoots were rooted

with 100% success on half-strength MS liquid medium supplemented with 9.8 µM

IBA, 2.85 µM indole-3-acetic acid (IAA), 2.68 µM naphthaleneacetic acid (NAA),

and 3% sucrose.

Bisht et al., (2010) developed a complete procedure for the regeneration of

plantlets of Gigantochloa atroviolaceae through axillary shoot proliferation. They

obtained axillary bud break in full strength liquid MS medium fortified with 25.0 µM

BAP and these axillary shoots produced were multiplied on semi-solid MS medium

supplemented with BAP(20.0 µM) + NAA (3.0 µM) giving a multiplication rate of

2.39. Optimal rooting was achieved on medium supplemented with 35.0 µM IBA

which were successfully hardened and acclimatized with 80% survival.

Negi and Saxena (2011) developed an efficient and reproducible protocol for

large-scale multiplication of Bambusa nutans using nodal segments collected from

field-grown clumps and cultured on Murashige and Skoog (MS) medium

supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kn). They



obtained multiplication in MS liquid medium supplemented with 13.2 μM BA,

2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every

3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-

fold. Shoot clusters containing three to five shoots were successfully rooted with

100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA,

2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthalene acetic acid (NAA), and 3%

sucrose.

Thiruvengadam et al., (2011) developed an efficient and reproducible

protocols to induce shoot multiplication from nodal cultures of aseptically raised

seedlings and and 1 year old plants of Bambusa oldhamii Munro using nodal

segments. Maximum multiple shoots were induced in MS medium supplemented with

4.4 μM 6-benzylaminopurine (BAP) in both seedlings and 1 year old explants.

Highest rooting (85.0%) was obtained on half MS basal medium with a combination

of 9.84 μM IBA and 2.69 μM NAA.

Sharma and Kalia (2012) developed a protocol of Bambusa nutans using nodal

segments taken from 40 yr old clump, inoculated on medium supplemented with 7.5

µM BAP for bud break (79.16 ± 7.22) and multiplication on medium supplemented

with 5.0 µM BAP for 4-5 subculture cycles. During multiplication phase, BAP alone

proved to be superior to kinetin. Effective IBA concentration was found to be 10.0

µM on which 72.22% of propagules rooted.

Singh et al., (2012b) studied the effect of season, media type, carbon source,

growth regulators and transplanting media on micropropagation of Dendrocalamus

asper, an important bamboo species. They found that the season of explant collection

played an important role in axillary bud sprouting and spring (February–April) to be

the best period for explant collection. Among the different media, MS was found to be

the best for micropropagation in which maximum numbers (4.83/explant) of shoots

were initiated supplemented with 15 μM BAP. Maximum shoot multiplication was

observed on MS medium supplemented with 10 μM BAP and 75 μM adenine

sulphate. Optimal rooting was achieved in shoots cultured on ½ strength MS medium

supplemented with 5 μM each of IBA and NAA.



Beena et al., (2012) studied the effect of carbohydrate sources and sucrose

concentrations for mass clonal propagation of Bambusa pallida. They found sucrose

in MS liquid medium consisting additives (ascorbic acid, 50 mg/l + citric acid, 25

mg/l + cysteine, 25 mg/l) to be the best carbohydrate source for shoot induction and

shoot multiplication. NAA 0.25 mg/l in combination with TDZ 0.25 mg/l in the

medium exhibited high frequency shoot induction and NAA 0.25 mg/l with BAP 1.0

mg/l helped for further multiplication of quality shoots. IBA pulse treated shoots were

rooted in the MS half strength agar gelled medium fortified with sucrose (2%) and

glucose (1%).

Sharma and Sarma (2013) developed an efficient micro propagation protocol

from nodal explants from field grown culms of Bambusa tulda. In vitro auxiliary

shoot formation was highest in Murashige and Skoog basal medium supplemented

with 1.0 mg/l 6-Benzyle Adenine (BA). Clumps of at least 3 shoots were used for root

induction in MS medium with Indole-3- Acetic Acid (IAA), Indole-3- Butyric Acid

(IBA) and naphthalene acetic acid (NAA). Response of rooting was found more in 5.0

mg/l naphthalene acetic acid with 100 percent survival in field condition after in vitro

hardening.

2.2.2. ORGANOGENESIS

Clonal propagation via organogenesis is a two-staged process involving the

proliferation (axillary meristems) or induction (adventitious meristems) of unipolar

shoots on explants followed by shoot excision and induction of root meristems. It is

generally agreed that plants regenerated from shoot tips or nodal buds are genetically

stable and free from somaclonal variations associated with plants differentiated from

callus. Therefore, a lot of studies are available in which enhanced axillary branching

has been utilized for micropropagation of bamboo species using juvenile and mature

tissues (Table 1).

Plant regeneration from cultured tissues can also be achieved by culturing

tissue sections lacking a preformed meristem (adventitious origin) or from callus and

cell cultures (de novo origin). Whether adventitious or de novo in origin, plant

regeneration can occur by one of two processes, organogenesis or somatic

embryogenesis.



Huang and Murashige (1983) worked in detail on nutrient requirement and

environmental conditions for callus cultures of Bambusa oldhamii, Bambusa

multipled, Sasa pygmaea and Phllostachys aurea. The shoot tips were used as

explants for culture initiation. They found that 2, 4-D in the range of 1.0 – 3.0 mg/1

was the optimal concentration for callus formation.

Huang et al., (1988) established liquid suspension cultures of Bambusa

multiplex, Bambusa oldhamii, Phyllostachys aurea and Sasa pygmaea. They used

shoot apices derived calli to start the liquid suspensions in MS medium containing 3.0

mg/1 2, 4-D with continuous shaking at 150 rpm.

Dekkers and Rao (1989) reported callus differentiation in seeds of

Dendrocalamus strictus, which were germinated on MS medium supplemented with

1.0 – 10 mg/1 2, 4-D.

Huang et al., (1989) investigated organogenesis leading to the formation of

adventitious shoots and subsequently plants from excised shoot apices of 4 bamboo

species i.e. Bambusa oldhamii, B. Multiplex, Phyllostachys aurea and Schizostachyum

pygmaea on MS medium supplemented with NAA and BAP.

Tsay et al., (1990) reported micropropagation of Dendrocalamus latiflorus

through node and callus culture. Callus establishment from ground corms was

achieved for Bambusa vulgaris, Gigantochloa asper, G. levis and Schizostachyum

lumbago.

Sood et al., (1994) achieved micropropagation of Dendrocalamus hamiltonii

using single node cutting taken from seedling plants. Direct shoot bud regeneration

lead of multiple shoot initiation or callus formation on MS medium containing 1.0

mg/leach of BA and 2, 4-D.

Wu-Yimin et al., (2000) established bamboo cell suspension culture. They

initiated callus from apical bud explants and lateral nodal bud explants of Bambusa

spp. (B. Glaucescens, B. Oldhamii and B. Eludes) in response to the combinations of

growth regulators: 2, 4-D, NAA, BA and 2, 4-D/ Kn.



Ramanayake and Wanniarachchi (2003) reported organogenesis from callus

induced on shoots, spidelets and roots from proliferating axillary shoots derived from

an adult giant bamboo Dendrocalamus giganteus.

Lin et al., (2004) reported somatic embryogenesis in Bambusa eludes from

callus cultures derived from nodal and intermodal tissues of in vitro plantlets on MS

medium supplemented with 9.2 µM Kn, 13.6 µM 2,4-D, 0.1% (v/v) coconut milk and

6% (w/v) sucrose.

Kalia et al., (2004) successfully induced organogenic callus from pre-injured

single shoots derived from in vitro multiplying cultures of Bambusa nutans.

Combinations of 2,4-D (5 µM), BAP (2.5 µM) and ABA (1 µM) proved to be more

efficient in inducing callus, in 79.93% of cultures with an average of 346.13 mg of

fresh weight callus, than auxins used alone or in combination with cytokinins.

Efficient regeneration of shoot buds and their conversion into shoots was recorded on

MS medium supplemented with BAP (5 µM) and NAA (1.25 µM) on which 18.11

buds were induced which proliferated into 10.31 shoots. Spontaneous regeneration of

roots on shoots was evidenced on regeneration medium itself.

Ogita (2005) reported callus and cell suspension culture of bamboo plant,

Phyllostachys nigra on a modified half strength MS medium supplemented with 3 µM

2,4-D The calli could be maintained on both solid and liquid media.

Macro-proliferation, a method of plant multiplication by separating the rooted

tillers has been used by many workers for enhancing the rate of multiplication of in

vitro raised plants and for continuous supply of plantlets. Splitting of rooted tillers

could double the production of Dendrocalamus asper plants (Singh et al., 2011) while

three-fold increase was achieved in and Bambusa balcooa (Mudoi and Borthakur

2009), .B tulda (Mishra et al., 2011)

Micropropagation has been widely used for rapid mass multiplication of

bamboos; however, its application on commercial scale is restricted often due to high

rate of plant loss when transferred to natural or ex vitro conditions. Only few reports

are available regarding successful field transfer of micro propagated bamboos. Arya et

al., (1999) reported 95% field survival of Dendrocalamus asper and transferred 6000



plants raised through seed tissue culture to the field. Sood et al., (2002) and Agnihotri

et al., (2009) reported a survival percentage of 70% in the field for the plants of D.

hamiltonii. Mishra et al., (2011) reported 91% survival of the plants of Bambusa tulda

in the green house.

Negi and Saxena (2011) have successfully produced 2500 plantlets with

95.83% hardening rate up to nursery stage and transferred 12 plants with 100%

success in the field. Singh et al., (2011) transferred 2500 and 3000 plants of

Dendrocalamus asper and D. hamiltonii respectively to the Forest Department land in

Yamunanagar, Haryana under the DBT’s Bamboo Mission. They reported a success

rate of 92.34% and 100% for D. asper and D. hamiltonii in the green house while

79.76% and 85% success was achieved in the field. Morphological growth variations

were not observed among these plants over a period of 1-2 years. Few other reports

also document good field performance of the tissue culture raised plantlets (Nadgir et

al., 1984; Saxena 1990; Mudoi and Borthakur 2009; Agnihotri et al., 2009). Besides

evaluating the morphological parameters, physiological parameters like

photosynthesis, transpiration, water use efficiency, etc have also been compared with

mother plants in Dendrocalamus hamiltonii (Agnihotri et al., 2009). Many groups

have tested the genetic fidelity of the tissue culture raised plants using molecular

markers also.

2.2.3. SOMATIC EMBRYOGENESIS

Micropropagation via somatic embryogenesis offers another easy and reliable

method for mass propagation as both the root and the shoot primordia are produced in

a single step. It can be used for large scale propagation of bamboo at minimum cost in

a relatively shorter time and with lowest labour inputs. Encapsulation of somatic

embryos in alginate beads to produce synthetic seeds holds great promise for

establishment of bamboo plantations. Intensive research on tissue culture of bamboos

related to somatic embryogenesis was initiated by Mehta et al., (1982) with the

production of plantlets of Bambusa arundinacea. After that, somatic embryogenesis

and plantlet regeneration has been reported in several bamboo species. In a recent

study, Bag et al., (2012) have reported somatic embryogenesis in Dendrocalamus

hamiltonii using nodal explants collected from 45 and 10-year-old clumps.



Generally, embryogenic tissue is initiated on a medium containing low

concentration of auxins, usually in the form of 2, 4-D and NAA, and cytokinins (BA

and TDZ). Mostly MS medium has been used for embryogenesis in bamboos,

however, in few studies other media such as B5 and N6 have also been used for

somatic embryogenesis. The generation of morphologically developed somatic

embryos does not guarantee satisfactory post-embryonic performance. Embryo

development in bamboos is initiated by arresting cell proliferation through the

removal of auxins and cytokinins and putting them on PGR free medium (Godbole et

al., 2002). Although, Rout and Das (1994) reported development, maturation as well

as germination of somatic embryos on (MS) basal medium supplemented with Kn, 2,

4-D and AdS. In general, maturation of somatic embryos is achieved on agar

solidified media, however, as demonstrated by Hassan and Debergh (1987) somatic

embryos can also be obtained in liquid medium. Mature somatic embryos germinate

and convert to plantlets in a growth regulator free medium (Godbole et al. 2002).

Although in some studies, cytokinin has been found to be an essential component in

germination of bamboo somatic embryos. Kn was used to promote the germination of

Bambusa oldhamii, B. beecheyana and Sinocalamus latiflora somatic embryos (Yeh

and Chang 1986a,b; 1987) while Lin et al., (2004) used TDZ for somatic embryo

germination in Bambusa edulis.

Rao et al., (1985) successfully obtained somatic embryogenesis and plant

regeneration of Dendrocalamus strictus by culturing seeds on B5 basal medium

containing 10 µM or 30 µM 2, 4-D.

Vongvijitra (1988) induced embryogenic callus using nodes of seven year old

aseptic seedlings in Dendrocalamus membranaceus on MS medium containing 1-

1.5x10 5 µM 2, 4-D and 0.2x10.5 µM BAP. Embryogenic callus was formed after 3-4

months.

Mukunthakumar and Mathus (1992) produced artificial seeds of

Dendrocalamus strictus by encapsulating somatic embryos in calcium alginate beads,

which were obtained on MS medium containing 3.0 mg/1 2, 4-D and 0.5 mg/1 Kn.



Jullien and Tran Thanh Van (1994) micropropagated Bambusa glaucescens

through embryoids formation on young leaves derived from in vitro grown plants on

MS medium supplemented with 2,4-D and BAP with 15 g/1 sucrose.

Rout and Das (1994) regenerated plants via somatic embryogenesis in callus

cultures derived from nodal explants of in vitro grown seedlings and excised mature

zygotic embryos of Bambusa vulgaris, Dendrocalamus strictus and D. Giganteus on

MS medium supplemented with 0.5 mg/1 Kn, 2.0 mg/12,4-D and 10.0 mg/1 adenine

sulphate. About 95-98% somatic embryos germinated into normal plants.

Chang and Lan (1995) reported embryogenesis and subsequent formation of

plantlets from callus cultures derived from the roots of Bambusa beecheyana on MS

medium consisting of 2.0 mg/1 Kn, 3.0 mg/12, 4-D and 6% sucrose.

Saxena and Dhawan (1999) reported callus induction and embryogenesis from

sterilized seeds of Dendrocalamus strictus cultured on semisolid MS medium

containing 3x10-5 µM 2,4-D and 3% Daurala sugar.

Godbole et al., (2002) reported plantlet regeneration via somatic

embryogenesis in Dendrocalamus hamiltonii. They used new sprouts from the nodal

segments of mature, elite bamboo for the induction of embryogenic callus.

Ogita (2005) successfully achieved callus induction from bamboo shoots of

Phyllostachys nigra Munro var. Henonis using a modified 1/2 MS medium

supplemented with 3 μM 2,4-D.

Gillis et al., (2007) reported mass propagation via somatic embryogenesis in

Bambusa balcooa using pseudospikelets. They induced callus on MS medium

containing 4.5 µM 2, 4-D and 3% sugar.

Arya et al., (2008c) reported somatic embryogenesis in Dendrocalamus asper.

Somatic embryos were induced on MS medium supplemented with 30 uM 2, 4-D and

2% (w/v) sucrose in dark conditions. These somatic embryos were further developed

on 5 µM ABA and 6% sucrose.

Kalia and Sharma (2009) achieved callus induction on pre-injured explants.

Performance of explants derived from in-vitro multiplying cultures proved better than



those from mature plants in which 2, 4-D proved to be superior in inducing callogenic

response than NAA or IBA. Enhancement of callogenic efficiency of in-vitro derived

explants was observed when BAP (2.5 μM) was supplemented to auxin-enriched

medium. Induced callus was multiplied on multiplication medium (5μM 2,4-D + 1.25

μM BAP) for varying periods but optimal multiplication rate for shoot callus (1.69)

and leaf callus (1.39) was obtained after subculture cycle of three weeks. Spontaneous

root regeneration occurred at frequency of 61% on shoots regenerated on

multiplication medium.

2.3. LOW COST OPTIONS FOR TISSUE CULTURE

The major application of tissue culture lies in the production of true-to-type

high quality planting material that can be multiplied under aseptic conditions on a

year round basis irrespective of seasonal variations and weather conditions. Tissue

culture is an expensive technology to propagate plants compared to other methods of

propagation such as by seeds and stem cuttings. The need for low cost plant tissue

culture systems, applicable for micropropagation and in vitro conservation of plant

genetic resources has been emphasized to allow the large scale application and

adaptability of such technology in developing countries (IAEA, 2004). Several

research and development projects have been undertaken to improve the productivity

of agricultural, horticultural and forest trees by the European union under Co-

operation in the field of Scientific and Technical Research (COST). Under this

program, coordinated and funded by the European Union, one of the primary aims has

been to reduce micropropagation cost. For example, the objective of ‘COST 843’

action has been the innovation of low-cost plant propagation methods that enhance

sustainable and competetive agriculture and forestry in Europe (COST Action, 2001).

The high costs of labour of micropropagation are a major bottleneck in the EU to fully

exploit in vitro culture technology. In the EU, labour currently accounts for 60-70%

of the costs of the in vitro produced plants. In another program, the large scale

production and induction of bamboo in the EU using tissue culture technology has

been undertaken with the main objective of reducing the costs of micropropagation

(Savangikar, 2002).



High costs involved in micropropagation are a major constraint to their

popular use (Pierik, 1988; Cassels, 1989; Ghosh, 1996; Sluis, 2006 ). Several

alternatives such as Automation, shifting of production activities to countries with

with low wage regime (Chu and Kurtz, 1989), substitution of costly media

components (Ghosh, 1996), enhanced multiplication rates (Cerevelli and

Senaratna,1995; Sluis, 2006) have been suggested to reduce costs of tissue culture

plants. Cost reduction in tissue culture can also be achieved using liquid nutrient

medium to exhausted agar media (Maene and Debergh,1985), use of industrial robots

(DeBry, 1986) and nutrient mist generation in special culture enclosure (Weather and

Giles, 1988). Levin et al.,(1988) reported to have developed an automated propagated

system which has substantially eliminated manual tasks, resulting in as much as 85%

savings in labour costs.

Using low cost tissue culture technology, unit cost of micropropagule and

plant production can be reduced that ultimately supports different sectors like forestry,

silviculture and cultivation of medicinal and economically important plants. Low cost

options should lower the cost of production without compromising the quality of the

micropropagules. Better utilization of resources and improved process efficiency can

make a breakthrough.

Generally tissue culture laboratories are having facilities of sterile airflow

rooms, expensive autoclaves for sterilization of media and tools and equally

expensive glasshouses, temperature and day-length to harden and grow plants. All

these facilities cost high. In the developed countries, there is no financial problem in

supply of regular electricity, chemicals, equipments and instruments used in

laboratories. But in developed countries such facilities are not easily ensured due to

financial crisis. Therefore, there is an urgent need to find out some low cost

alternatives to replace the costly inputs and infrastructures in micropropagation.

There are many factors which influence the cost of tissue culture raised

plantlets like labour cost (including skilled and unskilled), infrastructure including

facilities of electricity supply, culture maintenance and of acclimatization, cost of

culture containers and cost of plugs, media components (gelling agents, plant growth

regulators, micro and macro nutrients and carbon source) etc.



Cost per plantlet can be reduced by reducing electricity consumption by

designing the growth rooms in such a way that sunlight provides light without

interfering optimum temperature and using efficient explants sterilization procedures,

otherwise establishment culture costs very high. Use of low cost gelling agents and

carbon source will also help in lowering the cost of plantlets. The composition of

culture media used for shoot proliferation and rooting has a tremendous influence on

production costs. The main components of most plant tissue culture media are mineral

salts and sugar as carbon source and water. Other components may include organic

supplements, growth regulators and gelling agents (Gamborg et al., 1968). Proper

choice of media and containers can reduce the cost of micropropagation. The

replacement of expensive gelling agents, use of low cost carbon source and some

other medium components can reduce cost of production.

Plant production through micropropagation, the media chemicals cost less than

15% of the total cost (Prakash et al., 2004). One of the medium components- the

gelling agents such as agar contributes 70% of the cost of production. Other

ingredients in the media- salts, sugar and growth regulators have minimal influence

on production cost and are reasonably cheap.

2.3.1. GELLING AGENTS

Gelling agents are usually added to the culture medium to increase its

viscosity as a result of which media gets solidified. This semi solid media provides

support to the explants. Growth and development of explants is influenced by quality

and quantity of the gelling agent in media. Several kinds of gelling agents are

available in market like agar, gelrite, phytagel, agarose, gellan gum etc. agar is the

most frequently used gelling agent for preparation of most of the plant tissue culture

media because of the desirable characteristics of high gel clarity, stability and thought

to be biologically inert but later on a number of reports on its adverse effects have

been reported (Romberger and Tabor, 1971; Debergh et al., 1981; Debergh, 1983),

including batch-to-batch variability, inhibition of growth, presence of impurities and

impairment of vitrification.



Debergh (1983) reported that agar contributes to the matrix potential, the

relative humidity and affects the availability of water and dissolved substances in the

culture containers. Various brands and grades of agar are differing in the amounts of

impurities and gelling capacity. Agar is available in market with varying price, level

of purity and gelling capacity. Which kind of the agar grade should be used depends

on one’s target and on the plant species. It is usually unnecessary to use high grade

purity ager for large scale micropropagation; cheaper brands of agar have been

successfully used for industrial scale micropropagation (Boxus, 1978). To solidify the

media lowest concentration of agar depends on the purity and brand. Usually 0.6-

0.8% (w/v) agar is used to solidify the media.

The use of liquid media eliminates the need of agar. Other options include

white flour, laundry starch, semolina, potato, rice powder and sago etc. 70-82%

reduction in cost of gelling agents has been reported by using laundry starch, potato

starch and semolina in a ratio of 2:1:1 (Prakash, 1993). A number of substitutes for

agar have been tried out including methylcellulose and alginate (Adaoha Mbanaso

and Roscoe, 1982), starches from barley, corn, potato, rice and wheat (Bornman and

Vogelmann, 1984; Calleberg et al., 1989), isabgol (Babbar and Jain, 1998), gelatine,

pectin and a number of other support systems such as agitated liquid medium, filter

paper, cotton wool, polyester fleece and glass beads etc.

Differences in the agar medium and gelled media have been attributed to

limited diffusion of media components and water (Romberger and Tabor, 1971; Stolz,

1971), the National Research Development Corporation, India (NRDC, 2002) has

listed low cost agar alternatives which are worth evaluating for routine use in

commercial micropropagation.

However, the addition of such low cost gelling agents to the medium may

have some disadvantages. Some gelling agents contain inhibitory substances that

hinder morphogenesis (Powell and Uhrig, 1987) and reduce the growth rate of

cultures. Sometimes toxic exudates from the cultured explants may take a longer time

to diffuse. These gelling agents may influence availability of mineral ions and plant

growth regulators due to adsorbance of these molecules. Use of cheaper alternatives



to agar may give a dark colour to media, which makes it difficult to take observations

regarding contamination and rooting. These low cost alternatives to agar may create

problem during dispersion of media into culture vessels. Again these solidifying

agents may take more time and energy to clean the culture containers.

Zimmerman (1995) and Stanley (1995) have used a combination of 50.0 g/l

corn starch with 0.5 g/l gelrite for the propagation of fruit trees, such as apple, pear,

banana, raspberry, ginger, sugarcane and turmeric. The corn starch medium proved to

be better for shoot proliferation than on agar. However, it became difficult to detect

the contamination because the corn starch medium turned greyish-white.

Rooting of chick pea was found to be better on tapioca with 66.7% than on

agar with 40%. Addition of 80 g/l tapioca starch to the MS medium was found to be a

good substitute for ‘Bacto-agar’ for potato culture (Getrudis and Wattimena, 1994).

The results reported by Gebre and Sathyanarayana (2001) showed the poissibility of

using the tapioca as an alternative cheaper gelling substance (40 X cheaper than agar

at equal concentration) in micropropagation of potato through production of plantlets

or micro tubers. Maliro and Lameck (2004) reported cassava flour (even without

processing into pure starch) as a substitute to agar and improved growth of shoots of

Uapaca kirkiana and Faidherbia albida.

Barley starch (6.0 g/l) has also been used for culturing potato tuber discs and

for anther culture of barley (Sorvari, 1986; Sorvari and Schieder, 1987). Sago

(obtained from the stem pith of Metroxylon) at 13% concentration was substituted for

agar in MS medium for the multiplication of chrysanthemum through shoot tip culture

.The number of shoots and leaves and root length were significantly higher on sago

than on agar.

Isabgol is dried seed-husk of plantago ovata. It is an alternative gelling agent

because of its polysaccharidic and colloidal nature, good gelling ability, resistance to

enzymatic activity and better clarity than agar in gelled form, has the potential to

become a universal gelling agent for plant tissue culture media. However, its higher

melting point (70.6 o C) necessitates adjustment of pH and dispensing quickly (Jain



and Babbar, 2002). Isubgol at 3% in MS medium has been used for the propagation of

chrysanthemum (Babbar and Jain, 1998).

Babbar et al., (2005) has reported guar gum as a cheaper alternative to agar.

Seed germination response of two species Linum usitatissimum and Brassica juncea

was found to be similar on both guar gum gelled medium and on agar gelled media.

2.3.2. CARBON SOURCE

It is well known that the carbon in the culture medium is an essential

component of the medium as a source of energy and for maintaining the osmoticum

(Sul and Korban, 1998; Cuenca and Vietiez, 2000). Sometimes sucrose has some

distinct morphogenetic effects also. Generally sucrose is used as a source of energy

for in vitro cultures because under tissue culture conditions, tissue has a very low rate

of photosynthesis or remains non-photosynthetic. The highest dry weight of cell

suspension culture of Acer pseudoplatanus was recorded when sucrose concentration

ranged from 4% to 6% in the media (Bonga and Aderkas, 1992). Similar results have

been reported in suspension culture of Pinus elliottii (Treat et al., 1989). Sucrose is

not always most effective carbon source for shoot induction. Sorbitol has been found

to be better than sucrose in Malus robustua (Pua and Chong, 1984), while dextrose

was satisfactory substitute of sucrose in tumor cell culture of Picea glauca (Risser and

White, 1964). There are a few reports whereby glucose and/or fructose have been

found to be better sources of carbon than sucrose for inducing adventitious shoots or

axillary buds (Hsia and Korban, 1996; Sul and Korban, 1998; Cuenca and Vietez,

2000). Sucrose was better than both glucose and fructose in inducing shoot

organogenesis in Pinus pinea (Sul and Korban, 2004).



2.4. A GENERAL OVERVIEW OF WORK DONE IN BAMBOOS Table-1: Micropropagation of bamboos through enhanced axillary branching using juvenile and mature explants Species Explant Medium + PGRs References Induction Rooting 54 Bamboo species Node MS + BAP MS + NAA Prutpongse and

Gavinlertvatna 1992

Bambusa balcooa, Node MS + BAP +NAA

MS + IBA/NAA

Rathore et al., 2009

B. bamboos NAA B. bamboos Node MS + BAP MS + NAA Arya and Sharma

1998 Embryonic

axis of caryopsis

MS + BAP MS + BAP +GA3 + NAA

Kapoor and Rao 2006

B. nutans Node MS + BAP MS + IBA Yashoda et al., 1997 B. oldhamii Shoot apices MS + TDZ Lin et al., 2007 Node MS + BAP +

AdS MS + IBA +NAA

Thiruvengadam et al., 2011

B. tulda Shoot apices MS + BAP +Kn

½ MS + IAA +Cou

Saxena 1990

B. ventricosa Node MS + BAP +NAA + AC

MS + BAP +NAA + AC

Dekkers and Rao 1989

Shoot apices MS + BAP MS + BAP +NAA

Huang and Huang 1995

Dendrocalamus asper

Seed MS + BAP MS + BAP +NAA

Arya and Arya 1997; Arya et al.1999.

D. brandisii Seed MS + BAP +CW

MS + IBA Nadgauda et al.,1990

D. giganteus Node MS + BAP + Kn

½ MS + IBA + Cou

Ramanayake and Yakandawala 1997

Node MS + BAP + NAA

MS + IBA Agnihotri et al., 2009

Node MS + BAP MS + IBA/ IAA/ NAA + Cou

Sood et al. 2002

D. membranaceus Node MS + BAP MS + IBA Yashoda et al., 1997 D. strictus Shoot apices MS + BAP +

CW MS + IBA Nadgir et al.,1984

Node ,Coleoptile

½ MS + BAP MS Shirgurkar et al., 1996

Node MS + GA3 + Kn

MS + GA3 + Kn

Maity and Ghosh 1997

Shoot apices MS + BAP +triacontanol

MS + NAA + rice bran extract

Mishra et al., 2001



Shoot apices, node

½ MS + TDZ ½ MS + IBA Singh et al., 2001

D. strictus, D. giganteus

Node MS + BAP +AdS

½ MS + IBA Das and Rout 1991

Phyllostachys meyeri

Node ½ MS ½ MS Ogita et al., 2008

Thamnocalamus spathiflorus

Zygotic MS + BAP + IBA

MS + IBA Bag et al., 2000

Mature

explants

Bambusa balcooa Node MS + BAP MS + BAP + NAA

Mudoi and Borthakur 2009

Node MS + BAP + Kn

½ MS + IBA Das and Pal 2005

B. balcooa,B. nutans, B. salarkhanii,B. vulgaris

Node MS + BAP ½ MS + NAA + IBA

Nurul Islam and Rahman 2005

B. bambos Node MS + BAP MS + NAA Arya and Sharma 1998

B. edulis Inflorescence MS + NAA + IBA + 2,4- D

Lin et al. 2005

B. glaucescens Node MS + BA + AC

MS + BA + NAA + AC

Banik and Alam 1987

B. oldhamii Node MS + BAP MS + IBA + NAA

Thiruvengadam et al., 2011

B. tulda Node MS + Glut + IAA + BAP

MS + Cou Mishra et al., 2008

B. vulgaris, B. arundinacea

Node MS + BAP + Kn + CW

½ MS + IBA Nadgir et al., 1984

B. vulgaris Node MS + BAP + AdS

MS + BAP + IBA

Das and Rout 1994

Node MS + BAP MS + IBA Ramanayake et al., 2006

B. wamin Node MS + BAP + Kn

½ MS + IBA Arshad et al., 2005

Dendrocalamus asper

Node MS + BAP MS + IBA + NAA

Arya et al. 1999

Node MS + BAP MS + IBA Banerjee et al., 2011 Node MS + BAP +

AdS MS + IBA + NAA

Singh et al., 2011

D. giganteus Node MS + BAP + Kn + CW

½ MS + IBA + Cou

Ramanayake and Yakandawala 1997

Node MS + BAP Ramanayake et al., 2006

Node MS + BAP MS + IBA + NAA

Arya et al., 2006

D. hamiltonii Node MS + BAP +2,4-D

½ MS + IBA +NAA

Sood et al., 1994

Node MS + BAP MS + IBA Agnihotri and Nandi



+NAA 2009 Node MS + BAP

+NAA MS + IBA Agnihotri et al.,2009

Node MS + TDZ + AA

MS + IBA + CC

Singh et al., 2012a

D. longispathus Node MS + BAP+ Kn

½ MS + IBA +Cou

Saxena and Bhojwani 1993

D. strictus Node MS + IAA +AdS

MS + IBA + NAA+ Phloroglucinol

Chaturvedi et al., 1993

Node MS + BAP + Kn

Ravikumar et al., 1998

D. strictus Node MS + BAP + Kn + CW

½ MS + IBA Nadgir et al., 1984

Guadua angustifolia

Node MS + BAP MS + BAP Jimenez et al., 2006

Pleioblastus pygmaeus

Node MS + BAP MS Watanable et al., 2000

Pseudoxytenanthera stocksii

Node MS + BAP + NAA + AA +Cyst + Glut

½ MS + BAP +IBA +AA Cyst + Glut

Sanjaya et al., 2005

Thamnocalamus spathiflorus

Node MS + BAP + IBA

MS + IBA Bag et al., 2000

AA = Ascorbic acid; AC = Activated charcoal; AdS = Adenine sulphate; BAP = 6-benzylaminopurine; CC = Choline chloride; CW = Coconut water (milk); Cou = Coumarin; Cyst = Cystein; 2, 4-D = 2, 4 Dichlorophenoxy acetic acid; GA3 = Gibbrelic acid; Glu = Glutamin; IAA = Indole-3-acetic acid; IBA = Indole-3-butyric acid; Kn = Kinetin; NAA = α-Napthalene acetic acid; PGR = Plant growth regulator; PVP = Polyvinylpyrolidone; TDZ = Thidiazuron

chapter - 2 review of literature -...

Documents