Micropropagation of Bamboo

William Weldon
UFID# 1736-6507
Introduction

Bamboo is found naturally in Eastern Asia and has many economic uses. It is used as a

building material, an ornamental plant, and for food. While bamboo is an extremely versatile

crop, it is also very difficult to propagate. The plants themselves grow very quickly, but the seeds

are difficult to germinate for several reasons. The seeds are often sterile, non-viable, and

underproduced (Singh and Dahwan, 2013). Due to these complications, micropropagation the

most practical solution for the breeding of bamboo plants. The micropropagation methods vary

throughout the papers. There are even successful micropropagation techniques in use.

The main reason micropropagation is used for bamboo is the increased ability to create

different varieties, or genetic variability. In nature, it is very time consuming to obtain different

varieties of bamboo due to the extended period of time between flowering which makes it nearly

impossible to improve genetically (John and Nadagauda, 1999). When the plants are propagated

at a faster rate, they reproduce more quickly, allow for more genetic variation and make heartier

plants. This also allows more plants to be transferred to soil in order to increase the number of

viable subjects to study when they inevitably do bloom (Saxena and Bhojwani, 1993)

Stage 0

Much of what determines the success of micropropagation is tied to the health of the

initial donor plant. Donor plants that are more susceptible to disease or infection may pass those

traits along to their explants. Furthermore, if a donor is already infected there is a high likelihood

that the explants will also be infected and the experiment will be doomed from the start. In the

majority of the papers reviewed, the location of the donor plant was listed but little else in

regards to the donor plant was discussed. The health and environment of the plants was not
given. Saxena and Bohjwani (1993) excised nodes from Gwal Pahari in Haryana State. Rathore

and Rai (2004) used “superior genotypes in the field” from Nallal, Karnataka State Forest

Department, Bangalore, India and were chosen based off of their culm girth, rooting potential,

and leaf blade size. Negi and Saxena (2011) used node segments from plants on the TERI

campus in Gwal Pahari, Haryana, India from March to December. This is the only paper that

states a time of year where the explants were collected. Gupta et al. (1991) simply showed a

chart with the locations and sources of their explants. They received their bamboo cultures from

seedlings in Pune, India. Ramanayake et al. (2001) used explants from a single mother clump

grown in the field. They were able to harvest 83 stumps and 58 culms. This was the only paper

that specifically stated the number of explants they received. Singh et al. (2013) used explants

grown vegetatively from the Centre for Plant Biotechnology in Hisar, India. Qiao et al. (2013)

chose explants from field grown plants in Zhangzhou, Fujian Province, China. Kaur et al. (2011)

use explants grown from the CSIR-Institute of Himalayan Bioresource Technology in Palampur.

The other authors did not state where they received their explants.

Stage 1 Establishment of Aseptic Cultures

The main objective of Stage 1 is to make sure that the explants have been properly

surface sterilized and are free of microbial contaminants when they are placed into their culture

medias. It is also important to show how the explant was surface sterilized to make sure that

none of the outside bacteria survive. If the plants are not correctly surface sterilized, there is a

risk of losing the plant to infection before it ever gets a chance to grow. Of the ten papers cited,

only five stated their surface sterilization methods. Saxena and Bhojwani (1993), and Negi and

Saxena (2004) both used the same surface sterilization method of rinsing in 70-95% ethanol,

washing in tap water for 20 minutes, washing in 0.1% Mercuric Chloride for 10 minutes, then
washing with sterile distilled water for 10 minutes. Rathore and Rai (2004) also used Mercuric

Chloride but they only used 0.075% for 4-5 minutes and then washed thoroughly 6-7 times with

sterile distilled water. Kaur et al. (2011) sterilized with a 30% Tween 20 solution for 15 minutes

followed by a rinse in 70% ethanol for 1 minute. After the ethanol, they rinsed in a 0.04%

Mercuric Chloride solution for 5 minutes, and ended with a thorough rinse with distilled water.

Qiao et al. (2013) differentiated themselves from the others with an initial use of 70% alcohol for

30 seconds, 2% sodium hypochloride for 15 minutes, and then 3-4 rinses with autoclave water.

There are many different kinds of media that can be chosen to help a plant survive in a

culture. Which media is chosen depends highly upon the needs of the explant and the state which

the explant is in. The media being used is dependent upon the goal of the research being done.

This seemed to be the main difference in this set of papers. Eight of the ten papers reviewed used

the base MS medium (Murashige and Skoog, 1962). This just proves to show how effective the

MS media truly is when it is accepted by such a large portion of the scientific community. John

and Nadgauda (1999), Saxena and Bhojwani (1993), Negi and Saxena (2011), and Ramanayake

et al., (2001) all supplemented their medium with sucrose and 6-benzylaminopurine. The sucrose

and cytokinins are being used as a form of “bud-break” to increase the rate at which the explants

will grow. Qiao et al. (2013) supplemented their MS media with BA (1.33, 2.21, or 3.11µM),

NAA (2.69, 5.37, or 8.06µM) and PAA (73.45, 110.17, or 146.89µM) while Singh et al. (2013)

used thidiazurone. Baldwin et al. (2009) also used thidiazurone, but at various concentrations

(0.001,0.01, 0.1, 1.0, or 10µM). Rathore and Rai (2004) used a combination of 6-

benzylaminopurine (BA: 0.0 – 11.09µM) and 1-naphthaleneacetic acid (NAA: 0.0 – 5.37µM).

These various media show the wide range of choices that are available and how versatile a media

can be when it comes to changing the outcome of the explant.

 The conditions the explants are kept in are also important to note. There are many

variations in the amount of light provided for the explants. Negi and Saxena (2011) and Singh et

al. (2013) both used 16:8 photoperiods under 47µmol m-2s-1 of light. They also maintained

similar temperatures at 25±2ºC and 26±2ºC respectively. Qiao et al. (2013) kept their explants at

25ºC and in the dark for 7-8 weeks. They were the only paper to keep their explants entirely in

the dark during Stage 1. On the other end of the spectrum, Baldwin et al. (2009) exposed their

explants to 200-400µmol m-2s-1. This is by far the most intense amount of light provided in the

papers sampled. Both Rathore and Rai (2005) and Saxena and Bhojwani (1993) opted for 12

hour photoperiods. Throughout all ten of the papers reviewed, none of them mentioned indexing

their explants in their methods.

Stage 2 Multiplication of the Propagule

Once the Stage 1 process is finished and the plants have had time to stabilize in their

cultures, it is time to move on to Stage 2. It is important to make sure that the plants have had

sufficient time to stabilize. The disruption this multiplication causes stress to the plant. While

half of the papers did not mention a time period in which the explant was allowed to acclimate,

there was some information from the other four papers. The acclimation period ranged from 2-3

weeks. After the stabilization period is over, it is time to begin Stage 2.

This is the stage where the propagule is multiplied, usually through the increase of

axillary shoots. The type of media can change between Stage 1 and Stage 2 as the purpose of the

explants also change. The explant has become stable and now must be multiplied. The media is

changed to represent this difference. Saxena and Bhojwani (1993) added more cytokinins

(kinetin and 2-ip) and auxins (IAA, IBA, and 1-naphthaleneacetic acid). Rathore and Rai (2004)

added in various acids including ascorbic and citric acid along with glutamine. Negi and Saxena
(2011) added BAP (2.2 to 11µM) or Kn (1.16 to 3.48µM). They also tested the optimal

concentration of BAP (6.6µM) with different concentrations of Kn (1.16 to 3.48µM).

Ramanayake et al. (2001) and Singh (2013) both replaced the 6-benzylaminopurine with

thidiazuron. Ramanayake et al. (2001) also went one step further by removing the coconut water

growth regulator from some of their samples and increasing the levels of sucrose in varying

doses from 2% to 4%. Qiao et al. (2013) changed the concentrations of everything but PAA

when moving to Stage 2. They increased BA (4.44, 8.89, or 13.32µM), added KT (2.32, 4.65, or

6.97µM), and lowered the NAA (0.54, 1.08, or 1.61µM).

Stage 3 Pre-Transplant or Rooting

Stage 3 highlights the importance of preparing the plant for transplant to ex vitro

conditions. Explants are subjected to much less stressful conditions in vitro than they will be

when they are transplanted. The conditions in vitro are almost all controlled. There is a set

amount of light that does not generate too much heat for the plant. The temperature itself is

regulated so the plant is not unnecessarily stressed. The explant has also grown in a media that is

filled with nutrients and housed in a small humid container that allows the explant to spend less

energy remaining hydrated. All of these factors are immensely important when considering the

steps to take before transplant. If any one of these variables is unaccounted for, it could cause a

failure of the transplant and a loss of the explant. This is why it is important to prepare the plant

by beginning to change some of these variables while still in vitro. Unfortunately, most of the

papers did not mention a stage III. Of the papers that did mention a stage III, only one of them

mentioned a time frame for their changes. Saxena and Bhojwani (1993) gave their explant a

pulse treatment with IAA or IBA alone. Rathore and Rai (2004) reduced their media to half

strength and added indole 3-butyric acid. Negi and Saxena (2011) supplemented their explant
with growth hormones in an attempt to increase rooting. They also increased the levels of

cytokinin in their media. Singh et al. (2013) added IBA to their solution while Qiao et al. (2013)

added in B5 vitamins, macronutrients, and iron salts to increase rooting. The concentrations of

the additions are not stated.

Stage 4 Transfer to the Natural Environment

Acclimatization to the ex vitro environment is the primary goal of Stage 4. Being able to

survive in their environment under much more harsh conditions is the most difficult part of the

process. Unfortunately, bamboo has a relatively low success rate in regards to transition unless

under very specific situations. Saxena and Bhojwani (1993) used a mixture of potting soil

containing soil, soilrite, and farmyard manure (vol/vol) in black polythene bags in equal

quantities. They kept the greenhouse at 26±2ºC and the plants experienced a 12 hour

photoperiod. The amount of light is not listed. Negi and Saxena (2011) kept their explants under

greenhouse conditions of 28±2ºC and a humidity of 80-85%. The explants were kept near

cooling pads and moved towards exhaust fans over the period of one week. The potting soil was

a mixture of equal parts agropeat (Varsha Enterprises, Bangalore, India) and soil (v/v). The

explants were also kept under shade that halved the amount of light received for 20 days. Qiao et

al. (2013) came up with similar results after removing plant growth regulators from the media.

They experienced a 90% success rate after two weeks of hardening.

Many of the researchers did not go all the way to stage four or simply did not include

their findings other than success rates. There were no studies done for extended periods of time

after the explants had been transplanted to see if they were still thriving. For one of the most

important steps of a micropropagation experiment, Stage 4 conditions were the least described.
Conclusion

After reviewing all ten papers I found only two of them to be possible to replicate. Many

of the papers simply did not progress beyond certain steps, whether that was because they were

merely interested in the flowering rates or the possible increase in genetic variations of bamboo.

The lack of exact information is unsettling when a scientific article can state all five stages in just

one paragraph. There was very little information on the size of the initial explants, how they

were prepared or the process of inoculation. While these details may seem tedious and

unnecessary, they can be vital for someone interested in replicating the experiment. After reading

the articles from the early 90’s and the early 2010’s the difference in knowledge of bamboo

micropropagation has increased drastically. The information being provided is also more specific

and encompassing. It just proves to show that as more scientists begin to take their results and

articles more seriously, the scientific community as a whole benefits.

Proposed Protocol

Stage 0

Bamboo can be acquired in many locations such as local hardware stores and flower

shops. The species of bamboo received will differ greatly but this can be mitigated by purchasing

specific species online. Online purchase will ensure that the plants are all coming from the same

place so long as the distributer acquires them from reliable sources. Once the bamboo is

acquired, it will need to be cut into individual node explants and surface sterilized. Bamboo is a

very difficult species of plant to surface sterilize and extreme measures must be taken to avoid

contamination. The individual pieces should be no longer than 2cm. This will ensure that each

node has enough plant matter on each side to survive the extensive sterilization process.
Stage I

Once the desired number of explants have been separated from the donor, they should be

washed in a small container covered with a cheesecloth. They should be allowed to sit under

running tap water for 15-20 minutes. After they have been rinsed, they should be placed into a

70% ethanol solution for 1 minute while shaking gently. After the ethanol has been decanted

from the container, the explants should be placed into a solution consisting of 25% sterilized

distilled water and 75% bleach and two drops of Tween 20. The solution containing the explants

should then be placed on a shaker for 15 minutes. Make sure to check the explants for signs of

bleaching at 5 minutes, 10 minutes, and 12 minutes to make sure the bleach is not destroying the

sample. Once the explants have been properly bleached, the solution should be decanted and the

explants should be rinsed 3-5 times with sterilized distilled water. The explants should have their

bleached tissue removed before inoculation. Once the bleached tissue is removed, the explants

should be inoculated using sterile tools under a hood to minimize the risk of contamination. The

explants should be inoculated onto a solid MS media. The media should contain 3% sucrose and

be supplemented by 6-benzylaminopurine (6.6µM) to maximize successful bud growth. The

explants will be safe to subculture after 30 days depending on success. The cultures should be

kept at 26±2ºC.

Stage II

Any subcultures taken from the original explants should be placed in the same media as

the originals.
Stage III

To induce root growth the media should be supplemented with increased amounts of BA

and NAA in various amounts.

Stage IV

Explants should be moved into potting medium and kept in greenhouse conditions for 4

months. For best results, freshly transplanted explants should be kept near a cooling pad to

increase relative humidity. Once they have had time to acclimate, they can be moved towards

exhaust fans. The relative humidity of the greenhouse should be around 80% and the temperature

should 28±2ºC. The photoperiod should be 16:8 with 47µmol m-2s-1 light provided.
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