EFFECT OF NAPHTHALENE ACETIC ACID, INDOLE ACETIC ACID, GIBBERElLIC

ACID, BENZYLAMINOPURINE AND CUTTING TYPE ON ROOT AND SHOOT

FORMATION IN STEM CUTTING AND GRAFT SUCCESS IN SELECTED CULTI-
VARS OF Ferocactus pottsii & Ferocactus horridus

A Research Proposal
presented to
The Faculty and Staff of
MSU-IIT Integrated Developmental School

In Partial Fulfillment of the Requirement of

Research in Daily Life

Aianna Isobel Zoe C. Abragan

Jacinth Junell S. Arjona
John Benny L. Saguin
Researchers

April 2019
 CHAPTER 1
INTRODUCTION

Background of the Study

The first things to realise is that all cacti are succulent but not all succulents are cacti. The

development of succulence has forced the plants to solve, firstly, geometric and, secondly, chem-

ical problems. The plants’ first need was to adopt shapes offering the least surface to the sun for a

given area with the maximum ability to catch, absorb and retain any moisture available in the

form of dew and condensation when no rain was available.

It will be found that the majority of succulent plants are either spherical (globe shaped),

rhomboid (barrel shaped), columnar or rosette shaped. There are three families of succulent

plants which beginners find particularly difficult to separate, however, only the cactus is a peren-

nial plant having areoles which are not found on any other plant. The fruit are berries with no

divisions or partitions between the seeds and the ovary is always single celled. The hallmark of

all cacti is therefore the existence of the aeroles which look like miniature pin cushions. (Roan,

H.M., 1946)

Cacti are often used as ornamental plants, but many are also cultivated as crop plants.

They are almost exclusively ‘new world’ plants. This means that they are native only to North

America, South America, and the West Indies.

The high water use efficiency of cactus is attributed to Crassulacean Acid Metabolism

(CAM) which is present in rapidly growing cactus species such as Opuntia ficus-indica, O.
megacantha, and O. amychlea (Cactaceae) that produce forage for animals, vegetables, and fruits

with 14% glucose (Inglese, et al., 1996).

In India, an important part of the population is settled in rainfed dry areas which need

perennial vegetation to protect them from erosion using drought hardy and economically viable

plants. Cacti seem to be an option to sustain livelihood, reduce poverty and generate employment

opportunities. Cactus is drought tolerant due to its CO2 fixation capacity (CAM), it is well suited

to dry areas, where it can be used as an alternative food and fodder, as well as live fence to pro-

tect agricultural fields. In the seventh century the British introduced the cactus to India for

cochineal dye production but these plantations gradually disappeared due to pests and flooding

of the areas. Recent attempts to introduce cultivated cactus pear started late in the 1980’s (Singh

G., 2006).

Ferocactus horridus belong to the Cactaceae family. It is autochthonic to Mexico (South-

ern Baja California), but short spined forms, along with other variations, are presumably of gar-

den origin (Nursery selected form). F. horridus is a solitary fast growing little barrel cactus with

quite short and stocky central spine one, not enough to call it curved.

By contrast, Ferocactus pottsii has comparatively few spines which are of modest length,

so that the chubby plant body is in full view. It is a summer bloomer, usually coming into flower

in July and August. F. pottsii is native to the mountainous region along the border between Sono-

ra and Chihuahua states in northwestern Mexico, and southward into Sinaloa state.

Plant propagation is the process of creating new plants and asexual propagation is the

best way to maintain some species, particularly an individual that best represents that species. It
involves taking a part of one parent plant, specifically the vegetative parts of a plant: stems,

roots, or leaves, and causing it to regenerate itself into a new plant.

The resulting new plant is genetically identical its parent. The major methods of asexual

propagation are cuttings, layering, division, budding and grafting. Cuttings involve rooting a

severed piece of the parent plant; layering involves rooting a part of the parent and then severing

it; and budding and grafting is joining two plant parts from different varieties. (David C.

Sorensen)

Statement of the Problem

Progress towards understanding the evolutionary physiology of succulence has been

made in recent years. This study aims to provide a comprehensive portrait of the integrative bi-

ology of this large and diverse functional group as it is a long-term goal that will improve our

understanding of plant evolution and support successful exploitation of succulence in applied

contexts. The natural diversity of succulents may be utilized through plant progation techniques,

such as cutting and grafting, and the use of plant growth hormones to identify key regulatory

factors involved in the induction and development of succulence as a means to facilitate efficient

engineering in selected cultivars of Ferocactus pottsii and Ferocactus horridus.

Objectives of the Study

The main objective of the study is to assess the effects of Naphthalene Acetic Acid, In-

dole Acetic acid, Gibberellic Acid, Benzylaminopurine on root and shoot formation in stem cut-
tings and graft success in Ferocactus Pottssi and Ferocactus Horridus. This study specifically

aims to:

1. determine the number of roots, shoots, leaves and stems with shoots of the stem cuttings;

2. measure the root and shoot growth of stem cuttings in terms of: length of roots, length of

shoots, diameter of shoot,, length of leaves, and width of leaf blade;

3. compare the effects of the cuttings with treatment (with and without the shoot tip) to the cut-

tings withheld of treatment

4. determine which plant growth regulator and stem cut type stimulates profound increase in

growth and development parameters.

5. determine and quantify the effects of different concentrations of plant growth hormones and

soaking durations to the cuttings.

Significance of the Study

This study will delve into the world of horticulture to equip us with a higher knowledge

and understanding on the processes behind the propagation of the selected Ferocactus cultivars.

In the past, members of the Cactaceae family were mostly propagated by seeds. However, seeds

do not guarantee genetic stability and in some cases, seeds are often difficult to obtain and so, in

this study, the propagation techniques to be applied are to be tested out in the hopes of producing

large numbers of new, high-quality plants in a relatively short time and space. This is especially

important for endangered and desirable species such as cacti.

Scope and Delimitation

This study is limited to the evaluation of the effects of Naphthalene Acetic Acid, Indole

Acetic acid, Gibberellic Acid, Benzylaminopurine and cutting types on root and shoot formation

in stem cuttings and graft success in Ferocactus Pottssi and Ferocactus Horridus. The aforemen-

tioned cultivars will be limited to those in ACE and PP Backyard Succulent Nursery, Pala-o Ili-

gan City. For Graft success, only one type of auxin will be used and either Ferocactus Pottssi or

Ferocactus Horridus will be used as root stocks.

The environmental conditions such as temperature, amount of sunlight, and soil type will be uni-

form for all samples, and only the cuttings without treatment will be controlled. The results will

only be applicable to the selected cacti species because they may differ depending on the species

experimented on.

Definition of Terms

Grafting – the cutting and joining together of plants of different varieties or species so they

would grow as a single plant

Cutting – refers to a process used during vegetative propagation whereby a piece of the plant’s

stem is rooted into a growth medium such as moist soil.

Shoot – A young branch springing from the main stock of a tree or other plant.

Stock - a root system of a plant, often with a portion of the stem, to which a shoot or bud is

grafted.
Cultivar — a plant variety that has been produced in cultivation by selective breeding.

Rootstock — An underground part of the plant or a rhizome where new plant growth is possible.

It is often used to facilitate plant cuttings, grafting, and budding.

Scion — The top part of a grafted plant that will produce the plant’s shoots.

Plant growth hormones – They promote cell division, cell enlargement, flowering, fruiting and

seed formation.

Napthalene acetic acid - a synthetic plant hormone in the auxin family and a rooting agent and

used for the vegetative propagation of plants from stem and leaf cuttings.

Indole acetic acid - a plant growth regulator known to be an inducer of cell divisions and elon-

gation.

Gibberellic acid - a very potent growth hormone which promotes cell growth and development.

 CHAPTER 2

REVIEW OF RELATED LITERATURE

Plants in the genus Ferocactus are also well-known for their stout, strong spines, and on

some species, these are so dense that it is difficult to even see the plant within. By contrast, Fe-

rocactus pottsii has comparatively few spines which are of modest length, so that the plant body

is in full view. The ribs, the spine-bearing ridges which extend down the sides of the plant), are

narrow at the top, with deep furrows between them, but they bulge with age to become more

wide and rounded. Plants are usually globular, though old specimens may become short-cylindric

and up to 3 feet or so in height (1 m). F. pottsii is a summer bloomer, usually coming into flower

at the Ruth Bancroft Garden in July and August. As with other plants in the genus, the flowers

are produced in a ring around the center. They do not open all at once, but instead come a few at

a time over a period of weeks. The flowers are nearly an inch and a half across (35 mm), with

yellow petals and a thick bundle of red filaments at the middle, each topped by a yellow pollen-

bearing anther. The long-lasting fruits of F. pottsii are light yellow and round, with the dried re-

mains of the flower perched on top as a brown tuft. They add a decorative touch to the plant for

months after the flowering has finished. F. pottsii is native to the mountainous region along the

border between Sonora and Chihuahua states in northwestern Mexico, and southward into

Sinaloa state. (Ruth Bankroft, 2015)

Ferocactus horridus is a solitary fast growing barrel ribbed cactus that differs from the

type species in having a much shorter but stocky hooked central spine. It is suited for any rich,
well drained soil such us clay, pumice, lava grit, and only a little peat or leaf-mould. If potted,

repot preferably in the spring, if their roots become cramped. Generally, they should be repotted

every other year in order to provide fresh soil. However, this doesn't necessarily mean they'll

need larger containers. Fill about a quarter of the pot with broken crocks, gravel, etc. to promote

good drainage. After repotting, do not water for a week or more. Water regularly during the aes-

tival growth cycle and also needs to be avoided wetting the body of this plant while it is in sun-

light. A wet cactus in the sun light can cause sun burning which can lead to scares or even fungal

infections and death, keep rather dry in winter. No water should ever be allowed to stand around

the roots.

Ferocactus generally get to be very large plants, particularly when grown in the ground;

fortunately, they are fairly slow growers. When grown in pots, they will stay at a comfortable

size for many years. Although there are only about 40 species, many of them are fairly variable,

with widely varying spine lengths, shapes, textures and colors, depending on the specific locality.

Ferocactus are easily raised from seed. Planting is most successful when done in April or

May. The seedlings grow slowly at first, but soon take off. There are several advantages to grow-

ing Ferocactus from seed. The first is that the selection of type localities and spine variations is

far greater than you will ever see at a plant show or sale. The second and most important is that

you get to grow the plant through its awkward juvenile growth. During the second year Ferocac-

tus spines grow completely out of proportion to the Ferocactus body. A Ferocactus that will even-
tually be a foot in diameter, and 4 feet tall with 3 or four inch spines, will have as a seedling a

body less than an inch in diameter, with spines an inch or more long.

In a research conducted by Yan et al. (2014) on effect of naphthalene acetic acid (NAA)

on adventitious root development and associated physiological changes in stem cutting of

Hemarthria compressa, the researchers examined the effects of NAA concentrations and soaking

durations on IAA-oxidase (IAAO) activity, peroxidase (POD) activity, and polyphenol oxidase

(PPO) activity in the rooting zone during the induction phase of adventitious root formation and

rooting response in whip grass stem cuttings. The increased in POD and PPO activity and de-

creased in IAAO activity were caused by the lowering of NAA concentrations. The researchers

indicated that one of the critical factors to improve rooting is the increase of POD and PPO activ-

ity and decrease of IAAO activity.

Saifuddin et al. (2009) investigated the effects of naphthalene acetic acid and gibberellic

acid (GA3) on bract longevity under exposed sunlight conditions and six months of observation

of Bougainvillea spectabilis. The results show that bract longevity was prolonged after applying

NAA (50, 100, 150 ppm) at all stages of flower development. Application of 100 ppm GA3

showed the shortest duration of (20 days) flower life compared to the other treatments. However,

bract longevity in all the stages was prolonged by applying NAA (50, 100, 150 ppm) and GA3

compared to the control. When single treatments of NAA or GA3 were compared with combined

treatments (NAA and GA3), the prolonging effect of NAA and GA3 was significantly better.

Among the different concentration of combined treatments of NAA plus GA3, the best result was
exhibited by GA3 (100 ppm) plus NAA (100 ppm). It was concluded that the concentration (100

ppm) of GA3 plus NAA (100 ppm) could to protected unwanted early senescence and discol-

oration. It was also concluded that NAA plus GA3 were more effective than NAA and recom-

mended for exposed sunlight condition and for using on potted Bougainvilleas being transported.

In another research by De Klerk et al. (1997) on effectiveness of indoleacetic acid, in-

dolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus

‘Jork 9’, the researchers examined in vitro rooting of apple ‘Jork 9‘ shoots exposed for three

weeks to each of the three auxins commonly used for ex vitro rooting: indole-3-acetic acid

(IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). During the initial five

days of the rooting treatment, the cultures were incubated in darkness. In this period, the root ini-

tials are formed. Then, the cultures were moved to the light. NAA resulted in a low (ca. 8 roots),

and IAA or IBA in a high (ca. 15 roots) maximal root number. The maximal root number was

reached at a wide range of IAA concentrations (10-100 µM) but at only one concentration of IBA

(10 µM) or NAA (3 µM). With NAA and IBA, growth of roots and shoots was much more inhib-

ited than with IAA. For these reasons, IAA is the preferable auxin for in vitro rooting of apple

‘Jork 9’ shoots.

Ortolá et al. (1991) studied the use of naphthalene acetic acid as a fruit growth enhancer

in Satsuma mandarin: a comparison with the fruit thinning effect. In this research, naphthalene

acetic acid (NAA) increases the growth rate of the fruit of Satsuma mandarin (Citrus unshiu

Marc.), which results in a bigger fruit size at harvest without any undesirable reduction in yield.
In a high-yield regular bearer orchard, the effect of this treatment on the sizing of the fruit is sim-

ilar to that obtained when NAA is used as a thinning agent at an earlier stage of development of

the fruit. The apparently higher response obtained through thinning as determined by the mean

weight of the fruit at harvest, is mainly owing to the selective removal of the smallest fruit rather

than to a higher stimulation of the growth rate of the remaining fruits.

Plant grafting is an ancient and a widely used technique (Hartmann et al., 1997) that po-

tentially can combine the advantages of rapid in vitro multiplication with the increased produc-

tivity (Gebhardt and Goldbach, 1988). It is commonly used to propagate rare ornamental species

like cactus species (Estrada-Luna et al., 2002). Elimination of viruses, rejuvenation of mature

tissues, year round plant production, make specific genotypic combinations to increase plant

productivity and extend ecological limits of a particular plant species to tolerate edaphic condi-

tions are several mentioned advantages of micrografting (Richardson, 1996; Hartmann et al.,

1997; Estrada-Luna et al., 2002). Several authors have defined the sequence of events during a

compatible graft union formation: Formation of the union, development of a necrotic layer and

proliferation of callus bridge at the graft interface prior to the binding of vascular cambium

across the callus bridge, differentiation of new vascular cambium, forming a continuous cambial

connection between rootstock and scion (Moore, 1984; Hartmann et al., 1997; Estrada-Luna et

al., 2002, Hartmann, 2002). Production of new xylem and phloem thus permits the vascular con-

nection between the scion and rootstock (Aloni et al., 2010). Grafting is a common practice to

propagate cacti species. Grafted cacti are now regarded as one of the most admired ornamental

indoor plants worldwide.

 Based on the fact that hormones are involved in rootstock–scion interactions, in many

studies various plant growth regulators have been used for grafting improvement. Auxin is one of

the most effective hormones on growth, differentiation and development.

CHAPTER III

RESEARCH METHODOLOGY

A. Plant Materials and Surface Sterilization

The cactus species Ferocactus pottsii and Ferocactus horridus will be collected from

ACE and PP Backyard Succulent Nursery, Pala-o, Iligan City. After washing the plants with tap

water for 30 sec, it will be immersed in a solution of 30% insecticide for 5 min, distiller water for

one minute, and in 50% fungicide for secs. It will then be thoroughly rinsed with sterile distilled

water. The plant will be cut into desired lengths.

B. Hormone Application

Stem Cuttings

The cuttings (with shoot tip and without shoot tip) will be treated with four different

hormones (IAA, NAA, GA3, BAP) in five different concentrations (0 ,100, 200, 300, 400 mg/L)

at three soaking durations (10, 20, 30 minutes) respectively, and cuttings without treatment will
be considered as control. The treatments are subject to change based on the results from prelimi-

nary trials in the laboratory except for Ferocactus pottsii, treated cuttings will be air dried for at

least 5 days before planning in pots filled with a sterilized mixture of sand and soil-less medium

(1:2 v:v). The pots will then be incubated in a controlled growth chamber for 30 days (25 C, 16 h

photoperiod with cool, white fluorescent lamps and 65% relative humidity).

To quantify the effect of root growth promoters available in local stores, cuttings (with

shoot tip and without shoot tip) will be soaked into undiluted ANAA at three soaking durations

(10, 20, 30 minutes). ANAA contains synthetic growth hormones that imitate auxins such as in-

dole butyric acid. Indole acetic acid or naphthalene acetic acid.

C. Grafting Implementation:

Basic Steps

First, Prepare the needed materials (rootstock/stock, scion, knife/razor blade/ scalpel

blade, tape/rubber bands/ used stockings, clear plastics, alcohol, chopping board, tissue paper,

etc. Disinfect a sharp knife (to reduce risk of infection). Cut off the top of the rootstock. Trim the

edges of the rootstock so that its edges slope downward. Cut off a thin layer of the bottom of the

scion. Place the scion on top of the stock. Make sure at least some of the vascular cambium

(“vascular ring”) of both stock and scion is in contact. Let the scion sit on the top of the stock a

bit off- center. Lastly, hold the two cacti together with rubber bands/ tape (don’t make the rubber

bands too tight or too loose).

\Graft Success
 In this study, only one type of auxin will be used and Ferocactus horridus will be used as

rootstocks. Rootstocks with 9 and 2.5 cm height and diameter, respectively will be used in con-

trolled conditions. IAA will be used as an auxin. Grafted plants will be treated with four different

concentrations including 0, 100, 200, 300, 400 mg/L at grafting time. The treatments are subject

to change based on the results from preliminary trials in the laboratory. In addition to auxin

treatment at grafting time, IAA treatment will be repeated three different times at 3, 9, and 15

days after grafting. In each auxin concentration, it will be repeated once (3 days after grafting),

twice (3 and 9 days after grafting), and three times (3, 9 and 15 days after grafting). For example,

in 100-1, 100-2 and 100-3 treatment groups, this auxin treatment will be repeated once, twice

and three times, respectively at 3, 9, and 15 days after grafting. The same procedure will be done

for 200, 300 and 400 mg/L. Therefore, plants will be treated in 15 different treatment groups in-

cluding control ( C ). 100-1, 100-2, 100-3, 200-1, 200-2, 200-3, 300-1, 300-2, 300-3, 400-1, 400-

2, 400-3). The grafted plants will be maintained in controlled conditions (humidity 45% and 20

C).

D. Data Collection

Stem Cuttings

Randomized Block Design (RBD) will be followed in the experiment in an arrangement

with three replications having 10 cuttings in each replication under every treatment. 30 days after

soaking, various growth parameters will be evaluated against the treatments. For that purpose,

cuttings will be carefully uprooted and medium particles adhered to roots will be removed care-

fully by hand. Measurements will be done with 0.01 mm precision. The length of roots will be
measured using ImageTool Software while others will be measured with the use of Vernier

caliper in for each treatment and the mean will be calculated. Data will be collected on growth

parameters I.e. rooting percentage (RP), number of roots (RN), length of root (RL), number of

shoots (SN), and length of shoots (SL), diameter of shoots (SD), and number of stems with

shoots (SSN). For FEROCACTUS POTTSII, additional parameters such as the number of leaves

(NL), length of leaves (LL), and width of leaf blade (WL) will be considered.

Graft Success

Thirty days after the last treatment, the scion height and diameter, cambial layer diameter,

areole numbers, activated areole numbers and graft take percentage will be measured.

Cross- Section

Histological studies, such as cambial layer diameter, will be performed with cross-sec-

tion. Forty-five days after the last auxin treatments, plants will be harvested for cross section.

Ethanol 80% was used as a fixator. Handy cross sections will be done in about 2.5 mm around

graft zone.

E. Statistical Analysis

A one-way analysis of variance (ANOVA) will be used to identify if there is a significant

difference between the cutting types (with and without shoot tip), the four hormone types (NAA,

IAA, GA3, BAP), the five hormone concentrations (0, 100, 200, 300 and 400 mg/L), and three

soaking durations (10, 20, 30 minutes) in terms of growth parameters, followed by Tukey’s HSD

tests to determine the significant differences among treatments.

 Time Table

Dates

Feb Mar Apr May June July

Activities
20 27 6 25 13 25 30 17 Whole Whole
month month

Topic Approved ✔

Submission of Introduction ✔
and Related Articles

Submission of Review of ✔
Related Articles

Submission of First Graft #1 ✔

Submission of Revised ✔
Graft #1

Submission of Revised ✔
Graft #2

Submission of Revised ✔
Research Proposal

Final Research Proposal ✔

Defense

Preparation of Materials ✔

Experiment ✔
APPENDIX
Line-Item Budget

Supplies & Materials Estimated Prices Subtotal

Naphthalene Acetic Acid Php 600.00 Php 600.00
(25g)
Indole Acetic Acid (25g) Php 1,200.00 Php 1,200.00
Gibberellic Acid (25g) Php 900.00 Php 900.00
Benzylaminopurine (25g) Php 800.00 Php 800.00
Soil – less medium (5kg) Php 500.00 Php 500.00
Insecticide (25mL) Php 300.00 Php 300.00
Fungicide (40g) Php 200.00 Php 200.00
Ferocactus pottsii Php 200.00/pc x 30 Php 6,000.00
Ferocactus horridus Php 250.00/pc x 30 Php 7,500.00
Ethanol (250mL) Php 400.00 Php 400.00
Honey Php 350.00 Php 350.00
Cinnamon powder Php 90.00 Php 90.00
Plastic basin Php 130.00/pc Php 260.00
ANAA (5g) Php 400.00 Php 400.00
Knife Php 50.00 Php 50.00
Rubber bands Php 20.00 Php 20.00
Clear plastics Php 80.00 Php 80.00
Alcohol (250mL) Php 300.00 Php 300.00
Slicing board Php 150.00 Php 150.00
Tissue paper Php 100.00 Php 100.00
Printing Php 50.00 Php 50.00
Binding Php 300.00 Php 300.00
Total Estimated Balance Php Php 20,550